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J» /{/O\§‘/.>3 PRODUCTION OF D-ERYTHROASCORBIC ACID BY YEAST By Jung-Hsiang Tai A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1983 ABSTRACT PRODUCTION OF D-ERYTHROASCORBIC ACID BY YEAST By Jung-Hsiang Tai Production of ascorbic acid analogues by yeast was‘ investigated. A strain of gaggidg 5293;; producing a high yield of an ascorbic acid analogue was screened from 57 yeasts. The analogue produced by Q. 322531 was identified as D-erythroascorbic acid. ‘ * Investigation of nutrient requirements for production /// of D-erythroascorbic acid by 9. 323121 indicated a medium containing 5% glucose, 1% NHuN03, 0.2% phosphate buffer.and 0.05% MgSOu-7H20 was optimal. Vitamins and other minerals had no effect on the production of the analogue. The fer- mentation was optimal when carried out at 23 00, pH 6.0 with high aeration rate and 15% inoculum. Under these con- ditions, the fermentation was complete after 7 days. Ethylmethanesulfonate was used as mutagen to select high productivity mutants. Two mutants which showed im- proved productivity (1h0% and 160%) were isolated. An L-ascorbic acid sensitive mutant was also isolated.' ACKNOWLEDGMENTS The author is deeply indebted to his major professors, Dr. Haruo Momose and Dr. James Pestka for their guidance, understanding and encouragement throughout this work. Appreciations are also expressed to Dr. Everett Benefie, Dr. Pericles Markakis and Mr. Sterling Thompson for their helpful suggestions as members of the graduate committee. Special thanks to Marguerite Dynnik for her assistance in the laboratory. Finally, I wish to thank my wife, Yi-ding, for her help in preparing the final manuscript. ii TABLE OF CONTENTS LIST OF TABLES ....................................... LIST OF FIGURES .....................o..o............. INTRODUCTION ......................................... LITERATURE REVIEW ..............oo...............o.... Historical Aspects of Ascorbic Acid Analogues ... Ascorbic Acid Analogues and the Antiscorbutic ActiVity eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeo Occurrence of Ascorbic Acid Analogues in Organisms eoeeeeeeeeeeeeeeeeeeeeeoeeoeeeeoeeeee Bicsynthesis of Ascorbic Acid Analogues ......... Synthesis and Production of Ascorbic Acid Analogues eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeoe Uses of Ascorbic Acid Analogues ................. MATERIALS AND METHODS 000......OOOOOOOOOOOOOOOOOOOOOO. Madia eeeeeeeeeeeeeeeeeeeeoeeeeeeeeeeeeeeeeeeeeee YOEBU Cultures eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Cultivation Of YOBSUS eoeeeeeeeeeeeeeeeeeeeeeeeee GrOWth Pgrameter eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Eatimation Of D'ErYthrOBBCOPbio A01d eoeeeeeeeeee Mutation and Selection eeeeeeeeeeeeeeeeeeeeeeeeee RESULTS AND DISCUSSION 00....OOOOOOOOOOOOOOOOOOOOOOOOO Screening of D-Erythroascorbic Acid Producing Yeast eeeeeeeeeeeeoeoeooooeeeoeeeeeeeeeeeeeeeee Media COMPOSition eeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Physical Conditions eoeeeeeeoeeoeeeeeeeeeeeeeeeee Time Course for D-Erythroascorbic Acid PrOduction eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Genetic Improvements of D-Erythroascorbic Acid PrOduction eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee SUMMARY AND CONCLUSION 0......OOOOOOOOOOOOOOOOOOOOOOO. LIST OF REFERENCES 00..0.000....OOOOOOOOOOOOOOOOOOO0.0 iii Page Table 2. 3. 6. 7. 9. 10. LIST OF TABLES Physical properties and antiscorbutic activity of ascorbic acid analogues .................... The occurrence of ascorbic acid analogues in various Speciaa or yeast eeeeeeeeeeeeeeeeeeeeee The availability of carbon compounds in ascorbic acid analogues production by 9e krusei and g3 0§;if0rnifi eeeeeeeeeeeeeeeeeee The availability of nitrogen compounds in ascorbic acid analogues production by Q, kruse; and fl. Qflli£Q£B1£ eeeeeeeeeeeeeeeeeee Effect of trace elements on D-erythroascorbic acid production by Q, grass; at 30 C .......... Effect of minerals concentration on D-erythro- ascorbic acid production by Q. 323311 at 23 c . Effect of vitamins concentration on D-erythro- ascorbic acid production by Q. 322311 at 23 C . Effect of amino acids on D-erythroascorbic acid production by Q. krusei .................. D-Erythroascorbic acid productivity among mutants derived from wild type 9. gggggi ...... D-Erythroascorbic acid productivity among revertants derived from amino acids requiring mutant eeeeeeeeeeeeeeeeeeeeeeeeeeeeee iv Page 3.0 3h 35 us as h? 57 57 Figure 1. 2. 3. 10. 11. 12. LIST OF FIGURES Structures of ascorbic acid analogues. ........ Paper chromatogram of Q, krugei fermentation broth. eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee The effect of phosphate concentration on the D-erythroascorbic acid production by Q. gzgggi. The effect of carbon concentration on D-erythroascorbic acid production by Q, 323121. The effect of nitrogen concentration on D-erythroascorbic acid production by Q, kznggi. Glucose concentration and D-erythroascorbic 301d production. eeeeeeeeeeeeeeeeeeeeeeeeeeeeee The effect of aeration on D-erythroascorbic acid prOductione eeeeeeeeeeeeeeeeeeeeeeeeeeeeee The effect of initial pH on D-erythroascorbic 861d prOdUCtione eeeeeeeeeeeeeeeeeeeeeeeeeeeeee Temperature effect on D-erythroascorbic acid prOdUCtione eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee The effect of inoculum ratio on D-erythro- ascorbic 801d.pPOductiOne eeeeeeeeeeeeeeeeeeeee Time course of D-erythroascorbic acid fermentation. eeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeee Growth curves and D-erythroascorbic acid . productivity of wild type yeast and mutants. .. Page 38 to no he he so 52 53 5 3 SS 59 INTRODUCTION The common feature of ascorbic acid analogues in their chemical structures is the five-atom lactone ring containing the dienolic systems. The dienolic systems in these acids cause their acidity, reducing properties, and instability in alkaline solution. Synthesis of ascorbic acid analogues has consisted in devising methods for the formation of 2-keto and 3-keto hydroxy acids followed by their enoli- zation and lactonization. Bioconversion of D-sorbitol to L-sorbose by Aggtgpggtgr subogydang in the L-ascorbic acid synthesis and the direct fermentation in D-araboascorbic acid production from D-glucose by Penicilligm,sp. are two examples of biochemical synthesis of these compounds. The biosynthesis of ascorbic acid analogues by yeasts has also been reported. Bleeg (1966) reported on L-ascorbic acid from L-galactono-Y-lactone and D-glucose by figgghgzg: (mace; gazexigigg and its mitochondria. Yasuda (1967) reported on D-erythroascorbic acid from D-xylose by angigg sp. Heick et a1. (1969, 1972) also reported on L-ascorbic acid formation from D-glucose by many strains of yeasts. Microorganisms can grow in a wide range of physical and chemical enviroments; their growth and other physio- logical activities are a response to their physiochemical 1 2 enviroment. Development of a microbiological process begins from a test tube or flask scale fermentation where basic medium composition and physical variables are screened. Genetic techniques are then applied to obtain high yield of the product. The most common means of improving yield after physiological variables have been optimized is by the process of mutation. The objectives of this study were to establish apé propriate nutritional and physiological conditions for high yield D-erythroascorbic acid production by Qangigg gzgggi. In this report the role of carbon and nitrogen sources, the effects of inorganic salts, vitamins and amino acids, aeration, temperature, and pH on D-erythroascorbic acid production have been investigated. Ethylmethanesulfonate mutation and selection of analogue-resistant mutants as means of increasing yield were also investigated. LITERATURE REVIEW Ascorbic acid analogues (Figure 1) are the group of compounds that have a five-atom lactone ring containing the dienolic systems. Their chemical properties and ab? sorption data closely resemble to those of L-ascorbic acid, the well-known antiscorbutic vitamin C (Smith, 19h6). The unique feature of this group of compounds lies in the di- enolic system and it is the system which is responsible for the remarkable reducing properties displayed by these substances. The discovery of this group of compounds has been closely related to scurvy. Historica As ects of Ascorbic Acid Analogues Scurvy is one of the oldest diseases known to mankind. It was the major cause of death among the early sea voyagers who could not get the fresh supply of fruit and vegetables. The value of citrus fruit for preventing scurvy has been recognized for centuries. The first clinical experiment on scurvy was carried out by Lind in 17k? that showed con- clusively the therapeutic