Indian Journal of Microbiology Vol 46, No. 4, December 2006, pp 307-324
Review
Fungi as cell factories: Hype, reality and hope
R. Maheshwari
Formerly, Department of Biochemistry, Indian Institute of Science, Bangalore 560012
The extant diversity of fungi and their intraspecies genetic variability offers the scope of finding useful strains producing
enzymes, antibiotics or metabolites of scientific or practical importance. Genetic and molecular techniques allow a gene from
one organism to be transferred to a safe domesticated fungal species and engineer precise changes in its regulatory DNA
sequences to increase expression of the transgene. Cultivation of fungus in industrial-sized fermentors facilitates a desired
substance to be produced for commercial use. A few species produce homologous proteins in significant amounts but the
expression of heterologous proteins of mammalian origin for practical use is generally low. Among the perceived uses of
fungi as cell factories is production of human antibodies. For increasing the secretion of a heterologous protein, an understanding
is required of its structure and of the regulatory genes controlling expression of the protein. Furthermore a detailed study of
the architecture of the fungal cell wall over period of mycelial growth is required since proteins finally exit through the cell
wall.
Keywords: Fungi, protein secretion, recombinant protein, antibody production, biotechnology.
Fungi have evolved as chemical factories,
secreting in their environment several classes of
enzymes which break down polymeric constituents
of dead organic matter into soluble forms for
absorption and utilization as sources of carbon and
energy. An adjunct to the success of their absorptive
mode of nutrition is the production of antagonistic
chemicals which enable fungi to survive amidst
microflora and microfauna comprising of competitors,
parasites and predators. For example, yeasts produce
alcohol - a toxic end-product of anaerobic metabolism
- not for human use but to arrest the fouling of their
“sugary habitats” by microbial competitors. The
diversity of fungi, estimated to comprise over 1.5
million species1, and the intraspecies genetic variation
offers a huge resource for finding a potentially useful
strain that produces a required enzyme for
biotransformation of a particular substrate or a
metabolite of value. Genetic and recombinant DNA
techniques allow a strain to be improved by specific
alterations to overexpress the transgene and the
product to be produced for commercial application.
This review will assess fungi as cell factories and
consider on anvil the issue of hype, reality and hope
from the perspective of fungal biology.
*e-mail: [email protected]
Tel: 91 80 23341045
Why fungi?
In biotechnology unicellular yeasts and multicellular
filamentous molds, rather than plant and animal cells,
offer greater possibilities for production of a desired
substance by fermentation - the term fermentation in
biotechnology refers to the process of growing
microorganisms in large-scale to produce a
biochemical product. The reasons for the selection of
fungi are:
1. Their adaptability to diverse environmental
conditions provides greater opportunity of finding
and selecting an appropriate strain for use.
2. Scaling-up of process is easier because of their
rapid growth rate. For example Saccharomyces
cerevisiae (budding yeast) has a doubling rate
(0.5 h) in liquid medium almost matching the
bacterium E. coli; and the molds Aspergillus
and Neurospora double their mass in about 2.22.7 hours at 25ºC. Thus a fungus-based process
is completed in a shorter time (usually one week
or less) compared to animal cells (usually weeks)
or plant cells (months).
3. Selection of exceptional high-producing variants
among hundreds or thousands of colonies is
facilitated by colour tests or replica plating on
308 Indian J Microbiol, December 2006
selective media. Special media also allow colonial
growth of some filamentous fungi for plate assays.
4. Transformation of cells is easier. In yeast, the
transforming DNA is integrated at homologous
site in the chromosome although it is commonly
ectopic in filamentous molds.
5. The large surface area of plasma membrane
provides for increased sites for protein secretion.
6. Expression cloning combines the advantageous
features of unicellular yeast and the multicellular
molds, facilitating construction of strains secreting
higher levels of a desired enzyme.
7. Post-translational processing (glycosylation,
phosphorylation or acetylation) allows active
heterologous proteins to be produced 2.
8. Large-scale cultivation in defined media
containing simple carbohydrate and nitrogen
compound eliminates unknown influence of
constituents of complex substrates.
9. Inducible synthesis of some proteins allows their
production with minimum background proteins.
10. Useful strains can be permanently preserved as
spores. The spores also serve as material for
mutagenesis for further strain improvement.
