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My. ., ;<-:;;':/.».-,zf,+.scér, f / , “" ,th ’3’ H MWJ’ZSJ ’3’"! "'3’ /.:‘ 9: . ..' f'J'I.‘.""’ IN: ' 3"}. u‘ ' H“ '3. ' - . n4/121"../. y,- '//('..';’:I. " #7 "a?” I HA :1749';' . . / I, 14.4:1'1‘.4;.::E: $2.5? ‘ ‘-fi{é§u{£& n1£% . ' v ’ . w.:§:¥/!/; m a $7 I 'I’Jf'lb‘lq u" A.” 3‘ 31.14 , 1.49,, I m'f':w‘ '.‘p... .44 ~ ' : LIBRARY Michigan State University This is to certify that the dissertatipn entitled . Molecular Cloning of ngnln Peroxxdase cDNAs and Genes from a White-rot Basidiomycete Fungus Phanerochaete chgysogpprium presented by Yi-Zheng Zhang has been accepted towards fulfillment of the requirements for Ph - D degree in MPH Date Nana“? [/1 /787 MSU i: an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ««-w- rm..." ,-‘.,‘3 9- us "|“t I / ’ " .‘r‘ I; 1‘ .I‘ T r b\vi : k. IL. II» F M ("‘T L ’ ‘1‘ \'4 Q" '- (NU! MOLECULAR CLONING OF LIGNIN PEROXIDASE cDNAs AND GENES FROM A WHITE-BOT BASIDIOHYCETE FUNGUS WW BY YI-ZHENG ZHANG A DISSERTATION Submitted to Hichigen State University in pertiel fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1987 ABSTRACT MOLECULAR CLONING OF LIGNIN PEROXIDASE cDNAs AND GENES FROM A WHITE-ROT BASIDIOHYCETE FUNGUS {HANEEQQEAEIE £HBX§Q§£QBIEN BY YI-ZHENG ZHANG Ihgggxgghggsg ghzzgggpgzigm, a white-rot basidiomycete fungus, produces extracellular, HZOZ-dependent, glycosylated heme proteins called lignin peroxidases. There has been much recent interest in lignin peroxidases because these enzymes not only play a central role in lignin degradation, but have also been implicated in the detoxification of recalcitrant xenobiotics. Lignin peroxidases are elaborated by the fun- gus under nitrogen-limited conditions only during secondary metabolism. To better understand the expression, regulation and organization of lignin peroxidase genes in this fungus, molecular cloning procedures have been used to isolate lignin peroxidase cDNAs and genes. Two dif- ferent types of lignin peroxidase cDNA clones (represented by pCL64 and pCLGS) were identified in a cDNA library of 1. ghgygggpggigm using the synthetic oligodeoxynucleotide probes whose sequences were deduced from one lignin peroxidase (H8). Northern hybridization analyses demonstrated that the poly(A) RNA homologous to the above cDNAs was present in 6-day- old (idiophasic) ligninolytic cultures. Immunoassay showed the presence of expressed product of the cDRA clone in 3. 9911. Sequence analyses showed that the cDNA inserts of pCLGk and pCLGS (designated 6L64 and CLGS) contain open reading frames encoding lignin peroxidase proteins (designated L66 and L65) containing 372 and 371 amino acid residues, respectively. Both mature lignin peroxidases L64 and L85 contain 344 amino acid residues with Mr of 36,540 and 36,607, respectively, and are preceded by typical leader sequences for secretion. Although CLG4 and CLGS did not show cross hybridization, they had relatively high nucleo- tide homology of 71.5% and amino acid sequence homology of 75%; Six lignin peroxidase genomic fragments (named GLGl to GLGG) have been isolated from the gene library of 1. ghxygggpggigg using 0L64 and CLGS as probes. Further characterization showed that GLGl and GLGZ correspond to 0L64 and CLGS, respectively, whereas the other four genomic fragments represent four separate lignin peroxidase genes. The location of the lignin peroxidase gene in each cloned fragment and the transcriptional orientation of each gene have been determined. Sequence analysis of the lignin peroxidase gene in GLGZ showed that this gene has a CAAT box and a typical TATA box sequence. The comparison of the GLGZ sequence with that of CLGS showed that this gene contained nine small introns whose sizes ranged from 50-62 base pairs. The consensus sequence GTRNGY--- YTGAY---YAG is present in all introns. To my parents, my wife and my son iv AKNONLEDGHENTS I would like to thank Dr. C. A. Raddy for his guidance and support over the past six years. I would like to thank the members of my graduate committee: Dr. R. L. Anderson, Dr. J. B. Dodgson, Dr. P. T. Hagee and Dr. H. Sadoff. I am thankful to Dr. S. B. Dass in Dr. Reddy's laboratory for purifying lignin peroxidases of {hangggghagtg enzygggpgxigm and making available to me the N-terminal sequences of two of the proteins. I am grateful to all my other colleagues in Dr. Reddy's laboratory for their cooperation and support throughout my stay at ‘ Michigan State University. page List of Tables ................................................... ix List of Figures .................................................. x Introduction ..................................................... 1 Literature Review ................................................ 4 I. General structure of lignin ................................ 4 II. Microbiology of lignin degradation ......................... 6 l. Fungi .................................................... 6 2. Bacteria ................................................. 7 III. Physiology of lignin biodegradation ....................... 7 1. Oxygen and agitation ..................................... 7 2. Nutrient concentration ................................... 9 3. Induction of ligninolytic system by lignin and veratryl alcohol .................................................. ll 4. Metal ions ............................................... 13 IV. Role of M202 in lignin degradation ......................... 16 V. Enzymes involved in lignin degradation ..................... l7 1. Lignin peroxidases ....................................... 17 A. Purification procedures ................................ l7 8. Properties ............................................. 19 0. Multiple forms of lignin peroxidase .................... 20 D. M chanism of reaction catalyzed by lignin peroxidase... 23 2. Mn -dependent peroxidase ................................ 26 3. H20 -producing enzymes ................................... 26 A. G osa oxidase ........................................ 26 8. Mn -dependent peroxidase .............................. 28 c. Fatty acyl GoA oxidase ................................. 28 D. Glyoxal oxidase ........................................ 28 4. Other enzymes ............................................ 28 VI. Genetics and molecular biology of lignin biodegradation... 29 1. Genetic studies .......................................... 3O 2. Molecular biology ........................................ 33 A. Development of cloning system .......................... 34 3. Molecular cloning of lignin peroxidase cDMAs and genes ........................................ 35 REFERENCES .................................................... 38 vi CHAPTER 1 CHARACTERIZATION OF LIGNIN PEROXIDASE cDNA CLONES OP {EANERQQflAEIE Qflfixgggggglgy ............................. 48 Abstract .................................................... 49 Introduction ................................................ 50 Materials and Methods ....................................... 51 Results ..................................................... 54 Discussion .................................................. 69 Acknowledgments ............................................. 72 References .................................................. 73 CHAPTER 2 SEQUENCE ANALYSIS OF TWO cDNAs FOR LIGNIN PEROXIDASE OF {haggggghgggg ghgygggpggigg ............................. 76 Abstract .................................................... 77 Introduction ................................................ 78 Materials and Methods ....................................... 80 Results ..................................................... 82 Discussion .................................................. 95 Acknowledgments ............................................. 97 References ................................................ ,. 98 CHAPTER 3 MOLECULAR CLONING OF A FAMILY OF LIGNIN PEROXIDASE GENES FROM W W AND SEQUENCE ANALYSIS OF A GENE ENCODING THE MAJOR LIGNIN PEROXIDASE ......... 101 Abstract ................................................... 102 Introduction ............................................... 103 Materials and Methods ...................................... 105 Results .................................................... 109 Discussion ................................................. 117 Acknowledgments ............................................ 126 References ................................................. 127 APPENDIX A. IDENTIFICATION OF cDNA CLONES FOR LIGNINASE PROM PHANEBQQHAEIE QHRXSQ§£QRIEM ........................... 131 Materials and Methods ...................................... 132 Results and discussion ..................................... 133 Acknowledgments ............................................ 137 References ................................................. 137 APPENDIX B. USE OF SYNTHETIC OLIGONUCLEOTIDE PROBES FOR IDENTIFYING LIGNINASE cDNA CLONES ................................. 139 Principle .................................................. 139 Isolation of poly(A) RNA ........ q ........................... 140 Construction of cDNA library ............................... 141 vii Differential hybridization ................................. 142 Identification of ligninase cDNA clones .................... 146 Acknowledgments ............................................ 149 References ................................................. 150 viii W TABLE Page LITERATURE REVIEW 1 Effects of medium additives on production of lignin peroxidases ............................................... 15 2 Characteristics of extracellular enzymes purified from ligninolytic cultures of [hanggggngggg ghgysgspgxigg ...... 18 CHAPTER 2 1 Codon usage in lignin peroxidase cDNAs CLG4 and CLGS ....... 90 2 Comparison of amino acid composition in L64 and LGS ..... ... 92 CHAPTER 3 1 Comparison of codon usage in 2. ghngggpnggn (P), Agpgggillng (A), flgnggsngza (N) and yeast (Y).. .......... 122 2 Comparison of the positions, phases and the lengths of nine introns of lignin peroxidase gene in GLG2 ................... 124 I OF G FIGURE Page LITERATURE REVIEW 1 Nomenclature of carbons in lignin monomers and three precursors of lignin, p-coumaryl, coniferyl and sinapyl alcohols ....... 5 2 Structure of beech lignin ................................... 5 3 HPLC profile of extracellular fluid from a 5-day ligninolytic culture of 2. ghxyggspgxign ................... 22 4 Catabolic cycle of lignin peroxidase of I. ghzygggpggigm... 24 5 Scheme of 60-6 oxidative cleavage of diarylpropane catalyzed by Iggnin peroxidase of 2. enzygggpgzigm ......... 25 CHAPTER 1 1 Hybridization of lignin peroxidase cDNA clones with three synthetic oligonucleotide probes.. ......................... 56 2 Cross-hybridization between two groups of cDNA clones ...... 57 3 Restriction maps of the cDNA inserts in pCLC4 and pCLGS.... 59 4 Northern hybridization analysis ............................ 61 5 Immunoblotting assay for detecting lignin peroxidase production by different cDNA clones ........................ 64 6 Southern hybridization analysis of strain REM-F 1767 ....... 67 7 Southern hybridization analysis of strain ME 446 ........... 68 CHAPTER 2 1 Strategy for determining the sequences of lignin peroxidase cDNAs CLG4 and CLGS ........................................ 81 2 The complete nucleotide sequences of the lignin peroxidase cDNAs CLG4 (A) and CLGS (B) ................................ 83 Comparison of the hydropathy plots of two lignin peroxidases LG4 (A) and LGS (B) ........................................ 88 Comparison of amino acid sequences of LG4 and LGS .......... 93 Comparison of nucleotide sequences of CLG4 and CLGS ........ 94 CHAPTER 3 Sequencing strategy of lignin peroxidase gene in GLG2 ...... 108 Identification of lignin peroxidase genomic clones using cDNAs CLG4 and CLG5 as probes .............................. 110 Restriction maps of six lignin peroxidase genomic clones... 112 Nucleotide sequence of lignin peroxidase gene in GLGZ and the predicted amino acid sequence .......................... 115 Conservation of intron/exon junction and internal sequences in lignin peroxidase gene of 2. ghxygggpggjnl .............. 125 APPENDIX A Differential hybridization and identification of cDNA clone for ligninase .............................................. 134 Hybridization of plasmid from ligninase cDNA clones with the synthetic oligonucleotide probes ....................... 135 APPENDIX.8 Differential hybridization and identification of a ligninase cDNA clone ................................................. 145 Hybridization of ligninase cDNA clones with three oligonucleotide probes ..................................... 148 xi INTRODUCTION Lignin is a complicated, stereochemically complex, aromatic heterogeneous biopolymer and is a major component of vascular tissues in terrestrial plants (103). Lignin biodegradation has been an intensive area of research in the last few decades for several reasons. First of all, lignin is the second most abundant organic polymer in the biosphere, next to cellulose. Worldwide an estimated 25 billion metric tons of lignin are annually biosynthesized by plants. Lignin itself and its degraded products are renewable raw materials potentially useful in various biotechnogical processes. Secondly, lignin is intimately associated with cellulose and hemicellulose in woody plants, limiting the efficiency of bioconversion of these polysaccharides to useful products. Furthermore, since the lignin subunit contains 50% more carbon than cellulose and is recalcitrant to biodegradation, lignin biodegra- dation plays an important part in the carbon cycle on the earth. Although the complicated structure of lignin (see Fig. l in Literature Review) implies difficulty in biodegradation, a limited number of micro- organisms are known to oxidatively degrade lignin to carbon dioxide (16). Among them, one white-rot filamentous fungus, Ehgggggghgggg ghrysggpggigm, is known to degrade lignin more rapidly than most other organisms (see 43 and 44). This organism has become a model organism to study lignin biodegradation because of its great ability to degrade lignin, capacity for rapid growth in both complex and chemically defined media, prolific conidiation, relatively high temperature optimum for growth of about 40°C, and low level of phenol oxidase activity. Lignin degradation in this fungus has been shown to be a secondary metabolic 2 event and is triggered by nitrogen, carbon or sulfur starvation. Lignin peroxidase (ligninase), an extracellular, H202-dependent, glycosylated heme protein from R. ghrysosporigm, has recently been purified and characterized (26,32,106,107). Lignin peroxidase is elabo- rated by the fungus under nitrogen-limited conditions during secondary metabolism but not during primary growth (19). The enzyme catalyzes oxidative cleavage of a variety of alkyl side chains of lignin-related compounds including Ca-C cleavage of the propyl side chains, a major 8 reaction in fungal depolymerization of lignin (32,107). Lignin peroxi- dase activity is conveniently measured as veratryl alcohol oxidation to veratraldehyde (107). More recently, at least six proteins (H1, H2, H6, H7, H8 and H10) with lignin peroxidase activity were observed in the extracellular fluid of ligninolytic cultures of R. ghrysggpggigg (60). Proteins H2 and H8 constitute the major lignin peroxidases both in shaken and static cultures of R. ghxysgspgxigm (60). Lignin peroxidases have a molecular weights of 39 to 43 kilodaltons, have similar spectral and catalytic properties and antibody raised against lignin peroxidase H8 cross reacts with the other lignin peroxidases demonstrating at least partial homology among these proteins (58,60). However, some differences in peptides produced upon protease digestion have been noted. Multiple forms of lignin peroxidase have been identified by several other inves- tigators (71,85,101). In fact, Leisola et a1. (71) identified as many as fifteen lignin peroxidases from the extracellular fluid of lignin- degrading cultures of 2. ghxysgspggium. All these proteins catalyzed H202-dependent oxidation of veratryl alcohol to veratraldehyde (71). Lignin peroxidases are important enzymes with many potential practical applications. These include upgrading of lignocellulosic biomass, gig delignification, for the efficient production of feeds, fuels and chemi- 3 cals; biobleaching of pulps; increasing efficiency of wood pulping; treatment of industrial wastes; controlled modification of lignins to produce aromatic chemicals; cracking of petroleum; and detoxification of dangerous and recalcitrant environmental pollutants such as dioxins, polybrominated biphenyls, DDT and benzopyrenes. Numerous efforts have been made to improve the efficiency of lignin biodegradation and the production of lignin peroxidase. These include isolation of lignin peroxidase hyper-producing strains and mutants (7,13,32,60), optimization of culture parameters (7,19,46,60,70,73,99) and use of different kinds of fermentors for large scale production of lignin peroxidases (60,75,76,85). However, the ligninolytic activity and the concentration of the lignin peroxidase enzyme in the extracellular fluid is still quite low even under the best of conditions. To obtain a better understanding of the nature, organization, expression and regulation of the lignin peroxidase genes and to develop the full bioprocessing potential of these enzymes, initial studies to clone and characterize cDNA and genomic sequences for lignin peroxidase have been done and a primary report on the isolation of cDNA clones has been recently published (114). This dissertation will describe my studies on the isolation and characterization of cDNAs and genes for lignin peroxidase from 2. ghxysggngrigm. LITERATURE REVIEW Lignin is the second most abundant component of plant biomass and its biodegradation plays a very important role in the global carbon cycle. Lignin is a complicated, stereochemically complex, heterogeneous aromatic renewable biopolymer. Studies on lignin biodegradation have accelerated greatly in the past ten years, especially after the discoveries that hydrogen peroxide was found to play a central role in 202’ dependent enzyme, lignin peroxidase, was purified and characterized from lignin degradation (86,95, 96) and the first extracellular H Phanerochagtg ghrygggpgrium (26,106). The accumulated knowledge on the microbial degradation of lignin has been summarized in several recent reviews (3,14—16,42-44,54,58,66,69,94,115). This literature review will focus primarily on lignin biodegradation by 2. ghrysgspgzigm. I. GENERAL STRUCTURE OF LIGNIN The lignin polymer is formed through the peroxidase-mediated dehydrogenative polymerization of three cinnamyl alcohol derivatives: p- coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (Figure 1; 14). The three alcohols are present in different ratios in lignins from different plant species and often in different tissues of the same plants (16,54,103). A variety of linkages of the 6-0 and 0-0-0 type are the major linkages in lignin as seen in the schematic formula of a representive portion of the beech lignin (Figure 2; 83). The C-O-C bonds, including Ca-O-Ca, Cp-O-C5 and 04-0-05’ account for about 60% of the linkages in spruce lignin and for about 74% of the linkages in birch lignin (54). C II .. H ‘3: C 20" CHZOH CHZOH “C, I I I 6 2 5 3 0C ‘ H3 H3C0 OCH3 OH OH OH p-coumeryl alcohol coniferyl alcohol sinapyl alcohol Figure 1. Nomenclature of carbons in lignin monomers and three precursors of lignin, p-coumaryl, coniferyl and sinapyl alcohols (from Buswell and Odier, ref. 14). _ "29°" l "2‘,“ ca '9' C." ”25°" co Hzcou "i’ H con @ uc/ °\cu cue to 2' 9 0 “"3 "‘2 tn Cu u can -91 NICO ' n 2 l to _ -o .. o e "‘3' cu "E och3 "22°" “'5 aim—”_ij.‘ "a?" "‘ H usco Ht} 0 5,. "3“ 0c”; at 0c .3 ' 0 cu a cow 00' °" ° H to c"2°“ | 3 "25:00 3 i u J‘co chm—Q “"3 law“— Figure 2. Structure of beech lignin (from Nimz, ref. 83). II. MICROBIOLOGY OF LIGNIN BIODEGRADATION Although lignins are highly complex recalcitrant polymers, a few fungi and bacteria have been reported to degrade lignin to various extents. However, none of the microorganisms have been unequivocally shown to utilize the lignin polymer as a sole carbon or energy source for growth (54,59). 1. Fungi White-rot fungi, including several hundred species of basidiomycetes and a few species of ascomycetes, are believed to be the main organisms responsible for lignin biodegradation in nature (16). White-rot fungi are known to simultaneously degrade all the major components of wood: lignin, cellulose and hemicellulose (16,54,57). One species of white-rot fungus, E. ghryggspgrigg (11,88), is known to degrade synthetic lignin (lac-DHPs that are dehydrogenative polymerizates of coniferyl alcohol) and native wood lignins to C02 more rapidly than most other organisms and has become somewhat of-a model organism for studies on lignin biodegradation. Soft-rot fungi include different ascomycetes and fungi imperfecti and are characterized by softening wood tissue accompanied by significant weight loss. Species of Allgsghgrig, graphigm, Mongdictys, Eaegilomyces, Papglgspgra and Ihlglggig were shown to degrade lignin, although they appear to preferentially attack the wood polysaccharides (l7). Brown-rot fungi include numerous species of basidiomycetes which mainly degrade the polysaccharides in wood but cause only a limited degradation of lignin. The major reaction catalyzed by these fungi is 7 the demethylation of lignin, resulting in the formation of 0-diphenolic units (56) which undergo autooxidation to yield quinone-type chromophores that give the brownish discoloration to the degraded wood. The main difference between brown-rot and white-rot fungi is believed to be the inability of the former to attack aromatic rings or the aliphatic products of aromatic ring cleavage. 2..Bacteria Many genera of eubacteria (Acinsssbastsr. Aersmenas. fiasillss. Pseudemcnoas and Xanshemenas) and actinomycetes (Eicrsmencsnera. Regardia. Strantgmxces and Thermcnssnera) have been reported to degrade different types of extracted lignin and 14C-labeled DHPs (14,94). However, lignin is not known to be mineralized rapidly or extensively by these bacteria. III. PHYSIOLOGY 0F LIGNIN BIODEGRADATION Lignin biodegradation in 2. ghxysgspgrigm has been shown to be a secondary metabolic event (10,53) and is influenced by a number of factors, such as pH, oxygen, agitation and the concentration of various nutrients in the medium. Some of these factors may directly affect the enzyme activity while the others may play a role in affecting the gene expression. 1. Oxygen and agitation Oxygen is an important requirement for lignin degradation by 2. ghzygggngxigm as lignin decomposition is basically an oxidative process. Kirk et al. (61) compared the rates of 14C02 release from 14C-(ring)-DHP 8 when 2. ghgysgspgzigm was grown under different oxygen partial pressures and showed that the cultures grown under 5% 02 released only 1% of the label in 1[‘C-(ring)-DHP as 14002 after 35 days of incubation, whereas those grown under 21% and 100% 02 released 47% and 57%, respectively, of the label as 14COZ. These results indicate that the lignin-degrading system is functional under low oxygen concentration but at a much lower efficiency. 