value of lemon Juice in curing the disease. Nearly half a century elapsed before his proposals were adopted in 1795. A dramatic fall in the incidence of the disease then ensued, and by 1800 the conquest of scurvy 3 nonwoasnu new can mace oanhoona no monopoanamua.w .me ewes efinhoouw 6338586 mommw mouwum meuw.m $6.0m Am. efio< ewes ofinhooms cannooma uquhwonhaouo Ionahmnn meow mcmmm $5.6m mo..oum Amy Adv UH04 ownhooma -ooeamuq mammw . Mignon m-o.om Am. Ufio¢ awakened IQUhKoonto mum muouom .mv vwo< ownhoons -oeseemuq mum msouom mumuom Aha cwom ownhoomdnq N mo mw m-o.om “my cwod .ownhooma IoosMuA mun . muwuom mouo-m may ease cannooma nosnthMun momma «P. cwo¢ cannoomm IOOH—HGIAH mommw mouwsm mououm an S owed canaoond uoaadln mmo $6.6m . muouom aowv cwo4 ownpooma 532$ MG momma mumnom Amav vfiod ownhooma IOHSéIA N m6 we _ mIOIom mol...m AmPV UHO4 ownpoomm 6376 mcmmw monoum A E .fwh/wo mo\ ./om wand ownhoowm 13033.6 mommw mo 4.61m m..o.om Adrv dwo< ewnhoomw IOfiflHhhmIA no mo 8: 32.5983 -... P .mwm was practically complete. Modern work on scurvy began when Holst and Frolich produced the disease in guinea-pigs by giving them a res- , tricted diet in 1907. In 1912 Hopkins showed that animals could not thrive on purified isolated food constituents and he introduced the concept of "accessory food factors"; the same year Casimir Funk postulated the existence of "vitamins" and subsequently suggested the absence of different vitamins was responsible for the onset of deficiency diseases. In 1917 Zilva and Wells described an antiscorbutic agent in lemon juice for preventing scurvy. The antiscorbutic agent was named as water-soluble C at first; in 1920 Drumond re- named this substance as vitamin C. In 1922 Sherman and his co-workers devised a bioassay method for vitamin C. This method was based on the determination of the minimum amount of samples needed to prevent signs of scurvy in guinea-pigs. In 192k Zilva and his colleagues extracted a BOO-fold con- centration of the active compound from lemon juice. In 1928 Szent-Gyorgyi isolated what he called "hexuronic acid" in crystallin form, from cabbage and adrenal cortex. During the course of this work, he established C6H806 as the molecu- lar formula, the presence of an acidic functional group, and the facile and reversible nature of its oxidation. In 1932 Waugh and King isolated the vitamin from lemon and showed it to be identical with the "hexuronic acid" of Szent- Gyorgyi; they also showed that the antiscorbutic activity of hexuronic acid was similar to that of vitamin 0 obtained 7 from orange juice. In 1933 Haworth determined the structure of this vitamin, Reichstein and other researchers synthesized this vitamin, Szent-Gyorgyi and Haworth changed the name of hexuronic acid to L-ascorbic acid. In 1965, the trivial name L-ascorbic acid was recognized by the IUPAC-IUB Com- mission on Biochemical Nomenclature as an acceptable name for vitamin C (King, 1961; Sebrell and Harris, 1967: Sharman, 197k: Cameron and Pauling, 1979; Hodges, 1980; Crawford and Crawford, 1980). Ascorbic Acid Analogues and the Antiscorbutic Activity Investigation by Reichstein et al. (1933a, 1933b, 193k) made it possible to study the antiscorbutic action of synthetic L-ascorbic acid and some of its analogues by feeding the vitamin C-exhausted guinea pigs the diets supplemented with compounds to be tested for their anti- scorbutic activities. Although L-ascorbic acid is the most active antiscorbutic substance, some other analogues (Table 1) also show this feature with less activity, some of the analogues do not possess the antiscorbutic activity. A comparison of the structures and the antiscorbutic activity of these compounds reveals that for antiscorbutic activity the asymmetric carbon must lie to the left of lactone oxygen, when their formulas are written according to the convention in Figure 1 (Harris, 1967), no exception to this rule is yet known. Table 1.--Physical properties and antiscorbutic activity of ascorbic acid and its analogues -..—...— .5 Compound M- P - (oc) (“113” Aotivityb D-Erythroascorbic Acid (1) - - - L-Ascorbic Acid (2) 192 +23 1 6-Deoxy-L-ascorbic Acid (3) 168 +37 0.3 D—Araboascorbic Acid (h) 17M -17 0.05 6-Carboxy-L-ascorbic 206-210 - - Acid (5) ' L-Fucoascorbic Acid (6) - - 0.02 L-Rhamnoascorbic Acid (7) 199 +28 0.2 L-Glucoascorbic Acid (8) 1h0 +2h 0-025 (hydrate) . D-Glucohe toascorbic Acid (9) - - 0.01 L-Erythroascorbic Acid (10) 161 +9 0 D-Xyloascorbic Acid (11) 192 '23 0 L-Araboascorbic Acid (12) 170 +17 0 D-Gl bi Acid 1 1&0 - ucoascor c ( 3) (hydrate) 22 0 D-Galactoascorbic Acid (1h) 13h -6 O L-Guloascorbic Acid (15) 18k -22 0 O L-Alloascorbic Acid (16) 177 +29 aRotation in water. bAntiscorbutic activity relative to L-ascorbic acid. 9 Occurrence of Ascorbic Acid Analogues in Organisms L-Ascorbic acid, D-araboascorbic (isoascorbic) acid and Dberythroascorbic acid have been found in organisms. L-Ascorbic acid is present in most higher plants (King, 1973). and some algae (Talpasayi, 1967; Aaronson, et al., 1977). and is most highly concentrated in the more actively growing regions of plant tissues, as in root tips, seed sprouts, flowering and fruiting parts and green leaves. Only a few species of animals need to consume L-ascorbic acid in the diet, whereas most other species are able to synthesize their own. The biosynthetic ability is lost in the primates, guinea pig, flying mammals, late birds, fish, insects, invertebrates (Kutsky, 1981). Herein lies one of the great genetic mysteries, which is still incompletely solved (Chatterjee, 1973). Bourne and Allen (1935) suggested that vitamin C-like reducing substance is distributed in lower organisms. Geiger- Hubber and Galli (19h5) found the presence of an ascorbic acid analogue in Aspergillus sp.. Kalyanasundaram and Saraswathi- Devi (1955) found it in Fusarium. Bleeg (1966), Humpers (1967) and Heick et a1. (1969, 1972) reported the occurrence of L-ascorbic acid.among yeasts. Shigeoka (1977a) reported the occurrence of L-ascorbic acid in Eu lens, a protozoa. However, extensive studies in relation to lower organisms are needed to develop the information more fully. D-Araboascorbic acid has been rarely found in biological materials or products. Isherwood et a1. (1953. 195k) found 10 it in cress seedling in D-altrono-Y—lactone solution and in the rat injected with manno-V—lactone. Takahashi et a1. (1960) found its presence in various Penicillium sp.. Yagi et a1. (1967) screened about 5000 strains of fungi and bacteria, only Penicillium, but no other genera, was obtained as D-araboascorbic acid producers. D-Erythroascorbic acid was found in Candida sp. by Yasuda (1967). Biosynthesis of Ascorbic Acid Analogues L-Ascorbic acid is biosynthesized by a wide variety of plants and animals. It has been shown (Loewus, 1971) that in the biosynthesis of L-ascorbio acid, D-glucose is con- verted into L-ascorbic acid by two separate pathways. One in which C-1 of Dbglucose becomes C-6 of L-ascorbic acid, and one in which C-1cfi'D-glucose becomes C-1 of L-ascorbic acid. Those animals which cannot synthesize this compound contain no L-gulonolactone oxidase activity (Chatterjee et al., 1975). the terminal enzyme of L-ascorbic acid bio- synthesis. A thorough exploration of L-ascorbic acid synthesis by the simpler form of life, such as bacteria, yeasts and molds, has not been made. ' Animals synthesize L-ascorbic acid apparently from D-glucose via the D-glucuronic acid pathway of metabolism (Isherwood, Chen and Mapson, 195M). In this pathway (Reaction 1) glucose is oxidized to D-glucuronic acid, which is reduced to L-gulonic acid. L-gulonic acid is then lactonized to L-gulonolactone, which is oxidized to L-ascorbic 11 acid through the intermediate 2-keto-L-gulonolactone. The step from 2-keto-gulonolactone follows by rearrangement without requirement of a specific enzyme (King, 1973). Reaction I: D-glucose -9 D-glucuronic acid -9 L-gulonic acid -9 L—gulonolactone -9 (2-keto-L-gulono- lactone) -+ L-ascorbic acid Synthesis of L-ascorbic acid in plants is accomplished by a greater variety of reactions than those identified in animal tissue. Mapson and his co-workers (1956) demons- trated that L-gulonolactone, L-galactonolaotone, D-glucurono- lactone, and methyl-D-galacturonate were converted to L- ascorbic acid in cress seedlings. From their studies, they proposed two pathways for L-ascorbic acid synthesis in plants. One of these is the same as that of animals. The other pathway (Reaction II) began from galactose is briefly summarized as follows: Reaction II: D-galactose -e methyl-D-galacturonate -+ L-galactonolactone -9 L-ascorbic acid The experimental results of Loewus (1961) in strawberry did not unequivocally support the above mechanisms as major pathways, he proposed the third pathway (Reaction III): Reaction III: D-glucose -+ D-glucose 6-phosphate -+ D-gluconate 6-phosphate —» (3-keto-D- gluconate 6-phosphate ~9 L-ascorbic acid L-ascorbic acid synthesis in lower organisms is not yet clear, however, Bleeg (1966), Nishikimi et a1. (1978) found an enzyme in yeast which is similar to a key enzyme 12 for L-ascorbic acid biosynthesis in animals. Shigeoka and his co-workers (1977b) also proposed a pathway (Reaction IV) in Euglena. Reaction IV: D-glucose D-galactose UDP-glucose e———————- UDP-galactose 1 UDP-glucuronic acid -—5 UDP-galacturonic acid 1' D-gltcuronic acid D-galacturonic acid D-glucurono-Y-lactone L-galactonic acid L-gulono-Y-lactone L-galictono-Y—lactone \ L-ascorbic acid The biosynthetic pathway of D-araboascorbic acid in Penicillium notgtum has been elucidated by Takeshi and Mitsumoto (1961, 1965). This compound was synthesized from D-glucose, D-gluconate, D-glucono-Y-lactone, D-glucono-S- lactone, sucrose, maltose, and starch, but not from other substances such as D-glucuronate, 2-ketogluconate and glycerol. The proposed pathway is shown in Reaction V: Reaction V: Déglucose -+ D-glucono-S-lactone -91D- glucono-y-lactone —9 D-araboascorbic acid An indophenol reducing substance formed by Serrgtig mazgggggps was reported (Bereusi and Illenyi, 1938) as L- ascorbic acid. Takahashi et al. (1976) demonstrated this substance to be D-erythroascorbic acid while S. mazggsggng was cultured in 2% xylose. Other analogues from D-galactose and D-glucose were identified as D-xyloascorbic acid and D-araboascorbic acid respectively. The biosynthetic pathway of D-erythroascorbic acid (Reaction VI) in Candidg utilis 13 was proposed by Murakawa et al. (1977) as follows: Reaction VI: D-arabinose -9 D-arabono-S-lactone D-arabono-Y-lactone -+ D-erythroascorbic acid Sygthgsis and Productigp of Ascorbic Acid Analogues Four main methods are available for the synthesis of ascorbic acid analogues containing the characteristic five- membered unsaturated dienolic ring (Smith, 19u6: Theander, 1980: Hay, Lewis and Smith, 1967aL The synthetic methods based on the formation of 2-keto or 3-keto hydroxy acids followed by their enolization and lactonization are as follows: 1. Cyanohydrin synthesis from glycos-2-uloses. 2. Enolization and lactonization of glyculosonic acids or esters. 3. Condensation of hydroxy aldehydes with ethyl glyoxylate or mesoxalate. h. Condensation of esters of hydroxy acids. Among the analogues, L-ascorbic acid and D-arabo- ascorbic acid productions have been sold commercially. The most important starting-material for the synthesis of L- ascorbic acid is D-glucose. Commercial production of L-ascorbic acid uses the Reichstein-Grussner synthesis (Hay, Lewis and Smith, 1967b: Crawford and Crawford, 1980). In this procedure (Reaction VII) D-glucose is chemically hydrogenated to produce D- sorbitol. Non-isolated sorbitol thus produced is then 1h biochemically dehydrogenated by Acetobgcter subogydgns to L-sorbose. The isolated sorbose is then condensed with acetone to form diacetonesorbose. This is chemically oxi- dized to diacetone-Z-keto-L-gulonic acid, which, after hydrolysis, enolization, and lactonization yields L-ascorbic acid. This method would be further simplified if the problem of direct oxidation of sorbose to 2-keto-gulonic acid were solved chemically or biochemically. Reaction VII: D-glucose -q D-sorbitol -+ L-sorbose -9 diacetonesorbose -9 diacetone-Z-keto-L- gulonic acid -9 L-ascorbic acid The strains with powerful ability of L-sorbose forma- tion from D-sorbitol are Gluconobactgz zgsgum and Acgtg- bacter suboxydgns. In this fermentation, 18% of D-sorbitol is added into medium, after 30 hours fermentation, 95% of D-sorbitol can be converted to L-sorbose. Newer processes of L-ascorbic acid production (Kulhanek, 1970) based on two stage fermentations (Reaction VIII). Glucose is converted by biochemical dehydrogenation in the presence of calcium carbonate to calcium S-keto-D—gluconate by Acetobacter suboxydgng. Calcium S-keto-D—gluconate was catalytically hydrogenated to a mixture of calcihm D- gluconate with calcium L-idonate in a 1:1 ratio. A second fermentation with Psegdgmongs fluorescens or other bacteria oxidize the calcium L-idonate to 2-ketoidonic acid, which, can be transformed into L-ascorbio acid by enolization and lactonization. The Dugluconate processed analogously yields 1S ‘D-araboascorbic acid. Therefore the reaction mixture has to be processed further in order to separate out either hexonate, or, at least, to isolate the L-idonate component from the mixture. 1 Reaction VIII: D-glucose (D-gldconic acid) S-keto-D-gluconate L-idonate D-g uconate 2-ketg-idonic acid 2-ke88-gluconic acid L-ascorbic acid D-araboascorbic acid The newer procedure cannot compete with the Reichstein- Grussner synthesis. Preparation of L-ascorbic acid by this procedure would become more interesting if successful realization of stereospecific hydrogenation of S-keto-D- gluconic acid to L-idonic acid could be done under com- mercially acceptable conditions. L-Ascorbic acid has been identified as a metabolite of a variety of microorganisms. The direct fermentation has not been developed yet, at the present time, L-ascorbic acid is not produced in high yield by any microorganisms (Crawford and Crawford, 1980). A chemical process is used to produce D-araboascorbic acid by converting from methyl-D-arabino-hexulosonate (Miles Laboratories, 196a). In this process, methyl-D-arabino- hexulosonate is mixed with H20 or alcohol, and sodium carbonate. The mixture is refluxed in an inert atmosphere, then acidified with Hasou after cooled down, to give a 16 .87-95% sodium D-araboascorbate. The direct fermentation of glucose to give D-arabo- ascorbic acid has been developed (Yagi, et al., 1967). The Pgnigiiiigm spp. are used in this fermentation to give h0% yield. Over 80% yield could be obtained when washed mycelium is used in dilute glucose solution (about 1% glucose). This fermentation is carried out at 28 °c for 5 to 7 days. The control of pH is most important and main- tained at pH 3.8 to h.5 in this fermentation. D-Erythroascorbic acid was synthesized by Prince and Reichstein (1937) from D-arabinose (Reaction IX). In this synthesis a bioconversion from D-arabitol to D-xyloketose by sorbose bacteria was involved. Reaction IX: D-arabinose ~9 D-arabitol -9 D-xyloketose ~9 acetone-D-xyloketose -» 2,3«monoacetone- D-xylosonic acid -9 2,3-acetone-D-xylosonic acid -+ D-erythroascorbic acid Usgs o: Ascorbic Acid inglegueg Massive information on the uses of ascorbic acid analogues (mainly L-ascorbic acid and D-araboascorbic acid) can be found in the Chemical Abstracts. They are extensively used as food additives during the processing of foods and beverages (Bauernfend and Pinkert, 19783 Birch and Parker, 1978) to enhance nutritional values, or to replace the vitamin C loss during harvesting, processing, storage or home preparation, or as an oxygen carrier or water-soluble d‘ 17 ‘antioxidant, whose role in the oxidation/reduction reactions inherently results in improved keeping quality, color, flavor and texture, and improved processing of the food product. Ascorbic acid analogues are also widely used for non- food purposes, such as, the electroplating of alloys: the detection of metal ions, nitrate and nitrite, phosphorous: the catalyst in polymerization of vinyl chloride, acrylo- nitrile, cyanoacrylate adhesives: photographic emulsion for iron spot prevention: pharmaceutical uses: and many others. The U.S. market for L-ascorbic acid was 90 million pounds per year in 1981(Hochhauser, 1983). The direct fermentation methods to produce ascorbic acid analogues will be desirable if the yield can be increased dramatically through genetic manipulation such as mutation-selection or gene-splicing techniques. In this research, the high yield ascorbic acid analogue producing yeasts will be selected to study the nutrient requirements and optimal physical conditions for fermentation, then the mutation-selection will be applied to improve the production. MATERIALS AND METHODS .Hadia Media mainly used in this study were complete yeast extract-peptone-dextrose (YEPD) medium, Yeast Nitrogen Base (YNB) with carbon compounds, Yeast Carbon Base (YCB) with nitrogen compounds, minimal medium, and storage medium. Media for petri plates were prepared in 2-liter flasks, each flask containing 1 liter of medium (enough for about 50 plates). After autoclaving for 15 minutes under 15 pounds pressure at 250 oF, these media were poured into petri plates and the surface of the agar plates was allowed to dry for 2 days before use. These plates could be stored in plastic bags at 10 °c for over 3 months. Media for liquid cultures were prepared in beakers and dispensed into 13.5x 150 mm test tubes or 500 mL flasks by syringes. The tubes were stoPPered with steel caps, the flasks with cotton plugs, autoclaved them for 15 minutes. These media were ready to use after cooling to room temperature. 