11. Spontaneous hyphal fusions between geneticallyrelated strains allow construction of heterokaryotic
mycelium for large-scale production of therapeutic
antibody.
Architectural and functional marvels of hypha
A fungal factory is comprised of longitudinally
joined hyphal compartments. Inside these
compartments, extending over considerable physical
distance, are longitudinal arrays of actin bundles that
act as tracks for the intracellular movement and
positioning of organelles. The plasma membrane has
transmembrane protein pumps for uptake of nutrients
inside against concentration gradients. The
compartments are replete with nuclei, endoplasmic
reticulum and Golgi which bud out thousands of
secretory vesicles every second for fusing with the
plasma membrane for exocytosis. The hypha is
polarized, i.e., it is spatially coordinated into an apical
region characterized by rapid uptake of ions
(phosphate, potassium and ammonium) and nutrients
(sugars and amino acids) and different sites for release
of proteins, toxins, antibiotics and pigments. The
hyphal extension is driven by hydrostatic (turgor)
pressure of 4-5 bar3. To contain this internal pressure,
the hypha is surrounded by a rigid wall, yet is elastic
enough for it to extend rapidly. It is selectively porous
to allow digestive enzymes to exit4,5, but at the same
time retain some molecules.
From the viewpoint of fungal based fermentation
process, a feature of hypha that strikes as of special
significance is the presence of septa (Fig.1). Culturing
aerobic fungi requires that air be bubbled through the
culture medium with constant vigorous stirring. This
subjects hyphae to shear; however, like rungs of a
ladder, septa provide strength to elongated hyphae
(generally 5-10 µm in diameter), limiting shearing and
reducing the ‘bleeding’ of protoplasm from any
severed ends. Not surprisingly, most fungi used in
fermentation are septate. However, the septum is
centrally perforated; the pore being minute,
approximately 0.5µm. The perforated septa allow aged
or dysfunctional organelles to be replaced by migration
of healthy (functional) organelles from other
compartments. It also allows aged organelles to be
partitioned into separate (distal) region of hypha for
recycling. With protoplasm rapidly moving forwards
or backwards (approximately at a rate of 4-6 cm per
hour) through septal pores, the entire mycelium
comprising of hyphae interlinked by short lateral
branches (bridges) is converted into one single
intercommunicating unit in which metabolic activities
are synchronized. The interconnection (cell fusion)
among hyphae allows related strains to be fused into
a single heterokaryotic mycelium containing genetically
distinct nuclei. Heterokaryotic mycelium proffer the
production of human antibody (described later).
The septal pores can be plugged by a
proteinaceous material called Woronin body, enabling
parts of a hypha or parts of mycelium to take up
specialized functions. For example, parts of single
hypha or portion of mycelium can be isolated and
their metabolism shifted to pathways for generating
and maintaining a reducing environment for housing
and functioning of redox-sensitive enzymes6 and/or
formation of special products by shunt metabolic
pathways.
Finally, a puzzling feature of fungi that merits
comment is the unique multinuclear condition of
Fungal cell factory 309
Fig. 1. Enlarged view of hyphae. The hypha is divided by perforated
transverse walls (septa). The compartments (cell) contain several haploid
nuclei.
molds. Although the role of nucleus in heredity is
well established, the supernumerary nuclei do not
contribute to phenotype7. Rather, since in nature fungi
grow under limiting conditions of nutrients;
supernumerary nuclei may be a repository of scarce
phosphorus and nitrogen in the protected form of
nucleotides in DNA. When nutrient availability is
limited, the supernumerary nuclei, partitioned into
separate hyphal compartments, can be degraded by
regulated autophagy 8 and DNA recycled by
phosphodiesterases9,10, making available phosphorus
and nitrogen for translocation to apical, metabolic
region for synthesis of membrane and organelles, and
the hypha to prolong its functional state.
Thus over the course of evolution hypha has been
shaped into a factory of great metabolic potential,
with fine coordination of its component compartments,
and minimization on expenditure of energy.