2. ghrysggpgxigm grown under 100% oxygen concentration was found to produce more H202 and higher lignin peroxidase activity than that grown in air (19). The highest lignin peroxidase activity was observed in cultures initially (i.e. during primary growth) grown under air and then shifted to pure 02 atmosphere (73). Furthermore, the synthesis of veratryl alcohol, a typical secondary metabolite in 2. ghgygggpgxiun which is an inducer or mediator of the ligninolytic process, was enhanced by elevated oxygen concentrations (105). Agitation facilitates oxygen transit from the gas to the liquid phase and would be expected to result in increasing lignin decomposition. However, agitation was shown to completely inhibit lignin degradation in two wild-type strains (BKM-F 1767 and ME 446), but had only a moderate effect on lignin degradation by mutant 8026 (60) derived from BKM-F ‘ 1767, and two other wild-type strains (13,32). Leisola and Fiechter (70) later showed that the inhibition of lignin degradation due to agitation by cultures of BKM-F 1767 could be eliminated if veratryl alcohol was added to the growth medium. Addition of detergents, such as Tween-20 or Tween-80, to the cultures also prevented inhibition of ligninolytic activity in agitated cultures (46,99). Lignin peroxidase production in the latter cultures was still dependent on the speed of agitation because too high agitation rate led to complete inhibition of ligninolytic activity (70). 2. Nutrient concentration Limitation of nitrogen, carbohydrate and sulfur is known to trigger ligninolytic activity in B. ghrysogporium (47,53,61). Nitrogen starvation appears to be critical for sustained metabolism of the lignin polymer. This effect of nitrogen limitation on lignin degradation in 2. ghgysggpgrigm was first observed by Kirk et a1. (61) using 14C-labeled synthetic lignin (ring-14C DHP) and was later confirmed by a number of other workers (12,13,98). Reid (97) also demonstrated onset of ligninolytic activity following N starvation using 14C-lignin labeled aspen wood lignin as a substrate. Kirk et a1. (61) first found that B. ghlyggspgrium grown in low- nitrogen (0.6 mM NHANO3 and 2.4 mM L-asparagine) medium degraded l4t4% and 27:4% of [ring-IAC]-lignin to 14002 after 9 and 15 days of incubation, respectively, whereas this fungus grown in high-nitrogen medium (24 mM nitrogen) released only 5&1% and 7&2% of labeled lignin as 14602 after the same period of incubation. Appreciable differences in lignin degradation were not seen when L-asparagine, ammonium tartrate, urea or sodium nitrate was used as a nitrogen source. They also showed that addition of NH“+ to cultures immediately prior to the time of appearance of the ligninolytic system delayed its appearance, whereas addition of NH“+ to ligninolytic cultures resulted in an eventual, temporary decrease of ligninolytic activity (61). To understand how nitrogen affects the ligninolytic activity, Penn and Kirk (21) determined intracellular amino acid profiles and the total protein concentrations during onset of ligninolytic activity (measured as degradation of ring-U-laC-lignin by B. ghzysggpgxigm in low nitrogen medium). It was found that the total amino acid pool increasd to about 10 580 nmol per culture just before the onset of lignin degradation, then decreased to approximately 400 nmol per culture during and after onset of ligninolysis and the subsequent changes were slight. In both cases, variations in glutamate levels accounted for over half of these changes. Arginine showed a dramatic decrease from 52 to 8 nmol during the same period. In contrast, the changes in intracellular protein concentration followed a manner roughly opposite to that of the concentration of amino acids and protein turnover was rapid (5-7%) during the transition period. This indicates that a number of new enzymes involved in the secondary metabolism are being synthesized. Effects of various nitrogen sources on cell protein concentration and repression of ligninolytic activity in cultures of 2. ghxysggpggign were shown to be different. Glutamate suppressed ligninolytic activity by 83% but protein concentration increased by about 50% in comparison to the control, whereas histidine repressed ligninolytic activity by 76% but no protein increase was observed. Addition of NH“+ and glutamate showed different effects on the intracellular concentrations of glutamine and arginine, which play pivotal roles in nitrogen uptake and storage. However, both nitrogen compounds produced a similar increase (about 80%) in the intracellular glutamate levels although repression of ligninolytic activity remained in effect even when the glutamate levels returned to values observed in ligninolytic mycelia (22). Accumulated data indicate that glutamate plays an important role in the regulation of secondary metabolism in 2. ghxygggpgxigm based on the following observations. A new strain 2. ghzyggspgrium, INA-12 (13), produced lignin peroxidase under non-limiting nitrogen conditions (L- asparagine and NHaNO3 as nitrogen source) and the highest lignin peroxidase enzyme activity was obtained when glycerol was used as the 11 carbon source. However, when glutamate was used as nitrogen source, no lignin peroxidase activity was detected in the extracellular fluid (13). More recently, the lignin peroxidase enzyme activity in ligninolytic cultures was shown to be strongly repressed after adding NH4+ or L- glutamate (19,50,95). It has been shown that the onset of ligninolytic activity and idiophasic metabolism (secondary metabolism) in B. ghxyggspggium was preceded by a 10-fold increase in intracellular cAMP (79). Addition of L-glutamate repressed CAMP levels by 50% within 4 h, and cAMP levels remained low for 12 h in such ligninolytic cultures (80). Three enzymes involved in glutamate metabolism demonstrated different levels under different nitrogen concentrations (12). Relatively high levels of NADP-glutamate dehydrogenase, which serves a biosynthetic role, and glutamine synthetase activity could be observed under low nitrogen conditions, whereas NAB-glutamate dehydrogenase which probably functions in glutamate catabolism showed low levels under the same conditions. However, the levels of the three enzymes under high nitrogen showed opposite pattern to those observed under low nitrogen conditions (12). At variance with these results, Fenn et a1. (22) found that the specific activity of NADP-glutamate dehydrogenase increased at least 2- fold after adding NH“+ and glutamate. NAB-glutamate dehydrogenase, which was not detected in control cultures, increased with time following the + 4 . the various nitrogen repressors acted through biochemical repression of addition of glutamate and NH These results led them to propose that key enzyme(s) catalyzing lignin degradation. 3. Induction of the ligninolytic system by lignin and veratryl alcohol Previous studies showed that the ligninolytic enzyme system is 12 produced irrespective of the presence or absence of lignin in low- nitrogen cultures of 2. ghrysgspgzium (53). However, Ulmer et al. (110) showed that the presence of high concentrations of lignin in the growth medium greatly increased the ligninolytic activity by E. ghgygggpgrium, whereas a low concentration of lignin in the medium did not appreciably increase the ligninolytic activity. Maximal rates of degradation were also directly related to the amount of lignin added at the time of inoculation. This stimulation of ligninolytic activity by lignin did not become apparent until 24 h after the addition of lignin to the ligninolytic cultures previously grown in the absence of the polymer. The large size and the insolubility of lignin preclude its crossing the cytoplasmic membrane to directly react with the regulation system within the cells, suggesting that the inducer(s) may be the product(s) of lignin decomposition rather the lignin polymer itself. Faison and Kirk (l9) determined the effect of different lignin monomers, dimers and lignin degradation products on the production of lignin peroxidase (measured as veratryl alcohol oxidation to veratraldehyde) and ligninolytic activity (degradation of 1l‘C-lignin-—>]'4C02) and found that some lignin dimers and lignin metabolites gave higher induction than the monomers. Veratryl alcohol is a typical secondary metabolite synthesized a; 3939 by 2. ghxygggpgxiun after cessation of primary growth (78,105). Also, it is a substrate for the lignin peroxidase which oxidizes it to veratraldehyde (78). Veratryl alcohol is recently reported to be integrally involved in lignin degradation (40,74). Veratryl alcohol is synthesized 215 phenylalanine, 3,4-dimethoxycinnamyl alcohol and veratrylglycerol (105). Production of veratryl alcohol, similar to lignin decomposition, was also shown to be suppressed when glutamate 13 was added to ligninolytic cultures (21). A high concentration of oxygen was shown to increase the synthesis of veratryl alcohol (105). Veratryl alcohol has been shown to be a better inducer of lignin peroxidase and ligninolytic activity than lignin (20). Lignin peroxidase activity increased about four-fold 8 h after the addition of veratryl alcohol to 5-day ligninolytic cultures; this increase was prevented by adding cycloheximide, an inhibitor of protein synthesis, to ligninolytic cultures (20). Further studies showed that addition of 0.4 mM veratryl alcohol into the medium at the beginning of incubation resulted in an increase in specific activity and total activity of lignin peroxidase in the total heme-containing proteins in the extracellular fluid (60). However, the amount of increase in the synthesis of each lignin peroxidase isozyme was different (see Table 1). These results suggested that veratryl alcohol may act as an inducer of some of the lignin peroxidase genes, however, more direct evidence would be needed to prove this. Two mutants were recently isolated by Liwicki et a1. (77), which did not produce veratryl alcohol but still degraded 1l‘C-lignin-wheat ligninocellulose, suggesting that veratryl alcohol may not be required for lignin degradation as suggested by other A investigators (40,74). The fact that lignin peroxidase and ligninolysis do not appear during primary growth (tropophasic cultures) even in the presence of veratryl alcohol suggests that the mere presence of this alcohol is not sufficient to trigger the synthesis of lignin-degrading enzyme complex. 4. Metal ions Metal ions appear to be important for several enzymes involved in ligninolysis. For example, Mn++ is absolutely required for the Mn++- l4 dependent peroxidase activity whereas Fe++ is required for all heme- containing lignin peroxidases as well as the Mn++-dependent peroxidases (see Section V). A significant increase in ligninolytic activity as well as some of the lignin peroxidases was observed upon the addition of trace metal ions to ligninolytic cultures of B. ghxyggspgrigm (60). For instance, addition of 6 times the basal level of trace metals to the growth medium produced 2.8- and 2.5-fold increase in lignin peroxidases H1 and H2, respectively, but had little effect on the production of H6, H7 and H8 (60) (see Table 1). To determine which component(s) in the trace metal mixture is the stimulator, individual metal ions were added separately to a medium containing basal level of trace metals. The results showed that either Cu++ or Mn++ caused an increase in total lignin peroxidase activity equal to that observed with the complete trace metal mixture (60). The differences in patterns of production of different lignin peroxidases upon addition of veratryl alcohol and metal ions suggest that the expression of lignin peroxidase genes may be regulated differently. The inductive effects of veratryl alcohol and metal ions on different lignin peroxidases are presented in Table l. Stimulation of production of lignin peroxidases H1 and H2 was much higher than that of the other four lignin peroxidases when increased levels of trace metals were added individually or in combination with veratryl alcohol. Furthermore, the stimulation resulting from the addition of trace metals and veratryl alcohol appeared additive for H1 and H2 but not for others. These results together with the fact that H1 and H2 showed almost identical peptide patterns upon V8 peptidase digestion suggest that H1 and H2 are related proteins and that the expression of genes encoding these proteins might be controlled by the same regulator. 15 Table 1 Effects of medium additives on production of lignin peroxidases Basalc+ Basal + 5 x Basal + 0.4 mM _ 0.4 mM basal level veratryl alcohol Lignin a b veratryl of metals + 6 x basal level peroxidase Control alcohol of metals Hl 1.6 2 6(1 6) 4.5(2.8) 8 6(5 4) H2 8.7 11 7(1 3) 21.5(2.5) 32 9(3 8) H6 4.7 6 7(1 4) 5.1(l l) 5 5(1 2) H7 9.2 9 3(1 0) 11.2(1 2) 9 4(1 0) H8 71.1 75 3(1 1) 77.9(1 l) 64 9(0 9) H10 4.7 9 2(2 0) 6.4(1 4) 5 7(1 2) a Extracllular fluid from the four media was concentrated 20-fold and analyzed by HPLC. Areas under 409 nm absorbing peaks representing heme proteins were integrated and these values are shown above. The number in the parentheses gives the relative areas of each peak from the four samples normalized to the sample designated control. b The control medium contains a basal medium, 1% glucose, and 10 mM 2,2- dimethylsuccinate pH 4. 5. c The basal mediiaim contained. 1.08 x 103 ‘ M ammonium tartrate, 1.467 x 10-2 MKHPO, 2. 03 x 10 MMgSO‘. 7H20, 6. 8 x 10 M CaClz. 2H20, 2. 97 x 10 M thiamine. HCl and 10 m1 of a trace element solution. The base} trace element solution (called basal level of metals)3 contained. 7. 8 x 10 _124 nitriloacetic acid, 1.2x102 MMgSO .7H0, 2.9x10 MMnSO .H0,1.7x10 MNaCl, 3.59x 10" useso .7110, 7.7‘5x10" MCoCl, 9.0xio" MCaCl, 3.48x_1o" M ZnSO‘. .,7H20 a f; 10‘5 M CuSO .5820, 2.1 x 10 5 A1K(SO ) .12112 0, 21. 6 x 10" M 1131303 and 4.1 x 10 M NaMoO .2n‘o. Data taken from Kirk etzal. (60). 16 IV. ROLE OF H202 IN LIGNIN DEGRADATION Hydrogen peroxide is believed to play a central role in lignin degradation (86,95,96). Koenigs (64,65) first found that a number of wood-rotting fungi produced hydrogen peroxide (H202) from glucose in culture or from native substrates in wood and suggested the possible involvement of H202 in lignin degradation by white-rot fungi. Forney et a1. (24) later showed that when 2. ghgysgspgxium was grown in low nitrogen medium, an increase in the specific activity for H202 production was observed to coincide with the appearance of ligninolytic activity and both activities appeared after the culture entered the stationary phase. The ultrastructural studies of ligninolytic cells of 2. ghxyggspgxigm demonstrated that hydrogen peroxide production appeared to be localized in periplasmic "microbodies" of cells from ligninolytic cultures grown for 14 days in low nitrogen medium but not in cultures grown for 4 days in the same medium or in 14 day cultures grown in high nitrogen medium (23). This correlation between H202 production and ligninolytic activity was also observed by other groups (8,34). Besides, high oxygen concentration and the presence of lignin in the medium, which are known to stimulate ligninolytic activity in 2. ghrysggpggigm, were shown to stimulate the production of hydrogen peroxide (18,19,34). The involvement of hydrogen peroxide in lignin decomposition by 2. ghxygggpgriun has been firmly established since lignin and Mn++- dependent peroxidases, whose activities are dependent upon the presence of H 0 were recently purified from ligninolytic cultures of this 2 2’ fungus (see next section). 17 V. ENZYMES INVOLVED IN LIGNIN DEGRADATION Tien and Kirk (106) and Glenn et a1. (26) simultaneously discovered an extracellular H202-dependent enzyme which catalyzed the Ca-C cleavage B of lignin model compounds and limited depolymerization of lignin polymers. Since then, different kinds of enzymes which are believed to be involved in lignin biodegradation have been purified and charac- terized from cell extracts or extracellular fluid of ligninolytic cultures of 2. ghxysgspgxlum. The data on purified extracellular enzymes from ligninolytic cultures of B. ghzygggpgrigm are summarized in Table 2. 1. Lignin peroxidases AW Relatively simple procedures have been developed for purifying lignin peroxidases from 2. ghzyggspgzium (32,72,84,107) and these are briefly described here. The extracellular fluid from cultures showing peak lignin peroxidase activity are first separated from the mycelia by filtration of the culture through cheese cloth followed by centrifu- gation to remove fine particles. The supernatant is then concentrated using either Amicon membrane filtration technique or acetone precipi- tation (32). The concentrated fluid is dialyzed against appropriate buffer and then purified by HPLC, FPLC or other chromatographic procedures (see Table 2) to separate different enzymes. Purity of the protein is determined by the use of sodium dodecylsulfate polyacryamide gels (SDS-PAGE) and the protein bands are visualized using the silver staining technique. 18 Table 2 Characteristics of Extracellular Enzymes Purified from Ligninolytic Cultures of W W Enzyme Synonym Strain purification M. H. heme carbohydrate cofactor(s) reference procedure (I) Lignin Ligninase BKM-F1767 DEAE-Bio-Gel A 42,000 + 13 8202 106,107 peroxidase (Bio-Rad) fl Ligninase El DEM-F1767 FPLC Mono 0 MD + ND 6202 60 32, HS, H7, (Pharmacia) E8 and 310 Ligninase-l REM-F1767 —PBE-94 42,000- + 21 3202 85 (Pharmacia) 43,000 Diarylpropane Gold DEAE-Sepharose 41,000 + ND 8202 32 oxygenase Sephadex 6100 (Sigma) Diarylpropane Gold DEAE-Sepharose 39,000 + 6 3202 101 oxygenase I Sephadex 6100 (Sigma) II Gold 41,000 + 6 H 0 101 2 2 III Gold 43,000 + 6 8202 101 Diarylpropane ME446 Blue agarose 41,000 + ND 8202 26,67 oxygenase Sephadex 6100 Lignin ME446 DEAE-Sepharose so + up 3202 5 peroxidase CL-CB (Pharmacia) Fr-II and Fr-III "3"" ++ """""""""""""""""""" ll """"" Mn - 1h -dependent, ME448 DEAE-Sepharose 46,000 + ND H O , m 26.67 dependent lactate-activated Blue agarose m-hydroxy acids peroxidase peroxidase Sephadex 6100 proteins ++ MADE-peroxidase ME446 DEAR-Sepharose 46,000 + ND 8202, Mn 6 CL-CB (Pbarmacia) ++ Vanillyacetone HEM-F1767 DEAE-Dio-Gel A 45,000- + 17 3202, Mn 85 oxidase (Bio-Rad) 47,000 Peroxidase-M2 * ND-not determined. l9 B-Pmerfles Lignin peroxidase (ligninase) is an H202-dependent, heme-containing, glycosylated extracellular protein which catalyzes the Ca-C cleavage of 19 different lignin model compounds as well as a variety of other aromatic compounds, and depolymerizes lignin polymers. This enzyme contains one protoheme IX as the prosthetic group and has one atom of iron per molecule. The spectral analyses from EA (Electronic Absorption), EPR (Electronic Paramagnetic Resonance) and RR (Resonance Raman) spectroscopy showed that the native enzyme contained high-spin iron and the reduced enzyme contained a high-spin, pentacoordinate ferrous iron (5,100). Although lignin peroxidases purified from different strains appeared very similar to one another, some differences have been noted among them. For example, the absorption spectrum of native lignin peroxidase from the BKM strain used by Kirk and coworkers was not changed upon the addition of dithionite (107) whereas the enzyme from the strain used by Cold and coworkers displayed the red shift (32). The optimal pH of these two enzymes for the oxidation of fl-ether dimers is also different (32,107). The differences between the above two lignin peroxidases may reflect the facts that they were purified from different strains and different conditions were employed for growing these strains. Variation in culture condition is known to have a profound effect on the types and amounts of lignin peroxidase produced (60). Lignin peroxidases are known to catalyze the following reactions (26,32,36,45,67,72,85,101,106,107): (1) Ca-C cleavage of fi-l and 8-0-4 lignin model compounds; I9 (2) Hydroxylation of some benzylic methylene groups, and Ca and Cfi of the Ca-C olefinic bond in styryl structures; 6 20 (3) Oxidation of phenols, methoxybenzenes and benzyl alcohol; (4) Intradiol cleavage of phenylglycol; (5) Partial depolymerization of methylated spruce and birch milled wood lignin as well as 14C-ring-DHP; and (6) Ethylene generation from 2-keto-4-thiomethyl butryic acid (KTBA) in the presence of veratryl alcohol. C. u t o o e 0 da e Renganathan et al. (101) described three lignin peroxidases present in the extracellular fluid of ligninolytic cultures of 2. ghgysgspgrium, called forms I, II and III; form II accounted for 85% of the lignin peroxidase activity based on the oxidation of veratryl alcohol to veratraldehyde. Absorption maxima of the native, reduced, and a variety of ligand complexes of three lignin peroxidases were essentially identical and the same products were produced from different lignin model compounds by the three enzymes. However, the specific activities for veratryl alcohol oxidation were different. Later, Kirk et a1. (60) identified six lignin peroxidases present in the extracellular fluid of the ligninolytic cultures of E. ghgysggpggium strains BKM-F 1767 (Figure 3). The HPLC profile of the extracellular fluid from a 5-day flask ‘ culture of this fungus showed that there were at least thirteen proteins and ten of them (named from H1 to H10) contained heme (Figure 3). Six of the ten heme proteins (H1, H2, H6, H7, H8, H10) displayed veratryl alcohol oxidation activity. The lignin peroxidases H2 and H8 is the major proteins; H2 and H6 showed higher specific activities than that of H8 but the quantities of these proteins were relatively small. The V8 protease analyses showed that H1 and H2 produced almost identical peptides, H8 produced similar peptide pattern to H1 and H2 but lacked at least two major peptides and H6 and H10 produced very different peptide 21 patterns from those of H1, H2 and H8, indicating that the lignin peroxidases are related but different proteins. However, polyclonal antibody raised against H8 could react with the other five lignin peroxidases, indicating that there should be amino acid sequence homology present among these lignin peroxidases. The multiple forms of lignin peroxidase were confirmed by other groups as well (6,71). Leisola et a1. (71) more recently identified as many as fifteen lignin peroxidases in the extracellular fluid of ligninolytic cultures of g. Warm. 22 IOOr- s ' 1 Relative peok height Time (min) Figure 3. HPLC profile of extracellular fluid from a 5-day ligninolytic culture of 2. ghxysggpgxium. The sample of 20-fold concentrated extracellular fluid was used for HPLC analysis. Full line, A4 9; broken curve, A 80' Lignin peroxidase activity is indicated as positgve (+). The sloping line shows the acetate gradient (From Kirk et al., ref. 60) 23 D. Mechanism of reaction cgtglyzed by lignin peroxidasg A two single-electron oxidation mechanism has been proposed to explain the Ca-C cleavage of lignin model compounds and other reactions 8 catalyzed by lignin peroxidase (37-39,63,81,104). When diarylpropane is oxidized by lignin peroxidase, the enzyme reacts with H202 to form a two-electron peroxy intermediate compound I (Figure 4). This high potential oxy-ferryl intermediate extracts one electron from the aromatic ring of the substrate (Figure 5), resulting in the fomation of a substrate cationic radical and the one-electron oxidized form of the lignin peroxidase, compound II. The cationic radical cleaves at the Ca- 06 bond to form a cation from the o-carbon moiety and a benzyl free radical in the fi-carbon moiety. The cation immediately deprotonates to produce veratraldehyde as one product. The second radical (fi-carbon moiety) can undergo a variety of reactions including a further one- electron oxidation to yield a cation which subsequently hydrates with water to produce phenylglycerol. The second radical may also react with molecular oxygen to form a peroxy radical. This is consistent with the result of labeled-02 inserting onto the fl-carbon (102,107). Two molecules of peroxy radicals could interact to yield one molecule of phenylglycerol and one molecule of ketol (39). The compound II of lignin peroxidase could catalyze one-electron oxidation of either the second radical (fl-carbon moeity) or aromatic nucleus of another diarylpropane molecule, and then return to the native state. The presence of different forms of the enzyme and substrate was demonstrated during the oxidation of various lignin model compounds (5,37,39,63,81,86,104). 24. 2 Native enzyge H 0 214+ Ligninox . . Lignin 1 o 1 M Degradation Products 'Compound 11' O A" 'Compound I' Lignin Lignin l 1 Degradation OX Products Figure 4. Catabolic cycle of lignin peroxidase of 2. Chrysospgrium (from Kirk, ref. 55). 25 H o \ I cup" cup" omen c ’ ' ocu, ' OCH, | OCH: an _‘i". n 0 . H - cos, c... H -c cow, ——9 H - c ocn, . 