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TRANSFORMATION 0F_MHCQR_BY A PLASMID SHUTTLE
VECTOR WITH A DOMINANT SELECTABLE MARKER -
THE BENOMYL RESISTANCE GENE °

presented by

Juili L. Lin

has been accepted towards fulfillment

of the requirements for
Food Science &
Master

 

 

 

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4___=_._—x

TRANSFORMATION OF EQCOB BY A PLASHID SHUTTLE VECTOR NITH A
DONINANT SELECTABLE MARKER : THE BENONYL RESISTANCE GENE

by

Juili L. Lin

A THESIS

submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree

of

MASTER OF SCIENCE

Food Science and Human Nutrition

1989

ABSTRACT
TRANSFORMATION OF @5313 BY A PLASMID SHUTTLE VECTOR WITH A
DOMINANT SELECTABLE MARKER : THE BENOMYL RESISTANCE GENE
BY

Juili Lillian Lin

A transformation system was developed in the filamentous
fungous Mucor racemosus by complementation of the leucine
auxotrophic mutant strain R78 with the Leul gene from wild
type cells. To apply this transformation system to

industrially important species of Mucor, a dominant selectable

 

marker, the Benitgene, which encodes a benomyl resistant 3-
tubulin protein from u; crassa was inserted into the plasmid
shuttle vector pLeu4. The correct construction of pMBen
plasmids was confirmed with restriction endonuclease analysis.
These constructs were transformed into protoplasts of L
ragemosus R7B. The presence of unaltered pMBen plasmids in
Mg; transformant cells was confirmed with Southern analysis.
No significant increase in the resistance level to benomyl was
observed in cells transformed with pMBenl and pMBenz when
compared with resistance levels of the non-transformed host

strain. Further studies will focus on the regulation of BenR

gene expression in Mucor cells.

DEDICATION

To Lin, T. C. and Lin, J. - my dear parents

ii

ACKNOWLEDGEMENTS

I would have been unable to complete this research
without the help and encouragement of many people, and I
should like to express my most profound thanks to all of them.

First, of course, my tremendous gratitude lies with Dr.
John E. Linz. It was he who introduced me into the
fascinating world of molecular biology. He supported my
research with the facilities, valuable experience, patient
guidance and encouragement. I am also very grateful to the
members of my Master's program committee, Dru JamesiJ. Pestka,
Dr. Adinarayna C. Reddy and Dr. Denise M. Smith for their
helpful suggestions.

The most sincere appreciation is extended to Dr. William
L. Casale and Jason Horng for their assistance in research as
well as valuable personal advice. A heartfelt thank you goes
to Roscoe Warner for'his aid, especially in the photograph and
slide production and to Yun-Yuu Chen for her superior typing
help. Special thanks is extended to my laboratory coworkers:
Fumin Chiu, Yih-Jihn Lee, Yi-Yu Yen and Cheng-Shaun Chen for
their special friendship throughout my study.

Last, but not least, I want to express my love and thanks
to Mr. and Mrs. Lin, my parents, and Daniel F. Goerke, my
fiance, for their constant support, understanding and

encouragement during the study.

iii

LIST OF
LIST OF

I.

II.

III.

TABLE OF CONTENTS

TABLES
FIGURES

INTRODUCTION

HISTORICAL REVIEW

1. BIOTECHNOLOGY AND THE FOOD INDUSTRY
Food Processing Enzymes and Food
Additive;
Genetic Engineering

Industrial Applications
2. HETEROLOGOUS GENE EXPRESSION / SECRETION

SYSTEMS
Prokarvotgs
EEEAEYQEES
3. FUNGAL GENETIC SYSTEM
Sexual and Parasexual Recombination
Mglecular Genetics
Transformation Systems-overview
Selectable Markers
Agxotrophic Markers
Dominant Selectable Markers
ggngal Transformation Systems
Yeast
Filamentous fungi
4. MUCOR - PILAMENTOUS FUNGI OF THE CLASS
ZYGOMYCETES
Mndtgtrv
Morphogenesis
Transforggtion
5. BENOMYL RESISTANCE GENE PROM N; QRASSA AS
A DOMINANT SELECTABLE MARKER IN
TRANSFORMATION OF E; RACEMOSUS

 

MATERIALS AND METHODS
1. MICROBIAL STRAINS
2. PLASMIDS
3. CHEMICALS
4. GROWTH MEDIA AND GROWTH CONDITION FOR E;
COLI AND M; RACEMOSUS
Sporangiospore Preparations
5. THE MINIMUM INHIBITORY CONCENTRATION OF
BENOMYL OR COPPER FOR MUCOR
6. TRANSFORMATION AND SELECTION or E; COLI
iv

PAGE

U‘lb

1O
13
14
17
17
20
21
21
24
24
24
24
25

28
28
29
29

31
35
35
35
38

43
44

44

VI.

VII.

VI.

TRANSFORMANTS
7. THE IDENTIFICATION OF RECOMBINANT PLASMIDS
IN E; QQLI
8. ISOLATION OF PLASMID DNA FROM E; COLI
Minipreps

Large Scale Plasmid DNA Isolation
9. RESTRICTION ANALYSIS OF PLASMID DNA

10. PROTOPLAST FORMATION AND TRANSFORMATION
OF MUCOR

11. SINGLE COLONY ISOLATION OF MUCOR
TRANSFORMANTS

12. ISOLATION OF GENOMIC AND FLASMID DNA FROM
MUCOR

13. SOUTHERN ANALYSIS

RESULTS
1. SENSITIVITY OP MUCOR RACEMOSUS TO BENOMYL
OR COPPER
2. THE RONOLOGY OF THE 5 - TUBULIN GENES or g;
RACEMOSUS TO THE N; CRASSA BENOMYL
RESISTANCE GENE
3. RESTRICTION ANALYSIS AND SOUTHERN ANALYSIS
OF PLASMID PMCUPI-A
4. PNBEN PLASMID VECTORS; CONSTRUCTION AND
ANALYSIS
5. TRANSFORMATION OF NUCOR RACEMOSUS
PLeu4 Vector
PNBgn Vectors
6. SOUTHERN ANALYSIS OP 5; RACENOSUS
TRANSPORMED WITH PLASMIDS

pLeu4 Transformants

pMBen Transformants
7. RESISTANCE OP PRBEN TRANSPORMANTS TO

BENOMYL
DISCUSSION

 

SUMMARY

REFERENCES

45
46
47
47
48
48
50
SO
52
54

54

56
59
62
69
74
75
76
76
79

84
86

93

94

LIST OF TABLES
Table

1. World market value of selected Biotechnology-based
products

2. Some proposed genetic engineering solutions to
problems in food production

3. Food processing enzymes and food additives used in
the 0.8. food industry that benefit from genetic
engineering technology

4. Various processes of genetic recombination

underlying construction of new recombination
strains of industrial micro-organisms

vi

page

11

12

18

LIST OF FIGURES

page

23
32
36
39

41

57

60
63
66
67
70

72

77

80

figure

1. Microbial transformation procedures

2. Chemical structures of antifungal Benzimidazoles

3. Plasmid pLeu4

4. Plasmid p8T3

5. Plasmid pMCupl-A

6. Southern analysis of u; racemgsus strains of 12168
and R78 with Ben“ gene probe

7. Southern analysis of plasmid DNA with 1.3 kb
fragment from pMCupl-B

8. Construction of plasmids pMBenl and pMBenZ

9. Colony hybridization with the Bena gene

10. Restriction analysis of pMBenl and pMBenz

11. Plasmid pMBenl; restriction map

12. Plasmid pM8en2; restriction map

13. Southern analysis of genomic DNA of M; raggmgggs R78
and 3 transformants of pLeu4 with pUC9 probes

14. Southern analysis of pMBen plasmid, and genomic
DNA of M. racemosus R78 and 3 pMBen transformants
with Bear gene probes ‘

15. Southern analysis of pMBen plasmid, and genomic

DNA of fig racemosus R78 and 3 pMBen transformants of

with pUC19 probes

vii

82

INTRODUCTION

flucor, filamentous fungi of the class of Zygomycetes,
have received the attention of academic researchers because
of their ability to undergo cellular morphogenesis (Cihlar,

1985). Various species of Mucor are important to the food

 

industry because of the production of extracellular enzymes
which are used world wide in food processing (Crueger &
Crueger, 1982). Hydrolytic enzyme production also results in
this group of organism being leading causative agents of food

spoilage.

Although a large quantity of biochemical research data

has accumulated on Mucor, the absence of an efficient sexual

 

recombination system for genetic analysis and manipulation
(Gauger, 1965; Schipper, 1978) made it desirable to develop
a transformation system and introduce molecular genetics to
enhance the study of these organisms. One species, £399;
a emosus, has a haploid vegetative phase and a small genome
(lOJpr) which made it particularly convenient to target for
molecular genetic study. Recently a transformation system
was developed in this organism, based on the complementation
of a leucine auxotrophic mutant strain, 5; racemosus R78, by

1

2

a homologous gene (Leul) from wild type cells. This
transformation system has limitations in practical
application, because it can only be used for transformation
of this particular mutant strain. A transformation system
useful for other Mggg; species is necessary at this stage.

A dominant selectable marker for transformation is highly
desirable because it can be selected in a wide variety of
genetic backgrounds. For research on morphogenesis, this
approach can save the time of generating complex mutant
strains ordinarily needed for studies involving gene
complementation, gene disruption and gene replacement. To
the food industry, this approach will provide a simpler method

to utilize the tools of molecular genetics to study the

control of spoilage organisms (1L circinelloides, L

mm. M... W) and an opportunity to do genetic

engineering on enzyme producing strains or protein engineering

to alter the functionality of enzymes used in food processing.

Benomyl is a fungicide which has been used for a long
time (Davidse, 1988). Recently, a benomyl resistance gene,
Ben", which is a mutated 8- tubulin gene from a Neurospgra
grassa benomyl resistant strain, was cloned and its nucleotide
sequence determined. This benomyl resistance gene has been
used as a dominant selectable marker for the transformation
of fungi including L crassa and Gaeumannoyces g_r_aminis

(Henson, 1988). In preliminary experiments conducted in our

3
laboratory, two strains of L racemosus 12168 - wild type and
R78, were found to be sensitive to benomyl at a minimum
inhibitory concentration (M.I.C.) of 50 pg benomyl/ml for
cells grown in the dark and 100 ug benomyl/m1 for cells grown

in the light.

Based on these preliminary data, our goal for this
project was to utilize the benomyl resistance gene (Benn) from
Neurospora crassa as a dominant selectable marker to transform
M299; racemosus and to measure the functional expression of
this heterologous gene in Mm transformants. We hypothesize
that the expression of the L crassa Bena gene in L racemosus

will increase their resistance to benomyl.

HISTORICAL REVIE'

1. BIOTECHNOLOGY AND THE FOOD INDUSTRY

rocess n e nd o t ve

The food processing industry is the oldest and the largest
industry using biotechnological processes. Biotechnology can
be described as the controlled and deliberate application of
simple biological agents, living or dead cells, or cell
components, in technically useful operations, either of
productive manufacture or as service operations (Bu'lock,
1987). The use of biotechnology started more than 8000 years
ago with alcoholic beverage, vinegar, sourdough and cheese
production by "natural" microbial and enzyme processes (Knorr,
1985) . Modern biotechnology began with Weizmann's development
of a practical acetone-butanol fermentation process in 1915
(8u'lock, 1987) aided by the development of microbiology in
the late 19th century. Biotechnological processes are used
to mass-produce other fermented products like ethanol, food
and bakery products, animal feed, and food additives like
antibiotics, organic acids, nucleic acids, vitamins, single-
cell protein, and processing aids including enzymes like
hydrolyases, proteolyases, carbohydrases, and lipases

4

S
(Godfrey, 1983). From an economic point of view, in 1985,
United Kingdom (UK) consumers spent $ 36 billion on food,
which is 20% of their total consumption. The output of the
food industry in that year was about 17.5% of total gross
manufacturing output (King and Cheetham, 1987) . The food
processing industry, having annual sales of $ 30 billion in
the UK, and $ 300 billion in the United States, is the largest
user of biotechnological processes (Table 1, pg.6) (Knorr, et
a1, 1985). The application of biotechnology can reduce the
manufacturing cost and improve the quality of products in the

food industry (Newell, 1986).