Exploiting genetic variation
RFLP or RAPD or protein polymororphism
analyses have revealed high degree of intraspecies
variation in fungal populations, providing for selection
of genetic variants for specific uses. A relevant
example is Trichoderma viride QM-9414 isolated
from moldy cotton fabrics in Solomon Islands. It was
selected as the best cellulase-producing strain and its
productivity improved by further mutagen
treatments11. A mutant named T. reesei (to honour
Elwyn T. Reese for his pioneering research on
enzymatic degradation of cellulose) secreted 30- 40
gram protein per liter culture medium, of which 60%
310 Indian J Microbiol, December 2006
is just one specific cellulase protein, namely
cellobiohydrolase12. This yield of enzyme protein has
not yet been matched for any protein by recombinant
DNA methodology – a pointer that classical methods
of selection and improvement of strain by random
mutagenesis remain the mainstay of biotechnology.
Although the genetic changes behind cellulase
overproduction by the mutant Trichoderma reesei
strain are not known, nonetheless this work has had
much impact: (1) It indicated the amount (or the
limit) of protein that a fungus is capable of producing/
secreting. (2) It provided a major impetus for microbial
culture collections, particularly for isolation and
recognition of some potent polysaccharide-degrading
enzymes13. (3) It galvanized research on bioconversion
of lignocellulose - “the most abundant biological
material on earth”. This was an impetus for research
on enzymatic bioconversion of lignocellulosic material
into glucose to produce fuel ethanol by yeast
fermentation14. (4) It led to identification of several
other sources of polymer-degrading enzymes, their
purification and investigations of mechanisms of their
action. (5) It recognized T. reesei as a potentially
efficient expression host for recombinant proteins and
stimulated research on production of foreign
(heterologous) proteins by fungal fermentation process.
Its genome has been sequenced (http://
www.genencor.com/wt/gcor/pr_1059584144) and,
hopefully, the reasons for the improved production of
cellulase will be discerned.
The ‘perfect host’ system
A fungal factory capitalizes on simple requirements
of raw materials - a carbohydrate as source of carbon
and energy, inorganic salts as sources of nitrogen,
phosphorus and sulphur, trace minerals as
micronutrients, and sometimes vitamins. These are
easily satisfied by commercial grade compounds such
as sucrose and urea, or materials available in bulk
such as sugar cane or beet molasses, corn steep
liquor, starch, soybean meal. Recombinant DNA
techniques allow regulatory properties of a strain to
be modified for overproduction (>10 -15 % of total
cellular protein) of a particular protein. Secretion is
important - the product can be recovered for
downstream processing by simple filtration. The
brewing industry is attempting to genetically engineer
yeast strains which will aggregate (flocculate) and
settle down, yielding bright beer and wine. Fungi have
been regarded as the “perfect hosts” for production
of proteins and useful metabolites15.
Fungal fermentation
Since the hypha is only a single cell thick, even
small changes in culture conditions influence its
physiology and affect the yield. J.W. Foster, a pioneer
investigator of fungal metabolism, cautioned16: “Indeed
it is a common event to have an organism produce no
detectable amount of a particular metabolic product,
and yet under different cultural conditions, produce
that very substance abundantly. Finally, there is the
situation in which, on the one hand, one kind of product
is produced, and, on the other hand, a totally different
product.” Different trials in the same laboratory can
affect reproducibility of results. For example, in a
pioneering study of penicillin production by Penicillium
notatum, Backus and Stauffer17 found antibiotic yield
by surface-grown cultures to be markedly affected
by factors of which the worker may be ignorant of,
such as the “tightness of cotton plug” in the culture
flasks/bottles (implying aeration). In 1960, E. W.
Buxton18 sounded a warning that fungi are “a mutable
and treacherous tribe”. This allegation could have
been due to lack of strict control of physical and
chemical conditions of growth rather than their
inherent finicky behaviour. Fungi are increasingly
grown in stainless-steel vessels fitted with ancillary
machinery for in situ sterilization of culture media;
for adding inoculum and antifoaming agents; for adding
nutrients at desired rate and time; for supply of sterile
air; exhaust for carbon dioxide; stirrer for good-mixing;
constant measurement and control of pH, dissolved
oxygen concentration, of temperature, and for
intermittent removal of samples for monitoring process,
in hygienic and aesthetically pleasing surroundings
(Fig. 2).