1.” -¢ ° I I cum, , l (bi OCH, H - c“- on 9 I (._.1 fi ?. IN fi‘ ocn, ocH, ocn, I can, | l O C‘ {ram EA : electron acceptor CH,OH I CH,OH - 0 (EA) c‘J OCH. *—; H - C OCH. '1';- u | - 0 OH ‘1 2 Figure 5. Scheme of Ca~Cfl oxidative cleavage of diarylpropane catalyzed by lignin peroxidase of R. W (from Schoemaker et a1. , ref. 104) . 26 2. Mn++-Dependent peroxidase The same procedures as described for lignin peroxidases were used to purify Mn++-dependent peroxidase from ligninolytic cultures of P. ghzysggpggium. This enzyme is also an extracellular, glycosylated heme protein. The enzyme requires not only hydrogen peroxide but also Mn++ for its activity to catalyze the oxidation of a variety of phenol derivatives (25) and a number of dyes including Poly B-4ll and Poly R- 481 (84,85). Unlike lignin peroxidase, this enzyme cannot oxidize veratryl alcohol at pH 5.0 (45). The addition of a-hydroxylic acids and/or exogenous protein (such as egg albumin) to the reaction system stimulated the activity of the Mn++-dependent peroxidase from strain ME 446 (67) but not of that from another strain BKM-F 1767 (85). This enzyme also catalyzes the oxidation of NADH or NADPH to produce H in 202' the presence of Mn++ and 02 (6,85). Unlike lignin peroxidases which are extracellular, Mn++-dependent peroxidase appears to be associated closely with the fungal mycelia (85). All the peroxidase activity was lost upon dialysis against 20 mM Na-succinate, suggesting the loose binding of Mn++ to the enzyme (25). The enzyme rapidly oxidized Mn++ to Mn+++ which plays a role in the enzyme mechanism (25). More recently, Leisola et al. (71) showed that six Mn++-dependent peroxidases appeared and reached their maximal activity earlier than the lignin peroxidases in ligninolytic cultures of B. chrysggpggium. Peptide mapping, amino acid analysis and reaction against specific antibodies showed that all the Mn++-dependent peroxidases were probably products of one gene (71). 3. H202-Producing enzymes SWIM 27 Fungal glucose oxidase (fi-D-glucosezoxygen oxidoreductase, EC 1.1.3.4) catalyzes the oxidation of D-glucose to H202 and 6-D-gluconolactone which is nonenzymatically hydrolyzed to D-gluconic acid, in the presence of molecular oxygen. Glucose oxidase appears to play an important role in the production of H202 for lignin degradation in 2. chrysospozium according to the following observations (50). Glucose supported the highest level of oxygen-dependent H202 production in cell extracts of ligninolytic cultures compared to a number of other substrates. Glucose- dependent H202 production was exhibited by a single protein band from cell extracts, but not from extracellular fluid, of ligninolytic cultures. This protein band was not seen in cell extracts of non- ligninolytic cultures. Glucose oxidase activity, like ligninolytic activity, was shown to be a secondary metabolic event and was triggered in response to the starvation for nitrogen or carbohydrate. More recently, several glucose oxidase-negative mutants (ggx') were isolated and were shown to have neither the glucose oxidase activity nor the ligninolytic activity, whereas the revertants regained both glucose oxidase and ligninolytic activity (51,90). Glucose oxidase was purified from the cell extract of ligninolytic cultures of R. ghxyfiggpggiun ME 446 by the following steps: preparation of cell extracts, DEAE-Sephadex chromatography, Sephacryl chromatography and DEAE-Sephadex chromatography (49). The purified enzyme is a flavoprotein with a native molecular weight of 180,000 and a denatured molecular weight of 80,000, suggesting that the native form may consist of two identical polypeptide chain subunits covalently linked by disulfide bonds. No carbohydrate was found in the enzyme. The enzyme activity has an optimal pH between 4.6 and 5.0. Glucose is believed to be the primary substrate for the enzyme based on the Vmax/Km, whereas 28 the other carbohydrates, such as L-sorbose, D-xylose and D-maltose, are relatively minor substrates. B. EnH-deeendentmmae Mn++-dependent peroxidase described above was also shown to produce H202 by the oxidation of NADH or NADPH (6,25,85). One function of this enzyme may be to produce H202, which is essential for lignin peroxidase activity, from the reduced pyridine nucleotides which were shown to be secreted by 2. chgysgspgxium into the growth medium (68). However, the very low amounts of pyridine nucleotides present in extracellular culture fluid cannot account for the large amounts of H202 produced in ligninolytic cultures. C. W Greene and Gould (35) found that mycelia of lignin-degrading cultures of 2. ghxygggpgxium grown in low nitrogen medium consumed O2 and produced extracellular H202 when incubated with fatty acyl-60A substrates and suggested that peroxisomal fatty acyl oxidase may be an important enzyme for the production of H202 for lignin biodegradation in this fungus. However, the activity of this enzyme was too low to account for the level of H202 produced in lignin-degrading cultures. Also, no correlation between the regulation of production of this enzyme and lignin degradation system has been shown. D- Wicca: More recently, Kersten and Kirk (52) demonstrated an extracellular H202-producing enzyme, glyoxal oxidase in ligninolytic cultures of P. ghxysggpgxiun. This enzyme can oxidize several a-hydroxy carbonyl- and to H O . dicarbonyl compounds, coupled to the reduction of 02 2 2 4. Other enzymes 29 Cellobiose-quinone oxidoreductase (111) may play an indirect role in lignin biodegradation by preventing polymerization of lignin degradation products. The enzyme was purified from the cell extract of primary growth culture of R. ghgyggspgxium and has a molecular weight of 58,000 (112). Cellobiose-quinone oxidoreductase is a flavoprotein with a FAD prosthetic group. Huynh and Crawford (45) found that concentrated and unfractionated extracellular fluid of 2. ghxysggpgrign BKM-F 1767 could catalyze the conversion of 2-methoxy-3-phenylbenzoic acid (M1) to 2-hydroxy-3- phenylbenzoic acid (aromatic methyl ether demethylase), the conversion of methyl 2-hydroxy-3-phenylbenzoate (M3) to 24hydroxy-3-phenylbenzoic acid (aromatic methyl ester esterase) and the oxidation of vanillylacetone [4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, M2] 0 and MnH. 2 2 However, they were able to purify only one of these three enzymes, i.e. (vanillylacetone oxidase) in the presence of both H vanillylacetone oxidase (45). VI. GENETICS AND MOLECULAR BIOLOGY OF LIGNIN BIODEGRADATION Numerous attempts have been made to improve the ligninolytic activity of B. ghzyggspgxinn. These include changing the concentrations of media components (7,60), employing agitation to improve oxygen supply to cells (l9,46,70,73), adding different detergents and veratryl alcohol to the media (60,99), isolating different strains including mutants capable of higher lignin peroxidase production (7,13,32,60) and developing different types of fermentors for scaling up growth (60,75,76,85). By these manipulations, lignin peroxidase activity has been increased from about 5 U/L (107) to 1250 U/L (7). However, genetic and molecular 30 biological studies on E. ghrysgspgrium will be useful not only to obtain better understanding of the nature of lignin degradation but also to greatly improve the ligninolytic activity of g. Chrysospogigg so that the organism or its products could be used in a variety of biotechnological processes. 1. Genetic studies Gold and coworkers have developed some basic approaches for the study of the genetics of 2. chrysgspgrium. Since 2. chrysgspgrium produces asexual spores prolifically and has diffuse unrestricted growth on ordinary agar media, Gold and Cheng (27) designed a medium containing L- sorbose and sodium deoxycholate, which induces colonial growth of this fungus. The restricted size and heavy conidial production of colonies permits high plating densities and the use of a replica-plating technique. Gold at al. (30) isolated a number of auxotrophic mutants by using UV and X rays as mutagens. These auxotrophs have been characterized by employing complementation procedures described below (30,82). They spread mycelia from two of different auxotrophs onto solid minimal medium to force complementation (30). The results showed that vigorous growth ensued after a short lag time, suggesting that heterokaryon formation occured in the vegetative mycelial stage. Further studies showed that the vigorous growth was not due to cross feeding but due to complementation between the two different auxotrophs (30). Upon cytological study it was found that the wild-type conidia were 60% mononucleate, whereas the conidia from heterokaryotic mycelia ranged from only 1-10% mononucleate (30). Another approach, protoplast fusion, was also used to demonstrate complementation between auxotrophs (29). The prototrophs from protoplast fusion were found to be readily 31 homokaryotized and revert to the parent phenotypes, indicating that the prototrophic mycelia were heterokaryotic rather than diploid. Since classical basidiomycete crosses require the development of fruiting bodies, Gold and Cheng (28) demonstrated that fruit body formation in 2. ghgygggpggigm was controlled by glucose and nitrogen catabolite repression and that Walseth cellulose was the best source of carbon for the induction of fruit body and consequent basidiospore synthesis in this fungus. Alic and Gold (1) isolated recombinants with different phenotypes, including wild-type and double mutant phenotypes, from various crosses after inducing the formation of fruiting bodies from heterokaryons. The results indicate that the genetic recombination does occur. Cytological studies demonstrated that more than 90% of the basidiospores from wild-type and auxotrophic strains as well as from forced heterokaryons were binucleate. The ratio of recombinants to parental types is about 1:1:1:l, suggesting that the binucleate basidiospores are homokaryotic and that the two nuclei arise from a postmeiotic mitotic event. Further studies showed that the binucleate basidiospores were homokaryotic but not heterokaryotic and that E- ghrysgspgrigm had a primary homothallic mating system (2). More recently, Tien et al. (109) proposed a potential strategy for the lignin peroxidase-dependent selection of lignin-degrading microorganisms. This strategy involves covalently bonding amino acids to lignin model compounds in such a way that lignin peroxidase-catalyzed cleavage of the models produces the amino acids for growth. This procedure may also be used to select mutants which overproduce lignin peroxidase(s) or are defective in production of lignin peroxidase. Johnsrud and Eriksson (48) used classical mutagenesis and cross- breeding techniques to isolate several cellulase-negative mutants from 32 E. ghzygggpgrium K-3, one of which was later shown to have higher lignin peroxidase activity than the parent strain (62). Such lignin-degrading, cellulaseodeficient mutants may be very useful for biopulping and biobleaching applications in paper industry. A mutant (SC26) from BKM-F 1767 (60) showed the highest total ligninolytic activity, the highest total and specific lignin peroxidase activity compared with the parent strain and other two wild-type strains (ME 446 and K3) as well as three cellulase-negative mutants (62). Furthermore, this mutant degraded lignin under agitation conditions and could adhere well to the plastic discs of a rotating disk fermentor, which is potentially useful for scaling up lignin peroxidase production (60). Two phenoloxidase-negative mutants (4,31) were isolated and one of them was shown to be pleiotropic lacking lignin degradation and several other idiophasic characteristics as well (33). The revertants from this mutant recovered all lost functions. These results suggested that it may be a regulatory mutant (33) lacking phenoloxidase activity and several other secondary metabolic characteristics. Four classes of phenoloxidase-negative mutants were recently isolated by Liwicki et al. (77). Studies with these mutants indicated that mutations resulting in loss of phenol oxidase and ligninolytic activity were not necessarily pleiotrophic for other idiophasic functions, such as intracellular cAMP levels, sporulation, extracellular glucan production and veratryl alcohol synthesis. Several glucose oxidase-negative mutants have recently been isolated (51,90). These mutants were shown to be deficient not only in their ability to produce hydrogen peroxide but also in lignin degradation, lignin peroxidase activity, and decolorization of the dye poly-R 481 (51,90). These mutants retained, albeit at a lower level, the capacity 33 to produce veratryl alcohol (a typical secondary metabolite in B. ghgysgspgxium), and produced conidia at a level comparable to that of the wild type. The revertants recovered all the missing characteristics. These mutants may be another kind of regulatory mutant. Such mutants may be useful for the identification of genetic control elements using molecular cloning procedures. 2. Mblecular biology Although there has been some progress in developing classic genetic procedures for 2. ghzyggspggigm as described above, the available genetic information for this fungus is meagre compared to other filamentous fungi such as Aspgrgillgg and Nguzggpgna. Use of recombinant DNA procedures is another alternative to obtain a better understanding of the structure, expression, regulation and organization of genes involved in the whole ligninolytic system. A number of molecular biological procedures which have been shown to be successful in cloning and expression of heterologous genes in bacteria, yeasts and other filamentous fungi can be modified and used for cloning and characterizing genes encoding lignin peroxidases and other enzymes in 2. Warm. Reddy and coworkers have developed a number of different procedures suitable for molecular biological studies on 2. ghrysgspgrium. These include the isolation of chromsomal DNA and RNA from E. ghxyggspggium (92,113), preparation of spheroplasts from conidia and DNA transformation for this fungus (91). Rao and Raddy (92,93) constructed a YIpS-kanr (kanamycin-resistant) vector which is useful for isolating ags (autonomous replication sequence) from yeast and other eukaryotes and isolated an ax; sequence from this fungus. By inserting the isolated grs 34 from 2. ghgysgspggium into plasmid YIpS-kanr, a shuttle vector for E. ggli/fi. ggrgyigiag/B. ghxysgspgrium was constructed (91). This vector can transform wild-type strains of B. ghxysgspgrigm to 6418 resistance (91). Different cDNA clones for lignin peroxidase have been isolated and characterized (114). Broda and coworkers have also developed procedures for isolating mRNA and chromosomal DNA from this fungus (41,89) and showed that its estimated genomic size is about 4-5 X 107 base pairs (88). AW As a first step in the construction of a cloning vector suitable for transformation of 2. ghxyggspgnigm, an £15 sequence was isolated from this fungus. Rao and Reddy (92) constructed a gene library in the 5. ggrgngjgg integration vector YIpS which contains selection markers for E. 9911 [Apr (ampicillin-resistant) and Tcr (tetracycline-resistant)] and yeast (U353) and a replication origin (211) for E. 9911 but not for S. egregisigg. If k. ghxyggspgrium genomic sequences cloned into YIpS confer on the latter the ability to autonomously replicate in yeast, one should obtain high frequency of transformation of 31;- to 313+. Several g1; sequences have been isolated and characterized by using this approach (92). This fungus was later shown to be sensitive to the antibiotic G418 (91), which inhibits protein synthesis in prokaryotes as well as many eukaryotes. This suggested that the kanamycin-resistant gene from bacterial transposon Tn903, that encodes an aminoglycoside phosphotransferase which inactivates both kanamycin and 6418, could be used as a selection marker in 2. ghxysggpgxiun. Randall et al. (91) recently constructed a shuttle vector containing the 5;; sequence from R. ghzygggpgrium, the kgnr selection marker from Tn903, 211 sequence for replication in E. 9211, and the QEA3 marker for selection in yeast. This 35 shuttle vector should be very useful for molecular genetic studies of lignin biodegradation and for studying the regulation of secondary metabolism in 2. ghrysgspgzium. Randall et al. (91) have developed a transformation procedure for introducing this shuttle vector into 2. ghgysosporium. The shuttle vector, designated pRR12, could transform two wild-type strains of 3. ghgygggpggium to 6418 resistance with a frequency of about 20 transformants per microgram DNA (91). Although the transformation frequency was relatively low the transformation efficiency was quite 3 to 1.1 X 10.2 transformants/regenerated spheroplast). high (3.3 x 10' The transforming vector pRRl2 present at a low level in the transformants, could be consistently recovered by E. 9911 transformation. The recovered vector has been shown to be identical to pRR12. The vector is stably maintained in the transformants under selective conditions and can be recovered even after 18 months (91). This is the first demonstration of a transformation system for B. ghgygggpgrium and the first demonstration of a shuttle vector for a filamentous fungus. Molecular cloning procedures have been well established for E. 9911 and S. ggrgxigigg and a number of eukaryotic genes have been cloned by using these two systems. Therefore, either of them can be used to clone lignin peroxidase gene(s) from B. ghrysgspgxigm although yeast may be a better molecular cloning system for this fungus (9). Different strategies have been employed to clone genes for lignin peroxidase (87,108,114). Since some of the genes isolated from other filamentous fungi have already been shown to contain several small introns, the inability to 36 correctly process introns from foreign genes is a potential problem with the yeast system. Besides, the lack of functional expression of some foreign genes in either yeast or 5. £911 is another problem due to the differences in promoter sequences. Hence, it might be better to clone lignin peroxidase cDNA(s) first and obtain its expression in a suitable eukaryotic or prokaryotic expression system. These cDNA(s) can also be used as probe to screen the genomic library to isolate lignin peroxidase genes. Since ligninolytic activity in E. ghgygggpgxigm (BKM strain) appears after the cessation of primary growth and reaches a peak around the 6th day of incubation, the assumption was made that the messenger RNAs for lignin peroxidase proteins may also be present in 6-day-old cells. Therefore, the strategy to clone lignin peroxidase gene should be: 1) constructing a cDNA library from the ligninolytic cultures of E. ghzysggpgxigm into an expression vector (pUC series;ll4) or phage (Agtll;108); 2) screening the library either with antibody raised against the purified lignin peroxidase (108) or with synthetic oligodeoxynucleotide probes whose sequences were deduced from the partial amino acid sequences of lignin peroxidase (114); 3) characterizing the isolated cDNA clones and sequencing them to make sure that the correct clones were obtained; 4) using the cDNA(s) as probe to screen the genomic library to isolate genomic lignin peroxidase gene(s). Furthermore, the cDNA(s) can be cloned into suitable expression vectors to get maximal production of functional lignin peroxidase, whereas the genomic genes can be used to study the gene structure, regulation of gene expression as well as organization of lignin peroxidase gene(s) on the chromosomes. By using the above strategy, two different lignin peroxidase cDNA clones have been isolated and sequenced (114 and see chapter 1 and 2 in 37 this dissertation) and one other cDNA clone which is different from ours was also identified (108). All three cDNA clones showed very high homology in the nucleotide and amino acid sequences (see chapter 2). Northern hybridization showed that only poly(A) RNA from the ligninolytic cultures hybridized with cloned cDNA probes, indicating that the regulation of ligninolytic activity may be at the mRNA level (see chapter 1). Using two cDNA clones as probes, six lignin peroxidase genes have been isolated and characterized and one of them was sequenced (see Chapter 3). The sequence data from the genomic clone showed that this lignin peroxidase gene has typical promoter sequence TATATAA often found in higher eukaryotic genes and contains nine introns. These introns are relatively small (SO-62 bp) in comparison with those from higher eukaryotic genes (see Chapter 3). 10. ll. 12. 13. 38 REFERENCES Alic, M. and M. H. Gold. 1985. Genetic recombination in the lignin-degrading basidiomycete Phanerochaete abuse-searing. Appl- Environ. Microbiol. 50:27-30. Alic, M., C. Letzring and M. H. Gold. 1987. Mating system and basidiospore formation in the lignin-degrading basidiomycete Phanerochaete ghzxfiggngzium. Appl. Environ. Microbiol. 53:1464- 1469. Amer, G. I. and S. W. Drew. 1980. Microbiology of lignin degradation. pp67-103, in G. T. Tsao (ed.), Annual reports on fermentation processes, vol. 4. 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Role of molecular oxygen in lignin peroxidase reactions. Arch. Biochem. Biophys. 246: 155-161. Sarkanen, K. V. and C. H. Ludwig (eds.). 1971. Lignins: occurence, formation, structure and reactions. Wiley-Interscience, New York. 916pp. Schoemaker, H. E., P. J. Harvey, R. M. Bowen and J. M. Palmer. 1985. On the mechanism of enzymatic lignin breakdown. FEBS Lett. 183: 7-12. Shimada, M., F. Nakatsubo, T. K. Kirk and T. Higuchi. 1981. Biosynthesis of the secondary metabolite veratryl alcohol in relation to lignin degradation in Phanerochaete ghgygggpgrium. Arch. Microbiol. 129:321-324. Tien, M. and T. K. Kirk. 1983. Lignin-degrading enzyme from the hymenomycete Phanerochaete ghxygggpggigm Burds. Science 221: 661- 663. Tien, M. and T. K. Kirk. 1984. Lignin-degrading enzyme from Phanerochaete ghrygggpgrium: purification, characterization, and catalytic properties of a unique H 02-requiring oxygenase. Proc. Natl. Acad. Sci. USA. 81: 2280-22 4. Tien, M. and C. -P. D. Tu. 1987. Cloning and sequencing of a cDNA for a ligninase from Phgggzgghgggg ghzygggpgrium. Nature (London) 326:520-523. Tien, M., P. J. Kersten and T. K. Kirk. 1987. Selection and improvement of lignin-degrading microorganisms: potential strategy based on lignin model-amino acid adducts. Appl. Environ. Microbiol. 53:242-245. Ulmer, D. C., M. S. A. Leisola and A. Fiechter. 1984. Possible induction of the ligninolytic system of Phanerochaete ghxxsgspgzigm. J. Biotechnol. 1:13-24. Westermark, U. and K.-E. Eriksson. 1974. Cellobiose:quinone oxidoreductase, a new wood-degrading enzyme from white-rat fungi. Acta Chem. Scand. B28:209-214. Westermark, U. and K.-E. Eriksson. 1975. Purification and properties of cellobiosezquinane oxidoreductase from fipggggrighum nglxgzulgntgn. Acta Chem. Scand. B29:419-424. Zhang, Y. Z. and C. A. Reddy. 1988. Use of synthetic oligonucleotide probes for identifying ligninase cDNA clones. Meth. Enzymol. 168 (in press) Zhang, Y. 2., G. J. Zylstra, R. H. Olsen and C. A. Reddy. 1986. 115. 47 Identification of cDNA clones for ligninase from Phanerochaete ghzygggpggium using synthetic oligonucleotide probes. Biochem. Biophys. Res. Commun. 