In general, an industrial process can be implemented
either by chemical synthesis or bio-conversion which involves
the use of living cells or enzyme systems in chemical
modifications. Biocoversion is often preferable because of
high substrate specificity, regiospecificity (site
specificity), stereospecificity, and mild reaction conditions
(Crueger and Crueger, 1984). Today, with the aid of
biotechnology, the production of many food additives and food
processing enzymes is more economical and efficient. For
example, in 1954, world consumption of methionine was a few
million pounds at a cost of almost 3 3.00 per pound, while in
current years, methionine production has soared to over 200
million pounds per year, with a price of $ 1.70 per pound

(Paul, 1981).

 

 

 

Table 1. World Market Value of Selected Biotechnology-based
Products
Market size
(millions U.S. S)
Products _ Primary end use
1981 ”90
(estimated)
Amino acids l.9 x 103 2.2 X 103 Feed additive, food enrich-
l.8 X 103 ment and flavoring agent.
feed preservative
Citric acid Food additive, processing aid
Enzymes 310 to 400 1.5 x 103 Processing aid
Vitamins 668 Feed and food additive, food
1.] X 103 enrichment agent
Baker‘s yeast Food additive. enrichment
agent
Beer 27 X 10‘1 44 X 103 Beverage
Cheese Food
Fermented foods 3.5 x 103 6 x 103 Food
Misoll Food
Soy saucell Food

 

(Knorr and

Sinskey, 1985)

7

Genetic Engineering Modern biotechnology has entered
a new phase with the discovery of tools for genetic
engineering beginning in 1960. The ability to manipulate
genetic material by molecular biologists has made it possible
theoretically to construct DNA.molecules containing genes and
regulatory elements from any cell type and to clone and
propagate these new molecules in suitable host cells (8u'lock,
1987). Genetic engineering combines recombinant DNA
technology, which allows improved protein production, and
protein engineering, which allows the improvement of protein

properties and the creation of new products (Lin, 1986).

Recombinant DNA technology, which breaks and rejoins DNA
molecules from different species, has widened the range of
end-products that can be considered for commercialization to
include even those from unusual organisms, plants and animals
via heterologous gene cloning and expression (8u'lock, 1987;
Pitcher,1986). High yields can be achieved in desired host
organisms by increasing the copy number of the desired gene
in the host organism or by regulating the level of gene
expression (Pitcher,1986). The cost of end-product recovery
can be reduced by the utilization of a secretion system in the
host to generate a high production level of active
extracellular end-product. The production of unwanted
proteins, nucleic acids or other by-products can be eliminated

by specific deletion or by changing to a host organism without

8
these disadvantages. For example, bacterial secretion of calf
rennin was difficult to achieve, but scientists successfully
achieved the secretion of properly processed prochymosin, a
precursor of calf rennin, from filamentous fungi (Pitcher,

1986).

Protein engineering manipulates the primary structure of
a protein by synthesis of a novel DNA sequence or by site-
directed oligonucleotide mutagenesis (Craik,1985: Dalbadie-
McFarland, et al 1983; Zolkr and Smith, 1983). Modification
of the structure of a protein can improve the activities and
functionalities of’a protein including substrate specificity,
chemical stability, thermal stability, pH optimum, catalytic
activity or to create entirely new protein products (Pitcher,
1986; Kang, 1985; Lin, 1986). .Advances in protein engineering
require the continual convergence of several technologies,
such as protein crystallography, X-ray diffraction,
sophisticated computer graphic systems and systematic analysis
of protein folding and subunit association (Estell, 1985).
A seemingly minor alteration at the nucleic acid level can
have profound effects on the behavior characteristics of a
protein used in processing and can improve the processing
procedure or the texture, flavor, and color of the processed

food.

Several enzymes, some showing modified catalytic

9
properties, have been investigated using recombinant DNA and
site-directed mutagenesis techniques. A good example was
reported by Wells, et al. (1985) and concerned the improvement
in. chemical oxidation resistance of subtilisin. by site-
directed mutagenesis. Research on engineering of caseins,
which are functional milk proteins and serve as the basis for
a major segment of dairy industry, are progressing (King,

1987: Kang, 1985).

Industrial Applicgtiong The development of
biotechnology using the technology of genetic engineering may
lead this world to better health, more food, more economical
energy sources, cheaper and more efficient industrial
processes and reduced pollution (Prentis, 1984). The
application of genetic engineering can reduce the cost of
manufacturing by cloning an engineered gene into host cells
which can utilize cheap substrates, grow faster, and achieve
highly regulated production of a secreted end-product. Table
2. (pg.11) shows the development of genetic engineering to
solve some problems in food production. For example, CPC-
International (1984) has submitted to the Food and Drug
Administration (FDA) the first petition for generally
recognized as safe (GRAS) status for production of a food
grade enzyme, a-amylase, in an engineered strain of Bacillus
fightilis (Pitcher, 1986). Genecor is working on commercial

production of rennin by secreting filamentous fungi such as

10
Aspergillus and Trichoderma. This enzyme has received much
attention as a candidate for the commercialization of
recombinant DNA technology. Significant progress in
constructing amylolytic strains of yeast has demonstrated
their potential for ethanol or alcoholic beverage production
utilizing starch as a substrate (Tubb,1986). As shown in
Table 3. (pg.12), almost all food processing enzymes and
additives have been targeted by genetic engineering techniques
(Lin, 1986). The pharmaceutical industry has also utilized
genetic engineering successfully for years. Numerous
mammalian gene products including antiviral, antitumor and
antidiabetic agents and growth promoting factors have been

developed, expanded and are being commercialized (Lin, 1986).

2. HETEROLOGOUS GENE EXPRESSION / SECRETION SYSTEMS

Genetic engineering technology made possible the use of
microorganisms for large-scale synthesis of industrial
proteins from_ a ‘variety of sources including‘ mammalian,
animal, and plant cells or other microorganisms. Strain
selection and mutagenesis can be used to improve the
production of an endogenous (homologous) protein (Momos and
Furaya, 1980 ; Miyagawa, et al,1986). However, introduction
of a functional gene for a heterologous protein into a
suitable host microorganism, has the potential to achieve more

efficient production (Lin, 1986). While, every protein has

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12

Table 3. Food Processing Enzymes and Food Additives used in
the U.S. Food Industry that Benefit from Genetic

Engineering Technology

 

 

Category Example
Food Processing Enzymes
Starch processing a-Amylase
fi-Amylase

Dairy products

Brewing

VVme/fruit/vegetable processing
Fuel alcohol

Food Additives
Low-calorie products

Flavor enhancers

Human and animal diet
supplements

Stabilizing agents
Preservatives

Glucoamylase
Glucose isomerase
Pullulanase

Rennin
Lipase
Lactase
Amylases
Proteases

Pecfinases
Amylases
Glucoamylase

Aspartame
Thaumatin

Glutamic acid
5 '—Ribonucleotides

Amino acids

Vitamins

Xanthan gum
Cecropin

 

(Lin, 1986)

r1fi .v-v- *-

13
its own unique properties, so does every host / vector
secretion system have unique properties (Van Brunt, 1987).
The systems used for heterologous gene expression include E;
991i, gacillus (prokaryotic cells), yeast, filamentous fungi,

plant tissue and mammalian cells (eukaryotic cells).

Prokagyotas §;_ ggli, has been ‘used. as a host to
synthesize large quantities of recombinant proteins for a long
time. .E. 99;; cultures grow relatively rapidly by utilizing
cheap nutrient sources and can express complementary-DNA (c-
DNA) of foreign genes with the aid of constitutive or
regulated promoters (like the promoters from lac, trp genes,
Edens, et a1, 1982; Emtage, et al,1985) for over production
(Lin, 1986). But §_._ ggli does not have the ability to
properly perform the posttranslational modifications that many
eukaryotic proteins require to be fully functional.
Furthermore, 24, 991i, is a Gram-negative bacterium and
secretes end-products into the periplasmic space of the cell
(Hsiung, et al, 1986) if the proper g; ggli secretion signal
peptides are fused to the amino terminus of the protein. The
expression of rat proinsulin, human proinsulin, human
immunoglobulin light chain, and human growth hormone (hGH)
(Chang, 1987; Hsing, 1986) have been achieved, but the end-
products need to be recovered by osmotic shock and other

procedures. This increases the cost of product recovery.

14

The other commonly used prokaryotic host is gacillgs, a
Gram-positive bacterium. Bacillus, which has been used in
fermentation industry for a long time (Ehrlich, 1978), is a
GRAS strain, and is able to secrete the end-product into the
growth medium. This may reduce the end-product purification
cost. The expression of the a-amylase gene from 8;
gmyloliguefaci ens in 8; subtilis has improved production 2,500
fold over that level obtained previously (Wasserman, 1984).
But Bacillus also is not able to properly posttranslationally

modify preproteins translated from eukaryotic genes (Van Brunt

, 1986) .
Eukaryotes The yeast, Saccharomyces cerevis'a , a

lower eukaryote, is another microorganism which is widely used
in the food industry (Tubb,1986). iMany studies have been_done
on heterologous protein production in yeast (Kingsman, 1985).
Eukaryotic proteins including calf prochymosin (Mellor et a1,
1983), and interferon gamma (IFN-r) (Derynck, Singh. and
Goeddel, 1983) which are produced by E; ggli in an inactive
state, are produced as soluble, biologically active proteins
in _S_._ cerevisiag. Yeast cells apear to be very stringent
about the sequence-specific recognition of splice junctions
of Hn-RNA (Dobson, et a1, 1982). Yeast cells are not able to
correctly process the Hn-RNA of the Asperqillus glucoamylase
gene (Innis, et a1, 1985) , the tRNATyr gene of Xenopus oocytes

(Nishikura, 1982) or the Schigosaccharomyces tRNAser gene

15
(Greer, et al, 1987). So, a c-DNA copy of the foreign gene
must be cloned into yeast to make expression possible. S;
cerevisiae requires yeast derived expression signals; a
promoter region, which initiates and regulates gene
transcription; and a termination region, which stabilizes the
message, to generate efficient transcription. The copy number
of plasmid vectors in yeast was relatively low until the
discovery of the 2 pm plasmid, which leads to high copy
number. The secretion system of yeast cells is very similar
to that in higher eukaryotic cells. A functional secretion
signal sequence can be fused to the heterologous gene to
ensure the correct posttranscriptional modification and
cotranslational transport through the endoplasmic reticulum
resulting in secretion of a biologically functional end-
product (Kingsman, et al, 1985). Most of the heterologous
proteins produced in yeast are rich in mannose (Penttila, et
a1, 1987) and seem to be hyper-glycosylated, whereas
homologously produced mammalian glycoproteins contain a
variety of glycosyl residues with complex branching (Staneloni
and Lelorir, 1982). It is unlikely that mammalian proteins,
which require complex and specific carbohydrate modifications,
will be biologically functional when synthesized in yeast.
Intensive research. to solve the inherent obstacles of these

microbial hosts is continuing.

One group of organisms with great potential for synthesis

16
/ secretion of heterologous proteins is the filamentous fungi.
Filamentous fungi are eukaryotic cells which naturally secrete
vast quantities of extracellular products including amylase,
cellulase, protease, rennin, pectinase, lipase, antibiotics
and citric acid (Montenecourt, 1985; Cullen and Leong, 1986;
Crueger and Crueger, 1984). These products are widely used
in food processing and as food additives. For instance,
glucoamylase, one of the largest bulk enzymes in the world,
is produced by industrial strains of Aspergillus nigger. These
strains can naturally produce up to 20 grams of enzyme per
liter of growth medium from a single copy of the glucoamylase
gene (Van Brunt, 1986). Another group of industrially useful
filamentous fungi are Mgggr, which produce a variety of
extracellular enzymes (Van Heeswirck, 1986). Strains of M;
pgsillus Var. Lindt and 11; miehei are used world wide for
production of rennin, which is second on the list of
industrial enzymes in terms of volume of sales (Crueger and

Crueger, 1982).

Filamentous fungi are also good at the expression and
secretion of biologically active mammalian proteins like
bovine. chymosin (Cullen, et a1, 1987), human interferon
(Gwynne, et al, 1987) and human tissue plasminogen activator
(Upshall, 1987). The filamentous fungi can correctly perform
posttranscriptional processing of m-RNA, and often

posttranslationally modify heterologous polypeptides to

17
functional proteins. The development of a heterologous
protein secretion system in GRAS strains of filamentous fungi

is one significant approach to advance 'modern biotechnology.