1. Complete (YEPD) Medium This medium contained 1% yeast extract, 2% peptone, 2% glucose and 2% agar. It was used in the propagation of yeast cells from stock cultures, surface plate count, replica plating, and maintaining the mutants andkrevertants 18 f 19 ‘before examing the D-erythroascorbic acid productivity. The agar was omitted for liquid medium, this was used for routine growth to harvest large amount of cell mass. The harvested cells were then used for the inoculation or mu- tation process. 2. ENE This synthetic medium contained 6.7 g Bacto-Yeast Nitrogen Base (Difco, 1953) and different carbon compounds in one liter solution. This medium contained all vitamins and minerals required for yeasts growth, 2% glucose was added as carbon source while used as screening medium, 2% of various carbon compound was added while used in the study of carbon assimilation ability. ' 3. YCB This medium contained 13.1 g Bacto-Yeast Carbon Base (Difco, 1953) and different nitrogen compounds'in one liter solution. The inclusion of vitamins in this base was necessary for the utilization of nitrogen compounds by certain yeasts which cannot assimilate these compounds in the absence of vitamins. This medium was used in the study of effect of nitrogen source on D-erythroascorbic acid production by adding 0.5% various nitrogen compounds to the medium. 8. Minimal Medium This medium contained only carbon source, nitrogen source, phosphate salts, and magnesium sulfate. The amounts of these compounds were changed to study the media 20 'composition for Daerythroascorbic acid production. Supple— mental nutrients were added to study the growth requirements of mutants and the resistant effect of analogues. The pH was adjusted by adding 1 N NaOH or 1 N HCl: 2% agar was added for use in petri plates. A concentrated phosphate buffer (10% w/v) suggested by Fink (1970) was used as stock solution. To prepare this buffer, 87.5 g KHZPOu and 12.5 g KZHPOM were dissolved in water to make a total volume 1 liter. To prepare minimal medium, this buffer was diluted to the desired concentration with other components. 5. Storage Medium (2xYEPD) This medium contained double concentration of all components in YEPD except for agar (2%). Cultures main- tained on this medium and stored at u °C could be kept for one year (Fink, 1970). ' IsaaI_QulIa£a§ The yeast cultures used in the screening procedure were transferred from the stock cultures of Food Microbiolo- gy Lab., Department of Food Science and Human Nutrition, Michigan State University. Candida kzusgi was selected for the study of D-erythroascorbic acid production from 57 yeast strains because of its high yield in the production of ascorbic acid analogues. Wild type yeast cells were used to study the media composition and cultivation conditions. Mutants were obtained by treating the wild type.cells with 21 ~ethylmethanesulfonate (EMS). Revertants were obtained by mutating one of the mutants that showed very low erythro- ascorbic acid producing ability. An L-ascorbic acid sensi- tive mutant was isolated from the revertants. The re- sistant mutant from this sensitive mutant was obtained by EMS mutation. All of the wild type yeast cultures were maintained on the YEPD agar slants. All of the mutants and revertants were maintained on YEPD agar plates before the D-erythro- ascorbic acid productivity was examined. The valuable mutants and revertants were purified and maintained on YEPD agar slants. All of the cultures were stored at u 0C and were transferred to YEPD agar plates 3 days prior to use. Cultiygtion oi Yeggis 1. Broth Cultures Liquid media were prepared either in test tubes or 500 mL Erlenmeyer flasks two days prior to inoculation. A loopful of yeast cells were inoculated into 10 mL YEPD liquid medium in test tubes the day before experiments. The inoculated broth was then incubated at 30 0C on a Iwashiya refrigerator shaker overnight. The cells were harvested by an Damon IEC HN-SII centrifuge at the speed of about 1500 rpm and washed twice with sterile distilled water by recentrifugation and resuspension. The cells were then resuspended in the working medium to the same volume after washing. A 5% transfer of the cell suspension was made into c‘ 22 ~the working medium and then placed on the shaker under controlled temperatures to reach the stationery phase. 2. Plate Cultures (A Agar plates were made 2 days prior to use. After the agar surface was dried enough to absorb the liquid of inoculum quickly, 0.1 mL drops of adequate dilutions were dropped onto the agar surface with a calibrated sterile 1 mL pipet, spreading smoothly and uniformly with a flame- sterilized L-shape glass rod till the inoculum liquid was absorbed. The ends of the glass rod did not hit the flange of agar plate. The plates were inverted and incubated at 32 oC incubator till desired colony size formed. Dilution to obtain 30 to 300 colonies on one agar plate for surface count, and 200 to 300 colonies for replica plating were used. G o t ramete Turbidity was used for measuring growth. Yeast cells grown in YEPD liquid medium overnight were harvested by centrifugation, washed with sterile distilled water twice and resuspended in 0.1 N phosphate buffer. Duplicate surface plate counts of 15h, 155, 106_dilutions were made on YEPD agar plates and incubated at 32 0C for 2 days. At the same time, prepared a series of dilutions by 1:1, 1:2, 1:3, 1:u, et al. from the original cell suspension so that a range of turbidities at OD6oo from 0-0.8 with B&L 20 Spectrophotometer obtained. A standard curve ofRODé00 vs. 23 ~cell concentration (CFU/mL) was plotted. Routinely, the growth of yeast cultures in liquid medium were measured by determining the OD600 of the 26-fold diluted culture broth (0.2 mL broth + 5 mL distilled water, using 5.2 mL water as blank), and CFU/mL then determined from the curve. Estimation of nggxihroagcorbic Acid 1. Qualitative Estimation A paper chromatography method developed by Miki et al. (1962) was used to separate ascorbic acid analogues, the RF values were compared with Miki's (1962) and Murakawa's (1977). In this method, Whatman paper No. R, ZOxRO cm, was dipped into 3% (w/v) metaphosphoric acid solution to which 3% glycerol (v/v) was added for 10 minutes, dried in the air for 1 hour. Culture broth and standard ascorbic acid analogues were streaked along a line on the prepared paper. The paper was kept standing for 30 minutes in a moist atmosphere with a relative humidity of h0-70%. This paper was then placed in a glass chamber containing the mobile solvent, water-saturated methyl ethyl ketone (upper part) which had been stored for more than 2h hours after prepa- ration. Development was carried out at 25 0C for 5 hours by an ascending method. After completion of the develop- ment, the paper was takenout and air dried for about 10 minutes at room temperature. Then 0.013% 2,6- dichlorophenol indophenol (sodium salt) dissolved in water 2b ‘was sprayed. A white spot against pink background was indicative of an analogue and was marked before the back- ground faded away. 2. Quantitative Estimation The D-erythroascorbic acid in culture broth was measured by a modification of L-ascorbic acid analysis by Sullivan and Clarke (Omaye, Turnbull and Sauberlich, 1979). the dipyridyl method. Reagents used in this methods were 2,2'-dipyridyl, aqueous 0.5%: orthophosphoric acid, 85%: ferric chloride, aqeous 1% made fresh each 3 days: tri- chloroacetic acid (TCA), aqueous 10%, made fresh each use. To 0.5 mL of culture broth, 1.0 mL of 10% TCA was added and the samples centrifuged for 10 minutes. The following reagents were added in sequence to ShO‘pL of the supernatant: 160‘pL of 85% orthophosphoric acid, 2.78 mL of 0.5% 2,2'-dipyridyl, and 560‘pL of 1% ferric chloride. The samples were allowed to stand at room temperature for 30 minutes for the ferrous-dipyridyl chromophore to develop. Samples were then read at 00525 in B&L 2O Spectrophotometer. Standards of L-ascorbic acid ranging from 10‘pg/mL to 120 ‘pg/mL were used for a standard curve: the total amount of ascorbic acid analogues were measured as the L-ascorbic acid equivalent with the same reducing power. The produc- tivity of these analogues were expressed ae‘pg/mL equiva- lent. 11"? ° 25 .uutgtion and Seleciiog 1. Isolation and Purification of Mutants Mutants isolated from the master plates were purified by streak plate technique. The YEPD agar plates and sterile flat toothpicks were used. The mutant picked out from the master plate with a sterile toothpick was spread evenly over a small area (pool) toward the edge of the plate. Another sterile toothpick was used to make two strokes from the pool of the material. One of the strokes was made only to the center of the plate. Using the third sterile toothpick, a series of strokes were made at right angles to the first two strokes. A series of strokes were made at right angles to previous series with sterile toothpick, the strokes being made towards the pool. Care should be taken that the tooth- pick did not touch the pool at this stage. 