137:649-656. Zeikus, J. G. 1983. Lignin metabolism and the carbon cycle: polymer biosynthesis, biodegradation, and environmental recalcitrance. pp211-243, in M. Alexander (ed.), Advances in microbial ecology, vol. 5. Plenum Press, New York. 48 CHAPTER ONE CHARACTERIZATION OF LIGNIN PEROXIDASE cDNA CLONES FROH W W 49 ABSTRACT Characterization of two different classes of lignin peroxidase cDNA clones, pCLG4 and pCLGS, from Phanerochaete ghzygggpggium is described. The pCLGS group consists of only one cDNA clone (pCLGS) whereas the pCLG4 group consists of two other clones, pCLG3 and pCLG6. Three oligodeoxynucleotide probes, that correspond to amino acid sequences of tryptic peptides of lignin peroxidase H8, a major component of the multiple lignin peroxidases in 2. ghgygggpgxium, showed hybridization with pCLGS whereas only one of these probes hybridized with the members of the pCLG4 group. Northern hybridization analyses showed that the poly(A) RNA corresponding to the above cDNA clones was detectable in 6- day-old idiophasic cultures grown in low nitrogen medium (i.e. lignin- degrading cultures), but not in 2-day-old primary growth cultures grown in the same medium (i.e. nan-ligninolytic cultures). The expression of the lignin peroxidase protein, based on an enzyme-linked immunoblot assay using lignin peroxidase antibody, was obtained with the cDNA insert (named CLGS) in clone pCLGS. These results suggest that CLGS encodes lignin peroxidase H8. Southern hybridization analyses of restriction enzyme digested total genomic DNA of E. ghxxgggpggigg, utilizing labeled cDNA inserts of pCLG4 and pCLGS as probes, suggested the presence of a lignin peroxidase gene family in this fungus. 50 INTRODUCTION Lignin peroxidase, an extracellular, H202-dependent, glycosylated, heme protein has recently been purified and characterized from a filamentous white-rot fungus R. ghxygggpgrium (6,7,27,28). Lignin peroxidase is elaborated by the fungus under nitrogen-limited conditions during secondary metabolism but not during primary growth (5,16). The enzyme catalyzes oxidative cleavage of a variety of alkyl side chains of lignin-related compounds including Ca-Cfl cleavage of the propyl side chains, a major reaction in fungal depolymerization of lignin (1,7,10,12,28). Lignin peroxidase activity is conveniently measured as veratryl alcohol oxidation to veratraldehyde (28). Kirk et al. (16) recently reported that P. ghxysggpggigm elaborates at least six proteins (H1, H2, H6, H7, H8 and H10) with lignin peroxidase activity. H2 and H8 constitute the major lignin peroxidases both in shaken and static cultures of P. ghzygggpgxium (14,16). Antibody raised against lignin peroxidase H8 cross reacts with the other five lignin peroxidases demonstrating at least partial homology among the lignin peroxidases; however, some differences in peptides produced upon protease digestion have been noted (16). Renganathan et a1. (24) independently described three different molecular forms of lignin peroxidases, all of which are glycosylated and range in Mr from 39,000 to 43,000. These results suggest one or both of the following: 1) one gene may code for one lignin peroxidase protein; or 2) product(s) of a single (or multiple) lignin peroxidase gene(s) undergo various post-transcriptional and/or post-translational modifications. Lignin peroxidases are important enzymes with many potential practical 51 applications (4,13). These include upgrading lignocellulosic materials, gig delignification, for the efficient production of feeds, fuels and chemicals; biobleaching of pulps; increasing efficiency of wood pulping; treatment of industrial wastes; controlled modification of lignins to produce aromatic chemicals; cracking of petroleum; and detoxification of dangerous and recalcitrant environmental pollutants such as dioxins, polybrominated biphenyls, DDT and benzopyrenes (3,9,25). To obtain a better understanding of the nature, organization, expression and regulation of the lignin peroxidase genes and to develop the full bioprocessing potential of these enzymes, I initiated studies to clone and characterize cDNA sequences encoding lignin peroxidase. Four putative lignin peroxidase cDNA clones have been isolated and a preliminary report was published (34). Detailed characterization of these clones is presented in this chapter. MATERIALS AND METHODS WW 2. ghxyggfipgzium strain BKM-F 1767 (ATCC 24725) was maintained and grown as previously described (15,17) in 50 ml of low nitrogen medium (modified to contain 20 mM NaOAc, pH 4.5, instead of 10 mM 2,2-dimethyl succinate) in 500 ml Erlenmeyer flasks. Flasks were flushed with pure oxygen at the time of inoculation and reflushed every other day. A modified hot phenol extraction procedure was used for RNA isolation (33). Using this procedure, 10 to 20 mg of total RNA was obtained from 1 liter of culture and poly(A) RNA accounted for l to 28 of the total RNA (33). WW 52 The first strand cDNA was synthesized using AMV (avian myeloblastosis virus) reverse transcriptase and the second strand cDNA was synthesized utilizing RNase H and DNA polymerase I of E. 9211 (8). Double stranded cDNA was dC-tailed and then annealed with dG-tailed pUC9 (30) which was digested with restriction enzyme Pgtl. The annealed DNA molecules were transformed into E. 9211 JM83 (gr; lag-219 gtgA Eh; ¢80d1§gZAM15; 30) using the procedure of Hanahan (11). The transformed cells were spread on 2YT plates (1.6% Bacto-tryptone, 1% yeast extract, 0.5% NaCl and 1.5% agar, pH 7.0) supplemented with 100 pg/ml ampicillin and 40 pg/ml X-gal (5-bromo-4-chloro-3-indolyl-fi-D-galactoside; 20). Ten thousand white E. £211 colonies were picked and individually stored in each well of 96- well microtiter plates. 12W Lignin peroxidase has been shown to be produced in 6-day-old idiophasic culture of 2. ghrygggpggium grown in low-nitrogen medium; the enzyme was not detectable in l- or 2-day-old cultures in primary growth (5). Therefore, the differential hybridization technique allows isolation of the cDNA clones specific for the idiophase. It was much easier to screen such a mini-library of idiophasic clones than to screen the total cDNA library for isolating the cloned cDNA of interest. The synthesis of 2- and 6-day cDNA probes from the corresponding poly(A) RNA (2), the preparation of cDNA blots and the cDNA hybridization were previously described (33). Of the 10,000 cDNA clones in the cDNA library, 850 clones were shown to be specific to the 6-day cDNA probe by using this differential hybridization technique. d t o The preparations of blots from the 6-day cDNA mini-library and end- labeled oligodeoxynucleotide probes, and the hybridization were carried 53 out as previously described (33). e n b at o The 2-day and 6-day poly(A) RNA prepared as described above were treated with glyoxal (19) and electrophoresed on a 1.2% agarose gel. The RNA was then transferred from agarose gel to nitrocellulose paper (19) and hybridized with nick-translated probes prepared from the lignin peroxidase cDNA clones. W The cell extract of each potential cDNA clone was made, diluted with TBS buffer (10 mM Tris.HC1 pH 8.0, 150 mM NaCl) and blotted onto nitrocellulose filter paper by the use of Minifold equipment (Schleicher and Schuell, Keene, NH). The filter paper was treated with blocking solution (1% BSA in TBST, which contains TBS plus 0.05% Tween 20) for 30 min and the antibody prepared against lignin peroxidase H8 (1:200 in TBST) was bound to the antigen for 1 h. The filter was washed completely with TBST and then incubated with the anti-IgG alkaline phosphatase conjugate (Promega Biotech, Madison, WI; 1:7,500 in TBST) for 1 h. The filter was then washed with TBST, and was incubated for 15 min in a solution containing 66 pl nitroblue tetrazolium and 33 pl 5- bromo-4-chloro-3-indoly1 phosphate in 10 m1 of alkaline phosphatase buffer (100 mM Tris.HCl pH 9.5, 100 mM NaCl and 5 mM MgClz). Protein concentration was determined as described by Lowry et a1. (18). WW 2. ghxygggpgxium genomic DNA was isolated as previously described by Rao and Reddy (23) except that incubation with lysis buffer was at room temperature rather than at 65°C, and phenol extraction was done only once. The genomic DNA in TE buffer was digested with different restriction 54 enzymes (19), the fragments were separated by electrophoresis on a 0.7% agarose gel, and were then transferred onto nitrocellulose paper (19). The cDNA inserts of pCLG4 and pCLGS were isolated using low melting agarose gel procedure (22), nick-translated (19), and were hybridized with nitrocellulose filter paper containing genomic DNA. RESULTS a a e Three oligodeoxynucleotide probes were synthesized as previously described (34) on the basis of two amino acid sequences of selected tryptic peptides. The sequences of the tryptic peptides of lignin peroxidase H8 and those of the corresponding synthetic oligodeoxynucleotide probes used in identifying the cDNA clones are as follows: Leu-Gln-Lys-Pro-Phe-Va1-Gln-Lys Peptide l4 QUN-CAP-AAP-CCN-UUQ-GUN-CAP-AAP Corresponding mRNA GTQ-TTQ-GGN-AAP-CA Probe 14.1 AAP-CAN-GTQ-TTQ Probe 14.2 Leu-Val-Phe-His-Asp-Ala Peptide 25 QUN-GUN-UUQ-CAQ-GAQ-GCN Corresponding mRNA CAN-AAP-GTP-CTP-CG Probe 25 N - ACCT/U, P - AG, Q - CT/U Four lignin peroxidase cDNA clones (named pCLG3, pCLG4, pCLGS and pCLG6) were identified by screening 850 idiOphase-specific clones from the cDNA library with probe 14.1 and preliminary restriction analyses showed that pCLGB, pCLG4 and pCLG6 were similar to one another, but pCLGS was different from the others (34). 55 To confirm that the four clones contain lignin peroxidase cDNA, recombinant plasmids isolated from these clones were digested with different restriction enzymes and were hybridized with the three oligodeoxynucleotide probes (14.1, 14.2 and 25 shown above). The results (Fig. 1) showed that all three probes hybridized with the cDNA insert in pCLGS whereas only probe 14.1 hybridized with the cDNA inserts from pCLG3, pCLG4 and pCLG6. Each of the three probes hybridized with only one fragment from a given clone, indicating that the probe is specific for the sequence of the particular cDNA fragment showing the homology. Furthermore, the larger cDNA fragment of pCLG5 hybridized with both probes 14.1 and 14.2, which have overlapping sequences, whereas the smaller cDNA fragment hybridized only with probe 25 (Fig. 1, lane 3 in each panel). These results suggested that these four clones contain lignin peroxidase cDNA sequences and that the pCLG5 cDNA is different from the cDNA inserts in the other three clones and encodes the major lignin peroxidase H8. To prove this point further, recombinant plasmid DNA from each of the four clones was probed with 32P-labeled cDNA inserts of pCLG4 and pCLG5 individually. The results ( Fig. 2) showed that the pCLG4 cDNA probe did not show detectable hybridization with the cDNA insert from pCLGS, but showed strong hybridization with that from pCLG3, pCLG4 and pCLG6, respectively. Conversely, the pCLGS cDNA did not show hybridization with that from the other three clones. 56 B 123412341234 Figure 1. Hybridization of lignin peroxidase cDNA clones with three synthetic oligonucleotide probes. Clones pCLG3 and pCLG4 were digested with MRI and HindIII, pCLG5 was digested with MHI, _H_i_ndIII and MI, and pClG6 was digested with flingII and Eggl. Southern hybridization of these restriction digests with the three oligonucleotide probes 14.1 (panel A), 25 (panel B) and 14.2 (panel C) is presented. Different lanes contained: pCLGS (lane 1), pCLG4 (lane 2), pClGS (lane 3) and pCLGé (lane 4). 57 123412341234 Figure 2. Cross-hybridization between the two groups of cDNA clones. All the four cDNA clones were digested with EQQHI and filngII, fractionated on 1% agarose gel (Panel A), transferred onto nitrocellu- lose filter paper and hybridized with probe made from the pCLG4 cDNA insert (Panel B). After exposure to the X-ray film, the pCLG4 probe was removed and the filter was hybridized with the second probe made from the pCLGS cDNA insert (Panel C). Lanes 1-4 in each panel contained pCLG3, pCLG4, pCIGS and pCLG6, respectively. The size marker, flingII- digested lambda DNA, is shown in Panel A at the extreme left. 58 3W2; Detailed restriction maps of the cDNA inserts of pCLG4 and pCLG5 (Fig. 3) clearly show that these cDNA inserts are different from each other. The restriction maps of the cDNA inserts in clones pCLG3 and pCLG6 were identical to that of pCLG4. The estimated sizes of the cDNA inserts in pCLGB, pCLG4, pCLG5 and pCLG6 were 1.35 kb, 1.42 kb, 1.48 kb and 1.17 kb, respectively. To delineate which cDNA fragment is homologous to the synthetic oligodeoxynucleotide probe 14.1 or 25, pCLG4 and pCLGS were digested with appropriate restriction enzymes (based on the restriction maps and hybridization results), and the DNA blots were hybridized with the synthetic probes. The results showed that the flindIII-AXQI fragment of pCLG4 cDNA insert and Pstl-EQQRI fragment of pCLG5 cDNA insert hybridized to probe 14.1 and the Egtl-SstII fragment of pCLGS hybridized to probe 25 (Fig. 3). 59 pCLG4 H P PSBAE .A K v 0' 9‘6 l H Sm S I! P P Ho .aJEZ%- ‘V ‘V " ' *L4y '* S'""’ poly(A) tall Probel4.l pCLGS H P m PSBAE v e * H X 11 P - *6 “L136 *0 *Ho '10 SE poly(Ahoil a----., Probe4.l‘_____’ Probe25 Figure 3. Restriction maps of the cDNA inserts in pCLG4 and pCLGS. The wavy lines represent vector sequences and the straight lines represent cDNA inserts. The boxes and arrows represent the promoter of the LacZ gene of E. c011 and direction of transcription, respectively. Abbreviations used for the restriction enzymes shown in the figure are: A-Axal, B-BagHI, EeEQQRI, H-HindIII, Ha-Hggll, Hc-fligcll, K-KQQI, P- Eggl, S-figlI, SI-figgl, SII-fistll, Sm-fimgI and X-thl. 60 WW Previous studies showed that high levels of lignin peroxidase were produced in low nitrogen medium on the 6th day of incubation (i.e. during secondary metabolism), whereas little or no lignin peroxidase was produced on the second day of growth (i.e. during primary growth) in the same medium (5). Therefore, mRNA corresponding to the isolated lignin peroxidase cDNA should not be detectable in 2-day-old 2. ghgygggpgxium cultures grown in low nitrogen medium but should be present in otherwise identical 6-day-old cultures. To test this, poly(A) RNA isolated from 2- and 6-day-old cultures of 2, ghxysgspgxium grown in low nitrogen medium was probed with the four 32P-labeled lignin peroxidase cDNAs. Another cDNA clone (p33-B10), which was shown to hybridize with the cDNA probes made from both 2- and 6-day poly(A) RNA in the differential hybridization experiment (see Methods and Materials) was used as a positive control. The results showed that the four cDNA clones hybridized only with mRNA from the 6-day-old cultures (Fig. 4), but not with that from 2-day-old cultures, indicating that lignin peroxidase Synthesis is controlled at the level of mRNA production (or perhaps at the level of mRNA stability). The mRNA that hybridized with the plasmid DNA from each clone is very similar in size (~1.6 kb), consistent with the fact that molecular weights of different lignin peroxidases are comparable. 61 Figure 4. Northern hybridization analysis. Glyoxal-treated 2-day (lanes 1) and 6-day (lanes 2) poly(A) RNA were electrophoresed on a 1.2% agarose gel as described in Materials and Methods. The Northern blots were hybridized with p33-B10 (A), pCLG3 (B), pCLG4 (C), pClGS (D), pCLG6 (E), and pUC9 (negative control, F). The cDNA clone p33-B10 was used as a positive control, since it was shown to hybridized with 2-day and 6- day cDNA probes in differential hybridization experiments previously described (33). The 1 kb ladder DNA (BRL, Gaithersburg, MD) was used as the RNA size marker. 62 1W Since the cDNA was constructed into the E. 9211 expression vector, pUC9, the possibility existed that lignin peroxidase cDNA might be expressed in E. 9911 if the insert was in the right orientation and frame. To determine if there is expression of lignin peroxidase in any of the four lignin peroxidase cDNA clones, cell extracts and 50 X concentrated extracellular fluid from each clone were subjected to an enzyme-linked immunoblot assay as described in Materials and Methods. None of the extracts reacted with the lignin peroxidase antibody, indicating that the lignin peroxidase cDNA was not being expressed due to wrong orientation, and/or reading frame or due to some other reasons that are not clear at this time. To determine the orientation of the cDNA insert in clones pCLG4 and pCLG5, end-labeled oligo(dT)12_18 was used to probe the two cDNA clones, which were digested with different restriction enzymes. The results showed that the cDNA insert in pCLGS was in the opposite orientation to the promoter of the lggz gene in pUC9, whereas in pCLG4 it was in the correct orientation. To change the orientation of the cDNA fragment in pCLGS, the 1.48 kb figmHI-flindlll cDNA fragment was isolated and recloned into another expression vector pUC18 (21). Extracts of four new pUC18 cDNA clones (pCLG5-1, pCLGS-Z, pCLGS-3 and pCLG5-4) were then used in the immunoblotting assay to detect the presence of lignin peroxidase antigen.» The results showed that one of the new cDNA clones (pCLGS-l) produced antigen which gave a strong positive reaction with the lignin peroxidase antibody (Fig. 5; row D) whereas the other clones gave weak (pCLG5-2) or no reaction (pCLG5-3 and pCLGS-4). Comparison of the color intensity obtained with different concentrations of cell extract from 63 the new cDNA clone (pCLG5-1) with that of the purified lignin peroxidase showed that the antigen produced by pCLGS-l accounted for about 0.1% of the total protein. Identical results were obtained in five separate experiments. The reason for the lack of expression of lignin peroxidase in other clones is not known. 64 Figure 5. Immunoblotting assay for detecting lignin peroxidase production by different cDNA clones. Different lanes contain: purified lignin peroxidase H8 produced by 2. ghrxgggngzigm (row A); and cell extracts of cDNA clones pUC18 (negative control; row B), pClGS (row C), pCLG5-l to 4, which are the four new clones containing the insert from pCLGS and pUC18 (21), (rows D to G); and pCl£4 (row H). The purified lignin peroxidase concentrations in each lane of row A are: l) 0.05 ng; 2) 0.1 ng; 3) 0.2 ng; 4) 0.5 ng; 5) 1 ng; 6) 2 ng; 7) 5 ng; 8) 10 ng; and 9) 20 ng. The protein concentration in cell extracts of different clones was adjusted to contain in each lane: 1) 20 ng; 2) 50 ng; 3) 100 ng; 4) 200 ng; 5) 500 ng; 6) 1,000 ng; 7) 2,000 ng; 8) 5,000 ng; and 9) 10,000 ng. 65 u h d t 0 Anal sis The above results lead to the following question: do the poly(A) RNA species, which hybridized with the lignin peroxidase cDNA, represent a single transcript with post-transcriptional modification from one gene or transcripts from different genes? In other words, do the cDNA inserts in pCLG4 and pCLGS hybridize to different or identical segments of chromosomal DNA? To answer this question, the genomic DNA of E. Chrysgspgrium BKM-F 1767 was isolated and digested with different restriction enzymes, some of which have sites in the cDNA inserts of the clones of interest and some of which have no sites in the inserts. The Southern hybridization of chromosomal DNA of 2. ghxyggspgzium with 32P- labeled cDNA inserts of pCLG4 and pCLG5 showed that the cDNA inserts of pCLG4 and pCLG5 represent different lignin peroxidase genes (Fig. 6). The Southern hybridization pattern observed with pCLG4 cDNA was consistent with the restriction map of the latter except for the ngl and thl digestion (Fig. 6, panel A), which do not have a site in the pCLG4 cDNA (Fig. 3). It is possible that the gene corresponding to pCLG4 cDNA may have introns or the gene sequences on homologous chromosomes may be different somewhat. The most recent results (see Chapter 3) show that there is an thI site in one of the introns of a lignin peroxidase gene that showed strong hybridization with pCLG4 and had many similarities to the restriction map of pCLG4. In contrast to these. results, the Southern hybridization patterns shown by the different restriction digests of the chromosomal DNA with the pCLGS cDNA probe were different from what one would have expected from the bands on the restriction map of pCLG5 (Fig. 6, panel B). For example, there is no figmHI site in the pCLGS insert, but the fiamHI-digested chromosomal DNA 66 showed four hybridization bands with different intensities. These bands may be due to different lignin peroxidase genes which are homologous to the pCLGS cDNA insert, or to the existence of introns in the lignin peroxidase gene, or to restriction polymorphism on the two homologs. In addition, there were several weak hybridization bands in each lane (Fig. 6, panel B). Restriction digests of genomic DNA of P. ghgyggspggium strain ME 446 (another commonly used strain in lignin biodegradation studies) were also probed as described above. Both pCLG4 and pCLG5 cDNA inserts hybridized to distinct fragments of ME 446 genomic DNA, although once again hybridization patterns observed with the two probes were different, suggesting that pCLGS and pCLG4 indeed represent different lignin peroxidase genes (Fig. 7). Furthermore, the patterns of hybridization obtained with ME 446 genomic DNA (Fig. 7) were different from those observed with strain BKM-F 1767 (Fig. 6), indicating that there are differences between the genomic DNA of different strains of P. ghryggspgrigm. It is also of interest that ME 446 appears to contain at least two extrachromosomal DNA elements that hybridize strongly with the pCLG4 cDNA insert (Fig. 7, lane 1, panel A). These elements also hybridized strongly with pBR322 (data not shown). These plasmid-like elements are being studied further. 67 A B 123453333123456733 23.1— " 6.7—- 4.4—,, . . if)“ ‘ o - " ”g ‘! {ass—v i.” _9 .1- , , ~ ~1 Figure 6. Southern hybridization analysis of strain BKM-F 1767. The genomic DNA isolated from 2-day-old cultures of 2. ghgysgspgrium BKM-F 1767 was digested with figmHI (lanes A2 and B2), EggRI (lanes A3 and B3), ngI (lanes A4 and B4), zygII (lanes A5 and BS), figll (lanes A6 and B6), SmgI (lanes A7 and B7), ngI (lanes A8 and B8) and zhgl (lanes A9 and B9), and electrophoresed on a 0.7% agarose gel. The fractionated genomic DNA was transferred onto nitrocellulose paper and probed with the cDNA insert of pCLG4 (panel A). After exposure of the blot to the X- ray film, the probe was washed off and the blot was rehybridized with pCLG5 cDNA insert (panel B). Lanes Al and Bl contained uncut genomic DNA. The size marker used was flindIII-digested lambda DNA. 68 A 123456 7 8 9 "'1; 'fi-Y 46" 'wmr B _123456733 23.13— 3.42- 5.63—' Q P 4.35— 0 C «a . o . 4 0. ea. I C £23 : . . . 0.56— Figure 7. Southern hybridization analysis of strain ME 446. The genomic DNA isolated from 2-day-old cultures of 2. Chrysosporigg ME 446 was digested with BQQHI (lanes A2 and B2), EgQRI (lanes A3 and B3), Eggl (lanes A4 and B4), EEQII (lanes A5 and BS), §§ll (lanes A6 and B6), fimgI (lanes A7 and B7), XQQI (lanes A8 and B8) and thI (lanes A9 and B9), and electrophoresed on a 0.7% agarose gel. The fractionated genomic DNA was transferred onto nitrocellulose paper and probed with the cDNA insert of pCLG4 (panel A). After exposure of the blot to the X-ray film, the probe was washed off and the blot was rehybridized with pCLGS cDNA insert (panel B). Lanes Al and B1 contained uncut genomic DNA. The size marker used was HingII-digested lambda DNA. 69 DISCUSSION The results of this study strongly suggest that pCLG5 is a lignin peroxidase cDNA clone and contains the sequence coding for lignin peroxidase H8 based on the following lines of evidence. 1) The pCLGS cDNA insert showed hybridization with three synthetic oligodeoxynucleo- tide probes, the sequences of which were deduced from the amino acid sequences of selected tryptic peptides of the lignin peroxidase. Each of the probes used represents a mixture of 32 oligodeoxynucleotides, only one of which has perfect homology with unique sequence of the cloned cDNA; the other oligodeoxynucleotides in the mixture have at least one base mismatched with the cloned cDNA. Under high strigent hybridization conditions, only the oligodeoxynucleotide with perfect homology can hybridize with the lignin peroxidase cDNA. In the Southern hybridization experiments, the washing temperature employed was based on the equation presented by Suggs et al. (26). According to this equation, the estimated T which is the temperature at which one-half d’ of the duplexes are dissociated under the experimental conditions, is 36 to 44°C, 38 to 46°C and 23 to 36°C for probes 14.1, 25 and 14.2, respectively. The actual washing temperature used in the experiments was 42°C for probes 14.1 and 25, and 32°C for probe 14.2. Under these conditions, nonspecific hybridization could not exist because the duplexes with one mismatched basepair dissociate at a temperature about 10°C lower than the perfectly matched duplexes (26,31,32). Thus, the hybridization data, using the synthetic probes, strongly support the identification of the cDNA clones. 