3. FUNGAL GENETIC SYSTEM

Sexual and Parasexual Recombination In microbes,
recombination of DNA molecules can take place by a wide
variety of mechanisms, resulting in a complete chromosome at
one extreme to substitution of a single gene, or part of a
gene, at the other. The various recombination processes, with
examples of microbes to which they apply, (with an emphasis
upon industrial organisms), are listed in Table 4 (pg.18).
Sexual reproduction in eukaryotic microbes is akin to classic
breeding techniques in plants and animals which have resulted
in widespread successes in agriculture and horticulture. The
most notable commercial successes have been in genetic
improvement of yeast involved in baking, brewing, wine making
and single cell protein (Miwa, et al., 1978; Johnston &
Oberman, 1979; Spencer & Spencer, 1983; Snow, 1983). The
majority of eukaryotic micro-organisms of industrial
importance, however, do not appear to posses sexual
capabilities. Nevertheless, recombinants can be produced in
many filamentous fungi by exploiting 'parasexual' mechanisms

Ibased upon mitotic rather than meiotic events. An industrial

Table 4.

18

Various Processes of Genetic Recombination

Underl ying Construction of New Recombination Strains

of

Industrial

Micro-organisms

 

Type of microbe

Recombination process

Examples where applied

 

Eukaryotic

Eukaryotic and
prokaryouc

Prokaryotic

Sexual hybridisation
Parascxual breeding

Protoplast fusion

Native DNA transformation

Gene cloning, vector
transformation

Gene cloning, transfection
Transduction
Conjugation. plasmid transfer

Sacclzaromyccs yeast
Aspergil/us Spp.

A cremonium c/irOi'sogenum
Candida trapicalis
Bacillus spp.
Straplomyces spp.
Bacillus subtilis
Neurospora crassa
Bacillus
Streptomyces
Saccltarom'rces

Bacillus.
Sireptomtt'ces

 

(Johnston,

1985)

19
strain of Aspergillus gigg; used for the production of the
enzyme amyloglucosidase, is a parasexual recombinant of a high
yielding strain and a lower' yield strain ‘with superior
filtration characteristics (Ball, et al., 1978) . A self-
diploid strain obtained by inbreeding a production strain of
A; gigg; produces improved yields of citric acid (Das & Roy,

1978).

Notwithstanding some impressive results of sexual and
parasexual breeding, a major advance in the construction of
recombinant strains has ensued from the process of protoplast
fusion. The principal barrier to cellular or hyphal fusion
in many cases is the cell (or hyphal) wall, and its removal,
under appropriate conditions, frequently permits fusion
between normally incompatible strains, species, genera and
even phyla. Protoplast fusion has been used to produce new
strains of industrial microbes, e.g. Acremonium chrvsogenum
(Hamlyn & Ball, 1979), Penicillium ghrysogenum (Queener &
Baltz, 1979), Streptomvces (Hopwood, 1981), brewing yeasts
(Russell & Stewart, 1979) and distillery yeasts (Mowatt, et
al., 1983). Interspecific hybrids have included 2;
gngysogenum / P; cyaneo-fulvum (Queener 8: Baltz, 1979) and

intergeneric crosses including those of Candida tropicalis /

Saggharomycopsis fibuligera (Provost, et al., 1978) and
Candid; utilis / Trichoderma :eesei (Heslot, 1980).

20

o e a ice The major disadvantage of these
various genetic techniques, including protoplast fusion, is
the combining of large numbers of genes from both parental
strains into one cell. Thus if a range of strain
characteristics is of importance in the industrial process,
it.is very probable that.the majority’of recombinants obtained
is inferior to the parental production strain or strains.
Often, improvement of only a single characteristic is sought
and, in the simplest case, this will be controlled by a single
gene. It is therefore highly desirable to attempt addition
of only this one gene to the parental strain. The mechanisms
of molecular genetics including transduction, transformation
and transfection afford such a possibility. The procedures
of genetic engineering not only satisfy this objective but,
in addition, potentially allow transfer of a gene or genes

between any two organisms, whether related or unrelated.

Genes, normally located on chromosomes, are also found
extrachromosomally' as part. of DNA. molecules existing' as
plasmids or in organelles such as mitochondria. Genes may be
transferred between some micro-organisms by transduction and
transformation. In transduction, genes of a donor bacterium
are carried into a recipient strain by incorporation into
infective 'viruses (bacteriophages). Phage are: of’ great

importance as vector molecules in genetic engineering.

21
Transfogation Systems-Overview Genetic transformation
was used initially in bacterial cells and involves the uptake
and expression of genes from a donor strain in a recipient
organism. The preparation of protoplasts, however, improved
uptake in eukaryotic cells as did the discovery that PEG

promotes transformation in addition to protoplast fusion.

In transformation, vectors serve as a vehicle to carry
the foreign DNA into the host cell and help to propagate that
DNA fragment. ‘The essential steps for in vitro transformation
procedures are summarized in Figure 1 (pg.23, 24). Once
inside the host cell, fragments of DNA may either be
integrated into chromosomes or alternatively circularize to

form extrachromosomal molecules which behave as plasmids.

Selectablg Markers To facilitate selection of the
foreign DNA in transformed cells, the vector, generally a
plasmid, must carry'a gene whose expression in the host offers
a selective growth advantage. To date, two approaches used
for selection have been the complementation of a recessive
mutation in the auxotrophic host strain and, the use of
markers whose presence can be selected in wild-type cells, so
- called dominant transformation markers (Rine and Carlson,

1986).

22

Figure l. Microbial Transformation Procedures
(Prentis, 1984)

(l) fragmentation oflflUtextracted fromeaparticular'organism,

(2)

usually by digestion with 'restriction' endonuclease
enzymes.

joining of individual fragments into suitable vector DNA
molecules, such as plasmids or viruses, often by the
action of ligase enzymes.

(3) introductioncflfrecombinantlfifltmolecules:hfl13appropriate

(4)

cells in! transformation, transfection cnr transduction.
selection for cells, containing recombinant DNA by means
of selectable markers.

23

cut by San H l enzyme
(are for %
ampicillin / \\
resistance
(:1me
3 human cDNAs

mix and join

4; N
'w/

653

K
(amp')
human (:0 N A gene
// 1)
original plasmid /
recombinant
fl plasmid
mix plasmids with E. coli cells
E. coli'

 

 

\ t\,
@180 W (it D

chromosome \ fl Kl] E. Loli that his no plasmid

 

m

L j

 

 

culture medium plus ampiallin

Figure 1. Microbial Transformation Procedures
(Prentis, 1984)

24

Auxotrophic Markers Transformation of nutritional
mutants with wild-type genes has been the main approach. For
this procedure to be successful, it is necessary to have an
appropriate, prototrophic gene and a stable, auxotrophic
mutant (Turner and Ballance, 1986). Recessive auxotrophic
mutants are usually obtained by mutagenesis of the wild type
strain, and selection. of :mutants. by Lederberg's replica
plating technique (Crueger and Crueger, 1982). The wild type
prototrophic gene corresponding to the auxotrophic mutation
can be cloned by complementation of the mutant strain with a

DNA library generated from wild type DNA.

Dominant Selectable Marker; Dominant selectable
markers are ideal in any transformation system, because their
use avoids the need to construct complex mutant strains for
use as recipients. Host strain construction is relatively
tedious, and very time consuming, especially in organisms
lacking a sexual recombination system. Antibiotic resistance
genes are widely used as dominant selectable markers in
prokaryotic and eukaryotic transformation systems. The
antibiotic resistance gene may come from resistant mutants of

the wild strain, or from other resistant microorganisms.

Fungal Transformation Systems

Yeast In yeast, host strains that are auxotrophic

25
for amino acids such as leucine, tryptophan and histidine or
bases such as uracil, can be used with cloning vectors that
contain the appropriate yeast gene for complementation.
(Botstein, et al., 1979; Beach and Nurse, 1981; Beggs, 1978:
Hinnen. et al., 1978; Hitzeman, et al., 1980). However,
industrially-developed yeast strains are generally polyploid
(triploid or higer) and vectors used to transform them should
bear a positive selection characteristic (like, resistance to
toxic substances) that does not require prior genetic
modification of the host strain (Saunders, G., et al., 1986).
The use of the hygromycin B resistance gene, the G418
resistance gene and the copper resistance gene solved this
problem (Kaster, 1984; Butt, 1984). The CUPl vectors,
encoding metallothionein proteins which confer copper
resistance, have been successfully introduced into brewing

yeast strains (Henderson, 1985; Butt, 1987).

zilamentous zgngi The development of vectors for
transformation of filamentous fungi have, to a large extent,
lagged behind those developed in yeast, but in general have
followed the same basic strategies. In Neurospgra crass ,
the first vectors described used the N; crassa qga-z+ gene.
This gene encodes the catabolic enzyme dehydroquinase and can
be used to functionally complement a strain of N; crassa with
qa-z' and arom-9' mutations, (lacking both catabolic and

biosynthetic dehydroquinase activity) (Case, 1979). Later in

26
1984, another auxotrophic selectable marker, the am gene of
N; grassa, which codes for NADP - specific glutamate
dehydrogenase (GDH), was used to transform am mutant strains
to prototrophy (Kinsey, 1984). Recently the development of
benomyl resistance gene as a dominant selectable marker was
found to be ideal for transformation of N. crassa (Orbach,

1986).

The genetics of Asperqillus nidulans have been studied
in some detail since Pontecorvo, et al., (1953) first
described the system. A number of selectable markers has been
developed for transformation of the filamentous fungi
Aspergillus niggr and Aspergillus nidulans. In most cases
selection of transformants is based on complementation of
auxotrophic mutants. A plasmid carrying the N; grassa pyr4
gene (encoding orotidine - 5' - phosphate decarboxylase), was
used to functionally complement an A; nidulans pyrG mutant
(Ballance, 1983). Other auxotrophic mutants (from the same
organism) such as the trpC mutant, which lacks
phosphoribosylanthranilate isomerase activity (by the
trifunctional trpC gene) (Yelton, 1984); and the ArgB mutant,
which lacks ornithine transcarbamylase (Buxton, 1985) have

been successfully used as recipient hosts.

Some dominant selectable markers do not require special

mutant strains. For example, the amdS gene allows growth on

27

acetamide or acrylamide as the sole nitrogen source (Tilburn,
1983; Kelly, 1985). But this marker can be used only in
strains that have no requirement for nitrogen-containing
compounds interfering with the amds selection (Punt, 1987).
Another useful dominant selectable marker is the oliC31 gene,
which encodes an oligomycin resistant variant of the
mitochondrial ATP synthase subunit 9 and confers oligomycin
resistance (Ward.and.Turner, 1986; Ward” Wilkinson and'Turner,
1986). This gene should prove useful as a marker for gene
replacement or disruption experiments or transformation of
5 er ' us strains lacking auxotrophic lesions (Ward,
Wilkinson and Turner, 1986). However, this gene is probably
species - specific since it requires the formation of a
functional oligomycin - resistant ATP synthase complex (Punt,
et al., 1987). In 1987, a new dominant selectable marker used
for transformation of A; nidulans transformation was reported.
This marker, the _E_:_._ 9Q; hmB (hygromycin B) gene, encodes
phosphotransferase hph which phosphorylates and inactivates
the antibiotic, hygromycin 8 (Malpartida, 1983; Gritz and
Davies, 1983). Expression of the hph gene was controlled by
A; nidulans glyceraldehype -3- phosphate dehydrogenase gene
(gdh) and trpC expression signals in A; 1111911115
transformation (Cullen, 1987; Punt, 1987). Fusions of fungal
promoters with the hph gene have also been used to transform
§; cerevisiae (Gritz and Daves, 1983), and most, recently,

several filamentous fungi, e.g. Cephalosporium acremonium

28
(Queener, et al., 1985), Ustilago maydis (Cullen, 1987), and
Cochliobolus hoterostrophus (Yoder, et al., 1986) . Currently,
another prokaryotic dominant selectable marker, the neomycin
resistance gene, has been used successfully in transformation
of lower eukaryotes, animal cells an plant cells (Reiss,
1984). This gene encodes a neomycin phosphotransferase II
(NPT II) which confer the resistance to aminoglycoside
antibiotics like Kanamycin (Km), gentamycin and neomycin.
This gene has been successfully used in transformation of
fungi including Absidia qlauca (Wostemeyer, 1987) and
thcomvces blakesleeanus (zygomycetes) (Revuelta, 1986),
Cephalosporium acremonium (Skatrud, 1987), and the slime mold

Qictyostelium discordeum (Hirth, 1982).