0n incubation of plates at 32 oC, discrete single colonies were'picked from the streaked plates, and spread onto YEPD agar plates. After erythroascorbic acid production was examined, purified cultures were inoculated onto storage medium and stored at h 0C after good growth was reached on agar slants. 2. Replica Plating (Lederberg and Lederberg, 1952) A cylinder of wood with a diameter 3f§ inches was used as replica plating block to accommodate the petri plate. A metal ring 3% inches in diameter was used to secure the velvet cloth. The replica cloths were made from velveteen cut into 51 inches squares. These cloths were piled up in 2 a metal container, autoclaved for 50 minutes. In replica 26 ~plating, a sterile square was placed, nap up, on the cy- lindrical wood and held firmly in place with the metal ring pushed over the fabric and around the rim of the support. The YEPD agar plate carrying the initial colonies was inverted onto the fabric with slight digital pressure to transfer the growth. The imprinted plate then provided the pattern for transferring replica-inocula to subsequent minimal agar plates impressed in the same way. The minimal agar plates were then incubated at 32 °C for 2 days. After incubation mutants that showed growth on complete medium but not on imprinted minimal medium were isolated and purified. 3. EMS Mutation (Fink, 1970) Ethylmethanesulfonate was used as mutagen in the mu- tation process to give high yields of mutants in this study. The EMS was marked and stored in a freezer. The mutation process required 12 days per cycle and was as follows: Day 1. Wild type yeast cells from a YEPD agar plate were inoculated into 10 mL of liquid YEPD in an 13.5x150 mm test tube and grown at 30 °c overnight in a shaker. Day 2. The cells were harvested by centrifugation, washed three times with sterile distilled water and resus- pended in 10 mL sodium phosphate buffer at pH 8.0 (pH of buffer was very important in the EMS reaction), and 0.6 mL EMS was added. After shaking the tube with a vortex mixer, the mixture was incubated for 50 minutes at 32 °c without agitation. Immediately after incubation, the coils 27 ewere washed three times with sterile distilled water. Each time the cells were transferred to a new sterile culture tube. After the third wash, the cells were resuspended in 10 mL sterile water. Then 1.0 mL of the cell suspension was diluted into 10 mL YEPD liquid medium and shaken for 2 days at 30 °c. Day h. After the culture was grown up, the cells were 2, 103, 16“, 105-rold. Then 0.1 mL of each di- diluted 16 lution was spread over the entire surface of YEPD agar plate. These plates were incubated at 32 oC. Between 200 and 300 colonies appeared on each petri plate as desired. Diluted cell suspensions were stored at h 00. Day 6. The dilution that yielded agar plates between 200 to 300 colonies was selected to plate out on up to 100 plates and incubated at 32 °c for 2 days. Day 8. These plates were used as master plates for replica plating. Master plates were stored at h 00: im- printed plates were incubated at 32 °C for 2 days. Day 10. Mutants from master plate were picked with flat sterile toothpicks and purified by restreaking. Day 12. The YEPD agar plates were inoculated with a single colony from each of these mutant strains. The plates, each containing 30 mutants, were incubated at 32 0C for 2 days and stored at u °c. The mutants thus obtained were tested for the D-erythro- ascorbic acid productivity in minimal broth medium supple- mented with 5% YEPD liquid medium for growth. Thp mutants ( 28 .were tested for the growth requirements. h. Analogues Resistant Mutants The resistance of Q. kzusgi to several chemical ana- logues of D-erythroascorbic acid was tested. Each analogue was added into minimal agar plates at different concen- trations. Yeast cells grown in YEPD liquid medium were harvested and washed 3 times. The yeast cells were resus- pended in sterile water. 0.1 mL of 102, 103, 10a dilutions were spread both onto minimal agar plates and the plates supplemented with chemical analogues. After incubation at 32 0C for 2 days, colonies were observed for inhibition by chemical analogues. The analogues that showed inhibition were used to select the resistant mutants by EMS mutation. Mutants thus obtained were examined for the D-erythro- ascorbic acid productivity. $7" RESULTS AND DISCUSSION co b c c P oduc t. The screening of D-erythroascorbic acid producers among yeasts was carried out on a test tube scale. YNB with 2 % glucose as the sole carbon source was used as the screen- ing medium. The pH of the medium was adjusted to 5.0. A loopful of inoculum from each yeast culture was transferred into 2 mL sterile medium contained in a 13.5x150 mm test tube. The cultures were then incubated in a temperature- controlled shaker, continuously shaken (130 strokes per minute) at 30 °C for 5 days. The fermentation broth of each culture was then assayed for their ascorbic acid ana- logues content. The total amount of ascorbic acid analogues was expressed as‘pg/mL productivity for each yeast strain. Table 2 shows the results of screening study. There were 57 yeast strains examined in the screening procedure. These strains belong to two families, do- mxgetaggag (ascosporogenous yeasts) and Cryptggggggggag (anascosporogenous yeasts), 18 strains of yeasts in both families gave a positive result for ascorbic acid analogue production. Because of the limitation of analytical sensi- tivity, the medium used, and cultivation conditions, it is not possible to say that only these 18 strains ampng the 29 30 Table 2.--The occurrence of ascorbic acid analogues in various species of yeasts -.-.-_--. i .. -.. —.—--.o Yeast --.-— *- ”~--.-. ...—— . “..m—.“_—....._. o 9.. ......“u .. ... Ascorbic acid analogues content gag/mL) A. Ascosporogenous yeasts Dabaraaxsas 22222211 (YMA FPL 13) Hanssnula anamala Ii. annuals (U. of Calif.) H. aslifarais (NRRL Y 1u25) ii- aatumus fl, wingei (ATCC 1h355) 11. wingei (ATCC 114356) Bichiafamm Saccharomyceg boulardii (IZ 190R) carlsbergensis (ATCC 9080) carlsbergensis (IZ 210) carlsbergensis (IZ 626) carlsbergensis (IZ 1327) carlsbergensis (IZ 1h30) cgglsbeggegsis (IZ 1828) carlsbergensis (IZ 1831) carlsbergensis (IZ 183k) carlsbergensis (IZ 1973) W (ATCC 961:) aaraiisias (ATCC #126) erfikifiias (I2 299) var. turbidans QQEEYISLBE (IZ 310) ' magician (I2 629) asmisiae (I2 672) cgzeyisige (IZ 755) cerevisiae (IZ 765) . 2929113139. (I2 861:) var. allia- ungainlun O O O O O alumnu:kn| O IUJIUJIUJ O C C 103103 O 0 10210200 KB IUJ O ‘a’... ' a Neg Neg Neg 50 1S 15 10 10 Nee . 1S Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg 31 Table 2.--(Continued) ... - Ascorbic acid analogues Yeast content (ug/mL)- Saccharogyces cerevisiae (IZ 986) Neg S. cgzevisiae (IZ 987) Neg' S- careyisiaa (IZ 1716) Neg s. camisias (NRRL Y 129) Neg fie aaraYisiae (NRRL Y 568) Neg do midis: (NRRL Y 567) N98 S, cezevisiae (NRRL Y 635) 10 S. cepevisigg (NRRL Y 897) Neg S. ggzgxisiag (NRRL Y 898) Reg 5. scrutinise (NRRL Y 978) 10 S. adrenals: (NRRL Y 2031:) 10 S. cereyisins (ascorspore) 10 S. qugxisige (yeast cake) Neg So 9.91m winners (wine) N68 5. diastaticus (NRRL Y 201m) .Neg S. kluyveri (U. of Calif.) 10 §. kluyveri (strain 0 26) 15 g. oleaginosas (U. of Calif.) 15 s. rogxii (ATCC 2619) 10 §- uranium (NRRL Y 31:7) Nee S. uYarum (NRRL Y 131:?) N98 s. mum (NRRL Y 6001;) Neg Schizosaccharomyces japonicgs var.. Neg mulls Schizosaccharomyces octosponus 15 Schizosaccharomyces pombe Neg B. Anascosporogenous yeasts Brettanomyces claussnil (NRRL Y 1h1h) Neg 4?. A. f 32 Table 2.--(Continued) -.— m“-.-_ . ...- -Y.__.. .__.. - ...- .-_.—_. —. Ascorbic acid analogues Yeast content gag/mL)- mm Maui 30 mm (11111.3. (NRRL Y 900) 20 Bhodotomla tabla 1S Torulopsis sphaerica Neg .- ...-...- ...... ...—~_—__ .-..--n.... . 8No significant amount measurable. 3:: ‘. g I 33 ~yeasts used could produce ascorbic acid analogues. The two highest yielding strains, gaggigg Erase; and flanggnglg califggnig (NRRL Y 1&25) were selected for further screening. Requirements for carbon and nitrogen compounds by these two strains were examined. In the study of carbon compounds, 2% carbon source was added in YNB, 16 carbon compounds were used (Table 3), while other conditions were the same as first screening. In the study of nitrogen compounds, 0.5% nitrogen source was added in YCB, 8 nitrogen compounds were used (Table u), while other conditions were the same as first screening. 