2) Northern hybridization experiments showed that the cDNA from this clone hybridized only with 70 the 6-day poly(A) RNA from ligninolytic cultures of P. chrysosporium grown in low nitrogen medium but not with the poly(A) RNA from 2-day cultures (i.e. prior to the onset of lignin degradation) grown in the same medium. This is consistent with previous observations that lignin peroxidase is produced only during secondary metabolism (14,16). This result also suggests that the lignin peroxidase gene is regulated at the level of mRNA production although regulation at the level of mRNA stability cannot be ruled out. 3) The immunoblotting data showed that the product of the cDNA insert in pCLGS gave a strong reaction with the antibody raised against lignin peroxidase H8 purified from 2. ghxygggpgxigm BKM-F 1767. 4) The complete nucleotide sequence of pCLGS cDNA insert has now been determined and the amino acid sequence of the corresponding lignin peroxidase protein (designated L65) has been deduced (see Chapter 2). Mature lignin peroxidase LGS contains 344 amino acid residues and is preceded by a leader sequence containing 27 amino acid residues. Amino acid sequences of two tryptic peptides of lignin peroxidase H8 have exactly matching sequences in L65. The experimentally determined N-terminal sequence of Ala-Thr-Cys-Ser-Asn-Gly-Lys-Val-Val- Pro is found in the deduced N-terminal amino acid sequence of mature LG5 (Dass and Reddy, unpublished data). These results indicate that pCLGS cDNA encodes lignin peroxidase H8. 0n the basis of the hybridization with three synthetic oligodeoxynuc- leotide probes, restriction analysis, cross-hybridization and chromosomal DNA hybridization, clone pCLG4 represents a second group of lignin peroxidase cDNA clones consisting of pCLG3, pCLG4 and pCLG6. The cDNA inserts of this group of clones strongly hybridized with the synthetic oligodeoxynucleotide probe 14.1, and hybridized only with the 6-day poly(A) RNA from ligninolytic culture of P. ghxygggpggium grown in 71 low nitrogen medium, suggesting that pCLG4 may also contain the sequence coding for lignin peroxidase. Nucleotide sequence of pCLG4 cDNA has recently been determined and the amino acid sequence of the corresponding lignin peroxidase protein (designated LG4) has been deduced (Chapter 2). Mature LG4 also contains 344 amino acid residues with an Mr of 36,540, and is preceded by a leader sequence of 28 amino acid residues. The sequences of probes 14.1 and 25, except for one base pair mismatch, are found in pCLG4 cDNA. The experimentally determined N- terminal sequence of Val-Ala-Cys-Pro-Asp-Gly-Val-His-Thr-Ala-Ser-Asn found in lignin peroxidase H2 exactly matches the N-terminal amino acid sequence of mature LG4 (Dass and Reddy, unpublished data). Furthermore, pCLG4 cDNA has a high degree of homology (71.5%) to that of pCLGS cDNA. These data indicate that pCLG4 cDNA encodes another lignin peroxidase protein. The hybridization of chromosomal DNA with pCLG4 and pCLGS definitely shows that multiple lignin peroxidase genes are present in 2. ghgygggpgrigm and the cDNA inserts in these two clones are from different lignin peroxidase genes (Fig. 6, lane 2 in panel B). The genomic DNA, in addition to the one lignin peroxidase gene corresponding to pCLGS, appears to contain several lignin peroxidase genes whose sequences are homologous to pCLGS cDNA sequence since different restriction digests of chromosomal DNA showed several bands of hybridization of variable intensities with 32P-labeled pCLGS cDNA. This is best illustrated by the hybridization of BamHI-digested genomic DNA with pCLG5 cDNA. Tien and Tu recently reported on the isolation of another lignin peroxidase cDNA clone (AMLl) from a A-cDNA library of 2. ghxysggpggium (29). In agreement with the results of this study, Tien and Tu concluded 72 that lignin peroxidase gene expression is regulated at the level of mRNA production. The AMLl cDNA, comparable to pCLG4 and pCLG5 cDNA, encodes a mature lignin peroxidase that contains 345 amino acids and a leader sequence that contains 28 amino acids, which are predominantly hydrophobic. Tien and Tu (29) apparently were able to isolate yet another lignin peroxidase cDNA clone from 2. ghxygggpggium. These data offer further support to our idea that 2. ghxygggpggign contains multiple lignin peroxidase genes. ACKNOWLEDGMENTS We wish to thank T. K. Kirk and M. Tien for supplying lignin peroxidase antibody and the fungal strains used in this study. This work was supported in part by grant DE-FGO2-85ER13369 from the U.S. Department of Energy, Division of Basic Biological Sciences, and NSF grant DMB-841271. We acknowledge S. B. Dass for purifying lignin peroxidase H2 and H8 and providing us the partial N-terminal sequences. 10. 11. 12. 73 REFERENCES Andersson, L. A., V. Renganathan, A. A. Chiu, T. M. Loehr and M. H. Gold. 1985. 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G. Zeikus. 1978. Influence of culture parameters on lignin metabolism by Phagergghgegg ghgygggpgzium. Arch. Microbiol. 117:277-285. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Maniatis, T., E. F. Fritsch and J. Sambrook. 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Miller, J. H. 1972. Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Norrander, J. T. Kempe and J. Messing. 1983. Construction of improved M13 vectors using oligodeoxynucleotide- -directed mutagenesis. Gene 26:101-106. Perbal, B. 1984. A Practical Guide to Molecular Cloning. John Wiley and Sons, N.Y. Rao, T. R. and C. A. Reddy. 1984. DNA sequences from a ligninolytic filamentous fungus Phanereshaete £hII§2§EQIiBE capable of autonomous replication in yeast. Biochem. Biophys. Res. Commun. 118:821-827. Renganathan, V., K. Miki and M. H. Gold. 1985. Multiple molecular forms of diarylpropane oxygenase, an H 202- requiring, lignin degrading enzyme from Phenerashaete shrxs_snerism Arch. Biochem. Biophys. 241: 304- 314. Sanglard, D., M. S. A. Leisola and A. Fiechter. 1986. Role of extracellular ligninases in biodegradation of benzo(a)pyrene by Phanerochaete, ghxygggngxium. Enz. Microb. Technol. 8:209-212. Suggs, S. V., T. Hirose, T. Miyake, E. H. Kawashima, M. J. Johnson, 27. 28. 29. 30. 31. 32. 33. 34. 75 K. Itakura and R. B. Wallace. 1981. Use of synthetic oligodeoxyribonucleotides for the isolation of specific cloned DNA sequences. ICN-UCLA Symp. Mol. Biol. 23:683-693. Tien, M. and T. K. Kirk. 1983. Lignin-degrading enzyme from hymenomycete Ehgggxgghgetg ghrygggpgrigm Burds. Science 221:661- 663. Tien, M. and T. K. Kirk. 1984. Lignin-degrading enzyme from Phanergghagte ghrysgsngrium: purification, characterization, and catalytic properties of a unique H 0 -requiring oxygenase. Proc. Natl. Acad. Sci. U.S.A. 81:2280-2383. Tien, M. and C.-P. D. Tu. 1987. Cloning and sequencing of cDNA for e ligninase from Phanereshaete shrxseseerisa. Nature (London) 326:520-523. Vieira, J. and J. Messing. 1982. The pUC plasmids, a Ml3mp7- derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268. Wallace, R. B., M. J. Johnson, T. Hirose, T. Miyake, E. Kawashima and K. Itakura. 1981. The use of synthetic oligonucleotides as hybridization probes. II. hybridization of oligonucleotides of mixed sequence to rabbit fi-globin DNA. Nucl. Acid Res. 9:879-894. Wallace, R. B., J. Shaffer, R. F. Murphy, J. Bonner, T. Hirose and K. Itakura. 1979. Hybridization of synthetic oligodeoxyribo- nucleotides to ¢xl74 DNA: the effect of single base pair mismatch. Nucl. Acid Res. 6:3543-3557. Zhang, Y. Z. and C. A. Reddy. 1988. Use of synthetic oligonucleotide probes for identifying ligninase cDNA clones. Meth. Enzymol. vol.l68 (in press). (see Appendix B) Zhang, Y. 2., G. J. Zylstra, R. H. Olsen and C. A. Reddy. 1986. Identification of cDNA clones for ligninase from Ehgngzgghggtg ghgygggpgziun using synthetic oligonucleotide probes. Biochem. Biophys. Res. Commun. 137:649-656. (see Appendix A) 76 CHAPTER TWO ANALYSIS OF NUCLEOTIDE SEQUENCES OF TWO LIGNIN PEROXIDASE cDNAs FROM W W 77 ABSTRACT The complete nucleotide sequences of two types of lignin peroxidase cDNAs isolated from the white-rot fungus Phanerochaete ghrysosporigm, designated CLG4 and CLGS, are presented here. The amino acid sequences of the corresponding lignin peroxidase proteins, designated LG4 and LGS, respectively, have been deduced from the cDNA sequences. Mature lignin peroxidases LG4 and LGS are preceded by leader sequences containing 28 and 27 amino acids, respectively, and each contains 344 amino acid residues. The estimated molecular weights of mature LG4 and LGS are 36,540 and 36,607, respectively. Potential N-glycosylation site(s) with the general sequence Asn-X-Thr/Ser are found in both LG4 and LGS. Nucleotide sequence homology between the coding region of CLG4 and CLGS is 71.5% whereas the amino acid sequence homology between the two lignin peroxidases is 75%. The codon usage of lignin peroxidases is extremely biased in favor of codons rich in cytosine and guanine. Amino acid sequences of two tryptic peptides of lignin peroxidase H8 have exactly matching sequences in lignin peroxidase 185. Also, the sequences of the oligonucleotide probes, which correspond to the sequences in the tryptic peptides of lignin peroxidase H8 and which were used in isolating the lignin peroxidase clones from the cDNA library, have exactly matching sequences in CLGS. The experimentally determined N-terminal sequences of purified lignin peroxidases H8 and H2 are found in the deduced N- terminal amino acid sequences of mature LGS and LG4, respectively. These results indicate that CLGS encodes lignin peroxidase H8 and that CLG4 encodes lignin peroxidase H2. 78 INTRODUCTION Lignin is a major component of woody plants and is the most abundant and widely distributed renewable aromatic polymer on earth (20,23). The wood-decaying basidiomycete, Ehgggzgghgggg ghxygggpgrigg degrades lignin more rapidly and extensively than most other organisms (12,20). Lignin degradation by this organism is a secondary metabolic event that is triggered in response to nitrogen, carbon, or sulfur starvation (12,20). 3. ghgygggpgxiun produces multiple extracellular, glycosylated heme peroxidases named lignin peroxidases that oxidatively cleave lignin and related compounds in an H -dependent reaction (8,9,13,21,24). It has 202 been suggested that lignin peroxidases also detoxify recalcitrant xenabiotics such as dioxins, DDT and polychlorinated biphenyls (4,6). Lignin peroxidase activity is demonstrable in 6-day-old idiophasic cultures grown in low nitrogen medium but not in 2-day-old nonligninolytic cultures grown in the same medium (7,13). At least six different lignin peroxidases (H1, H2, H6, H7, H8 and H10) have been identified in the extracellular fluid of P. ghgygggpgziug (13). Proteins H2 and H8 constitute the major lignin peroxidases both in static and shaken cultures of this organism (13). The reported molecular weights of lignin peroxidases vary from 39,000 to 43,000 (9,21,24). A cDNA library has recently been constructed in the Pgtl site of E. 9211 vector pUC9 (29), representing 6-day-old lignin-degrading culture of 2. ghrygggpggigm. Two different types of lignin peroxidase cDNA clones, pCLG4 and pCLGS, have been identified by probing the cDNA library with a l4-nuc1eotide-long oligonucleotide probe that corresponds to the sequence of a tryptic peptide of lignin peroxidase H8. Clone 79 pCLGS, but not pCLG4, also hybridized to probe 14.2 (which has partial overlap with probe 14.1) and probe 25, that corresponds to a sequence in another segment of lignin peroxidase H8 (see Chapter 1). Northern blot analyses showed that the cDNA clones hybridized with the poly(A) RNA extracted from a 6-day-old ligninolytic culture but not with that from a 2-day-old non-ligninolytic culture, suggesting that lignin peroxidase production is regulated at the level of transcription. The two types of clones exhibit little cross-hybridization to each other and have different restriction maps. Furthermore, Southern blots of chromosomal DNA probed with the two types of cDNAs gave very different hybridization patterns. These results suggested that the cDNA inserts in pCLG4 and pCLG5 represent different lignin peroxidase genes and that a family of lignin peroxidase genes is present in 2. ghrygggngxium. In this chapter, the complete nucleotide sequences of these two lignin peroxidase cDNA clones and the predicted amino acid sequences are presented. 80 MATERIALS AND METHODS cDN e ue Lignin peroxidase cDNA clones pCLG4 and pCLGS were isolated from the cDNA library [cloned into the £§§I site of E. 9211 vector pUC9 (26)], representing poly(A) RNA from a 6-day-old lignin degrading culture of g. ghgygggpgrium strain BKM-Fl767 (ATCC 24725), as previously described (29). The lignin peroxidase cDNAs in clones pCLG4 and pCLGS are designated CLG4 and CLGS, respectively. The DNA sequence was determined by the dideoxy chain-termination method (22) and the sequencing strategy employed for the two lignin peroxidase cDNAs is shown in Figure 1. Specific fragments were isolated and subcloned in the appropriately digested M13 vectors. The plasmid pCLG5 was cut with ngl-figgl and the resulting fragments (219 bp, 388 bp, and 671 bp) were subcloned in Ml3mp18 or Ml3mp19 (18,28). The sequence of the distal parts of the longer clones was determined using several synthetic primers designed on the basis of determined proximal sequences. Based on the preliminary sequence of both clones, several primers were designed and used to sequence the entire complementary strand. The primers bind to pCLGS- and pCLG4-M13 derivatives at intervals of about 200 bases. Thus, sequencing ambiguities in both clones, resulting from compressions due to GC-rich regions, could be resolved by substituting dITP (16) or deoxy-7-deazaguanasine triphosphate in place of dGTP (17). 81 CLG4 A I - a MC: H S ; P P #- L 1 I“ 1 Jr 4 “11:5. a ':-—-“ t 1. e r: CLG 5 g r E = 1 ”A? f 1? "5° Figure 1. Strategy for determining the sequences of lignin peroxidase cDNAs CLG4 and CLGS. The straight arrows indicate sequencing from restriction sites and the arrows starting with a wavy line indicate sequencing from a synthetic primer. Abbreviations used for the restriction enzymes shown in the figure are: E-EQQRI, H-flindIII, P-zgtl, Sm-fimgI, and X-thl, MCS, multiple cloning sites. 82 RESULTS Nucleotide Sequences and nguced Amino Acid Seguences The complete nucleotide sequences of CLG4 and CLGS and the predicted amino acid sequences of the gene products (designated LG4 and LGS, respectively) are shown in Fig. 2. CLG4 and CLGS contain 1263 and 1285 bases, respectively, not including the terminal poly(A) sequences. The coding region of CLG4 is flanked by 33 base pairs (bp) in the 5'- noncoding region and 111 base pairs in the 3'-noncoding regions, whereas CLGS coding region is flanked by 33 bp and 133 bp in 5'- and 3'- noncoding regions, respectively. Both cDNA sequences contain a relatively high G+C content (60.2% and 65.5% for CLGS and CLG4, respectively) in the coding region, but a much lower G+C content (44.4% and 48.7% for CLGS and CLG4, respectively) in the 3'-noncoding regions. The sequence 5'-GACATGG flanking the ATG codon in CLGS is consistent with the translation initiation sequence of (A/G)NNATGG commonly seen in other eukaryotic genes (1). Genes from filamentous fungi generally have one or more polyadenylation sites. Higher eukaryotic genes possess the polyadenylation signal AATAAA (2) though this is rarely present in yeast genes or genes from filamentous fungi (1). However, the lignin peroxidase cDNA clones from 2. ghzygggpgrigm contain the sequence AATATA in CLG4 and AATACA in CLGS, 14 bp upstream of the poly(A) addition site (Fig. 2). 83 Figure 2. The complete nucleotide sequences of the lignin peroxidase CLG4 (A) and CLGS (B). The predicted amino acid sequences which are identical to those of tryptic peptides of lignin peroxidase H8 (29) are underlined (solid lines) while the nucleotide sequences complementary to the oligonucleotide probes 25 and 14.1 are noted with dashed lines. Nucleotide sequence of probe 14.2 is marked with a dotted line on the top. In panel A, the nucleotide sequences of probes 25 and 14.1 are identical to that in pCLG4 except at the positions marked with an asterisk. Wavy line in each panel represents putative signal peptide processing region. The possible glycosylation sites are boxed. The putative polyadenylation sequences near the 3' end are underlined. 841 gmm oo- son n-~ can owed eds who awn coo son nwm ofiu Own «AN nub om~ oo ‘0 361 man mm~ One c- mum om 00m so nmm om end OIIIIIIIIOIOIIIIlIIII!Illllllllllvll|lllllllllllillIOIIC'lIIII <<<<<<<<< ocoeuooaoouseuas=osmasuomcsummm14ogauom:soats:aoasonaogmusag~ouomaauomnanna=oanao=oaaaoauuuu«waszonaouaaaooumsn>=oaa~oamaa< <6ooaaoms=m seaos¢>onmouma scaoaosmuoa~=ae ouwhocubesoooe ososuu:aaa>assxaoc4a~aaesaa=osassmaaozauau6m¢aunassundeadHaaouauuuaa~oouaossuaauecaugaaaooaHaaoau:aoa.a~o~.>n~saoaa=naanaaasaaoaaeaosaso <0ooeoumonmauanaxaoamwuuomsosua1.5 (hydrophobic regions) in various portions of LG4 and LGS probably represent sequences passing through the interior 87 of the protein (14). The regions comprising amino acids 221-233 in LGS and 224-236 in LG4 are strongly hydrophilic, have identical sequences except in one position (see Fig. 4), and contain one proline residue and thus have features of a desirable immunogenic peptide (15) for potentially raising common antibodies against lignin peroxidases. Similarly, another eleven amino acid region, from residues 168 to 178 in LG4 and 165-175 in LGS, has identical sequence, is strongly hydrophilic, and therefore is also likely to be a suitable antigenic peptide for raising lignin peroxidase antisera. A high degree of amino acid sequence homology observed between stretches of CLG4 and CLGS flanking the above two regions is consistent with this idea. 4.0 30 88 .l "11'r " H 1” WW!“ LTh ‘ =J_— . - -.A_.__a 250 300 30 372 $1 _t—F— ‘ ’1... \1 '1'" 1 Figure 3. LG4 (A) and LGS ea- .10--- am Comparison of the hydropathy plots of two lignin peroxidases (B). 89 QLdsszLsazs The codon usage of both lignin peroxidases is extremely biased in favor of codons rich in C and/or G residues (Table l). The order of preference of the third (wobble) base within each codon family is C > G > T > A in both the lignin peroxidase cDNA clones. This rule is strictly obeyed except for the codons starting with CG and GG (Arg and Gly, respectively). In these two cases CGG and GGG are not used at all. In all codons ending with a pyrimidine, a C-residue is highly preferred, with the exception of ACT (Ser) and GCT (Ala) in LGS. Codons ending in purine preferably use a G- over an A-residue at the third position. Twelve codons, including CGG and CGG, are not used in either lignin peroxidase cDNA. The overall codon usage in OLGA and CLGS is not much different from that of the highly expressed genes of another eukaryote, 5. ggrgxisiag (3), in which biased codon usage occurs exclusively in the highly expressed genes. In Eggzggpggg and Agpgggillgg, there is a tendency to avoid usage of codons ending in A and to prefer using codons ending in pyrimidines (particularly C), especially in highly expressed or constitutive genes (see ref.l). Thus, the highly biased codon useage of CLGh and CLGS is likely to reflect their high expression levels as well. 90 Table 1. Codon Usage in Lignin Peroxidase cDNAs 0L64 and CLGS AAs Codons LGS LG4 AAs Codons LGS LG4 Phe TTT O 1 Ser TOT 8 2 TTC 28 26 TCC 11 14 Leu TTA 0 0 TCA O 0 TTC 1 1 TCG 6 7 CTT 4 A Pro CCT 6 l CTC 18 16 CCC 14 15 CTA 0 0 CCA 3 O CTC 3 5 CCG 10 15 Ile ATT l l Thr ACT 8 3 ATC 19 20 ACC 10 11 ATA 0 0 ACA 0 0 Met ATG 9 8 ACC 2 11 Val CTT 4 1 Ala GCT l6 8 CTC 17 17 GCC 15 17 GTA 0 0 GCA 2 7 GT6 2 3 GCG ll 12 Tyr TAT 0 0 Cys TOT 1 1 TAC 1 0 TGC 7 7 0C TAA 1 1 OP TGA 0 0 AM TAG O O Trp TGG 3 3 His CAT 2 0 Arg CCT 5 4 CAC 4 11 CGC 5 6 Gln CAA 2 l CGA 1 O CAG 18 22 CGG 0 0 Asn AAT l 0 Ser ACT 2 0 AAC 15 11 ACC 0 1 Lys AAA 0 O Arg AGA O O AAG 8 ll AGG O O Asp CAT 8 3 Gly GGT ll 9 GAO 15 19 GGC 16 22 Glu GAA 1 O GGA 1 0 GAG 17 14 CGG 0 0 91 C uc eo ide and educed Amino Ac d Se uences There are a number of similarities between the two lignin peroxidases. Both contain almost the same number of amino acid residues and both are very similar in amino acid composition (Table 2). A comparison of the amino acid sequences of the two lignin peroxidases shows a very high percentage of homology (75%) between the two lignin peroxidases (Fig. 4); the longest stretch of identical sequence between the two lignin peroxidases is a 13 amino acid region from residues from 275 to 287 in LG4 and 272 to 284 in LGS (Fig. 4). Furthermore, hydropathy plots of the two lignin peroxidases look very similar (Fig. 3). A 71.5% homology was observed between the nucleotide sequences of CLG4 and CLGS in the coding regions (Fig. 5). The longest stretch of homology spans 20 nucleotides from 724 to 743 in CLG4 and 715 to 734 in CLGS. However, there are some differences between the two lignin peroxidase cDNA clones. For example, CLG4 uses fewer T-residues and more C- and G- residues in the third base of various codons than CLGS (Fig. 2 and Table 1), resulting in very short stretches of identical nucleotide sequence between the two cDNA fragments. This may explain why no cross- hybridization is detectable between CLG4 and CLGS when nick-translated probes from the respective cDNA were used. Besides, the degree of homology is low in the 5'- and 3'-noncoding regions in CLG4 (1-33 and 1153-1153, Fig. 2A) and CLGS (1-33 and 1150-1285, Fig. 23). 92 Table 2. Comparison of Amino Acid Composition in LG4 and LGS AAs 1.9.4. 1.9.5. Number Percentage Number Percentage Ala 44 11.83 44 11.86 Arg 10 2.69 11 2.96 Asn 11 2.96 16 4.31 Asp 22 5.91 23 6.20 Cys 8 2.15 8 2.16 Gln 23 6.18 20 5.39 Glu 14 3.76 18 4.85 Gly 31 8.33 28 7.55 His 11 2.96 6 1.62 Ile 21 5.65 20 5.39 Leu 26 6.99 26 7.01 Lys 11 2.96 8 2.16 Met 8 2.15 9 2.43 Phe 27 7.26 28 7.55 Pro 31 8.33 33 8.89 Ser 24 6.45 27 7.28 Thr 25 6.72 20 5.39 Trp 3 0.81 3 0.81 Tyr l 0.27 0 0.00 Val 21 5.65 23’ 6.20 93 LG4 LGS LG4 LGS LG4 LGS LG4 MAFKKLLAVLTAALSLRAAQGAA--VEKRATCSNGKVVP---AASCCTWFNVLSDIQENLFNGGQ MAFKQLLAALSVALTLQVTQ-AAPNLDKRVACPDG-VHTASNAA-CCAWFPVLDDIQQNLFHGCQ CGAEAHESIRLVFHDAIAISPAMEPQASSVR- GADGSIMIFDEIETNFHPNIGLDEIVRLQKPFV CGAEAHEALRMVFHDSIAISPKLQSQGKFGGGGADGSIITFSSIETTYHPNIGLDEVVAIQKPFI QKHGVTPGDFIAFAGAVALSNCPGAPQMNFFTGRAPATQPAPDGLVPEPFHSVDQIIDRVFDAGE OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO AKHGVTRGDFIAFAGAVGVSNCPGAPQMQFFLGRPEATQAAPDGLVPEPFHTIDQVLARMLDAGG FDELELVWMLSAHSVAAANDIDPNIQGLPFDSTPGIFDSQFFVETQLAGTGFTGGSNNQGEVSSP OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO FDEIETVWLLSAHSIAAANDVDPTISGLPFDSTPGQFDSQFFVETQLRGTAFPGKTGIQGTVMSP LPGEMRLQSDFLIARDABIAQEHQQEEHHQSKLVSDFQFIFLALTQLGQDPDAMTDCSAVIPISK OOOOOOOOOOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOOOOOOOOOOOOOOOO LKGEMRLQTDHLFARDSRTACEWQSFVNNQTKLQEDFQFIFTALSTLGHDMNAMIDCSEVIPAPK PAPNNTPGFSFFPPGMTMDDVEQACAETPFPTLSTLPGPATSVARIPPPPGA 61 124 127 189 192 254 257 319 323 Figure 4. Comparison of amino acid sequences of LG4 and LGS. The colons and periods between two lignin peroxidase protein sequences represent identical and conserved amino acid residues, respectively. Sequences are aligned to maximize homology with a minimal number of gaps which are represented by dotted lines. The longest stretch of identical amino acid sequence is underlined. The single letter code representing amino acids was previously described (5). 9A OLOA OOTAOAOOTOAOCOTOOOOTOTOAOOAOOAOCAATOOOOTTOAAOOAOOTOOTOOOAOOOOTOTOOOTOO 70 CLGS *TO*O*OTOTO*AAOOOTTO*OTTTOOA***AO*****O******A?