4. NUCOR - FILAMENTOUS FUNGI OF THE CLASS ZYGOMYCETES

Food Industry Species of Mucor produce a variety of

 

extracellular enzymes including amylase (Adams, 1976), rennin
(Arima, 1968, Ottesen, 1970), cellulase (Somkuti,1974) and
lipase (Somkuti, 1968) . Strains of IL pusillus var. Lindt and
N; miehei are used in the production of rennin, which is the
second. most important enzyme used. in the food industry
(Crueger and Crueger, 1982). Species of NM are also
important to the food production industry because of food

spoilage (Pitt, 1985). Mucor is ubiquitous and grows rapidly

29
in nature. N; circinelloides has been reported to spoil
cheese (Northolt, 1980) and yams (Dioscoria sp.; Ogundana,
1972). N; piriformis was reported to be a destructive
pathogen of fresh straw berries (Lowings, 1956: Harris, 1980) .
N; racemosus is responsible for a spongy soft rot of cool

stored sweet potatoes, potatoes and citrus (Chupp, 1960).

Norphogenesig The biochemistry and molecular
biology of M rouxii and _Mu_co_; racemosus have been studied
because of their ability to undergo physiological alternations
between yeast and hyphae. It is a particularly attractive
system since the yeast - to - hyphae transition can be either
induced (Bartnicki - Garcia, 1962; Mooney, 1976) or inhibited
(Larsen, 1974; Ito, 1982) in a variety of ways. A large
quantity of biochemical and molecular research data has
accumulated about macromolecular biosynthesis, enzyme activity
and expression, and posttranslational modification of proteins

(Cihlar, 1985).

Transforggtion The absence of an efficient sexual

 

recombination system for genetic analysis and manipulation of
Nggg; (Gauger, 1965; Schipper, 1978) made it desirable to
develop a transformation system and introduce molecular
genetics to the study of these organisms. One species, N;
racemosus, has a haploid vegetative phase and has a small

genome (107 bp) which made it particularly convenient to

30
target for molecular genetic study. A high frequency
transformation system was developed in this organism using
protoplasts of a leucine auxotrophic mutant of N; ;9999999§
strain R78 and a shuttle vector pLeu4, which is able to
propagate in bacterial cells and in N; racemosus (Van
Heeswijck, 1984). The plasmid shuttle vector pLeu4 contains
a fragment of DNA from N; 99;; plasmid pUC 13 and a fragment
of homologous DNA containing the functional Leul gene from a
wild type strain of N; racemosus. The application of this
transformation system is very limited because the construction
of Leu' mutant strains for use as recipients in transformation
is tedious and very time consuming. This transformation
system cannot be currently used to study most other important
strains of N999; including the food spoilage causing strains
or enzyme producing strains which are less amenable to genetic
manipulation. Owing to the drawbacks and limitations in the
use of this transformation system, we sought to develop a

dominant selectable marker for use in Mucor, which can be used

 

in wild-type genetic background.

Resistances to antibiotics or fungicides are potentially
very useful dominant selectable markers. The development of
a dominant selectable marker for transformation of N;t_c_o; will
provide a mechanism to utilize the tools of molecular genetics
such as gene cloning by complementation, gene disruption and

gene replacement in the study of either dimorphism or food

31
spoilage in Hugo; species. This system also will enhance our
ability to genetically engineer the GRAS industrial strains
of Mucor. For example, microbial rennins produced by nugg;
are temperature stable, remaining active in the curd after
precipitation and subsequently causing harmful proteolysis
(Cruger and Cruger, 1982). The advances of biotechnology in
genetic engineering provides the opportunity to alter the heat

stability of this protein.

5. BENOMYL RESISTANCE GBNE PROM fl; CRASSA AS A DOMINANT

SBLECTABLB MARKER IN TRANSFORMATION OF H. RACEMOSUQ

 

Benomyl (Figure 2, pg.32) is one of the benzimidazole
fungicides which have been used in agriculture for a long time
(Davidse, 1988). The benzimidazoles bind to tubulin, which
is a heterodimer. Its subunits, usually designated a-tubulin
and fi-tubulin, each have molecular weights of ca 50,000
Daltons (Dustin, 1984). Benomyl binding to tubulin interferes
with the normal assembly of microtubules, causing their
gradual disappearance as the tubulins disassemble (Davidse,
1986). The inhibition of microtubule assembly disturbs a
great number of cellular processes that involve microtubules,
including mitosis, meiosis, DNA.synthesis (by inactivating the
spindle in animal and plant cells); intracellular transport

of molecules, particles, and organelles; maintenance of cell

32

H O H O
N H N ll
TNH—C—OCH; s '0 ' NH—C—OCHJ

coroendoznm nocodozole
(CEH) )3CH3
02C. o H
N
N " N “‘
m—NH-‘C‘OCHJ M
N N
benomyl unobendozme

Figure 2. Chemical Structures of Antifungal Benzimidazoles

(Davidse, 1988)

33
shape, and cell mobility through ciliar and flagellar action.
Benomyl induces nuclear instability in A; nidulans diploids
(Hastie, 1970). In.benzimidazole—treated germinating conidia
of;A idulans, spindle formation‘does not take place (Kunkel,
1980) . Under light and electron microscopy, the disappearance
of microtubules in carbendazim treated hyphal tip cells of
Egsarium acuminatum causes the displacement of mitochondria
from hyphal epics, reduces linear growth rate and arrests
metaphase of mitosis (Howard, 1980; 1977). Benomyl also
induces ‘multipolar' germination in.,N;, crassa due to the
inhibition of nuclear migration into multiple germ tubes.
The germ tubes stop growing, swell and emit several branches

(Can Caesar-Ton, 1988).

In 1978, Sheir-Neiss, et a1. suggested that the structure
of fi-tubulin is the major determinant for binding of
benzimidazoles. Recently, a benomyl resistance gene, Ben‘,
was cloned, and its nucleotide sequence determined (Orbach,
1986) . The Ben" gene is a mutated fi-tubulin gene from a
benomyl resistant strain of N; crassa and encodes a protein
of 447 - amino acid residues. The mutation responsible for
benomyl resistance comes from a single base change in the DNA
sequence (UUC to UAC), which results in a phenylalanine-to-
tyrosine change at amino acid position 167. This benomyl
resistance gene has been used as a dominant selectable marker

for the transformation of N; crassa at a frequency of >103

34
transformants per pg of DNA. The amino acid sequence of N;
crassa fi-tubulin has been found to be highly homologous to the
sequence of other tubulin proteins. Homology to $1
cerevisiae, §;, pgmbg, chicken, Trypanosoma brucgii, and
thamydomonas reinhardtii protein is 76, 77, 83, 78, and 79%,
repectively (Orbach,19860. This Ihigh level of sequence
conservation of fi-tubulins from.different organisms suggested
that the benomyl resistance gene might be used as a dominant
selectable marker in transformation of other organisms
including m. In fact, the BenR gene has been successfully
used in the transformation of Gaeumannoyces graminis (Benson,

1988).

In preliminary experiments conducted in our laboratory,
two strains of M1 racemosus 1216B - wild type and R78, were
found to be senstitive to benomyl at a minimum inhibitory
concentration (M.I.C.) of 50, 25 ug/ml for cells grown in the
dark and 100 pg/ml for cells grown in the light. Sensitivity
of the organism to moderate levels of benomyl suggests that
the benomyl resistance gene from N; crassa may be a useful
dominant selectable marker to improve the transformation
system of Hugo;. Our goal for this research project was to
utilize a vector containing the N; crassa benomyl resistance
gene to transform M299; cells, and to test the expression of

this heterologous gene in Mucor transformants.

MATERIALS AND METHODS

1. MICROBIAL STRAINS

Escherichia coli strain JM83 (asa (pro-lac) rpsL, thi
phi80 dlacz M15) g; 99;; strain LE392 (F-, hsd R514 supE44
supF58 lach gal K2 galT22 met 81 trpR55 ) and L 29;;
strain DH 50 (endAl hst17(r'Km*K) supE44 thi-1 recAl gyrA
relAl Q80lacz M15 (lacZYA-arthmw) were used as recipient
strains in bacterial transformation experiments and for

propagation of vector molecules.

flugg; racemosus ATCC 12168 R78 (Leu-) (abbreviated as M;
racemosus R78), a leucine auxotrophic mutant strain derived
from the wild type parent strain (M; racemosus
[circinelloides] ATCC 12168 (abbreviated as 11; racemosus
12168), VanIHeeswijckq A. ,1984), was used as recipient strain

in the Mucor transformations.

2. PLASMIDS

Plasmid pLeu4 (Figure 3, pg.36, 37) is a Mucor / E; coli
shuttle vector constructed from pUC13 containing the fi-

35

Figure 3.

Plasmid pLeu4

36

flme . Hwtulnw H H HHHQI PVfim

80mm” . HI: 1% H H Hutwl .GNNV

    

ommwuz:ce _umm.mmmv
ommm Hear
n mmmfi . H H H :1
mama _ fix an moan
V3041 15¢
..
mNHH.Hm§K
macro.

mam . 73¢ «flit
Gmm . HIQme

 

a .Zmu. Hamdemmm.
Hymn. .mmmm.
Hfisw mmon
qumw omon
_usm.mmon
HN fl... mmmm.

 

 

38
lactamase gene (AmpR) for the selection of ampicillin
resistance in §;_ggli, and a 4.4 Kb PstI restriction fragment
of H; racemosus genomic DNA which carries a leucine
biosynthetic gene (Leul) as a selectable marker for
transformation of M1 racemosus 1216B R78 (leucine auxotroph)
and a putative autonomous replication sequence (ARS) (Roncero,

et al., 1988).

Plasmid pBT3 (Figure 4 pg.39, 40) is a pUC12 vector which
contains a 3.1 Kb HindIII restriction fragment from the
genomic DNA of a benomyl resistant Neurospora crassa strain
Bm1511 (r)a (Orbach, et al, 1986), encoding a benomyl

resistant fi-tubulin protein.

Plasmid pMCupl-A (Figure 5, pg.41, 42) is a pUC8 vector
which contains a 3.3 Kb Eco RV - Nru II restriction fragment
from the genomic DNA of M; racemosus strain 12168 (Luis Sosa,
1987, personal communication) thought to encode a

metallothionein protein conferring copper resistance.

3. CHEMICALS

Chemicals used in these experiments were from Sigma
Chemical company with the exception of those specifically
described. Restriction ¢endonucleases, T4 DNA. ligase ,DNA

polymeraseI (Klenow fragment), 2'-deoxyribonucleotides, and

Figure 4.

Plasmid pBT3

39

 

40

mNNNHmumm
ammwhfln
Smw 2..qu
Emm Hzfim .
HHNNHEH...
mommsmm
mmwm HEM ,
Nsmm HHHHES

 

mmfia . Huron...
x180.“ . HIQmaw

mom. HE?
mqm. Hts?

mmfi H8
mmn HER
a... .g
@va HRH... HH mm .mvvm
own. H 9mm. H22

H H HHEE .mvmv
Hora mvmm

Figure 5.

Plasmid pMCupl-A

41

 

42

swam.HHanwm

Sam. H... u

vHsm.mem

.HH m HHmy

mamm Hm mwagx
BR H Sam

 

BGVN . H H330.

an ammo
¢-Hazoza

unmzo

88m . .pwtum

H .HHHE.~I

43
buffers for these reactions were obtained from Boehringer
Mannheim Biochemicals. [a-P”] dGTP (>6000 Ci/mmole) was
obtained from New England Nuclear (USA). Polyethylene glycol
4000 (PEG 4000) was obtained from Fluka AG (Switzerland).
Novozyme 234 (prepared from.Trichodermg_harzianum, containing

mainly a-1,3-glucanase activity) was from Novo Industries.

4. GROWTH MEDIA AND GROWTH CONDITIONS FOR E; COLI AND M;

RACEMOSUS

E; coli cultures were grown in Luria-Bertani (LB;

 

Maniatis, et a1, 1982) medium at 37°C with vigorous shaking
(rotary shaking platform,250 rpm; rotating wheel, 100 rpm).
E_,_ QLL strains which contained plasmids conferring ampicillin
resistance were propagated on LB medium supplemented with 100

ug/ml of ampicillin. L coli strains which produced 8-

 

galactosidase from ijC jplasmids were identified, by
supplementing LB media with the chromogenic substrate, 5-
bromo-4-chloro-3-indolyl-fi-D-galactoside (x-gal, Sigma) to a

final concentration of 40 pg/ml.