9. 52259; could assimilate citric acid, fructose, glucose, glycerol, mannose and sucrose for growth and bio- synthesis of ascorbic acid analogues. Sucrose was the poorest carbon source of this group. Although citric acid and mannose gave lower growth yield than fructose, glucose, and glycerol, the ascorbate productivity were about the same. Q. 323521 could not grow on arabinose, galactose, inositol, lactose, maltose, manitol, sorbitol, sorbose, D-(+)- and L-(-)-xylose. This yeast strain could grow on different nitrogen sources but not on KNOZ. The produc- tivity of ascorbic acid analogues in this yeast depends not on growth level but on nitrogen source. The best nitrogen source for growth was ammonium citrate, but'the productivity, was pretty low comparing with that of ammonium nitrate which was the best nitrogen source for ascorbic acid analogue production. Ammonium chloride and ammonium acetate also 39 Table 3.--The availability of carbon compounds in ascorbic acid analogues production by Q, krusei and , fl, cglifornia .... -..... . ---.... ...—v ..wo—-_._....._o---———.-. .. .———- — . ‘.. . .. .. +».—————w.——._ 5; california —-—- _. ...—..— Q; quaai Growth Productivity Growth Productivity Carbon source (0D600) (Mg/mL) (0D600) gag/mL) D-(J)-Arabinose N.G.a Negb N.G. Neg Citric acid 0.37 30 N.G. Neg Fructose 0.51 30 0.h6 50 Galactose N.G. Neg 0.13 Neg Glucose 0.u8 30 0.h9 50 Glycerol 0.53 35 0.51 55 i-Inositol N.G. Neg N.G. Neg Lactose N.G. Neg N.G. Neg Maltose N.G. Neg 0.31 20 Mannitol N.G. Neg O.h0 50 Mannose 0.35 30 0.33 US D-Sorbitol N.G. Neg 0.21‘ 20 L-Sorbose N.G. Neg 0.22 35 Sucrose 0.27 10 0.25 20 D-(+)-Xylose N.G. Neg 0.35 25 L-(-)-Xylose N.G. Neg N.G. Neg v 3No growth. b No significant amount measurable. 35 Table h.--The availability of nitrogen compounds in ascorbic acid analogues production by Q. grusei and - ii. califomia 0.3111311 5.. W Growth Productivity Growth Productivity Nitrogen source (0D600) gug/mL) (0D600) (pg/mL) NHuCI' 0.u5 Nega 0.u3 27 NHuNO3 0.h8 no 0.u6 Neg (NHu)2HP0h 0.u6 18 0.39 30 NHHOAc 0.52 Neg 0.51 Neg (NHu)2oCitrate 0.53 15 0.50 Neg KNO3 0.15b Neg 0.37 Neg KNO2 N.G. Neg 0.35 Neg (NHu)ZCO 0.25 Neg 0.u0 10 aNo significant amount measurable. bNo growth. «7". 36 ‘gave good growth, but no measurable amount of ascorbic acid analogues was found. fl, galifgggig could utilize fructose, glucose, glycerol, maltose, mannitol, mannose, sorbitol, sorbose, sucrose and D-(+)-xylose for both growth and ascorbic acid analogues production. It also showed slow growth on galactose, but no measurable amount of ascorbic acid analogues was produced in this carbon source. This yeast could not grow on arabinose, citric acid, inositol, lactose or L-(-)-xylose. It could assimilate all nitrogen compounds in Table h for growth, but only ammonium chloride, ammonium phosphate and urea could be used for ascorbic acid analogues production, the best source being ammonium phosphate. ' The accumulation of ascorbic acid analogues in Q. krusei seemed to be a constant metabolic pathway because it produced these compounds during growth on citric acid, a carbon source from which hexose must arise by glucon- cogenesis, however, 3. galifgznig,could not grow under the same condition. The study of nitrogen availability for both yeast strains showed that the nitrogen source is a critical factor in the ability of these cells to produce ascorbic acid analogues. It might possibly be the reason that many yeast strains could not produce measurable amounts of ascorbic acid analogues in YNB plus glucose. This as- sumption needs more investigation to prove, and is beyond the scope of this research. «x 37 A vitamin-free minimal medium containing only glucose, optimized nitrogen sources, phosphate salts and magnesium salt was devised for Q. £22321 and fl, galifgznig.’ in this medium, Q. Krnggi could produce up to 120‘pg/mL ascorbic acid analogues, n times as much as the screening medium, while the other strain could produce only 1/3 the amount in the minimal medium. The fermentation broth of Q. Kzgggi was streaked on Whatman No. h paper along with L-ascorbic acid and D-arabo- ascorbic acid. This paper was then developed in water- saturated methyl ethyl ketone. There was only one white spot for the fermentation broth on the Paper (Figure 2) after spraying with.0.013% 2,6-dichlorophenol indophenol. Comparing the RF value of this spot with that of Murakawa's et a1. (1977) showed this analogue to be D-erythroascorbic acid, a five carbon ascorbic acid analogue. This compound was the only ascorbic acid analogue in glucose fermentation by Q. unassi- Since there is no quantitative analysis for D-erythro- ascorbic acid available, Takahashi and his co-workers (1976) used the quantitative method for L-ascorbic acid analysis based on its reducing property to define the total amount of ascorbic acid analogues in fermentation broth. In this study, the 2,2'-dipyridyl method is used for the quanti- tative determination of D-erythroascorbic acid. The amount of this compound is therefore expressed as L-ascorbic acid equivalent (pg/mL). .x Rf value 38 1.0 1 0 0.5 — 0 0 0 L l 4‘ Ass Ara X Fig. 2.--Paper chromatogram of Q. kzusei fermentation broth. Developing solvent was methylethylketone saturated with water, and spraying agent was 0.013% 2,6-dichlorophenol indophenol. Asa, L-ascorbic acid: Ara, D-araboascorbic acid; X, Q, krusei fermentation broth. «'7‘ 39 ‘Madis_fiam22n1112n C, Erase; was the only strain selected for D-erythro- ascorbic acid production. A minimal medium was used to study the effects of carbon concentration, nitrogen concen- tration, minerals, vitamins, phosphate salts, amino acids on the D-erythroascorbic acid production. The media thus developed were used to study the cultivation conditions, time course, and genetic improvements of D-erythroascorbic acid production by the yeast. 1. Effect of Phosphate Concentration Phosphate plays important roles in physiological fun- ctions, such as energy transport in the form of ATPxphospho- lipids in cell membrane; skeleton in DNA and RNAstuffering and osmotic pressure of intracellular fluids. The concentration of phosphate buffer was varied from 0.05 to 0.5% (w/v) with 3% glucose, 1% NHhN03,'0.05% Mgsou- 7H20 in the minimal medium. It was found (Figure 3) that 0.2% (w/v) phosphate salts gave the optimal productivity. 2. Effect of Carbon and Nitrogen Concentration Glucose and ammonium nitrate were used as carbon source and nitrogen source respectively. The carbon con- centration in minimal medium was changed from 1% to 15% to study its effect on D-erythroascorbic acid production; 1% ammonium nitrate, 0.2% phosphate salts, 0.05% MgSOu°7H20 were also added. The results (Figure h) show that a between 5% and 9% glucose gave the highest productivity. The nitrogen concentration in minimal medium,was no N O O - / - on) U1 0 J ‘\ U1 0 ] ProductiV1ty Lug/mL) 8 ca I _ C////3 O 1 I 1 1 001 002 093 00“ 005 Phosphate buffer ( % ) Fig. 3.--The effect of phosphate concentration on the erythroascorbic acid production by.Q, kggse . 50- 0\o l l l I I I T 5 7 9' 11 13 15 Glucose ( % ) Productivity ()ug/mL) A~ U Fig u.--The effect of carbon concentration on erythro- ascorbic acid production by_§, h:ggg_. a?" u1 ,changed from 0.25% to 3% in the minimal medium with 3% glucose, 0.2% phosphate salts and 0.05% Mason-7H20 to study its effect on D-erythroascorbic acid production. The result (Figure 5) showed 1% ammonium nitrate was the optimal con- centration. In order to have more information on carbon concen- tration, an experiment was done by adding 20 mL minimal medium into 500 mL Erlenmeyer flasks with the glucose con- centration 5%, 7% and 9% respectively. The result of this fermentation at 23 0C is shown in Figure 6. It indi- cates that the growth in higher sugar concentration reached optimal earlier, the erythroascorbic acid level is the same. 3. Effect of Trace Elements In this study, 5 inorganic salts and 9 vitamins were individually added to minimal medium with 5% glucose, 1% NHuN03, 0.2% phosphate salts and 0.05% MgSOh-7HZO. The temperature of fermentation was controlled at 30 0C. In preparing concentrated stock solutions of inorganic salts for dilution into synthetic medium, each inorganic salt was stored at low pH to prevent precipitation. All these stock solutions were sterilized by autoclaving and distributed to minimal medium before use. The vitamin solutions were also prepared and sterilized before adding into synthetic medium. Folic acid, biotin, Ca-pantothenate, riboflavin, pyridoxin-HCI and i-inositol are heat resistant and thus sterilized by autoclaving; p-aminobenzoic acid, niacin, and thiamin-HCl were sterilized 4' 50 Productivity Lyg/mL) 0 Fig. 350 300 250 200 150 1 00 50 Productivity Qua/mL) Fig. #2 4 c/ l 1 l f r I r 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Ammonium nitrate ( % ) 5.