******T**T*TT**TA***O** 70 OLOA OOOTOAOOOTOOAOOTOAOOOAA---OOTOOOOOOAAOOTOOAOAAOOOOOTOOOTTOOOOOOAOOOO~- 135 OLOS *T**OT******OO*OTO*O**OGOT**O*** ------ O****O*******OOA*O***T*OA*****AA 13A OLGA -OTOOAOAOOOOOTOOAAOOOOOOO---TOOTOTOCATOOTTOOOOOTOOTOOATOATATOOAOOAOAAO 201 CLGS cwwc31*c** ......... ******TCT*****CA*c******AAc**T**cTCc*********c***** 195 OLGA OTOTTOOAOOOTOOOOAOTOOOOTOOOOAOOOCOAOOAOOOOOTTOOTATOOTOTTCOAOOAOTOOATOO 271 chs ******A*T**c********T**c********T**T**i1*0‘icritc*c************c****** 255 OLGA OTATOTOOCOOAAOOTTOAOTCOOAOOOOAAOTTTOOOOOOOOOOOOOOOOAOOOOTOOATOATTAOOTT 3A1 CLGS *******T***OOTA*OO**O******O**OT*OO*TOO*A---*****O**T**T**T*****O*T*** 332 OLGA OTOOTOOATOOAOAOOAOOTAOOAOOOOAAOATOOOOOTOOAOOAOOTOOTOOOOATOOAOAAOOOOTTO A11 onus toAtcAitttitttitiAc*T***T**ctiiwitttftititiiitAwtiitcatCiciitiiitwiiti 402 OLGA ATCOOOAAOOAOOOOOTOAOOOOTOOOOAOTTOATOOCATTOOOTOOTOOOOTOOOOOTOAOOAAOTOOO A01 CLGS c**CA*********T*****T*ccttT***********c********c**ctic*ccc*c**1*t***** 412 OLGA OOOOOOOOOOGOAOATOOAOTTOTTOOTTOOOOOOOOOGAOOOAAOOOAOOOOOOOOOOOAOOOTOTOOT 551 CLGS tc**1**1*********A*c******Ac***1**Tc*Tcc******r***6*A*****T***itCititt 542 OLOA OOOOCAOOOOTTOOAOAOOATOGATOAOOTTOTOOOTOGOATOOTTOAOOOTOOTOOOTTOOAOOAOATO 621 OLOS O**A************T*TG*T**O**AA*OA***AO**TO*OT*O**T**O****AA*****T***O** 612 OLGA OAOAOTGTOTOOOTOOTOTOTOOOOAOTOCATOGOOOOTOOOAAOOAOOTOOAOOOOAOOATOTOOOOOO 691 CLGS tweetc******A**********Ae*****c**********c***t*TA*********A****c50two: 5.2 OLGA TOOOOTTOOAOTOOAOTOOOOOOOAOTTCOAOIQQQAQIIQIIQQIQQAQAQOOAOOTOOOCOOTAOOOO 761 CLGS ****c********ctic*tttt?AT1*ifitTtii*iitiiiit*t**ti**f****fifcct**c****c 752 OLGA ATTOOOTOOOAAOAOTOOTATOCAOOOGAOOOTOATOTOOOOOOTOAAOOOOOAGATOOOTOTOOAOAOO 031 eggs c***A****iccTT**AAC*A*******CAG*trfccaiiiiiiifcCA**i**t********c***1*1 322 OLGA OAOOAOTTOTTOOOOOOTOAOTOOOOOAOOOOATOOOAOTOOOAOTOOTTOOTOAAOAAOOAOAOOAAOO 901 CLGS ***TT*c**A****T******c*******c**c**************c***************T*c**** .92 OLGA TOOAOOAOOAOTTOOAOTTOATOTTCAOOOOOOTOTOOAOOOTOOGOOAOOAOATOAAOGCOATOATOOA 971 OLGS accretcc********5*********c1ctwcaetga1c5*********cwitccwctrwwctwwtcwta 952 OLGA OTOOTOOOAOOTOATOOOOOOOOOOAAOOOOOTO---AAOTTOOOOOOOTOOTTO ------ TTOOOOOOO 1032 chs ******T*crttitit***51Cf*********c*ccatttAAtActitcccAt**TCCTTc*iiiicCit 1032 OLGA OOTAAOAOOOAOOOOOAOATOOAOOAOOOOTOOOOATOOAOOOOOTTOOOOAOOOTOATOAOOOOOOOOO 1102 chs itc*TiiiiAT6*A***Tc**********1****tCGAG*****C***i****1***fcc*i1crititw 1102 OLGA OTCOOTOTOOOTOOOTOOOTOOOATOOOOOOOOOOOOOTOOOOOAAOTAAOOTATOTOTATOOTOOAOAT 1172 CLGS *O***O*OA*O*****************T**T**T**TOGTO*T---*****AOOOATO*OA**TOOO** 1169 OLGA OOTC?OOOTTOTAOOTOOTOOOTATOOTOOOAOOOTTATOTOOOOTTTOOATOATOTATAOOTOOTOOTO 12A2 OLOS OAOAO*OOOGTATTOO*AA!**A*ATT*A*A***AAO***OTO*AO*OTTT*O*A***O*AA**TO*T** 1239 cnca OAATATAOAAAOTOOTOTATOAAAAAAAAAAAAAAAAAAAAAA 1205 chs T*O*O*OT***OA*O***T*TOAOOAAATAOAOTOTOATTTOOTOOAAAAAAAAAA 1295 Figure 5. Comparison of nucleotide sequences of OLGA and CLGS. Only the variant nucleotides are presented in CLGS. The asterisks represent the identical nucleotides. Sequences are aligned to maximize homology with a minimal number of gaps which are represented by dotted lines. The longest stretch of identical nucleotides between OLGA and CLGS is underlined. 95 DISCUSSION Previous studies (Chapter 1) suggested that the cDNA insert in CLGS encodes lignin peroxidase H8, the predominant lignin peroxidase in 2. ghrysggpgxium grown in low nitrogen medium under static conditions (13). The results of this study show that the amino acid sequence deduced from the complete cDNA sequence of CLGS matches the amino acid sequence of two tryptic peptides (#1A and #253) obtained from purified lignin peroxidase H8 (29). The experimentally determined N-terminal sequence of lignin peroxidase H8 (Ala-Thr-Cys-Ser-Asn-G1y-Lys-Val-Val-Pro) is found in the deduced amino acid sequence of LGS. Immunoblotting data showed that the protein expressed by the lignin peroxidase cDNA clone in g. 9211 is reactive with the antibody raised against lignin peroxidase H8. These data unequivocally demonstrate that CLGS represents one of the lignin peroxidase genes. Furthermore, the signal sequences and putative glycosylation sites in the deduced amino acid sequences of both LGA and LGS are consistent with the fact that lignin peroxidases are extracellular, glycosylated proteins. The high degree of homology in the nucleotide sequences of CLGA and CLGS and the remarkable similarities in the deduced amino acid sequences of these clones indicate that CLGA and CLGS represent two separate members of a lignin peroxidase gene family. Furthermore, the N-teminal amino acid sequence of another purified lignin peroxidase (HZ) from the same fungus exactly matched the N-terminal amino acid sequence of mature LGA deduced from CLGA sequence. These results provide additional support to the idea that CLGA represents another lignin peroxidase gene. A report on the isolation and sequencing of cDNA (AMLl) from 2. 96 ghgygggggzium was recently published (25). A comparison of AMLl with CLGA and CLGS shows that these represent related but different genes. The amino acid and nucleotide sequences of AMLl showed a high degree of homology to those of above two cDNA clones. The homology of the nucleotide sequence between CLGA and AMLl and CLGS and AMLl is 7A.l% and 81.2%, respectively, whereas the amino acid sequence homology is 78.5% and 85%, respectively. The mature lignin peroxidase of AMLl, similar to LGA and LGS, is preceded by a leader sequence; contains a Lys-Arg cleavage site, N-glycosylation and polyadenylation sites, and is regulated at the level of mRNA production. The estimated molecular weight of 37,072 for the AMLl lignin peroxidase is comparable to the molecular weights of 36,5A0 and 36,607, respectively, for LGA and LGS. The similarities in nucleotide and amino acid sequences suggest that the genes encoding LGA and LGS originated from one ancestral lignin peroxidase gene. Different Southern hybridization patterns were seen when the chromosomal DNA of E. ghzygggpgriun was probed with 32P-labeled CLGA and CLGS (see Chapter 1). For example, four bands appeared when figmHI-digested chromosomal DNA was hybridized with labeled CLGS, whereas no fiagfll site is found in CLGS itself. Restriction digestion of chromosomal DNA with other restriction endonucleases produced similar results. On the other hand, Southern hybridization of the chromosomal DNA using CLGA as the probe resulted in banding patterns which are in accordance with the restriction pattern observed with CLGA. These results suggest that after the ancestral lignin peroxidase gene was duplicated, mutations continously appeared in lignin peroxidase genes corresponding to CLGA and CLGS and these became different members of the same gene family. 97 ACKNOWLEDGEMENTS The author is indebted to D. H. de Boer and C. Collins at Genentech for helping in sequencing the cDNA clones. This work was supported, in part, by grants from the U. S. Department of Energy (DP-F GOZ-8SER 13369) and from the National Science Foundation (DMD-8A127l). 10. 11. 12. 98 REFERENCES Ballance, D. J. 1986. Sequences important for gene expression in filamentous fungi. Yeast 2:229-236. Birnsteil, M. L., M. Busslinger and K. Strub. 1985. Transcription termination and 3' processing. the end is in site! Cell Al:3A9-359. de Boer, H. A. and R. Kastelein. 1986. Biased codon usage: an exploration of its role in optimization of translation, pp225-286. In W. Reznikoff and L. Gold (eds), Maximizing gene expression. Butterworths, Boston. 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Res. Commun. 137:6A9-65A. 101 CHAPTER THREE MOLECULAR CLONING OF A FAMILY OF LIGNIN PEROXIDASE GENES FROM W W AND SEQUENCE ANALHSIS OF A GENE ENCODING TEE MAJOR LIGNIN PEROXIDASE 102 ABSTRACT Eleven lignin peroxidase genomic clones have been identified by probing a Phanerochaete ghrysospogium (strain BKM-F 1767) BQQHI genomic library with lignin peroxidase cDNAs CLGA and CLGS. These clones fall into six groups, designated pGLGl, pGLG2, pCLG3, pCLGA, pCLG5 and pCLG6, based largely on the size and restriction map of each genomic fragment (designated, respectively, GLGl, GLG2, GLG3, CLGA, CLGS and GLG6). The location of the lignin peroxidase gene in each cloned fragment and the orientation of transcription in each gene have been determined. Restriction maps of lignin peroxidase genes in GLGl and GLG2 are similar to those of CLGA and CLGS. The other four clones contain sequences homologous to CLGS. Sequence analysis of GLGZ showed that it encodes a mature lignin peroxidase with an Mr of 36,607 which is preceded by a leader sequence of 27 amino acid residues. A sequence similar to the TATA box and a possible CAAT box are located A5 and 90 bp upstream of the startpoint of CLGS. By comparing the sequence of this gene with that of cDNA clone CLGS, nine small introns whose sizes range from 50 to 62 bp have been identified. These introns are randomly distributed in the gene and all have the consensus sequence GTRNGY---YTGAY---YAG. A possible polyadenylation signal sequence AATACA is located 1A bp upstream of the termination point of cDNA. 103 INTRODUCTION Phanerochaete ghxygggpgrlum, a white-rot basidiomycete, is known to produce during idiophase an extracellular, glycosylated, heme-containing protein called lignin peroxidase that obligatorily requires H202 for activity (1A,15,39,A0). This enzyme catalyzes oxidative cleavage of lignin and a variety of lignin model compounds (1,1A,15,17,19,39,A0). Lignin peroxidase has also been implicated in the detoxification of recalcitrant xenobiotics such as dioxins, polychlorinated biphenyls and benzopyrenes (8,9,16,36). The activity of lignin peroxidase is routinely assayed by measuring HZOZ-dependent veratryl alcohol oxidation to veratraldehyde (A0). Several recent reports indicate that E. ghrysggpgxium elaborates not one but a family of related lignin peroxidases (2,22,26,3A), the number of which varies depending on the strains, culture conditions and other factors that have not been well defined (12,20,22,3A). Kirk et al. (22) reported that B. ghxyfigfiggxigm produced at least six heme-containing Aproteins (H1, H2, H6, H7, H8 and H10) with veratryl alcohol oxidation activity. Lignin peroxidases H2 and H8 are reported to be the major proteins in the extracellular fluid of ligninolytic cultures of E. ghgygggngrign strain BKM-F 1767. Polyclonal antibody raised against lignin peroxidase H8 was shown to cross react with the other lignin peroxidases, indicating some degree of homology among these proteins. Analysis of protease digestion patterns revealed that H1 and H2 produced identical peptides, whereas H8 produced a pattern that was similar but lacked at least two major peptides present in H1 and H2 (22). The peptide cleavage patterns of H6 and H10 were very different from the H1, 10A H2 and H8 patterns. These results indicate varying degree of relatedness among different lignin peroxidases. Renganathan et a1. (3A) independently described three different molecular forms of lignin peroxidases from another strain of E. ghxyggggggium, all of which are glycosylated and range in Mr from 39,000 to A3,000. Homology among multiple lignin peroxidases of 2. ghrysggpgrigm was also reported by Leisola et al. (26). These results raise the question whether the different lignin peroxidase proteins are encoded by different genes, or are encoded by the same gene but are the results of different post- transcriptional and/or post-translational modifications. We recently isolated two different lignin peroxidase cDNA clones (CLGA and CLGS) from the cDNA library of 2. ghxygggpgxigm BKM-F 1767 and presented the complete sequences of these cDNAs (AA, Chapter 1 and 2). These two clones did not show cross-hybridization but had a very high degree of homology in nucleotide sequences (71.5%) in the coding regions and in amino acid sequences (75%) of the predicted lignin peroxidases. The two mature lignin peroxidase proteins contain 3AA amino acid residues, are preceded by typical signal sequences, and have similar hydropathy (Chapter 2). However, the two cDNA clones have very different sequences in both the S'- and 3'-noncoding regions, suggesting that the poly(A) RNAs corresponding to the two cDNAs are from different genes rather than from the same gene that undergoes post-transcriptional modifications. A third lignin peroxidase cDNA isolated from E. ghrysggnorium, also showed high degree of homology in nucleotide and amino acid sequences to those of CLGA and CLGS but had low homology with those two cDNA clones in the noncoding regions (A1, Chapter 2). Furthermore, CLGA and CLGS showed very different hybridization patterns with the chromosomal DNA of 2. ghxyggfingxigm, indicating that the 105 sequences in CLGA and CLGS correspond to different lignin peroxidase genes (Chapter 1). Probing of restriction enzyme digested chromosomal DNA with CLGA showed a hybridization pattern very similar to the CLGA restriction map, suggesting that there may be only one lignin peroxidase gene corresponding to CLGA. However, when CLGS was used as probe in a parallel experiment, several different bands of hybridization of varying intensity were obtained, suggesting that, in addition to the lignin peroxidase gene corresponding to CLGS, there might be several other lignin peroxidase genes whose sequences may be partially homologous to CLGS. In this chapter, I describe the isolation and characterization of six lignin peroxidase genes including their restriction maps, gene boundaries and the transcriptional orientation of each gene. The DNA sequence analysis of one lignin peroxidase gene corresponding to the CLGS, is also presented. MATERIALS AND METHODS g1a§m1g§‘_§§1§13§_§ng_nggig The shuttle vector YRp12 (37) was used for construction of the genomic library of 2. ghxygggpgrium BKM-F 1767 (ATCC 2A725). This vector contains the entire pBR322 sequence and 132;, Egg; and 5;; sequences from Sagghgzgnyggg ggxegigigg. Vector pUC19 (30,A2) was used for subcloning and characterizing the cloned genomic fragments containing lignin peroxidase gene. Vectors M13mp18 and M13mp19 (30,A2) were used for DNA sequence analysis. E§§h§119h1§_g911 DHSa (BRL, Gaithersburg, MD) [F' m1 hggRlNrk' Ink“) mean 5111-1 mu mA96 gglAl (QIgF-lggzya)U169 ¢80dl§QZAM15 A'] was used as the host strain for library construction and for subcloning. E. 9211 JM107 [gngA1, gygA96, m. 11.141117. £42844. m1. A'. (ins-mm/F'. mass. are“. mfiz 106 AM15] (A2) was used as the host strain for sequencing. Malt extract medium (21) was used for growing 2. chrygggporium and LB (27) and YT media (29) were used for growing E. 9211 strains DHSa and JM107, respectively. DNA_1§21§§ign Plasmid DNA and M13 RF DNA were isolated by using rapid alkaline procedure (27). Isolation of chromosomal DNA from P. ghrygggggrigm was described previously (33). Different cDNA or genomic DNA fragments were isolated using low-melting temperature agarose gel (32). ggnggIuggign_g§_§gggmig_L1bxggy A BamHI site is not present in either CLGA or CLGS and fiamHI genomic DNA digests exhibit five bands of different sizes that hybridize with CLGA and CLGS. Hence, we made the assumption that each figmHI genomic fragment that shows hybridization with CLGA and CLGS probably contains one entire lignin peroxidase gene. Vector YRp12 was digested with figmHI, dephosphorylated with calf intestine alkaline phosphatase and then ligated with completely BamHI- digested chromosomal DNA of E. ghxygggngriug. The ligated DNA was transformed into E. 9211 DHSa using the procedure described by Hanahan (18). The transformed cells were spread on LB plates supplemented with ampicillin (100 pg/ml), incubated at 37°C for 18 h, and all the ampicillin resistant transformants (Apr) on the plates were pooled if the ratio of tetracycline sensitive transformants (Tcs) to Apr was higher than 50% and stored at -20°C until use. This genomic library contains about 19,000 recombinant clones. 0‘. 7c .9 0 ~41. ';,.L,da~- .1;- .1. 31.“, . Colony hybridization was used for screening lignin peroxidase clones from the genomic library (27). Nitrocellulose filters harboring A,000 genomic clones each were prepared as described previously (A3) and were 107 first hybridized with the cDNA probe CLGA. Then, the probe was washed off and the same filters were hybridized with the cDNA probe CLGS. The positive clones were streaked on LB plates for purification. The purified colonies from each plate were picked and spotted on a LB master plate and another LB plate on which nitrocellulose filter was placed. Both plates were incubated at 37°C for 1A h. The colonies on the filters were lysed in situ (A3) and then hybridized with the respective probe as described above. DN 0 d H d t To determine the location of lignin peroxidase gene in each isolated clone and their transcriptional orientation, the plasmid DNA from each recombinant clone was digested with different restriction enzymes, the fragments were separated on 1% agarose gel, transferred onto nitrocellulose filters (27), and then probed with various 32P-labeled fragments of the two cDNAs, CLGA and CLGS. DNA_§ggugnging Different restriction enzymes were used to produce various fragments for subcloning into the appropriately digested sequencing vectors Ml3mp18 or M13mpl9. The sequencing strategy employed is shown in Figure 1. The dideoxy chain-termination procedure (35) and 35S-dATP were used to sequence lignin peroxidase gene in GLG2. 108 Figure l. Sequencing strategy of lignin peroxidase gene in GLG2. Abbreviations for restriction enzymes are: AI-Agal, AII-AngI, E-EQQRI, Ea-EggI, Hc-hincII, K-Kpnl, P-£_§I, SI-figtl and Sc-figgl. 109 RESULTS 1 o t o d e t c o e ox dase Genomic lone The YRp12 genomic library of 2. chrysospogiug was probed with CLGA and CLGS to identify the lignin peroxidase genomic clones. One clone that showed hybridization with CLGA was designated pGLGl. Ten clones that hybridized with probe CLGS were digested with BamHI, EQQRI and zhgl and the restriction patterns and the size of each cloned fragment were compared. Based on these data, the 10 positive genomic clones could be divided into 5 groups and these were designated pGLG2, pGLG3, pGLGA, pGLGS and pGLG6. All clones except pGLG3 contained a BamHI site on either side of the genomic inserts, whereas only one figmHI site could be recovered in pGLG3; the other site was apparently lost during the library construction. To reconfirm that the six types of clones contain sequences homologous to CLGA or CLGS, the genomic inserts (named GLGl, GLG2, GLG3, CLGA, CLGS and GLG6) from these clones were isolated, purified and recloned into pUC19 and these new clones were used in the rest of the work described here. All the new clones were digested with figmHI (or figmHI-fligdill for GLG3 in new clone), fractionated on agarose gel and then probed with 32P-labeled CLGA and CLGS. The results showed that only GLGl hybridized with CLGA (Fig. 2B), consistent with the Southern hybridization analyses previously reported (Chapter 1). The other five cloned fragments showed hybridization with the cDNA probe CLGS but the intensity of hybridi- zation varied (Fig. 2C). The strongest hybridization was shown by GLG2, implying that this genomic clone may correspond to CLGS, whereas the other four clones showed less intense hybridization bands, suggesting that the sequences of these clones have partial homology to CLGS. 110 B 123456-123456 Figure 2. Identification of lignin peroxidase genomic clones using cDNAs CLGA and CLGS as probes. Panel A, agarose gel picture; Panel B and C, hybridization of the genomic clones with CLGA and CLGS, respectively. In each panel, lanes 1 to 6 contained CIGl, GLG2, GIG3, CLGA, GIGS and GLG6 in pUC19, respectively. The size marker, flingII-digested lambda DNA, is shown in panel A. 111 c t o L e o C o e Consistent with the above suggestions, the restriction maps of middle regions of GLGl and GLG2 were closely comparable to those of CLGA and CLGS, respectively. The restriction maps of the other four lignin peroxidase genomic clones were different from that of GLG2, suggesting that these represent different lignin peroxidase genes (Fig. 3). The estimated sizes of GLGl to GLG6 are 3.82 kb, 6.A9 kb, 3.03 kb, A.8l kb, 7.76 kb and 6.55 kb, respectively. The cDNA sequence data (Chapter 2) showed that the two cDNA fragments, including the coding region and the 5'- and 3'-flanking sequences, are smaller than any of the cloned genomic fragments. To define the limits of lignin peroxidase gene, each clone was digested with one or more restriction enzymes, fractionated on agarose gels and then hybridized with the entire 32P-labeled cDNA CLGA or CLGS. The heavy lines in Fig. 3 show the possible coding region in each gene. All the cloned fragments except CLGA are believed to contain intact lignin peroxidase genes because these regions showing hybridization are either in the middle of the cloned fragments, such as GLGl, GLG2 and GLG3, or far away from the cloning site, such as GLGS and GLG6. The figmHI site of GLGA is very close to the 3'-end of the coding region. Since the transcriptional orientation of CLGA and CLGS are known, different segments of each cDNA were used as probes to determine the transcriptional direction of each lignin peroxidase gene. The same filters which had been hybridized with the entire cDNA fragment were also used for successive hybridization with the 3' and/or 5' fragments of cDNAs and the orientation of each lignin peroxidase gene is shown in Fig. 3. 112 a if a L 454'"? 4'5? 1 GLG1 ——-> ._J—__,kb - 5 a L??? W '3 35st? 1‘? SH“? 51311 GLGE <-—— i s: i" 5:“:1‘ ’1'" a“ 1‘ 61.63 -——> a '- a 1 W??? ‘31"? 3 l OLGA <—-——- ' e ' ‘ a L ’31" ’4‘ H 51' 53”??? T4 ‘3"? l GlGS -——-+’ , 1kb I 3 s H P KSp sax Sm 51 xx: 5 x a 1 1 4 G H u A i 4 l GLGG 4....- Figure 3. Restriction maps of six lignin peroxidase genomic clones. The heavy lines represent the gene locations and the arrow with long line underneath each restriction map represents the transcriptional direction. The abbreivations for restriction enzymes are: B, MI; E, EEQRI; H. HindIII; K. E201; P. Earl; Sm. final; SP. finhl; 31. Earl; 811. §§§II; X, thl and Kb, ng1. 113 uence s o L Clone GLG2 appears to correspond to CLGS based on the intensity of hybridization of the former with CLGS and the many similarities in restriction maps of CLGS and GLG2. Since CLGS encodes the major lignin peroxidase (H8), the 2.6 kb figtl-Axgll fragment in the middle of GLGZ (see Fig. 3) was sequenced and the amino acid sequence was deduced (Fig. A). These data show that the lignin peroxidase gene in GLG2 codes for a protein containing 371 amino acid residues with an Mr of 39,Al7, and the mature protein (Mr-36,607) is preceded by a leader sequence of 27 amino acids, which is believed to be important for the secretion of this enzyme. The leader sequence has the characteristics of typical bacterial and mammalian signal peptides, i.e. have a few charged residues at the N-terminus followed by a hydrophobic core and then a more polar C-terminal region defining the cleavage site. The two basic residues, Lye-Arg at position 26-27, may represent a proteolytic cleavage site, suggesting the existence of a pro-form of lignin peroxidase. The mature lignin peroxidase has two potential N-glycosylation sites at positions 283 and 323 with the sequence Asn-Gln-Ser and Asn-Asn-Thr, which are consistent with the published consensus sequence of Asn-X-Thr/Ser. These results are consistent with the fact that lignin peroxidase purified from ligninlolytic cultures of P. ghrygggpgxigm is an extracellular, glycosylated protein. The sequence data showed that the promoter sequence TATATAA is AS bp upstream and a possible CAAT box sequence (CGACAATGC) is 90 bp upstream of the 5' startpoint of CLGS. Nine small introns are identified in the lignin peroxidase gene in GLG2 by comparing the genomic and cDNA sequences. The size of the introns ranges from 50 to 62 bp. Each intron llA has GT and AG at the 5'- and 3'-end, respectively. A possible polyadenylation signal sequence AATACA is located 1A bp upstream of the poly (A) addition site. Comparison of the sequences between lignin peroxidase genomic and cDNA clones showed that there were several minor substitutions in both the coding and 3'-noncoding regions (see Fig. A).: All substitutions in the coding region are located at the third nucleotide of each codon, with the result the same amino acid sequence is seen in spite of these substitutions. 