YPG (complete) medium (1 Liter) contained 10 g of bacto-
peptone, 3 g of bacto yeast extract and 20 g of glucose. The
pH was adjusted to 4.5 with sulfuric acid. YNB (minimal

defined) medium (1 Liter) contained 0.5 g yeast nitrogen base,

44
1.5 g ammonium sulfate and 1.5 g glutamic acid. The pH was
adjusted to 4.5 or 3.0 (depending on the different procedures)
with sulfuric acid. After autoclave sterilization, sterile
glucose (1%, final concentration), thiamine (1 ug/ml, final
concentration) and niacin (1 ug/ml, final concentration) were

added (Van Heeswijck, 1984).

Sporangiospore Preparations M; racemosus 12168

and M__._ racemosus 12168 R7B were grown on YPG media or YNB

 

media enriched with lmM leucine. Cells transformed with pLeu4
derived plasmids were grown on YNB (minimal) media without
leucine to select for the functional Leul gene on the
plasmids. Asexual sporangiospores were inoculated onto the
center of solid growth medium and incubated at room
temperature, under room atmosphere, with normal room light
for 7 days to generate large spore stocks. The sporangiospores
were harvested with ice-cold sterile distilled water by
scraping the surface of the mycelium with a sterile glass rod
(Paznokas and Sypherd, 1975). The spores were diluted with
YNB broth medium, quantitated with a hemacytometer, and used
to inoculate fresh cultures or stored at -20°c after addition

of glycerol (20%, V/V).

5. THE MINIMUM INHIBITOR! CONCENTRATION OF BENOMYL OR COPPER

FOR MQCOR

45

The minimum inhibitory concentration (MIC) of benomyl or
copper for M399; were determined with YNB media plus leucine
supplemented with various concentrations of the inhibitors.
Approximately 1 x 102 viable sporangiospores of MA racemosus
12168 and M; racemosus 1216B R78 were inoculated onto the YNB
medium. Duplicate plates were incubated at room temperature
under normal room light for 1 week, and individual colonies

counted.

6. TRANSFORMATION AND SELECTION OF E; COLI TRANSFORMANTS

Transformation of g; 99;; strains JM83, JM101 and DHSa
were performed as described in Maniatis et al (1982). Log
phase cells were resuspended in a solution containing calcium
chloride (100 mM). Exposure to calcium ions renders the cells
able to take up DNA (competent cells). Plasmid DNA (1-4 ug)
was mixed with 200 pl of competent cells for 30 minutes on
ice. The competent cells were then heat shocked at 42‘C for
2 minutes to allow the DNA to efficiently enter the cells. The
cells were grown 60 minutes at 37'C with shaking in 1 ml LB
media to allow cell recovery and the synthesis of plasmid-
encoded p-lactamase, which cleaves the B-lactam ring of
ampicillin and confers resistance to ampicillin. Cells were
then plated on LB media containing 100 ug/ml of ampicillin to

allow the selection of plasmid containing colonies.

46

7. THE IDENTIFICATION OF RECOMBINANT PLASMIDS IN E. COLI

£4,991; transformants containing the desired recombinant
plasmid were identified by colony hybridization (Maniatis,
T.,et al,1982 , Grunstein and Hogness 1975) with 32P-labeled
DNA fragments as probes. This procedure was used to screen
small numbers (100-200) of bacterial colonies. The colonies
were simultaneously consolidated onto a master agar plate and
onto a nitrocellulose filter laid on the surface of a second
agar plate. After growth overnight, the cells on the
nitrocellulose filter were lysed, denatured with Southern base
(1.5 M NaCl and 0.5 M NaOH) and neutralized with Southern
neutralizer (1 M Tris-Cl and 1.5 M NaCl, pH adjusted to 8.0
with HCl). The DNA liberated from these colonies was fixed
to the filter by baking at 80'C for 2 hours under vacuum.
Hybridizations were performed at 37°C in a hybridization
solution which contained 5X SSC (20X SSC contains 3M sodium
chloride and 0.3M sodium citrate, pH 7.0), 5X Denhardt's (1L
of 50X Denhardt's solution contains 10 g ficoll, 10 g
polyvinylpyrrolidone, 10 g bovine serum albumin [Pentax
Fraction V] in 1 L of distilled water), 50% formamide, 0.1%
SDS, 5mM EDTA.AND 100 ug/ml denatured salmon sperm DNA. After
two non-specific washes (at 2x SSC, room temperature, for 15
minutes) and one high stringency final wash (0.5 X SSC, at
65'C for 1 hour), the filters were analyzed by

autoradiography. Colonies, whose DNA gave positive

47
autoradiography results, were recovered from the master

plates.

8. ISOLATION OF PLASMID DNA FROM E. COLI

Migiprgpg Plasmid DNA was isolated by the alkaline
lysis method using a rapid, small-scale protocol described in
Maniatis (et a1, 1982). DNase-free RNase (20 ug/ml) was added
to digest contaminating RNA. The plasmid DNA was stored in TE
(10 mM Tris-Cl, 1 mM EDTA, pH 8.0) buffer at -20°C to prevent

DNase degradation.

Largg goals Plasmid DNA Isolation We used the large

scale plasmid isolation method described by Maniatis et al
(1982). A small volume of overnight culture (0.1 ml) was
added to 25 ml of LB containing ampicillin (100 pg/ml) in a
100 ml pyrex erlenmeyer flask, and incubated at 37'C with
vigorous shaking until the growth of cells reached midlog
phase (A.“,equals 0.4). This culture was added to 500 ml of
LB (containing 100 ug/ml ampicillin) in a pyrex erlenmeyer
flask (2 liter) for overnight incubation. The chloramphenical
amplification procedure was not used, because amplification
does not significantly improve in the recovery of pMB-l

derived pUC vectors.

After alkaline lysis, the plasmid DNA was purified by

48
equilibrium centrifugation on cesium chloride - ethidium
bromide density gradients. After isoamyl alcohol extraction

and dialysis, the plasmid DNA was stored in TE buffer at 4°C.

9. RESTRICTION ANALYSIS OF PLASMID DNA

Approximately 0.6 to 0.8 pg of plasmid DNA was digested
with restriction endonucleases according to directions
provided by the manufacturer. The DNA fragments were
separated according to size by electrophoresis through 0.8%
agarose gels together with Lamda-DNA/HindIII size marker.
Gels were stained with ethidium bromide (0.5 pg/ml) and

photographed under UV light.

10. PROTOPLAST FORMATION AND TRANSFORMATION OF MUCOR

Protoplast formation and transformation of Mucor
racemosus 1216B strain R78 were based on methods by Van

Heeswijck (1984,1984) with the following modifications.

Viable sporangiospores (107) of _M_._ racemosus 1216B
strain R78 were suspended in 50 ml of YNB broth (enriched with
1mM of leucine) and germinated at 28°C under vigorous shaking
for 5.5 hours until the germ tubes were 3 to 5 spore diameters
in length. Germlings were harvested by filtration through

nylon cloth (mesh size 35 pm), washed twice with 0.5 M

49
sorbitol, and resuspended in 0.5 M sorbitol, 0.01 M-sodium
phosphate buffer pH 6.5. Chitosanase (200 units) and Novozyme
(2 mg) were added to 103germlings in 1 ml of buffer. After
40 minutes of incubation at 28°C with mild shaking,
protoplasts were generated. Protoplasts were collected by
centrifugation (400 X g for 8 minutes at room temperature),
and washed twice with 0.5 M sorbitol and once with 0.5 M
sorbitol in MOPS buffer, then resuspended with 0.5 M sorbitol
in MOPS (3-N-morpholino propane sulphonic acid) buffer (10 mM
MOPS pH 6.3, 50 mM CaCl,) to a final volume of 1 ml. The
presence of protoplasts was confirmed by adding distilled
water to a small sample. Protoplasts were observed to burst

on a hemacytometer slide under a light microscope.

Plasmid DNA was pretreated with 1 mg of heparin in 20 pl
of 0.5 M-sorbitol in MOPS buffer for 20 minutes on ice.
Plasmid DNA (1 to 4 pg) was added into 0.2 ml of protoplast
suspension containing 20 pl of 40% (W/V) PEG 4000 in MOPS
buffer, and incubated on ice for 30 minutes . Then 2.5 ml of
40% PEG 4000 in MOPS buffer was added, and the mixture was
incubated at room temperature for 25 minutes to complete the
transformation. Cell suspensions were washed twice with 20
ml of 0.5 M sorbitol in MOPS and centrifuged at 400 X g at
room temperature for 5 minutes. The supernatant was
discarded, and the pellet.was resuspended.in 5 ml of YPG broth

(pH 4.5) with 0.5 M sorbitol, and incubated at room

50
temperature for 30 minutes to allow cells to recover. The
cells were washed once with 0.5 M sorbitol and resuspended in
5 ml of YNB broth pH 4.5 with 0.5 M sorbitol. Aliquots of
these cell suspensions (0.1 m1 and 1 ml) were added to a 9 ml
YNB (pH 3.0, with 0.5 M sorbitol) agar overlay (1% w/v agar).
Duplicate samples were plated on freshly made YNB medium (pH
3.0) with 0.5 M sorbitol. The pH value of the medium was

reduced to limit the colony size of transformants.

11. SINGLE COLONY ISOLATION OF MQCOB TRANSFORMANTS

Sporangiospores of putative transformants were harvested
with 200 pl of ice cold sterile distilled water by carefully
scraping the surface of mycelia of each isolated colony with
a sterile platinum loop. Dilutions of these spore suspensions
(1 and 10*) were plated on YNB plates, which were incubated
at room temperature in an anaerobic jar (BBL.GasPak-anaerobic
system) for 5 days to isolate single yeast colonies. One
well-grown single colony was selected from each plate and
reinoculated onto YNB medium to generate a sporangiospore

stock.

12. ISOLATION OF GENOMIC AND PLASMID DNA FROM MUCOR

DNA isolation from Mucor strains was based on methods by

 

Van Heeswijck (1984) with the following modification. Viable

51
sporangiospores (5 x 103 of each clone were inoculated into
250 ml of YNB minimum broth, and incubated at 28°C with
shaking for 10 hours under air (0.6 liters / minute). The
mycelia were harvested with filtration (Whatman 1mm), and
ground under liquid nitrogen in a mortar with a pestle. Then
8 mls of ice-cold TES (contains 100 mM Tris pH 8.0, 150 mM
sodium chloride, 100 mM EDTA, 0.1% SDS) was added to the
frozen powder and mixed for 1 minute. This suspension was
extracted successively with equal volumes of phenol saturated
with TES, phenol saturated with 100 mM Tris (pH 8.0), and
phenol / chloroform : isoamylalcohol (25 /24:1) solution for
1 hour, respectively, on a rocker mixing platform. The
aqueous phase from each extraction was separated from the
organic phase by centrifugation (6800 Xlg, 10 minutes at 4°C).
DNA was precipitated by addition of NaOAC (final concentration
is 0.25 M) and 2 volumes of 100% ethanol followed by storage
overnight at -20°C. The DNA was pelleted by centrifugation
(6800 X g, 10 minutes at 4°C), dried under vacuum, and
resuspended in.1 ml of TE with 50 pg/ml of RNase and 100 pg/ml
of proteinase K. The solution was incubated at 37°C for 2
hours. This DNA solution was then extracted with an equal
volume of phenol / chloroform :isoamylalcohol (25 / 24:1)
solution for 5 minutes. After centrifugation (14,000 X g, 4°C
for 20 minutes), the aqueous phase was removed. The DNA was
precipitated, pelleted by centrifugation and vacuum dried as

described above. The pellet was resuspended in 800 pl of TE.

52
The DNA concentration was measured with a spectrophotometer
(1 unit LAm] equals 40 pg DNA/ml). The concentration of a
small quantities of DNA was estimated by comparison of the
flouresence of DNA. bands with flouresence of known DNA
standards by agarose gel electrophoresis and staining with

ethidium bromide. DNA samples were stored frozen at -20°C.