--The effect of nitrogen concentration on erythroascorbic acid production by Q. krgsei. i l 1 l I l r" I ' l I v I 1 2 3 h 5 6 ‘7 8 9 10 11 Time ( days ) 6.--Glucose concentration and erythroascorbic acid production, 5% (C0, 7% (£0, 9% (D). h3 ~ by filtration through milipore filters. These solutions were then added to the separately autoclaved medium. The effect of trace elements on D-erythroascorbic acid production is shown in Table 5. None of these nutrients showed increase in yield at the given concentration, while ferric salt, cobalt salt, folic acid, niacin, p-aminobenzoic acid, pyridoxin-HCl and thiamin-HCl gave negative results. 9. gggggi can grow well without any of these elements in the medium. A further investigation with different concentrations of some trace elements was done. The fermentation was carried out at 23 oC, other conditions were not changed. Table 6 and Table 7 show the results of this investigation. No prominent increase in productivity could be found although some of these nutrients gave better growth response. When the magnesium salts was eliminated in the minimal medium, there was no difference in growth and productivity when compared with original medium. u. Effect of Amino Acid The natural occurring d-amino acids were used in this experiment a 1% stock solution of each amino acid was steri- lized by filtration and added separately to autoclaved minimal medium to the final concentration of 0.1% amino acid, 15% of yeast cells were transferred into these media. This fermentation was carried out in a shaker at 23 °C for 5 days. The results in Table 8 indicate the follpwing: uh Table 5.--Effect of trace elements on erythroascorbic acid production by Q, Knuggi at 30 oC -9"- .... o. ....-- We‘.-_ .-—.-—-.-— .—————..——..— .———— Concentration Productivity - Trace elements QPg/L) (fig/mL) None 0 210 CuSOuoSHZO no 200 FeCl3 200 160 MnsoLL too 200 ZnSOu-7H20 h00 210 00012-6H20 hOO 100 Biotin 2 180 Ca-Pantothenate u00 200 Folic acid 2 150 Inositol 2000 200 Niacin too 120 P-Aminobenzoic acid 200 150 Pyridoxin-HCI #00 120 Riboflavin 200 200 . Thiamin-HCI u00 150 ...—0‘ D“.“”.'wrr‘-p~--A- - --- g, hS Table 6.--Effect of minerals concentration on erythroascorbic acid production by Q. krngei at 23 0C a“ mun-...”... Productivity, Concentration Growth Minerals (Ag/L) (0D600) (pg/mL) None 0 0.6h 300' Cusou55H20 25 0.7h 310 50 0.7a 320 100 0.66 310 200 0.63 300 Znsou.7H20 500 0.6a 320 1000 0.70 .330 1500 0.70 330 2000 0.68 280 MnSOu 500 0.62 310 1000- 0.70 ‘310 1500 0.68 310 2000 0.68 320 ‘1’ M6 Table 7.--Effect of vitamins concentration on erythroascorbic acid production by C. 32312; at 23 °c -0--. .... . -o-c-.- Concentration Growth Productivity_ Vitamins (pg/L) (0D600) (pg/mL) None 0 0.6h 300' Riboflavin 250 0.65 300 500 0.65 300 750 0.65 290 1000 0.66 290 i-Inositol 2500 0.70 300 5000 0.68 320 7500 0.66 320 10000 0.6a 300 Biotin 15 0.62 260 30 0.63 ‘280 60 0.62 280 120 0.63 280 ‘2’». h? Table 8.--Effect of amino acids on erythroascorbic acid production by Q._kru§ei Productivity Growth Amino acid (0.1%) (0D600) (pg/mL) None 0. 68 300 Neutral amino acids Alanine 0.75 360 Valine 0.75 280 Leucine 0.6h 170 Isoleucine 0.70 200 Proline 0.75 300 Phenylalanine 0.68 1&0 Tryptophane 0.6M 170 Methionine 0.75 160 Glycine 0.85 360 Serine 0 . 80 3170 Threonine 0.80 310 Cysteine 0.66 80 Tyrosine 0.66 220 Acidic amino acids Aspartic acid 0.80 190 Glutamic acid (MSG) 0.90 260 Basic amino acids Lysine 0.75 hOO Arginine 0.75 330 Histidine 0.76 350 AB .(1) The 3 basic amino acids were effective nutrients not only for growth but for productivity, (2) the 2 acidic amino acids decreased the productivity although they gave high growth response, (3) the sulfur-containing amino acids group, methionine and cysteine gave low productivity, cysteine gave only about as much 20% productivity as that of lysine, (h) the amino acids containing phenyl group, phenylalanine, tryptophane and tyrosine, gave decreased productivity. (5) the amino acids with branched carbon chain, valine, leucine and isoleucine, showed less produc- tivity than those without branched carbon chain, alanine, glycien, serine and threonine. The highest productivity was obtained by lysine, the lowest by cysteine. Such' information might be useful in the study of biosynthetic pathway. Physical Conditions 1. Effect of Aeration and Agitation The aeration and agitation in this fermentation was accomplished by the reciprocating action of a shaker. The shaking speed was fixed on 130 strokes per minute in the study of D-erythroascorbic acid production. In order to change the aeration rate, a simple qualitative method was used. Different volumes of medium were distributed into test tubes or flasks; smaller volumes of medium resulting higher rate of aeration. ' The primary objective of shaking is to supply the A9 ~necessary oxygen to the yeast either for growth or p.3rythr0- acid production. A second function is to keep the yeast cells in suspension so that they could be surrounded by nutrients or oxygen uniformly. ' The result in Figure 7 show the D-erythroascorbic acid production favored high aeration. It implied that the biosynthstic pathway of D-erythroascorbic acid is an oxi- dative process, however, the formation of the dienol structure also suggested that some reductive reactions might be involved. The oxygen needed for this reaction is incorporated through the intermediate stage of the dissolved oxygen molecule. In other words, the yeast cells respond to the liquid phase oxygen concentration in regulation its over- all metabolic activities. The solubility of oxygen is extremely limited as compared to other nutrients, it is necessary to continuously supply the broth with oxygen in order to meet the metabolic demands of the yeast cells. 2. Effect of pH The effect of pH on D-erythroascorbic acid production was studied by changing the initial pH values in the medium from 2 to 8; the pH of the medium was.adjusted with 1N N303 or 1N HCl. Two inocula ratios of yeast cells (15% and 100%) were applied. With a 15% washed inoculum, after the cultures were shaken for u days, the final pH of all this cultures dropped to pH 2. The results in Figure 8 showed peaks intboth 50 .sofipmpnoewom . Apnea unwwav camom madam one Anson pmoHv oadom user who» no momssm.m ha Sofiaoauoam pace ownhoommohnahho no nofipmaom mo poshmmII.~ .wam A as v wonfimpcoo cw oasaor Suarez om o: om om or mmememmw _ . p p N N p . . ~ P b v» I C) U\ I oer I I O O O U\ N «- (qw/W) “Insomnia I omm r oom Productivity (,ug/mL) Productivity'(flg/mL) 51 00 _ 3 1 30 /O\ Q d ZOO-1 "20¢r 5‘ Ei‘ 100 \\\"‘ q; s - 10C) 2 E O I v I "“I 1"“ I"!”‘"""I"""0 1 2 3 h 5 6 7 8 Initial pH 150 - 100 - ‘A a a AF———&\\\\L\\\\A ' SO— / W‘“flfi/ O I I I l 1 I I‘ 1 2 3 II 5 6 7 8 Initial pH Fig. 8.--Effect of initial pH on erythroascorbic acid production by 9, 333331 with 15% transfer (upper part), growth (0), productivity (0); and 100% transfer (lower part), incubation time, 12 hr (A). 60 hr (A). 52 ~growth and productivity at pH h and pH 6 respectively. The 100% washed inoculum showed different results at 12 hours and 60 hours incubation. The data obtained from 1.5 hours, 3 hours, 6 hours incubation showed no difference from that obtained from 12 hours incubation. The pH of each tube dropped down to pH 2 after 60 hours incubation. 3. Effect of Temperature 6 Temperature can be expected to exert a profound effect on all aspects of growth, metabolism and yeast cell survial . In this study, Qandidakmai was incubated at 18 °c, 230°C, 30 °C, 37 00 or N3 0C. This yeast grew well at each temperature except #3 00 when the yeast cells coagulated and did not grow. The optimal temperature (Figure 9) for erythroascorbic acid production is 23 °C in this study. u. Amount of Inoculum This study was carried out on flask scale with 20 mL of minimal medium. The inoculum ratios were studied at 5%, 10% and 15%. Minimal medium with 5% glucose was used in this fermentation, the cultures reached optimal growth after 7 days cultivation at 23 °C. The higher inoculum favored the growth yield of yeast cells as well as the D- erythroascorbic acid production (Figure 10). Time Course for D-Egythroasgorbic Acid Productigg The time course of the fermentation by wild type yeast cells was studied. The fermentation medium was on minimal medium with 5% glucose. 20 mL of this medium waayplaced in a 53 Q ~250 -. / I. 25 § 8 ’ s 3331200 - o . 20 g: I? ' o—c 0:150 q P15 O or! "3 5 g 100 - , 10 3 pg): \ U 50 - - S O I 1‘ “b O 1 6 2'3 3TO 37 L13 Temperature ( °C ) Fig. 9.--Temperature effect on erythroascorbic acid production by Q. 3.22.821: growth (0), productivity (0). _ (3 500 P 50 G) u a . 9: \Llroo -' / F- “.0 g m) 3: a P1300 -1 . r- 30 o +3 -u '5‘ o :3 200 '1 " 20 a 3 ' g '3100 - - 10 v ‘4 9.. 