115 Figure A. Nucleotide sequence of lignin peroxidase gene in GLG2 and the predicted amino acid sequence. The start and termination points of the corresponding cDNA (CLGS) are indicated by asterisks (*) over the DNA sequence. Sequences homologous to CAAT box, TATA box, and the potential hexanucleotide polyadenylation signal (AATACA) are underlined. IVSl to IVS9 represent the nine intervening sequences (introns) identified in the lignin peroxidase gene. The boundary of each intron is marked by slashes. Bases in CLGS cDNA that are different from those in GLG2 are presented immediately above the corresponding genomic bases. 116 GAWWAMWWAWWTMWWAW - 5 7 O WIMWMWAWMAWWAWW - A 50 WTWTAWAMWW ' 1 I WWW/WWW 1 19 MetAlePheLyeLyeLouLmAiaVe1L0uThrA1aA1aLeu8uLouArfl1eA1calms 21 - r uroc 230 m1 1yA1aA1aVa101uLnAr¢A1¢ThtOye8¢rAnnOlyLera1Ve1PtoA1aAias.:CyIOyIThx-TerhoAaneueuSorAspI100 52 WMWWWWWIWMTWWWM/W 356 1:161uAsnLouPhoAsnG1yGlkaCthlyA1l61uA1aliIGI|ISO£IIOAI3LO m2 uVall‘holl 7A mmmummnmm/mammmmmlmm "A 1sAspAieIleAlaI1eSerProA1dht01uProG1nA1030280: m3 VaiAruclyAleAeptnyS 06 T mmmmmmmmmmmmwmmm 50A ammonium-Aspen!leGIuTthsnfluflilPx-oAInI1061yLeuAsp61uIleVaIAraLufilnLnProPheVaIGmLysliIalyValThrProOlyAepPhoI 10A 138 WWW/mmmmmmm 7 1 3 lemmfllyneVemaLanSuAauCnProGlyAhPuOlthePheMlyAxm MA 162 A . WIWWWIMWMWMATWWMIWN 030 lantelnl’roAiatroAlpOiyLouVaIPtoOluProPhofl ms iISQrVampGInne 100 AWWWWWIWWWWWIM 968 I10“prg7.1PhIAIpAlIGlyGluPhWflluLMluLouVaquflotLouSOrAl MB .310 202 WWTAWWWAWTWWW 10a SerVeiAlaAlaAlaAsnAspI 1mpPrernI1061:1617LeuProPheAepSorThtP1-061y110PhoAIpSuGInPhePhoVa161uThrc1nLouAleGlyThz-C1yl‘ho‘l'hr 2A2 O m/mmammm/WWWW 1166 01 m7 yobs.rAthfllyGlu'eiSerSex-Proboul’roclychflowunausea-pa: 285 WWWWWMWWW 1306 oLouIIoAlaAzmpAlaAuThrAlaCyIGIuTrpGlnSerPhoVemnAInGInSOflJILouVIISetAIpPheOhPhoIiePheLouAlaLeuThrGInLeuGIyGInAlpPr 305 mmmmmmmmmm I GTAO 1A2 5 oAspAIflotThtAapruSerAlaVail 10ml 1.80:1.ye PtoAlaProAlnAsnThzl’roG1yhdorPhoPhePronoGIyfletThtHompAepVemiw1M1- 3 A 3 mWWATWmAWMImWWWW 15“ mo Gym-GiurhtProPheProTtheusorThrLouProOIyProAiaThrSuVe1A1aA 36A T _O ourImmrmmunnmummnmmrmmmmflmam 1662 1:311 m9 erroProProPtofllyAlw 37 1 A T " wmmmmmmmwmmmrmmu 1702 flAMMWWWWW 1902 WWW 1033 117 DISCUSSION i n e o ida ene ami Six types of lignin peroxidase genomic clones have been isolated by screening the BamHI-library of 2. ghgysgsprgium BKM-F 1767 with the previously isolated lignin peroxidase cDNAs, CLGA and CLGS. Five of them represented by pCLG2 through pGLG6 showed hybridization with CLGS under high stringent conditions. These data are consistent with earlier results (Chapter 1) showing that figmHI-digested chromosomal DNA of 2. Chrysgspgriug gives four bands of hybridization with CLGS. The sizes of the five lignin peroxidase clones represented here (7.76 kb, 6.55 and 6.A9 kb, A.81 kb and 3.03 kb) are comparable to those of the fiégfll bands. Since lignin peroxidase gene in GLG2 showed the strongest hybridization with CLGS and has similar restriction map to that of CLGS, it appeared that this clone contained the genetic analogue of CLGS. A comparison of the sequence data of GLG2 with that of CLGS clearly shows this to be true. Clones pCLG3, pGLGA, pGLGS and pGLG6 displayed strong hybridization with CLGS but had different restriction patterns compared to that of CLGS or GLG2, suggesting that these are partially homologous to GLG2, but represent different lignin peroxidase genes. Partial sequencing of CLGA has shown that these two (GLG2 and CLGA) have much higher nucleotide homology than that seen between CLGA and CLGS (data not shown). One genomic clone (pGLGl) showed strong hybridization with CLGA (see Fig. 2B, lane 1) and its restriction map was similar to that of CLGA (Fig. 3), suggesting that the lignin peroxidase gene in this genomic clone corresponds to CLGA. Consistent with this idea, the previous results (Chapter 1) showed that 32P-labeled CLGA gave a single 118 band of hybridization with BamHl-digested chromosomal DNA. Restriction enzyme ngl cuts GLGl into two fragments, but does not have any site in CLGA, suggesting that this site may be located in an intron of this lignin peroxidase gene. Comparison of detail restriction digestions of CLGA and GLGl showed that final site is located in one intron (data not shown). These results indicate that the genomic clones represent a family of six lignin peroxidase genes. Tien and Tu (A1) isolated and sequenced another cDNA clone (AMLl) from P. ghrygggpgrium, which has a high degree of nucleotide and amino acid sequence homology with CLGA and CLGS (A1, Chapter 2). None of the six lignin peroxidase clones described here has restriction map similar to that of AMLl, implying that 2. ghzygggngxium may have one more lignin peroxidase gene corresponding to this cDNA. The results clearly show that six members of the lignin peroxidase gene family in B. ghzygggpgzium BKM-F 1767 have been isolated. HPLC profile showed that there were six primary lignin peroxidase proteins in the extracellular fluid of 2. ghxysggpgxium grown in low nitrogen medium (22). It remains to be proven that each isolated lignin peroxidase gene Aencodes one of these six lignin peroxidase proteins. The sequence data of CLGS and genomic clone CLG2 provide very strong evidence that GLG2 codes for lignin peroxidase H8, a major extracellular protein produced by 2. ghgygggngrigm in ligninolytic cultures. We recently determined amino acid sequence at the N-terminus of another purified lignin peroxidase, H2, and showed that this sequence (Val-Ala-Cys-Pro-Asp-Gly- Val-His-Thr-Ala-Ser-Asn) exactly matches with the first twelve amino acid residues of the mature lignin peroxidase deduced from cDNA CLGA. These data indicate that GLGl encodes another major lignin peroxidase H2. The sequencing of the other lignin peroxidase proteins and genes is 119 now getting under way. Ce e tructu e o Perox da e Gene GLC In high eukaryotic genes, two consensus sequences have been identified to be important for initiation of transcription: the CAAT box 70 to 90 bases and the TATA box 20 to A0 bases upstream of the major start point (5,25). In yeast, most genes have the TATA box A0-120 bp upstream of the mRNA initiation sites (11,38), whereas the CAAT box is generally either clearly absent or partially disguised (11). In filamentous fungi, some genes possess the similar canonical TATA sequence at approximately the expected position, while others simply have an AT-rich region 30-100 bp upstream of the 5' start point of mRNA (3). Sequences related to the CAAT consensus have also been described in some fungal genes, but this is often not very clear (3). The lignin peroxidase gene of 2. ghgygggpggium contains a typical TATATAA sequence which lies A5 bp upstream of the start point of cDNA CLGS. There is a sequence CGACAATGC, similar to the CAAT box, 90 bp upstream of the startpoint of cDNA, even though it does not contain the typical GGC/GCAATCT sequence found in higher eukaryotic genes. There are two other CAAT box-like sequences far upstream of the cDNA start point, at positions -A15 (GGCCAATAG) and -575 (CGACAATCT), respectively, and both have more conserved sequences with the typical CAAT box than the first CAAT box {-99 bp) in this gene. In both yeast and fungal genes, the mRNA start site is characterized by a preceding pyrimidine-rich sense strand (3,6,10) and this feature is present in lignin peroxidase gene GLG2, also. The ratio of CT to the total sequence between TATA box and start point of cDNA is 61.A% (27/AA). The sequence surrounding the initiator codon ATG in GLG2 is CAGACAIQG, 120 in which 7 of 9 nucleotides match with the eukaryotic translation initiation site consensus sequence CCA/GCCAIQG (25). Using point mutations flanking the initiator codon AUG, Kozak (2A) more recently identified ACCAIQG as optimal sequence for translation initiation by eukaryotic ribosome and a purine in position -3 is critical for efficient translation. In GLG2, there is a G located at -3 position of ATG. Similar to other filamentous fungal genes (3,7,31), GLG2 contains several small introns (see below for detail discussion). The codon usage in GLG2 is very biased in favor of codons ending with C- and/or G- residues. Comparison of codon usage of lignin peroxidase cDNAs and gene with that of different filamentous fungal and yeast genes is shown in Table l. The comparison shows that the codon usage in R. ghxysggpgxium is less biased than that in yeast but more similar to that in Neurogpoza than to that in AsngIgillug (Table 1). Some codons in S. ggxggigigg are extremely preferred but are not or very poorly used in lignin peroxidase gene. For example, the preferred codon for Arg in yeast is AGA (90%) while this codon is not used at all in 2. ghzygggpgzium. The same situation is found in Cys, Gln, Glu, Leu and Pro in which yeast prefers codons ending with A or T (Leu is exception) whereas in 2. ghzygggpggigm codons ending with C or G are preferred (Table 1). These data suggest that the translation efficiency for the lignin peroxidase cDNAs in yeast cells may be very low. Sequencing of many higher eukaryotic genes has revealed that AATAAA, which usually lies 10-30 bp upstream of the poly (A) addition site, is by far the predominant sequence directing cleavage and polyadenylation of pre-mRNA although minor nucleotide substitutions, such as AATTAA, AATACA and AATATA, are known to occur (6). However, the consensus sequence AATAAA is rarely present in either yeast or filamentous fungal 121 genes (3). In contrast to this, at the 3'-end of the lignin peroxidase gene GLG2, AATACA sequence is present 1A bp upstream of the polyadenylation site, although this sequence is believed to be relatively inefficient for polyadenylation in higher eukaryotic genes (6). A similar sequence AATATA was found in the same position in cDNA CLGA. However, the cDNA XMLl showed a different sequence AAATAT 13 bp upstream of the poly(A) addition site (A1). The consensus G/T cluster sequence found downstream of the poly(A) addition site in other eukaryotic genes is absent in GLG2 (6). 122 Table 1. Comparison of Codon Usage in 2. ghgygggpggium (P), Miles (A). Mam (N) and Yeast (Y). AAs Codons P A N Y AAs Codons P A N Y Phe TTT l 26 16 1A Ser TCT 16 20 19 A9 TTC 99 7A 8A 86 TCC A8 29 A3 A5 Leu TTA 0 3 0 7 TCA 0 9 3 3 TTC A 16 9 87 TCC 30 1A 13 l CTT 16 20 26 2 ACT 3 10 5 2 CTC 66 28 A7 1 ACC 3 l8 17 l CTA 0 6 2 A Pro CCT 17 29 23 16 CTC 1A 27 16 0 CCC A2 A0 57 1 Ile ATT 8 A2 28 51 CCA 5 13 9 83 ATC 92 53 70 A8 CCG 36 18 ll 0 ATA 0 5 2 1 Thr ACT 27 31 22 A6 Met ATG 100 100 100 100 ACC 50 A3 57 A6 Val CTT 12 29 22 55 ACA 0 16 12 5 CTC 78 AA 63 AA ACC 23 10 9 3 GTA 0 6 3 1 Ala OCT 28 35 28 72 CTC 10 21 12 1 COO 32 35 62 26 Tyr TAT 0 33 17 12 GCA '12 15 3 l TAC 100 67 83 88 GCG 28 15 7 1 His CAT 8 21 25 16 Cys TCT 21 36 15 96 CAC 92 79 75 8A TGC 79 6A 85 A Gln CAA 8 33 16 9A Trp TGC 100 100 100 100 CAG 92 67 8A 6 Arg CGT A5 27 36 7 Asn AAT 2 2A 9 5 CGC 52 32 38 0 AAC 98 76 91 95 CGA 3 13 5 0 Lys AAA 0 19 A 8 COO ' 0 13 6 0 AAC 100 81 96 92 ACA 0 7 A 90 Asp GAT 27 A9 35 32 ACC 0 8 ll 3 GAC 73 51 65 68 Gly OCT 35 3A A5 95 Glu CAA 6 36 10 96 CGC 63 36 A5 3 GAG 9A 6A 90 A CGA 2 21 7 1 CGG 0 9 3 l a Codon usage information for E. ghxygggpgxigm is based on the data obtained with the three lignin peroxidase cDNAs and one gene (A1, Chapter 2 and 3). b Codon usage information for Agpgrgillug (3), Ngugggpggg (3), and yeast (A) is based on data obtained with eight, eleven and ten genes, respectively. 123 Arrangement and Structure of Introns Nine introns have been identified in lignin peroxidase gene in CLGZ by comparing its sequence with that of the corresponding cDNA CLGS. The position, size and phase of each intron in this gene is shown in Table 2. All the introns are very small (50-62 bp) and are randomly distributed in the coding region. Thus, the number of introns of lignin peroxidase gene of 2. ghgygggpgrium is much higher than that found in yeast genes. For example, in S. ggrggigigg, only seventeen of several hundred sequenced genes were found to contain one or two introns (13) and the intron in each gene is located at the extreme 5'-end. The size of the introns in lignin peroxidase gene is very similar to that of genes from other filamentous fungi (7,23,28,31). For example, the glucoamylase gene of Agpgrgillgg nigg; contains five introns, four of them are 55 to 75 bp in length (7). The fl-tubulin gene of Neurgsporg grass; contains six introns and five of them are 57-7A bp in size (31). The phase of intron does not show any specificity (see Table 2). The comparison of axon/intron junctions with the consensus eukaryotic splice junctions (3) is shown in Fig. 5. All introns in lignin peroxidase gene in GLG2 seem to have the consensus sequence GTRNGY...YTGAY...YAG, similar to the introns of genes from other lower and higher eukaryotic organisms. Our results show that introns are probably a common occurence in lignin peroxidase genes. Preliminary sequencing data with CLGl and CLGA have shown that introns are present in these two genes also (data not shown). 12A Table 2. Comparison of the Positions, Phases and the Lengths of Nine Introns of Lignin Peroxidase Gene in GLG2. Position 21 71 89/90 161 175 201 2A3 3A3/3AA 365 Size 52 51 52 50 52 52 51 62 58 The positions are given in amino acid residue numbers in the lignin peroxidase protein. The sizes of introns are presented as base pairs (bp). Phases 1, II and III mean that the introns follow the first, second and third nucleotide of a codon, respectively. 125 IVSl AGG/GTGCGTCCCGGATGGCTACCGACGCATTGCACAA ----- CTAAC ------- AGCTACGCGATAG/GT IVS2 TCT/GTAAGCATACTGTTACTCGCCGCACAGTGCTTGCCTC--TTGAC ----------- ACGCCCTAG/CG IVS3 CGG/GTGTGTATCCCTCGGCTTCTGCACTGGCATGGAGC----TTGAC -------- CGTGAACGTTAG/TG IVSA CGG/GTACGTTGCAAAATGCGGAATTGAAACAATGTTA ----- CTCAT --------- TTCGAGGAGAG/CA IVSS TCC/GTACGCCGAATCATCCTGTGACCTCTCATCAGTATA---CTGAC --------- AAATCCTACAG/AC IVS6 TGC/GTTAGTGTCTCCTGGAGCCCTTGTTTTCATGCA ------ CTGAC ------ CAGTTTCGCTACAG/AC IVS7 TGG/GTTGCGTAACTCTATCAATTGCGCTGGACCGCGGG----CTGAT --------- CGTTCTCGCAG/CG IVS8 GCT/GTACGTTCCACCGTCCCCCCACCCTGATACAGGACTCC-CTGAC-TGACGATCCTTATGCTTAG/TT IVS9 CAT/GTGGGTACCTCTTGCCCTCTGGTGACGGTATATTAG---CTGAT---TACGAATGGATATCTAG/CC g. ghxygggpgrigm SK/GTRNGY 32-37 YTGAY 9-19 YAG/N High eukaryotes CAG/GTAAGT CTAAT (T/C) NCAG/G Yeast N/GTA'I‘GT TACTAAC nYYNYAG/N Filamentous fungi g/GTAYGTT TGCTAAC ACAG/g Figure 5. Conservation of intron/exon junction and internal sequences in lignin peroxidase gene of 2. ghxyggspgxigm. The conserved intron/exon junction and internal sequences of genes from high eukaryotes (28), yeast (23) and Filamentous fungi (3) are also presented for comparison. The boundaries of introns and exons are marked by slashes. Letters in upper case represent conservation in >70% of introns and lower case represents 50-70% for all filamentous fungi, including 2. ghzysggpggium. Abbreviations for single letter codes are: K-—G or T, N-—A or C or G or T, R—A or G, S—C or G and Y—C or T. 126 ACKNOWLEDGEMENTS This work was supported in part by grant DE-FG02-85ER13369 from the U.S. Department of Energy, Division of Basic Biological Sciences, NSF grant DMB-8A127l and a FEED grant from the State of Michigan. 10. ll. 12. 13. 127 REFERENCES Andersson, L. A., V. Renganathan, A. A. Chiu, T. M. Loehr and M. H. Gold. 1985. 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Kirk. 1983. Lignin-degrading enzyme from the hymenomycete Rhgngrgghgggg ghrygggpgrium Burds. Science 221:661- 663. A0. A1. A2. A3. AA. 130 Tien, M. and T. K. Kirk. 198A. Lignin-degrading enzyme from Zhangzgghgggg ghxyfiggpgxlum: purification, characterization, and catalytic properties of a unique H 02-requiring oxygenase. Proc. Natl. Acad. Sci. USA. 81:2280-22 A. Tien, M., and C.-P. D. Tu. 1987. Cloning and sequencing of cDNA for a ligninase from Phanerochaete. ghryggspgrigm. Nature (London) 326:520-523. Yanisch-Perron, C., J. Vieira and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the Ml3mp18 and Ml3mpl9 vectors. Gene 33:103-119. Zhang, Y. Z. and C. A. Reddy. 1988. Use of synthetic oligonucleotide probes for identifying ligninase cDNA clones. Meth. Enzymol. 168 (in press) Zhang, Y. Z., G. J. Zylstra, R. H. Olsen and C. A. Reddy. 1986. Identification of cDNA clones for ligninase from Ehgngggghgggg ghrysggpgxiug using synthetic oligonucleotide probes. Biochem. Biophys. Res. Commun. 137:6A9-656. APPENDICS APPENDIX A IDENTIFICATION OF cDNA CLONES FOR LIGNINASE FROM PHANEROCHAETE CHRYSOSPORIQ! USING SYNTHETIC OLIGONUCLEOTIDE PROBES By Y. Z. Zhang, G. J. Zylstra, R. H. Olsen and C. A. Reddy Published in Biochem. Biophys. Res. Commun. 137:6A9-656 (1986) 131 IDENTIFICATION or cDIA ctouas roe LIOMIIASB recs Phanerochaete chrysosporium uszao SYNTHETIC curse-notaorins PROBESI' r. 2. Zhang‘, G. J. Zylstraz, n. H. 01sen3, and c. A. Reddy.‘ . 1Department of Microbiology and Public Health, Michigan State University, 2 East Lansing, MI 1188211-1101 Cellular and Molecular Biology Program and 3Department of Microbiology and Immnology, University of Michigan Medical School, Ann Arbor, MI A8109 Received April 28, 1986 Four cDNA clones for ligninase were isolated from the cDNA library (constructed into the Pet! site of E. c_g__li vector .pUC9) representing 6 day- old lignin degrading culture of Phanerochaete OhUSOJOPIUII by the use of three synthetic oligonucleotide probes corresponding to partial amino acid sequences of tryptic peptides of the ligninase. ~Each of the‘ three probes, 111.1, 111.2 and 25, represents a mixture of 32 12- or 111-base long oligonucleotides. Three cDNA clones hybridized with probe 111.1 but not with probe 25 or 111.2, but one cDNA clone hybridized with all of the three probes. Differential hybridization studies showed that these clones are unique to 6— day poly(A) RNA, but not to 2-day poly(A) RNA. 0 1m Academic mu. m. Lignin is a highly complex, amorphous, aromatic polymer, which is a major component of woody plants, and is the second most abundant renewable organic resource on the earth (1). Research has intensified worldwide on ligninolytic microorganisms and their enzymes because of their possible industrial potential in biopulping and in the conversion of lignin and lignocellulosic materials to useful chemicals (1). Phanerochaete chrysosporium, a white-rot basidiomycete, is a rapid lignin degrader and is most widely studied from both the basic and applied standpoints. Lignin degradation in this organism is a secondary metabolic event and is triggered in response to R, O or 3 starvation (1-3). Tweenzymes, glucose oxidase and ligninase, which are believed to be involved in lignin degradation, have been purified and characterized from *Journal article no. 11926 from the Michigan Agricultural Experiment Station. a Corresponding author. 132 ligninolytic cultures of this _mngus (ll-6). Ligninase is a Rzoz-dependent, extracellular’ heme protein with a molecular weight of A1,000-A2,000, and catalyzes C c'CB cleavage of the propyl side chains of lignin, a major reaction in fungal depolymerization of lignin (5,6). Ligninase is an extremely important enzyme because of its possible application in pulping wood, biobleaching of pulp, treating wastes, conversion of byproduct lignins to useful chemicals, and detoxification of xenobiotics such as dioxins, DDT and polychlorinated biphenyls (1, 5-7). As a first step in obtaining a better understanding of lignin degradation by g. chrysosporium at the molecular level and to develop the full bioprocessing potential of this organism, we initiated studies to isolate and characterize the ligninase cDNA clones. We describe here for the first time the isolation of cDNA clones fer ligninase by screening the cDNA library of g. chgysosporium using three synthetic probes, corresponding to amino acid sequences of different portions of the ligninase in this organism. MATERIALS AND METHODS Poly(A) RNA extraction. g, chrysosporium strain BKM-F 1767 (ATCC 2A725) was maintained and grown as previously described (‘1) except that 20 mM sodium acetate (pH A.5) replaced 2,2—dimethylsuccinate in the medium. RNA. was extracted using a modification of the hot phenol extraction procedure (9). Oligo(dT)-cellulose was used to purify poly(A) RNA which usually accounted for 1-2$ of the total cellular RNA. Construction of cDNA library. Double stranded cDNA, synthesized from poly(A) RNA from 6-day cultures as previously described (10), was dO-tailed and then annealed with dC- tailed pUC9 (11) which was digested with restriction enzyme PstI. The annealed DNA molecules were used to transform E. _c__oli JM83 (ara Alec-pro strA thi d80dl_a__cz M15) using the procedure of Hanahan —(12). The transformed E. _c____oli cells were spread on 2 YT plates (13) supplemented with 100 uglml ampicillin and I10 ug/ml X-gal (13; 5-bromo-A-chloro-3- indolyl-B-D— galactoside). Ten thousand white, potential cDNA clones were picked and were individually stored in each well of 96-well microtiter plates. Differential hybridization. The HAT? filter containing cloned cDNA was prepared as previously described (9). The 2-day and 6-day cDNA probes were made from Z-day and 6-day poly(A) RNA, respectively, as described (1"). The filter papers were prehybridized and hybridized with Z-day probe at ”2°C for 36 h. After exposure to the X-ray film, the 2-day probe was washed off and the filters were then hybridized with the 6-day probe. Eggninase peptide seguenci_g and oligonucleotide probe construction. Tryptic peptides of ligninase (6) were prepared as described by Ciegel et a1. (15). Peptides were manually sequenced by the Edman degradation procedure as described by Tarr (16). Amino acids were identified as their phenylthio- hydantoin derivatives using an HPLC detection system as described by Swanson et al. (17). Oligonucleotides were synthesized using an Applied Biosystems 380A DNA Synthesizer utilizing the method of Caruthers (18). 133 Hybridization with oligpgucleotide probes. The HATF.filter paper containing cloned cDNA prepared as above or nitrocellulose paper harboring fragments of recombinant plasmids was prehybridized at 37°C overnight (9), and hybridized with the synthetic probe at 25°C for 1 h. The paper was then washed once in 6 X SSC/0.0S$ sodium pyrophosphate for 30 min at 25°C and once in the same solution for 10 min .at "2°C or 30°C (for probe 1A.2 only). The oligo- nucleotide probes were end-labeled with TA polynucleotide kinase. Other procedures. Plasmid isolation was carried out using the rapid alkaline procedure (19). The digestion of plasmid DNA, the electrophoresis of DNA on agarose gel and the transfer of DNA fragments from agarose gel to nitro- cellulose paper were carried out as described by Maniatis et a1. (9). RESULTS AND DISCUSSION Synthesis of oligonucleotide probes. Several tryptic peptide fractions of the main ligninase protein R8 (22) were sequenced to find a consecutive series of low-redundancy amino acids suitable fer probe construction. Two such peptide fractions, 1" and 258, were found (see below) and three oligonucleotide probes (1A.1, 1A.2 and 25) deduced from the amino acid sequences in these peptides were constructed. Due to the redundancy of the genetic code each probe consists of a mixture of 32 different oligonucleotide sequences. Peptide Fraction 1!: Leu-Oln-Lys-Pro-Phe-Val-Gln-Lys Amino Acid Sequence QUN-OAP-AAP-OCN-UUQ-GUN-CAP-AAP Corresponding mRNA GTQ-TTQ-GGN-AAP-CA' ' Probe 1n.1 Sequence AAP-CAN-GTO-TTQ Probe 1A.2 Sequence Peptide Fraction 253: Leu-Val-Phe-His-Asp-Ala Amino Acid Sequence QUN-OUN-UUQ-CAQ-OAQ-GCN Corresponding mRNA CAN-AAP—OTP-CTP-OO Probe 25 Sequence Differential hybridization. It has been known that ligninase activity is not detectable in 2-day cultures, but relatively high levels of activity are found in 6-day cultures of g. chrysosporium grown in low N medium (6-7). Thus, differential hybridization of the cDNA library with the 2-day and 6—day probes (see Materials and Methods) should allow us to isolate cDNA clone(s) specific I N = ACCT/U, p s as, q . cr/u ,..o .ueegvool 0... ..)