13. SOUTHERN ANALYSIS

Southern analysis of Hugo; genomic DNA and plasmid DNA
samples was based on the transfer technique described by
Southern (1975). DNA was separated according to size by
electrophoresis through a 0.8% agarose gel. The DNA in the
gel was denaturated, neutralized, transferred and immobilized
on a nitrocellulose filter (BA85, from Schleicher & Schuell
FRG) with the same solutions as described in colony
hybridization. DNA fragments to be used as probes were
labeled with [ex-”PJ-dGTP by the random primer method (Feinberg
and Vogelstein, 1983). The DNA attached to the filter was
then hybridized to the denatured 32P-labeled DNA probe.
Hybridization was carried out at 37°C - 40°C in hybridization
solution which contained 5X SSC, 5X Denhardt's solution, 50%
formamide, 0.1% SDS , 5 mM EDTA and 100 pg/ml of denatured
salmon sperm DNA for 18 hours. After two non-specific washes
(2X SSC, room temperature for 15 minutes) and the one final

wash (1 hour, at 65°C, 3X, 0.5x, or 0.1X SSC depending on

53
stringency needed), filters were autoradiographed to locate
the position of target bands complementary to the radioactive

probe.

RESULTS

1. SENSITIVITY OF Mucor racemosus TO BENOMYL OR COPPER

A prerequisite for the use of the benomyl resistance gene
(Benn) as a selectable marker in transformation of Mucor
racemosus is the sensitivity of the host strain to this drug.
Approximately 1x102 viable sporangiospores of M_.. racemosus
were inoculated on YNB medium (12168) and YNB plus leucine
medium (R78) respectively, containing different amounts of
benomyl (0, 10, 20, 50, or 100 pg benomyl/m1). Growth of M;
racemosus 12168 and M_. racemosus R7B was observed after 6 days
on all plates except those containing 50 pg benomyl/ml for
cells incubated in the dark, and on plates containing 100 pg
benomyl/ml for cells incubated in the light. These data
suggested the M.I.C. to benomyl of both the wild strain
(12168) and the leucine auxotrophic mutant strain (R78) of
Hugo; was between 20 and 50 pg benomyl/ml for cells grown in
the dark and was between 50 and 100 pg benomyl/m1 for cells
grown in the light. In order to be able to detect a small
increase in resistance level of pMBen transformants to
benomyl, it was desired to determine the M.I.C. more
accurately. Approximately 1x102‘viable sporangiospores of MA

54

55
racemosug were inoculated onto YNB medium (1216B) and YNB plus
leucine medium (R7B), containing 0, 50, 60, 70, 80, 90, 100,
110, 120 or 130 pg benomyl/ml for cells grown in the light,
and 0, 20, 23, 27, 30, 33, 37, or 40 pg benomyl/ml for cells
grown in the dark. Growth was observed after 6 days (and
maintained up to 15 days) on all plates except those
containing 100 pg or more benomyl/m1 for cells incubated in
light, and on plates containing 30 pg and more benomyl/ml for
cells incubated in the dark. These data indicated that the
M.I.C. of benomyl to both the wild strain (1216B) and the
leucine auxotrophic mutant strain (R7B) of M999; was between
27 and 30 pg benomyl/ml in the dark and.was between 90 and 100

pg benomyl/ml in the light.

The sensitivity of M; racemosus to copper was tested in
similar fashion on YNB (1216B) and YNB plus leucine (R7B)
media containing 0, 0.1, 0.5, 1, 2, 10, or 20 mM copper. The
data suggested that the M.I.C. of copper to both M; ;a9emosus

12168 and M; racemosug R7B was 2.0 mM after 6 days of growth.

The resistance of M; ;acemosus to hygromycin B was also
tested in similar fashion on YNB (1216B) and YNB plus leucine
(R7B) media containing 0, 100, 200, or 500 mg/ml hygromycin
B. The data indicated that these two m; strains were
resistant to hygromycin B at 500 mg/ml. Because these two

Mucor strains were resistant to this high dose of hygromycin

 

56
B, it was not suitable to use hygromycin B as a selectable

marker in transformation.

2. THE HOMOLOGY OF THE S - TUBULIN GENES OF M; BACEMOSUS TO

THE M; QRASSA BENOMYL RESISTANCE GENE

The amino acid sequence of the Ben" gene from N_,_ 9rassg
is highly homologous to genes encoding 8 - tubulins in other
organisms. To determine the degree of homology of this BenR
gene of N__._ crassa to that of _M_._ racemosus, a DNA fragment
containing the BenR gene from _N_._ crassa was purified,
radiolabeled and hybridized to a nitrocellulose paper carrying
genomic DNA of M; racemosus R78 and 12168 digested with 5.9M.
E9931, and MindIII. The nitrocellulose filter was washed at
medium stringency (2X SSC, 0.1% SDS, at 65°C for 1 hour), and
autoradiographed (Figure 6, pg.57). There were more than 3
DNA fragments in R78 and 1216B which hybridized to the benomyl
resistance gene. The Southern analysis data suggested that
the Ben“ gene from M; crassa has some (medium) homology with
several DNA fragments in the genome of M; racemosus. Medina,
etal., (1988) reported that there are 3,8-tubulin.genes in the
M; ;acemosus genome. Figure 6. (pg. 57) revealed several
restriction site polymorphoisms between the B-tubulins of' M;
;acemosus 1216B and M_. ;acemosus R7B ,the mutant strain

derived from.M; ;acemosus 1216B. The differences between the

fi-tubulins in these strains indicated that they differ from

Figure 6.

Panel A

57

Southern analysis of genomig DNA of M; racemosus
strains 12163 and R7B with P-labelled 2.6 kb
SalI fragment from plasmid pBT3, which contains

the BenR gene .

Genomic DNA (20-40 ug) was digested with different
endonucleases , then analyzed by electrophoresis on
a 0.8 % agarose gel. Lane 1, 3, 5 : genomic DNA of
M; racemosus 1216B digested with BamHI, EcoRI, and
HindIII endonucleases. Lane 2, 4, 6 : genomic DNA
of M; racemosus R7B digested with BamHI, EcoRI, and

HindIII endonuclease.

 

 

 

Panel B The genomic DNA on agarose gel was transferred onto
a nitrocellulose paper, hybridized with the BenR
probe, washed at medium stringency, and exposed to X-
ray film for 40 hours at -70°C.

58

Figure 6.

Panel A Panel B

 

59

each other in more regions of the genome than the Leul gene.

3. RESTRICTION ANALYSIS AND SOUTHERN ANALYSIS OF PLASMID

PMCUPI-A

The plasmid pMCupl-A is a pUC based vector, which
contains a 3.3 kb HindIII fragment from the genome of M;
racemosus thought to carry the gene encoding a metallothionein
protein. This protein binds copper ions and enhance resistance
to copper. Restriction analysis of pMCupl-A with MindIII,

SalI, and PstI endonucleases revealed that.the plasmid.was not

 

constructed as indicated by the supplier (L. Sosa, gift). A
3.8 kb fragment was generated with SalI endonuclease

digestion, while PstI only cut this plasmid once. Further

 

restriction analysis (data not shown) revealed that pMCupl-A
contains an unknown 500 bp DNA fragment between Sal; and

HindIII site.

We then obtained a second plasmid, pMCupl-B, a pUC based
vector, thought to contain a 1.3 kb EcoRV / NruII fragment
subclone of the original 3.3 kb Hind III fragment of pMCupl-
A. This 1.3 kb EcoRV / NruII fragment was isolated, radio-
labeled and used as a probe in Southern hybridization analysis
of pLeu4 and pMCupl-A (Figure 7, pg.60,61.) The 1.3 kb EcoRV
/ NruII probe hybridized to all bands containing pUC sequences

including a 7.1 kb fragment derived from pLeu4 in lane 1, a

60

Figure 7. Southern analysis of plasmid DNA with 32P-labelled
1.3 kb fragment from plasmid meupl-B

Panel A Plasmid DNA was digested with different endonucleases
, then analyzed by electrophoresis on 1 % agarose
gel. Lane 1 : plasmid DNA of pLeu4 digested with
SalI endonuclease. Lane 2 : plasmid DNA of pMCupl-
A digested with EcoRV and PvuII endonucleases. Lane
3 : plasmid DNA of pMCupl-A digested with SalI
endonuclease.

Panel B The plasmid DNA on the agarose gel was transferred
to a nitrocellulose paper, hybridized with 1.3kb
fragment from plasmid pMCupl-B, washed at high
stringency, and exposed to X-ray film for 20 hours

at -70°C.

 

61

Figure 7.

Panel A

Panel B

 

62
2.35 kb fragment derived from pUC 8 in lane 2, and a 2.7 kb
pUC derived fragment in lane 3. We have not confirmed that
pMCupl-A and pMCupl-B contained the M; racemosus Cup gene.
The use of the copper resistance gene as a dominant selectable
marker for M999; transformation was abandoned awaiting

verification of these two plasmids.

4. PMBEN PLASMID VECTORS: CONSTRUCTION AND ANALYSIS

The pMBen plasmid vectors were constructed as
described in Figure 8 (pg. 63, 64) A 2.6 kb §gl; restriction
fragment from pBT3 (Orbach,1986) containing the benomyl
resistance gene (BenR) from M; 9rassa was found to be
sufficient to transform M; crassa to benomyl resistance at

high frequency (Orbach, 1986). Translation of the Benthene

starts 352 base pairs downstream from the SalI site, and stops

 

300 base pairs upstream from the HindIII site. The 2.6 kb
figl; insert and a 3.1 kb HindIII insert which also contains
the benomyl resistance gene were generated from pBT3 by
digestions with _Sa_l_I_ and HindIII respectively. These DNA
fragments were gel purified by electrophoresis on a 0.8 %
agarose gel, and by electroelution (the electroelution
apparatus is from International Biotechnologies, Inc.). The
recessed 3'ends of the 3.1 kb HindIII fragment were filled in

with.dNTPs using the Klenow fragment of E; coli DNA polymerase

 

I (Maniatis, 1982) to«generate blunt ended DNA fragments which

63

Figure 8. Construction of Plasmids pMBenl and pMBen2

The plasmids. pBT3 and pLeu4 are described in
MATERIALS AND METHODS. The region representing particular
DNA fragments are labeled : Q , BenR gene; ai-
,Leul gene; ,4. ,AmpR: ampicillin resistance gene.

64

 

cutiuith SalI
cut with SalI

 

J,

 

J. 533 11 8 t1
5a 1
p Leu4 LBUI
en-R
5.11
FStI thl

 

 

65

were then combined with SmaI linearized pLeu4 DNA and T4 DNA

 

ligase. The vector pLeu4 was also digested with 9g and
combined with the 2.6 Kb §99I fragment. Ligation of vector
and insert was carried out at a ratio between insert fragments
and linearized vectors of 3:1 for the 9911 sticky-end ligation
and 5:1 for §99I blunt—end ligation. The newly constructed
plasmids were transformed into E; 9911 DH 5a to propagate the
vector molecules, and to screen for clones with the desired
construction. Recombinants containing the _N_. crassa BenR gene
were identified by colony hybridization with a radio-labeled
2.6 kb §9ll fragment from pBT3 as probe. As shown in Figure
9 (pg.66), 8 of 121 sticky - end recombinants (obtained upon

subcloning of the 2.6 kb SalI fragment) and 3 of 50 blunt -

 

end recombinants (ontained upon subcloning of blunt ended 3.1
kb MindIII fragment) were detected to contain BenR inserts.
After single colony isolation, the plasmid DNA of these
positive recombinants was isolated with the "miniprep" method.
Samples of the resulting plasmid were digested with XbaI an9
291:; restriction endonucleases and analyzed by electrophoresis

on a 0.8 % agarose gel. XbaI cuts 3 times in the pLeu4

 

vector, while 299; cuts twice in pLeu4 and once in the BenR
insert. The £99; digests were designed to reveal the
orientation of insertion by the generation of 3 DNA fragment
: 4.4 kb, 2.7 kb, 2.6 kb in one orientation and 3 DNA fragment
: 5.3 kb, 4.4 kb and a non-detectable 10bp fragment in the

other orientation. Restriction analysis suggested that of 8

66

\ li( k;\' I \~l) l [(3 \ ll()§§ N/‘ljl lil.LJF§’f—lzb§l) l l(} \ ll()5§ .1 ‘
Figure 9. Colony hybridization with the BenR gene

The a; 99;; cells grown on nitrocellulose paper were
lysed, denatured, neutralized and hybridized with the BenR
probes to identify the cells which has correct recombinant
plasmids. Filters were washed at high stringency and exposed
to X-ray film for 6 hours at room temperature. "+" represents
the colony containing pBT3 as a positive control. "-"
represents the colony containing pLeu4 as a negative control.