0 I I r I 0 5 1o 5 20 1 Transfer ( % ) Fig. 10.--Effect of transfer ratio on erythfibascorbic acid production by C. 15.12286 , growth (0), productivity (0). SM ‘500 mL Erlenmeyer flask, with 15 % inoculum and fermented at 23 oC. The growth of yeast cells and the D-erythro- ascorbate concentration began to increase quickly after about 1 day; both were maximal at about 7 days. The fermentation broth was qualitatively estimated by paper chromatography to show the identity of D-erythroascorbic acid in the broth. The pH of the fermentation broth dropped very quickly and reached pH 2 within the first #8 hours and gradually decrease to pH 1.8 thereafter'(Figure 11). Genetic Impzoxemegtg of D-Erythroascorbic Acid Productivity The usual genetic manipulation required in industrial process is for yield improvement. The most common way of obtaining yield improvement after physiological variables have been optimized is by the process of mutation. In this study, ethylmethanesulfonate (EMS) was used as mutagen. The EMS mutation procedure is convenient and gives high yield of mutants. EMS is an alkylating agent that causes transition and transversion of purine and pyrimidine bases on DNA molecules. which make it possible to obtain revertants from mutant by the same process. In this study, 3% and 6% of EMS (v/v) were applied. The killing effect of these two concentrations on wild type 9, kzuggi were 80% and 95% respectively, the remaining 20% and 5% survivals were grown in YEPD medium and plated on YEPD agar. All the mutants that could grow on YEPD agar but not on minimal medium were picked out and transferred (As/ml. ) Productivity 55 600-1 63-560 1 500- 5-505; 0 \O a (400" O ”#1403: 9s m a. O 300- 35.30% A V 200- ‘ , 2—20 I ‘ ‘NA A . a 100- 1-10 0 0 I I I I I I I 0 0).. to- 1 2 3 LI 5 6 7 Time _( days ) Fig. 11.--Time course of etythroascorbic acid fermentation by C. 1322.16.19 growth (0). productivity (0). PH (13). 1"," 56 ‘onto YEPD agar plates. The D-erythroascorbic acid producing power of mutants was examined by growing them in minimal medium supplemented with 1% YEPD liquid medium for growth. Among two hundred mutants examined, none of them could produce increasing amount of D-erythroascorbic acid, most of them possessed between 60 to 100% of the productivity as much as the wild type cells (Table 9). I Mutants that showed less than 20% of the productivity in minimal medium supplemented with 1% YEPD liquid medium. One of these mutants did show moderate growth. This mutant was selected for another mutation to obtain rever- tants. The nutritional requirements of this mutant were4 examined. The mutant was grown in minimal medium con- taining 0.1% of various amino acids in each tube and incu- bated for 3 days. It showed moderate growth in the medium with aspartic acid or glutamic acid (acidic amino acids), and also showed a little growth in the medium with methionine or cysteine (sulfur-containing amino acids). The amino acids requiring mutant was mutated with 6% EMS, the master plates were imprinted onto both minimal agar plates and minimal agar supplemented with 200’pg/mL L-ascorbic acid. 0f the 500 revertants growing on minimal agar plates only one could not grow on minimal medium supplemented with L-ascorbic acid; this mutant was defined as the L-ascorbic acid sensitive mutant. The D-erythroascorbic acid productivity of these 57 Table 9.--Erythroascorbic acid productivity among mutants derived from wild type C. kzgsei ' --fi—w. ... .. - ennuiaul~nb p- M *_._. _. - .. -..—l-..‘ --———— .- ~—-. -..--._'.._.._. % Productivitya % Mutantsb 80 - 100 25 60 - 80 27 #0 - 60 28 20‘- #0 11 0 - 20 9 8Wild type yeast as 100%. bTotal number of mutants was 200. Table 10.--Erythroascorbic acid productivity among revertants derived from amino acids requiring mutant vcro. —‘ _. — n»... % Productivity % Revertantsa > 100 20 BO - 100 50 60 - 80 19 #0 - 60 6.5 0 - #0 0.5 3Total number of revertants was 500. (1" 58 ‘revertants was tested, 20% of them (Table 10) produced.more D- erythroascorbic acid than wild type cells. Among these higher productivity mutants, the two highest were selected (mutant 282 and mutant 353) to study the fermentation time course along with wild type cells in the minimal medium at 23 00 (Figure 12 A&B). The revertant 353 grew faster and better than the wild type yeast, and the revertant 282 grew at about the same level as wild type yeast. D-Erythroas- corbate productivity were 160% and 1#0% respectively. The compound in the fermentation broth of the two mutants identified by paper chromatogram showed that both mutants produced the same compound as wild type yeast. The EMS mutation of yeast cells changed their metabolic pathways. The productivity of D-erythroascorbic acid was decreased among most of the cells either because of growth level or nutritional requirements after the first mutation. After the second mutation the productivity of most cells were restored. Some mutants even showed significant increase in both growth level and metabolic activity. Repeated mutation of the high productivity mutants could lead to better yield of D-erythroascorbic acid in future studies. 00 o o - es utants The selection of analogue-resistant mutants has proved to be a useful technique for increased production of vitamin (Matsui, et al., 1982) and amino acids (Tsuchida, et al., 1975) by bacteria. The over production of an essential Growth (x107 CFU/mL) Productivity (pg/mL) 59 600 -- 500 too 300 200 100 Time (days) Fig. 12.-~Growth curves ( A ) and erythroascorbic acid productivity ( B ) of wild type yeast (A), mutant 282 (O), and mutant 353 (O). ‘a 60 ~metabolite in a resistant bacterium in response to an anti- bacterial substance is already known (White and Woods, 1965). This mechanism.is based on the assumption that analogues or analogue-like antibacterial substances are_ available for altering genetically the control mechanism of metabolites synthesis. In this study, 7 structure related compounds were used, they were L-ascorbic acid, D-araboascorbic acid, D-glycero- L-manno-heptonic acid ~1-lactone, 06,,A-glucooctanoic acid lactone, w-D-glucoheptonic acid -1-lactone, D-(+)-ribonic acid, D-glucurono-3,6-lactone. None of these compounds showed inhibitory effect on wild type yeast growth even with concentration of these compounds up to 20,000 ug/mL in the minimal agar plates. Two structure-related fungal metabo- lites were also used, penicilic acid and pautulin. These showed no inhibition on growth at the levels up to 250 pg/mL. An L-ascorbic acid sensitive mutant was isolated from the back mutation of an amino acids requiring mutant. This L-ascorbic acid sensitive mutant could grow on minimal medium but not on minimal medium supplemented with L- ascorbic acid; in 500 pg/mL L-ascorbic acid concentration about 0.1% of the cells could grow, while in 1000 Pg/mL concentration about 0.01% of the cells could grow. When Ems mutation was applied to this sensitive mutants, many revertants were obtained which could grow on minimal medium plus 10,000 pg/mL L-ascorbic acid, however; 61 .no increase in D-erythroascorbic acid productivity was found. xii. g I SUMMARY AND CONCLUSION The screening of D-erythroascorbic acid producers was carried out on a test tube scale. 0f 57 Yeast strains examined, 18 showed the ability to produce ascorbic acid analogues. The two most potent producers, Candida krugei and fignagnnla gglifgzgig, were selected for further study as to the effects of carbon and nitrogen sources. A minimal medium was devised and the productivity of ascorbic acid analogues of these two strains were examined. 93 kzgggi was selected for paper chromatography and a single ascorbic acid analogue, D-erythroascorbic acid was found in fermentation broth. An optimal medium containing 5% glucose, 1% NHuN03, 0.2% phosphate buffer and 0.05% MgSOu-7H20 was devised for the study of physical variables and mutation-selection process. The study of physical variables showed that 23 00, high aeration rats, an initial pH of 6.0, and 15% inoculum were optimal for D-erythroascorbic acid production. EMS mutation gave two mutants which showed 160% and 1#0% productivity respectively of that for wild type yeast cells. The se- lection of analogue-resistant mutants was not succesful. The ascorbic acid analogues are widely usedgas 62 63 ‘antioxidants in industry because of their strong reducing power. D-Erythroascorbic acid could be used in bath for electroplating iron alloys to give good brightness and good adherence, or as a reducing agent in acrylonitrile polymeri~ zation to give good fiber color. The direct fermentation method would be desirable if the yield of D-erythroascorbic acid could be improved dramatically through genetic tech- niques. 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