...J U '. .‘.~..# I. ,v ‘5. .p‘ . 121‘s! 1‘ .\ IDQDCAO 0 e1. ”.5 .‘- C-v. O... O C ..;A-¢ i- /'e D A, 9 Figure . Differential hybridization and identification of cDNA clone for ligninase. Hybridization of cDNA clones with 2-day probe (A), 6-day probe (3), or synthetic probe 111.1 (C) was performed as described in Materials and Methods. A representative cDNA clone for ligninase in each hybridization is indicated by arrow. for the enzymes involved in secondary metabolism such as ligninase. Therefore, all clones of the cDNA library were hybridized with 2-day and 6-day cDNA probes and about 850 clones specific for 6-day mRNA were isolated. Identification of cDNA clones for ligninase. Four ligninase cDNA clones, designated pCLGS, pGLGA, pCLGS and pCLG6, were identified after screening the above mentioned 850 clones with the oligonucleotide probe 1A.1. These four clones were confirmed to be unique to 6-day mRNA by doing a second differential hybridization. One representative clone is shown in Fig. 1. Recombinant plasmid DNA from each of the clones was digested individually with restriction enzymes PstI or BamHI-HindIII. The fragments were electrophoretically separated on 11 agarose gel, transferred to nitrocellulose paper and were then hybridized with the synthetic probes 1A.1 and 25. Ethidium bromide stained agarose gels and the results of hybridization (Fig. 2) showed that there was a PstI site in the cDNA insert of each of the four clones and that each of the recombinant plasmids, except pCLGé, contained 135 l 2 3 A 5 5 7 8 9 Bl 2 3 9 5 ‘ 7 ' Cl 1 3 A 5 I 7 I I Figure 2. Hybridization of plasmid DNA from ligninase cDNA clones with the synthetic oligonucleotide probes. DNA from each clone was digested with PstI (lanes 1-A) and BamHI-HindIII (lanes 5-8), respectively, subjected to electro- phoresis on 11 agarose gel, stained with ethidium bromide (A), transferred onto nitrocullulose paper and hybridized with probe 1".1 (B) or probe 25 (C) as described in Materials and Methods. Different lanes contained: pCLGB (lanes 1, 5), pCLGA (lanes 2, 6), pCLGS (lanes 3, 7) and pCLGG (lanes A, 8). Vector pUC9, digested with HindIII, was loaded on lane 9 as a negative control. three PstI sites. Plasmid'pCLCG appears to have lost the Pet! site at the junction of the vector with the cDNA insert and, therefore, yields only two PstI fragments (Fig. 2A). Note that none of the cDNA clones were completely digested by restriction enzyme PstI even when 5-fold excess of enzyme was added. This incomplete digestion may be due to dO-dG tailing used in cDNA library construction. The PstI digestion resulted in the generation of one cDNA fragment from each clone which hybridized with probe 111.1 (Fig. 28). None of the four clones contained BamHI site in their respective cDNA inserts. Cloned cDNA insert in pCLG3, pCLCA and pCLG6 each contained one HindIII site, whereas that in pCLGS lacked this site (Fig. 2A). Plasmid pCLOé gave only two fragments on BamHI-HindIII digestion (the smaller fragment is not visible in Fig. 2A), suggesting that this plasmid has lost not only a Pet! site (see above) but also the BamHI site. Thus, BamHI-HindIII digestion resulted in the generation of three fragments from clones pCLG3 and pCLOII and two fragments from pCLCS and pCLGG. One of the BamHI-HindIII fragments from each clone hybridized with probe 111.1. Only one of the four cDNA clones, pCLGS, hybridized with probe 25 (Fig. 2, C). A comparison of the hybridization patterns of pCLGS with probes 131.1 and 25 showed that the small fragment of 136 the cDNA insert from PstI digestion hybridized with probe 25, whereas the larger one hybridized with probe 1A.1 (lane 3 in Fig. 28 and C, respectively). Probe 1A.2 showed the same hybridization pattern as probe 1A.1 for pCLGS but showed no hybridization with the other three clones (data not shown). These hybridization results indicate that‘ the ligninase cDNA clone, pCLGS, corresponds to the main ligninase protein H8 described above. Synthetic oligodeoxyribonucleotides are widely employed as hybridization probes for the isolation of desired cloned DNA sequences (20). Oligonucleotides are known to hybridize at specific sites in cloned DNA and mixtures of oligonucleotides are used to screen for the desired clone with greater confidence (20). In this study, we have,shown that specific synthetic oligonucleotide probes, corresponding to partial amino acid sequences of ligninase, hybridized strongly to different cDNA clones. The washing temperatures of 112°C (for probe 111.1 and 25) and 30°C (for probe 111.2) used for washing hybridized filters fall within the range of washing temperatures, predicted by the equation of Suggs et al. (21). These results together with the other data presented above lead us to conclude that pCLG3, '1, 5 and 6 are indeed ligninase cDNA clones. It has recently been shown that there are several proteins showing ligninase activity in the extracellular culture fluid of P. chrysosporium (22). Antibody raised against the main ligninase protein, 118, was shown to react with the other ligninase species as well (22). These data suggest that these ligninase species might either be the products of the same gene, but have undergone different post-transcriptional/post-translational modifica- tions, or are products of different genes with shared homologous sequences which are responsible for the ligninase activity and the antigen-antibody reaction. The size of the cDNA insert in each ~cDNA clone is comparable: 1.35 kb, 1.A2 kb, 1.A8 kb and 1.2 kb in pCLG3, pGLGA, pCLGS and pCLG6, respec- tively. However, their restriction enzyme digestion patterns are different. From our preliminary results (data not shown), mum is different from other three clones based on the restriction map and hybridization analysis. For 137 example, each of the restriction enzymes EcoRI, XhoI and SstI has a recogni- tion site in the pCLCS insert, but not in the inserts in the other three clones. 0n the other hand, HindIII and Sell have one site each in the inserts of pCLG3, pCLOA and pCLG6 but not in that of pCLCS. Nick-translated pCLC3 insert did not hybridize with pCLCS but did hybridize with the other three cDNA clones. Also, very different hybridization patterns were observed when nick-translated pCLG3 and pCLGS inserts were hybridized with the different restriction enzyme digests of ‘2. chgysosporium chromosomal DNA (data not shown). These results indicate that the cDNA sequences in pCLG3, pCLGA and pCLG6 are very similar if not identical and that pCLG3 and pCLGS represent different genes. Further characterization of the latter two clones, including sequencing, is in progress. ACKNOWLEDGMENTS This work was supported, in part, by grant DE-F602-85ER13369 from the 0.8. Department of energy, division of Basic Biological Sciences, and NSF grant DMD-8111271 to CAR, and a grant to R0 from the Michigan Biotechnology Institute. 02 was supported by a Horace H. Rackham predoctoral fellowship and by NIH (5-T32-GM07315-08). He wish to thank T. K. Kirk and M. Tien for supplying purified ligninase. D. Ciegel and C. Williams provided facility and advice for peptide sequencing. REFERENCES 1. Crawford, R. L. and Crawford, D. L. (198") Enzyme. Microbial. Technol. 6, 133-180. 2. Keyser, P., Kirk, T. K. and Zeikus, J. G. (1978) J. Bacteriol. 135. 790-797. ’ 3. Kirk, T. K., Schultz, E., Connors, H. J., Lorenz, L. F. and Zeikus, J. C. (1978) Arch. Microbiol. 11?, 277-285. A. Kelley, R. L. and Reddy, C. A. (1986) J. Bacteriol. (in press). 5. Tien, M. and Kirk, T. K. (198“) Proc. Natl. Acad. Sci. USA 81, 2230-228“. 6. Cold, M. H., Kuwahara, M., Chiu, A. A. and Glenn, J. K. (198'!) Arch. Biochem. Biophys. 23!, 353-362. 7. Bumpus, J. A., Tien, M., "right, 0., and Aust, S. D. (1985) Science 8. Forney, L. J., Reddy, C. A., Tien, M. and Aust, S. D. (1982) J. Biol. Chem. 257. 11N55-11A62. . 9. Maniatis, T., Fritsch, E. P. and Sambrook, J. (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, N.Y. 10. Gubler, U. and Hoffman, B. J. (1983) Gene 25, 263-269. 11. Vieira, J. and Messing, J. (1982) Gene 19, 259-268. 12. Hanahan, D. (1983) J. Mol. Biol. 166, 557-580. 13. Miller, J. H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1A. Berlin, V. and Yanofsky, C. (1985) Mol. Cell. Biol. 5. 8A9-855. 15. Giegel, D., Massey, V. and Hilliams, C. R. Jr. (198'!) Flavins and flavoproteins, p. 331-33", Halter deCruyter Co. 16. 17. 18. 19. 20. 21. 22. 138 Tarr, G. E. (1977) Methods Enzymol. I7, 335-357. Swenson, R. P., Hilliams, C. H. Jr. and Massey, V. (1982) J. Biol. Chem. 257, 1937-19AA. Caruthers, M. H., Beaucage, S. L., Becker, C., Efcavitch, 11., Fisher, E. F., Galluppi, C., Goldman, R. deHaseth, P., Martin, F., Matteucci, M. and Stabinsky, Y. (1982) in Setlow and Hollaender (eds.) Genetic Engineering, I, p. 1-17, Plenum Publishing Co. Timothy, J. C. and Rodriguez, R. L. (1982) Gene 20, 305-316. Itakura, K., Rossi, J. J., and Wallace, R. B. (198") Ann. Rev. Biochem. 53. 323-356. Suggs, S. V., Hirose, T., Miyake, T., Kawashima, E. H., Johnson, M. J., Itakura, x. and Hallace, n. a. (1981) ION-UCLA Symp. Mol. Cell. Biol. 23, 682-693. Kirk, T. K., Croan, S. C., Tien, M., Murtaugh, K. E. and Farrell, R. L. (1986) Enzyme. Microbial. Technol. 8, 27-32. PLEASE NOTE: Copyrighted materials in this document have not been filmed at the request of the author. They are available for consultation, however, in the author's university library. These consist of pages: 131-138 139-149 University. Microfilms International 300 N. ZEEB RD. ANN ARBOR, MI 48106 (313) 7614700 APPENDIX B USE OF SYNTHETIC OLIGONUCLEOTIDE PROBES FOR IDENTIFYING LIGNINASE cDNA CLONES BY Yi-zheng Zhang and C. Adinarayana Reddy Accepted to Publish in Methods in Enzymology Vol.168 (1988) 139 APPENDIX 3 USE OF SYNTHETIC OLIGONUCLEOTIDE PROBES FOR IDENTIFYING LIGNINASE cDNA CLONES Ligninase, an extracellular, HZOZ-dependent, glycosylated heme protein, has recently been purified from a white-rot basidiomycete, Bhgngxgghgggg ghgyggspgxigm (1,2). This enzyme is synthesized under nitrogen-limited conditions during secondary metabolism. This enzyme has many potential applications, such as upgrading lignocellulosic materials 21; delignification for the efficient production of fuels, feeds and chemicals; biobleaching of pupls; treatment of industrial wastes; controlled modification of lignins to produce aromatic chemicals and cracking of petroleum. To better understand the nature, organization, expression and regulation of the ligninase genes and to develop the full bioprocessing potential of this enzyme, we initiated studies to isolate and characterize the cDNA clones for ligninase. Principle Synthetic oligodeoxyribonucleotides are useful as specific probes for the detection and isolation of cloned cDNA or gene sequences of interest (3-5). As a general approach, a chemically synthesized mixture of oligonucleotides whose sequences represent all possible codon combinations, predicted from a partial peptide sequence within a protein, are employed. Therefore, one of the oligonucleotides in the mixture must be complementary to a region of DNA coding for the protein. 1A0 Since probes which form duplexes with a single base pair mismatch have significantly less thermal stability than their perfectly matched counterpart, appropriate choice of hybridization temperature or filter wash temperature would virtually elimilate the formation of mismatched duplexes without affecting the formation of perfectly matched ones. Hence, the use of stringent hybridization criteria would allow the selection of the single correct sequence from the mixture. The basic steps involved in cloning ligninase cDNA from P. ghgygggpgxium are as follows (6): 1) construction of cDNA library using poly(A) RNA from a 6-day-old lignin degrading culture; 2) isolation of cDNA clones specific for 6-day culture using differential hybridization; 3) synthesis of oligonucleotide probes, deduced from partial amino acid sequences of ligninase; A) use of these probes to screen, isolate and identify the ligninase clones from the 6-day specific cDNA mini-library. Isolation of poly(A) RNA 2. ghxysgfingrigm strain BKM-F 1767 (ATCC 2A725) is grown in 50 ml of low nitrogen medium (modified to contain 20 mM NaOAc, pH A.5, instead of 10 mM 2,2-dimethy1 succinate) in 500 ml Erlenmeyer flasks (6). Flasks are flushed with pure oxygen at the time of inoculation and reflushed every other day. A modified hot phenol extraction procedure is used for RNA isolation (7). In this procedure, mycelia from 1 liter of a 6-day- old culture are harvested by centrifugation, washed with 50 mM NaOAc (pH 5.2) andsuspended in 20 ml of extraction buffer (0.15 M NaOAc pH 5.2, 5% SDS and 2 mM EDTA). The mycelial suspension is then mixed with 10 ml phenol and 25 g glass beads (0.A5 mm in size) and blended in an Omni- Mixer (Sorvall) for 20 min. Ten ml of chloroformzisoamyl alcohol (2A:l) is added to this mixture and the blending is carried out for an 1A1 additional 10 min. The mixture is then heated at 60°C for 15 min while gently shaking and is chilled on ice. After centrifugation (10,A00 x g for 20 min at AOC), the upper phase is extracted with phenol-chloroform (1:1), the RNA is precipitated with ethanol and the pellet is dissolved in 10 ml of 2 mM EDTA. After heating at 65°C for 10 min, the RNA solution is chilled on ice for 10 min and 10 ml of 2 X loading buffer (1 M NaCl, 20 mM Tris.HCl pH 7.5, 2 mM EDTA and 1% SDS) is added. The RNA solution obtained is generally too viscous to pass through the oligo(dT)-cellulose column, hence, it is mixed with oligo(dT)-cellulose powder (BRL, Gaitherburg, MD) and the mixture is gently shaken for 30 min at room temperature. The oligo(dT)-cellulose with the bound poly(A) RNA is then spun down, washed with loading buffer and then packed in a small glass column (10 x 1 cm). The column is washed with loading buffer and the poly(A) RNA is eluted with TES buffer (10 mM Tris.HCl pH 7.5, 1 mM EDTA and 0.2% SDS). For isolating 2-day poly(A) RNA, the same procedure is used except that the RNA solution is directly loaded on the oligo(dT)-cellulose column. Using this procedure, 10 to 20 mg of total RNA is obtained from 1 liter of culture and poly(A) RNA accounts for l to 2% of the total RNA. Construction of cDNA Library The first strand cDNA is synthesized using AMV (avian myeloblastosis virus) reverse transcriptase and the second strand cDNA is synthesized utilizing RNase H and DNA polymerase I of 5. £911 (8). Double stranded cDNA is dC-tailed and then annealed with dG-tailed pUC9 (9) which is digested with restriction enzyme PstI. The annealed DNA molecules are transformed into E. 9911 JM83 (an; 1g§;p;g strA thi 80dlg§2 M15) using the procedure of Hanahan (10). The transformed cells are spread on 2YT 1A2 plates (1.6% Bacto-tryptone, 1% yeast extract, 0.5% NaCl and 1.5% agar, pH 7.0) (11) supplemented with 100 pg/ml ampicillin and A0 pg/ml X-gal (5-bromo-A-chloro-3-indolyl-fi-D-galactoside, 11). Ten thousand white E. 9911 colonies that are potential cDNA clones are picked and individually stored in each well of 96-well microtiter plates. Differential Hybridization figgignglg Ligninase has been shown to be produced in 6-day-old idiophasic culture of 2. ghgyggspgxium grown in low-nitrogen medium; the enzyme is not detectable in l- or 2-day-old cultures in primary growth. Therefore, differential hybridization technique allows isolation of the cDNA clones specific for the idiophase. It is much easier to screen such a mini-library of idiophasic clones than to screen the total cDNA library for isolating cloned cDNA of interest. Ergggguzg Each cDNA clone is replicaplated onto 137 mm HATF filter (Millipore, Bedford, MA) which is placed on LB plates (1% Bacto- tryptone, 0.5% yeast extract, 1% NaCl and 1.5% Bacto-agar, pH 7.2; 100 pg/ml ampicillin is added just before pouring plates) and then is grown at 37°C for 1A h. The filter paper is peeled off and dried on 3 MM Whatman chromatography paper for 20 min. One circular 3 MM paper (about 137 mm diameter) is placed in each of four Petri dishes (150 x 10 mm) labeled 1, 2, 3 and A. The 3 MM papers are saturated with 10% SDS, denaturation solution (1.5 M NaCl and 0.5 M NaOH), neutralizing solution (1.5 M NaCl and 0.5 M Tris.HC1 pH 8.0) and 2 x SSPE (0.36 M NaCl, 20 mM NaHZPO4 and 2 mM EDTA, pH 7.A) in Petri dishes 1, 2, 3 and A, respectively. The cDNA clones on the HATF filters are lyzed, denatured and neutralized in dishes 1, 2 and 3, respectively, by placing the filter in each of the plates for 5 min (7). The filter is then placed in 1A3 dish A for 5 min. The cDNA blots are then baked at 80°C for A h, wetted with 6 x SSC (7) and then washed in prewashing solution (3 x SSC and 0.1% SDS) at 65°C for 10 h with several changes of the same solution to completely remove all cell debris. After briefly blotting on a 3 MM paper, every eight blots are put in one hybridization bag and 16 ml of hybridization solution (50% formamide, 5 x Denhardt's solution, 1 M NaCl, 10 mM Tris.HCl pH 8.0, 1 mM EDTA, 50 mM NaHZPO4 pH 6.8, 10 pg/ml carrier DNA) is added. The blots are prehybridized at A2°C overnight, the synthetic 2-day cDNA probe (see below) is added at a final concentration of l x 106 cpm/ml and the hybridization is carried out at A2°C for 36 h. The hybridized blots are washed in high stringent solution (10 mM Tris.HC1 pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% NaAP207 and 50 mM NaCl) three times at room temperature and thrice at 65°C (each wash is for 15 min). The blots are then exposed to X-ray film for a suitable length of time and the film is developed (7). The probe is then washed off at 65°C for 2 h in distilled water and the bolts are hybridized with the 6-day cDNA probe (see below). The 2-day and 6-day cDNA probes are synthesized, respectively, from poly(A) RNA isolated from 2- and 6-day-old cultures using a modification of the procedure of Berlin and Yanofsky (12). The reaction mixture used for probe synthesis contains in 50 pl: 50 mM Tris.HC1 pH 8.3, 8 mM MgCl 40 mM x01, 2 mM DTT, 1 mM dATP, dTTP and dGTP, 1 pg oligo- 2. (dT)12_18, 1 pg poly(A) RNA, 50 U RNasin and zoo pCi a-32P dCTP (~600 Ci/mmole). The reaction is started by adding 300 U M-MLV (Moloney murine leuemia virus) reverse transcriptase (BRL, Gaitherburg, MD). The reaction is carried out at A3°C for l h and then 2 pl of 0.5 M EDTA, 10 pl of 10 mg/ml carried DNA and 7 pl of 20% SDS are added. The unincorporated nucleotides are separated from the labeled cDNA by 1AA passing the reaction mixture through a Sephadex-GSO column. The fractions showing the high radioactivity are combined and to this cDNA elute 1/10 volume of 2 M NaOH is added and the mixture is heated at 65°C for 5 min. After chilling on ice for 10 min, 2 M HCl (equal to 1/10 volume of the cDNA elute) is added to neutralize the probe. Of the 10,000 cDNA clones in our cDNA library, 850 clones are shown to be specific to the 6-day cDNA probe using the differential hybridization technique described above. A representative differential hybridization blot is shown in Fig. 1. 1A5 L_.Li 11 1 1 .7 . t .1, . 1.11___ Figure 1. Differential hybridization and identification of a ligninase cDNA clone. The cDNA clones in the library are hybridized with the 2-day cDNA probe (A), 6-day cDNA probe (B), or synthetic oligonucleotide probe 1A.l (C). Note that the ligninase cDNA clone indicated by arrow in each panel shows hybridization with the 6-day cDNA probe but not with the 2- day cDNA probe. From: Y. Z. Zhang, G. J. zylstra, R. H. Olson, and C. A. Reddy, Biochem. Biophys. Res. Commun. 137, 6A9 (1986). 1A6 Identification of Ligninase cDNA Clones gliggnuglgggjgg_pgghg§ Three oligonucleotide probes deduced from the amino acid sequences of selected tryptic peptides of ligninase H8 (13) are used for screening the mini-cDNA library. The sequences of these probes are shown below: GTQ-TTQ-GGN-AAP-CA PROBE 14.1 AAP-CAN-GTQ-TTQ PROBE 14.2 ' CAN-AAP-GTP-CTP-CG PROBE 25 N - ACCT/U, P — AG, Q - CT/U All probes are mixture of 32 oligonucleotides because of the redundancy of the genetic code. The oligonucleotides are end-labeled with polynucleotide kinase in a 50 pl total reaction mixture containing kinase buffer (50 mM Tris.HC1 pH 8.0, 10 mM MgCl and 15 mM DTT), 500 ng 2 synthetic oligonucleotide (dissolved in water), 100 pCi 1-32P ATP (A500 Ci/mmole, ICN, Irvine, CA) and 10 units TA polynucleotide kinase (IBI, New Haven, CT). The mixture is incubated at 37°C for 30 min and then cooled on ice until use. 2Igpgrggign_gfi_gpng_hlg§§ The cDNA blots are prepared as described above using the 6-day specific cDNA clones. H brid b w The cDNA blots are prehybridized at 37°C for A h in a solution containing 6 x SSC, 1 x Denhardt's solution, 0.5% SDS, 0.05% NaAP207 and 10 pg/ml carried DNA. The prehybridization solution is drained out and hybridization solution (6 x SSC, 1 x Denhardt's solution, 0.05% Na and 20 pg/ml tRNA), 4P2°7 along with probe 1A.l, is added. The hybridization is carried out at room temperature for 1 h. The hybridized blots are washed once at room temperature in a solution containing 6 x SSC and 0.05% NaAP207 for 30 1A7 min and once at A2°C in the same solution for 10 min. Four cDNA clones show strong hybridization with probe 1A.l. A representative clone is shown in Fig. 1C. To further identify these clones, the plasmid DNA isolated from these clones is digested with different restriction enzymes in such a way that each clone gives three unequal fragments, one from the vector and two from the cDNA insert. The fragments are transferred onto nitrocellulose paper using Southern blotting (7) and the DNA blots are hybridized with the above three synthetic probes as described above. The temperature for the second washing for probe 1A.2 is at 32°C. The results show that only the cDNA insert in clone pCLG5 hybridizes with all the three probes, whereas the other three clones (pCLG3, pCLGA and pCLG6) show detectable hybridization with probe 1A.l only (Fig. 2). Furthermore, each of the three probes hybridizes with only one cDNA fragment from a given clone, indicating that the probes are specific for unique sequences in the cDNA. Additional studies show that the product expressed by the cDNA insert of pCLG5 is immunoreactive with the ligninase (H8) antibody, indicating that the cloned insert in pCLG5 is ligninase cDNA. The sequence analyses show that the probe sequences used for identifying ligninase cDNA clones are present in both pCLGA and pCLG5. The procedure described above for isolating the ligninase cDNA of E. ghgysgspgxigm is potentially applicable for isolating the ligninase genes from other organisms. Besides, the procedure described can be used to isolate genes for other secondary metabolic enzymes such as glucose oxidase which has recently been purified from P. ghzygggpgrium (1A). 1A8 Figure 2. Hybridization of ligninase cDNA clones with three oligonucleotide probes. Clones pCLG3 and pCLGA are digested with BgmHI and HindIII, pCIGS is digested with BgmHI, flingII and PstI, and pCLGé is digested with flindIII and EggI. The DNA blots are hybridized with probe 1A.l (panel A), 1A.2 (panel B) and 25 (panel C). Different lanes contain: pCLG3 (lane 1), pCLGA (lane 2), pCLG5 (lane 3) or pCLG6 (lane A). 1A9 Acknowledgments This research was supported, in part, by grant DE-FG02-85#13369 from the U.S. Department of Energy, Division of Basic Biological Sciences, NSF grant DMB-8AA271, and a grant from the Michigan Agricultural Experiment Station. This is publication no.12063 from the Michigan Agricultural Experimental Station. 10. 11. 12. 13. 1A. 150 REFERENCES M. Tien and T. K. Kirk, Proc. Natl. Acad. Sci. U.S.A. 81, 228 (198A). M. H. Gold, M. Kuwahara, A. A. Chiu, and J. K. Glenn, Arch. Biochem. Biophys. 23A, 353 (198A). R. b. Wallace, M. J. Johnson, T. Hirose, T. Miyake, E. Kawashima, and K. Itakura, Nucl. Acid Res. 9, 879 (1981). J. W. Sozostak, J. I. Stiles, T.-K. Tye, P. Chiu, F. Sherman, and R. Wu, Meth. Enzymol. 68, A19 (1979). A. A. Reyes and R. B. 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