67

Figure 10. Restriction analysis of pMBenl and pMBen2

Plasmid DNA of pMBenl and pMBen2 was digested with
different endonucleases and analyzed by electrophoresis on
0.8% agarose gel. Lane 1, 3, 5, 7, 9, 11, 13 : plasmid DNA
of pMBen2 digested with KpnI, SstI, SalI, HindIII, EcoRI,
BamHI and PstI endonuclease . Lane 2, 4, 6, 8, 10, 12, 14 :
plasmid DNA ofijBenl digested with K nI, SstI, SalI, HindIII,
EcoRI, BamHI and PstI endonuclease

 

 

 

68

Figure 10.

 

69

§9l1 recombinants, 2 clones contained the 2.6 kb §9l;/ BenR
gene in one orientation (named pMBen1) and 5 clones contained
the 2.6 kb gay Bena gene in the opposite orientation (named
pMBen2). One clone did not show the predicted resistiction
map and was discarded.

One clone from each orientation was selected for detailed
restriction analysis with 7 different endonucleases including
E§_I. lfiflflflL. £2231. IEDELLLL. §éll. i232; (§§El). auui K221
(Figure 10, pg.67, 68). Based on these data, the predicated
restriction maps of pMBen1 and pMBen2 plasmids were confirmed

(shown in Figure 11, pg.70 and Figure 12, pg.72).

Plasmid DNA of recombinant clones containing pMBen1 and
pMBen2 was isolated from E; 9911 cells using large-scale
protocol and cesium chloride density gradients. This pure
DNA was then used to transform protoplasts of M; ;a9emosus

R78.

5. TRANSFORMATION OF MUCOR ggcznosug

Initial experiments were designed to demonstrate our
capability to transform the filamentous fungus M; ;acemosus.
Approximately 1 x 105 viable sporangiospores of M; racemosus
R7B were inoculated onto each of 20 YNB agar plates to test

the spontaneous reversion frequency of this Leu' strain to the

Figure 11.

Plasmid pMBen1

70

 

88V? . vajfi.

1!

         
  

m.m.xm;u. .99...
mmHmHHm..,d

71

an amnaH

2.9.... H H HES .mmHm

H36; .mvmm
Hxém anm.

.39 . H 3.... HS? .33
mm: Hg... .HHHSH .mmvm
H .HEnH . Hogmrwgm

HH mm, sang

Figure 12.

Plasmid pMBen2

72

73

@834 . mel

88 . 53m
99 . H3...
mNHH Huge

1'

          
  

m.x.m.m .8an

an amnaH
Ncomza

H6 mm .Nmam
Hanna mmmm
Haas anm

qurnnmmm
Hear mva

H I2 . mmmm
H.u swamhm a m

H H HEN}. .mhmm

H .mem H 3m. :51 .mmmfi

74
Leu' phenotype. No M; racemosus colonies appeared after 7
days. The spontaneous reversion frequency of this Leu' strain
is less than 1/2x106. This makes the detection of
transformants possible because the transformation frequency
of R78 with pLeu4 is 600 transformants/3.2x106 viable

protoplasts (Van Heeswijck, 1984).

Protoplasts of M; racemosus R7B were generated as
described in MATERIALS AND METHODS. Approximately 60 % of
the sporangiospores of M; ;acemosus R7B inoculated in YPG
broth germinated and produced germ tubes 3-5 spore diameters
in length after five and a half hours of incubation.
Approximately 50 % of the total protoplasts were generated
from germlings after 40 minutes of cell wall digestion with

Chitosanase and Novozyme 234. Because the mycelia of Mucor

 

are unicellular, not every protoplast generated has a nucleus.
Only the protoplasts with a nucleus are able to regenerate
cell wall and grow colonies on YNB plus leucine plates by the
second day. The regeneration efficiency of M; ;acemosus R7B
was 60 %, (ie., 60% of protoplasts grew under nonselective

conditions).

pLeu4 Vgctor Protoplasts were transformed with
the plasmid pLeu4. Prototrophic colonies appeared 2-3 days
after transformation and were capable of sporulation. The

transformation frequency was 50 colonies/pg DNA/106

75
protoplasts. No prototrophic colonies grew when no pLeu4 DNA
was added to protoplasts in the transformation experiment.
Sporangiospores of putative transformants were harvested and
reinoculated into YNB (minimal media). The ability of
transformants to grow suggested that the acquired Leu+
phenotype was stable under selective pressure. Spore stocks
were produced from 5 of the putative transformants. Three
stable putative transformants were then further characterized

by Southern analysis (see below).

pMBen Vecto;s In. order' to increase ‘the number' of
transformants generated, more protoplasts were used in
transformation experiments. To eliminate confusion due to
isolation of revertants, the spontaneous reversion frequency
of the R7B Leu' strain back to Leu+ was tested again and was
found to be less than 1/9x107. Approximately 2x106 protoplasts
of R7B were mixed with 1—3 pg pMBen1 and pMBen2 DNA.
Transformant colonies appeared 3-4 days after transformation.
Since cells in these colonies must synthesize their own
leucine, they grow more slowly than protoplasts regenerated
on medium containing leucine. Twenty four prototrophic
colonies were obtained with pMBen-1 and 12 prototrophic
colonies were obtained with pMBen-2. The transformation
frequency of pMBen1 was 30 colonies/pg DNA/2x106 protoplasts
and that of pMBen2 was 50 colonies/pg DNA/2x106jprotoplasts.

Increasing the number of protoplasts used in an experiment

76
increased the total number of transformants obtained but did

not increase the transformation efficiency.

Putative transformants were single colony isolated by
growing as yeasts under C02 (see Materials and Methods).
Sporangiospores of 5 pMBen1 and 5 pMBen2 transformants were
harvested and reinoculated onto YNB plates. All transformants
were able to grow and sporulateu Two clones containing pMBen1
, B1-3 and B1-5, and two clones containing pMBen2 , B2-1 and
B2-5, were chosen to conduct further analysis. Approximately
1x102 of viable sporangiospores of B2-1 were plated on YNB and
YNB plus leucine duplicate plates“ Only 5% of the spores were
able to grow in YNB plates while 99% of spores were able to
grow in YNB plus leucine plates. This result suggested that
the transformants were mitoticalLy or segregationally
unstable. Only 5% of spores apparently received at least one

copy of the plasmid and the Leul gene.

6. SOUTHERN ANALYSIS OF M_. RACEMOSUS TRANSFORMED IITH PLASMIDS

pLeu4 Transformants Genomic DNA of 3 transformants
(T1, T2, T3) and non-transformed cells (R7B) was isolated and
digested with different restriction endonucleases. The DNA
fragments were separated by electrophoresis on a 0.7% agarose

gel and transferred onto nitrocellulose paper for Southern

77

Figure 13. Southern analysis of genomic DNA of 3M; ;999mosus
R7B and 3 pLeu4 transformants with P-labelled
pUC9 plasmid probe.

Genomic DNA (20-40 ug) was digested with different
endonucleases, analyzed on by electrophoresis on a 0.7 %
agarose gel, transferred onto a nitrocellulose paper and
hybridized with 32P—labeled pUC9 probe. Filters were washed
at high stringency and exposed to X-ray film for 42 hours
(Panel A) and 22 hours (Panel B) at -80°C. Lane 1, 5 of Panel
A and Panel B : genomic DNA of M; racemosus R7B undigested,
digested with SalI, EcoRI, and ClaI endonucleases . Lane 2,
6 of Panel A and Panel B : genomic DNA of pLeu4 transformant
T1 undigested, digested with SalI, EcoRI, and glaI
endonucleases . Lane 3, 7 of Panel A and Panel B : genomic
DNA of pLeu4 transformant T2 undigested, digested with §a1I,
EcoRI, and ClaI endonuclease . Lane 4, 8 of Panel A and Panel
B>: genomic DNA of pLeu4 transformant T3 undigested, digested
with SalI, EcoRI, and ClaI endonuclease .

 

78

und igesfed Sal]

A 12345678

23.67-

4.26-

2.30-

 

 

EcoRl r ClaI
812212218
23. 97- ‘ . z '
9. 46-
6.66- " *-
4.29— . . ~ __

2.30-

 

79
hybridization with a radio-labeled pUC9 probe (Figure 13,
pg.77, 78). The pUC9 DNA is nearly identical to the pUC13
backbone of pLeu4, and has no homologous sequences in the
genome of the R7B host. The probe hybridized to the genomic
DNA of the transformants (Lane 2, 3, 4, 6, 7, 8 on panel A
and Lane 2, 3, 4, 6, 7, 8 on panel B), but not to DNA of non-
transformed R7B cells. Therefore, only cells containing the
pLeu4 plasmid hybridized to this probe as expected. These
results confirmed the entry of pLeu4 plasmid into R7B host
strain during transformation. The pUC9 probe hybridized to
3.8 Kb and 7.1 Kb M993; restriction fragments (Lane 2, 3, 4
on panel B) as predicted. This data suggested that there was
no DNA rearrangment after the plasmids were transformed and

propagated in the M; racemosus host.

pMng Transfozgangg Transformants B1-3, B1-5,
thought to contain pMBen1, and B2-1, B2-5 thought to contain

pMBen2, were chosen to do Southern Analysis. Genomic DNA of
these 4 clones was isolated from cells and digested with 9;a_I_,
9991, and Es_t_I_ endonucleases, separated on a 0.8 % agarose gel
and transferred to nitrocellulose paper. The nitrocellulose
was probed with the 2.6 Kb 591; Ben" insert and pUC 19, which
is nearly identical to the pUC12 backbone of the pLeu4 vector.
Filters were washed under high stringency conditions and
autoradiogrophed (Figure 14, pg.80, 81 and Figure 15, pg.82,

83). ClaI endonuclease was shown previously to have no

80

Figure 14. Southern analysis of pMBen plasmids DNA and
genomic DNA of M. racemosus R7B and 3 pMBen1 and
pMBen2 transformants with RP- -labeled 2. 7 kb §aa11
fragment (BenR gene) from plasmid pBT3 .

Panel A-l Plasmid DNA of pMBen1 and pMBen2 was digested
with different endonucleases and analyzed by
electrophoresis a on 0.8 % agarose gel. Lane 1, 2, 3
: plasmid DNA of pMBen1 undigested, digested with
SalI, and PstI endonucleases. Lane 4 : plasmid
DNA of pMBen2 digested with PstI endonuclease.

 

Panel A-2 Genomic DNA (20-40 ug) was digested with different
endonucleases, then analyzed by electrophoresis on
a 0.8% agarose gel. Lane 1, 4, 7, 10 : genomic DNA
of pMBen1 transformant 81-5 undigested, digested
with ClaI, SalI, and PstI endonucleases. Lane 2,
5, 8, 11 of : genomic DNA of pMBen2 transformant
B2-1 undigested, digested with ClaI, SalI, and Est;
endonucleases. Lane 3, 6, 9, 12 : genomic DNA of
pMBen2 transformant B2-5 undigested, digested with
QlaI, SalI, and PstI endonucleases. Lane 13 :
genomic DNA of M; racemosus R7B digested with 2s91
endonuclease. Lane 14 : genomic DNA of M; W
12168 digested with gstI endonuclease.

 

 

Panel B The plasmid and genomic DNA on agarose gel was
transferred onto a nitrocellulose paper and
hybridized with 3ZP-labelled 2. 7 kb Sal; fragment
(BenR gene) from plasmid pBT3. Filters were washed
under high stringency and exposed to X-ray film for
30 minutes at -70°C.

81

Figure 14.
Panel A—1 Panel A—2
Uncut ClaI SalI PstI
123434 SdlZJ436789IOIIIZI3I4Sd

23J3-

342
&56

L36-

p»
on
(OM

   

 

Panel B
Unclut ClaI SalI PstI
1.23 4 12 3 4 5 6 7 8 91011121314

     

Figure 15.

Panel A-l

82

Southern analysis of pMBen plasmid DNA and
genomic DNA of M; racemosus R78 and 3 pMBen1 and
pMBen2 transformants with SZP-labelled 2 .6 kb pUC19
probe.

Plasmid DNA of pMBen1 and pMBen2 was digested

with different endonucleases and analyzed by
electrophoresis on a 0.8 % agarose gel. Lane 1, 2, 3 :
plasmid DNA of pMBen1 undigested, digested with SalI,

and

PstI endonucleases. Lane 4 : plasmid DNA of pMBen2

 

digested with PstI endonuclease.

Panel A-2

Panel B

Genomic DNA (20-40 ug) was digested with different
endonucleases, then analyzed by electrophoresison
0.8% agarose gel. Lane 1, 4, 7, 10 : genomic DNA
of pMBen1 transformant B1-5 undigested, digested
with ClaI, SalI, and PstI endonucleases. Lane 2,
5, 8, 11 : genomic DNA of pMBen2 transformant
B2-1 undigested, digested with ClaI, SalI, and PstI
endonucleases . Lane 3, 6, 9, 12 : genomic DNA
of pMBen2 transformant B2-5 undigested, digested
with ClaI, SalI, and PstI endonucleases. Lane 13
: genomic DNA of M; racemosus R7B digested with
PstI endonuclease. Lane 14 : genomic DNA of M;
racemosus 1216B digested with PstI endonuclease.

 

The plasmid and genomic DNA on agarose gel was
transferred onto a nitrocellulose paper, hybridized
with pUC19 probe, washed at high stringency, and
exposed to X-ray film for 30 minuatesat -70°C.

83

Figure 15. Panel A-Z
Panel A-1
Uncut ClaI Sal! PstI
r—*—1r—*—1r-*-1r———4--n

1234Sd 541234567891011121314521

   

Panel 8

I SalI P tl
Uncut Cla §
12341234567891011121314

    

84
restriction sites in the pMBen plasmid (data not shown) but

was assumed to cut in the genomic DNA of Mucor. The SalI

 

endonuclease cuts the BenR insert out from pMBen plasmids.
299; was used to distinguish between pMBen1 and pMBen2 (see
restriction maps in Figure 11, pg.70, 71, and Figure 12,
pg.72, 73) . The BenR probe hybridized to DNA of each of the
BenR'transformants under high stringency conditions, but not
to that of the R7B host strain or to the M; racemosus 1216B
(wild type). The pUC19 probe hybridized to DNA of each of
the BenR transformants under high stringency condition, but
not to that of the R7B host strain or to the M; racemosu§
1216B (wild type). The ability of these putative
transformants to grow on YNB plates combined with the results

of Southern analysis confirmed the presence of pMBen plasmid

in the host strain after transformation.

The hybridization patterns of pure pMBen plasmid DNA and
pMBen plasmids in the transformants' DNA.were the same. 'These
data indicated no detectable gene rearrangement after the
plasmids were transformed and propagated in m; host. These
results were similar to those reported for other M; racemosus

plasmids by Van Heeswijck (1986).

7. RESISTANCE OF PMBEN TRANSFORMANTS TO BENOMYL

To determine if the BenR gene from M; crassa was

 

 

85
functionally expressed in M; ;acemosug transformants, we
measured the level of resistance to benomyl in 4 transformant
clones. Approximately 200 and 2000 viable spores of ;M;
;acemosus R7B, M; ;acemosus 12168, and 4 pMBen transformant
clones were inoculated onto YNB plus leucine medium (for M;
;acemosus R7B only) and YNB medium, containing benomyl at
final concentrations of 0, 80, 90, 100, 110, 120 or 130 pg
benomyl/ml. Growth of the 4 pMBen transformants, M; racemosgg
12168, and M; racemosus R7B was observed after 6 days (and
maintained to 15 days) on all plates except those containing
100 pg or more benomyl/ml (incubated in the light). These
data suggested that the M.I.C. of benomyl to M; racemosus
1216B, R78 and 4 chosen transformants is between 90 and 100
pg benomyl/ml when grown in light. There was no significant
increase in resistance to benomyl in.pMBen transformants. 'The
colonies of transformant 82-1 grown in YNB medium versus in
YNB plus leucine were also compared. There were consistantly
fewer colonies of 82-1 grown in YNB medium than that in YNB
plus leucine medium. This result suggested that the
tranformants were mitotically or segregationally unstable as

mentioned before in Result 5 (pg.77).

DISCUSSION

In this work, transformation of protoplasts of M;
;acemosus R7B, a leucine auxotrophic mutant was accomplished
using a shuttle vector, pLeu4, containing the Leul gene as a
selectable marker. In order to improve this transformation
system, our goal was toiconstruct.pMBen‘vectors containing the
Ben" gene from M; crassa as a dominant selectable marker and
to test the functional expression of this heterologous gene
in M999; cells transformed with the construct. The plasmids
pMBen1 and pMBen2 were constructed and used to transform M;
;acemosus R7B. Southern analysis of the genomic DNA of 4
transformants suggested that the BenR gene was present in the
cells with no obvious DNA rearrangements. Although the pMBen
plasmids were shown to be present in transformants cells,
there was no significant increase in the resistance level of
pMBen transformants detected when compared with the resistance

level to benomyl of the host strain (without pMBen),

indicating that the Ben3<gene was not functionally expressed

in Mucor.

The M.I.C. to benomyl of both the wild type strain
(1216B) and the leucine auxotrophic mutant strain (R7B) of

86

87
M999; was between 27 and 30 pg benomyl/m1 for cells grown in
the dark and was between 90 and 100 pg benomyl/ml for cells
grown in the light. To determine the reason for the observed
differences in M.I.C., two sets YNB and.YNijlus leucine media
containing 50 pg/ml benomyl were prepared. One set was stored
in the dark and the other in the light for 7 days. The media
were inoculated with spores of M;_;acemo§9§ 1216B and.R7B, and
incubated under the opposite conditions (ie. the media which
were stored in dark were incubated in light, and vise versa).
After 15 days of incubation, 32 colonies grew on the medium
incubated in light while there was zero growth on the set
incubated in the dark; These data suggested that the
breakdown of benomyl by light was not the reason why these two
M999; strains grew'better in light, but that light contributed

as an important factor in the improved growth of M. ;acemosus.

 

One possibility is that cells grow more slowly in the dark,
and are consequently more sensitive to benomyl. Since M999;
has the ability to adapt to antibiotics such as cycloheximide,
trichodermin, and amphotericin B (Linz and Sypherd, personal
communication), it is also possible that the Mm cells
adapted.to benomyl more rapidly in the light, so they appeared

to be more resistant to benomyl.

The transformation efficiency of M; racemosus R7B with

pLeu4 was 500 transformants/pg DNA/107 protoplasts: with

pMF25, 250 transformants/pg DNA/107 protoplasts were obtained;

88
with pMF29, 64 transformants/pg DNA/107 protoplasts were
obtained (Chiu, M.S. Thesis, 1988) and with.pMBen1 and.pMBen2,
150 and 250 transformants/pg DNA/107 protoplasts were
obtained. In our laboratory, the more protoplasts that were
added to the transformation reaction, the more transformants
were obtained. However, there was no significant change in

the efficiency of transformation.

The Leufjphenotype of M; racemosus R7B transformed with
pLeu4 or pMBen was stable after spores were reinoculated onto
YNB plates (ie, selective pressure). However only 5% of the
spores generated from cells growing on YNB were able to grow
when plated under this selective pressure. Transformants
tended.to lose their plasmids at an even faster rate when they
were grown on YPG (rich medium) containing leucine (without
selective pressure). These results suggested that there is
mitotic instability’ either' during' cell growth. or’ during
sporulation. One possible reason for this observation could
be that the plasmids do not integrate into genome of the host
cell, so they would not be segregated equally to daughter
cells during mitosis. Generally, there are at least three
possible fates of a plasmid which enters the host cell. A
plasmid may replicate autonomously. It may integrate into the
chromsomal DNA randomly by nonhomologous recombination or it
may integrate into chromosomal DNA at specific sites via

homologous recombination. The results of our Southern

89
analysis shown previously indicated there was no detectable
DNA rearrangement after plasmids were transferred into host
cells. This confirmed results reported by Van Heeswijck
(1984), who also demonstrated that the pLeu4 plasmid can be

rescued by transformation of E; coli with Mucor genomic DNA.

 

The plasmid obtained from 3; mm. is unchanged from the
original plasmid. These experimental data supported the
notion that pLeu4 derived plasmids replicate autonomously.
Another possible explanation of the results observed in the
Southern analysis is that the transformed plasmid integrates
into the genome at one location and duplicates tandemly. The
results seen in Southern analysis could be the same in this
situation. In support of this explanation, plasmid probes
used in Southern analysis of transformant DNA digested with
9191 endonucleases (which does not cut in the plasmid)
hybridized to to smaller DNA fragments than in undigested DNA,

suggesting integration of plasmid DNA.

It is possible that most of the pLeu4 derived plasmids
replicate autonomously in M999; while a few plasmid molecules
integrate into the genome of host cells. Under these
conditions, the low number of integrated plasmid may not be
detected in our Southern analysis. Our laboratory is
currently testing this hypothesis. Transformant cells were
grown without selective pressure for several generations, and

were transferred onto selective medium to generate isolated

90
colonies. This protocol was repeated several times. Since
there is a high level of segregational instability in M999;,
cells grown in the absence of selective pressure will rapidly
lose their plasmids. If any plasmid DNA integrated into
genome of host, it should be detectable after inoculation of
spores back onto YNB plates. Whether pLeu4 derived plasmids
integrate into the Mg; genome or not is an interesting
question. More future research is required to fully answer

this question.

The presence of pMBen plasmid in transformants was
confirmed by Southern analysis with the Benkigene and pUC19
probes. The Leul gene on pMBen vectors was expressed in R78
cells enabling them to grow on YNB media lacking leucine.
However, the resistance level of the transformant strains of
81-3, 81-5, 82-1, and 82-5 to benomyl was not improved when
directly compared with the sensitivity level of the R78 host.
The BenR gene from N_._ crassa has been expressed functionally
in fungi other than M; crassa. The functional expression of
a heterologous gene in a eukaryotic host entails a complicated
series of events. First, the gene must be transcribed to
produce a stable premessage (Hn-RNA), which requires
posttranscriptional modification to generate a stable m-RNA.
Then the m-RNA must be translated into a protein. In many
cases, further posttranslational processing is necessary to

produce a functional end-product.

91
The BenR gene was confirmed to be present in $99; cells
in apparently unaltered form, but was not functioning to
improve the benomyl resistance level. This could be due to
one of many possible reasons such as : 1) no transcription
of the Benll gene, 2) no stable message (m-RNA) produced,

because the enzyme system in Mucor could not properly process

 

the Hn-RNA, (there are 6 introns in the Ben" gene), 3. the m-
RNA was not translated, 4. lacking the translated protein,
5. the produced B-tubulin protein was not incorporated into
microtubules in M; ;acemosus. The first question to ask is
whether this heterologous Ben'l gene was expressed in M_.,
;acemosus transformant cells or not (eg., was there a stable
message transcribed from this gene?). Northern analysis of
RNA isolated from transformant cells using a Ben“ gene probe
will answer this question. If no stable message is detected,
then, presumably a homologous promoter, and a homologous
transcription terminator will produce a functional mRNA in M;
;acemosus. Currently, the promoter from M; racemosus TEF-l,
promoter from a gene which encodes elongation factor 1a (Linz,
et al.,1986), and the Leul promoter are being characterized

for possible use with the BenR gene.

If stable message is detected in Northern analysis, then
we must determine whether a functional B-tubulin protein is
translated from this message. Specific antibody to the 8-

tubulin protein will be useful in this regard.

 

,—

 

92

If the heterologous benomyl resistant protein can not
incorporate in the microtubules in M999; to improve benomyl
resistance in transformed cells, then a homologous benomyl
resistance gene needs to be cloned. This homologous
resistance gene could come from a benomyl resistant Mm;
strain (through spontaneous mutation or through.mutagenesis),
or by genetic engineering of a wild type fi-tubulin gene from

Mucor.

SUMMARY
Our goals for this research project were to insert the
N; crassa Ben'l gene, a dominant selectable marker, into pLeu4,

to transform Mucor cells with this construct, and to test the

 

expression of this heterologous gene in M999; transformants.
The correct construction of pMBen1 and pMBen2 was confirmed
with restriction endonuclease analysis. The plasmids pMBen1
and pMBen2 were used to transform M; racemosus R7B. Southern
analysis of the genomic DNA of 4 transformants suggested that
the Benll gene was present in transformants and that there were
no obvious DNA rearrangements in the BenR gene. Although
unaltered pMBen plasmids were present in the transformants,
no significant increase in the resistance level to benomyl of
pMBen 'transformants was detected ‘when. compared. with. the
resistance level to benomyl of the host strain (without

pMBen). Further studies will focus on the transcription of

 

the benR gene in Mucor cells to determine if the heterologous

M; 9;assa promoter drives transcription in M; racemosus.

93

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