The production of many enzymes is regulated by
temperature, dissolved oxygen or/and pH. As an
example, by maintaining pH at 6, recombinant
glucoamylase production was enhanced over 10-fold
compared to that without pH control19. A system for
the regulation of gene expression by ambient
(extracellular) pH has been identified. This system
consists of the products of the pacC and palA, B, C,
F, H, and I genes. While pacC encodes a zinc finger
Fungal cell factory 311
Fig. 2. Fermentor and auxiliary equipment for online control of process parameters. Photo
courtesy of Bioengineering /Spinco.
transcription factor, the pal genes encode components
of an ambient pH signal transduction pathway20. A
lesson from penicillin fermentation was that secretion
of a product is neither related to growth rate nor the
biomass yield: lactose-a poor carbon source- gave
higher titers of penicillin. The production of
recombinant glucoamylase by cultures of Aspergillus
niger was increased by continuous dilution of medium
with mineral salt medium and slow addition of peptone
and glucose21. Fermentation processes are becoming
increasingly sophisticated by programmed profiling of
medium addition/dilution, temperature, and pH and
dissolved oxygen to manipulate growth rate and
biomass formation, release enzyme and inactivate
protease22.
Impact of recombinant DNA technology
The small genomic size of fungi - approximately
10-40 Mb - has speeded up genome
sequencing23,24,25,26 in the expectation that genes will
be manipulated in systematic manner to create
superior alleles for improved yields (https://
fungalgenomics.concordia.ca/fungi/Anig.php). The
genetic material in both yeast and molds can be
modified by random or site-directed methods; mutants
can be isolated; genes can be cloned through their
ability to complement mutant phenotypes; and an
endogenous gene replaced with an engineered
derivative. Shuttle plasmid vectors were constructed
which can replicate both in E. coli, yeast or molds,
enabling genes cloned in a bacterium or yeast to be
returned for its high-level expression in a filamentous
fungus and secretion in greater quantity and higher
purity (see below).
Expression cloning
Expression cloning simplifies the time-consuming
process of purifying desired enzyme from the mixture
as enzyme is over-produced by an efficient strain of
filamentous fungus. Often concentration of culture
filtrates by simple freeze-drying may suffice practical
needs. This method combines the advantageous
features of unicellular yeast and multicellular,
filamentous mold, i.e., the ability of yeast to express
312 Indian J Microbiol, December 2006
heterologous fungal cDNA, its ability to be grown as
colonies on screening plates for visual detection of
enzyme activity27, 28 and the gene from a selected
clone to be expressed in mold for higher secreted
amount of enzyme. As an example, pectinases have
industrial applications for clarification of fruit juices
that contain highly methyl-esterified pectin. E. coli
shuttle vector was used to transform yeast as
intermediate host and the secreting colonies visually
identified by replica plating27. The gene encoding
enzyme from a selected yeast colony (donor) was
subcloned and transferred into a safe, filamentous
fungus for large-scale production (secretion) of
enzyme. The nonhomologous pectin methyl esterase
together with polygalacturonases caused a rapid
depolymerzation of pectin29.The steps in the expression
cloning are given in scheme shown in Fig. 3.
Examples of non-homologous protein production
The examples below illustrate how molds began
to be used for production of foreign proteins.
Chymosin
According to a legend, an Arab nomad with a
saddlebag of milk to sustain him on a journey was
mRNA
↓
cDNA library in E. coli
↓
50 pools (5000 transformants/pool)
↓
Transformation of yeast (25,000/pool) using a shuttle vector
↓
Screening sub-libraries (200 plates with 500 colonies)
↓
Rescreening of positive clones
↓
Isolation of DNA
↓
Transformation of E. coli
↓
Characterization of DNA by nucleotide sequencing for presence of cloned gene
↓
Cloning of gene in a filamentous fungus secreting protein in high amounts
Fig. 3. Scheme for expression cloning.
Fungal cell factory 313
crossing desert riding a horse. After several hours
when he stopped to quench his thirst, he found that
the milk had separated into a pale watery liquid and
solid white lump (cheese). Taking cue from this
legend, the ancient Romans recognized a link between
saddlebag made from the stomach of a suckling calf
and the transformation of milk into cheese. Therefore,
for making cheese, an enzyme preparation called
rennet, made from the lining of the stomach of suckling
calves began to be added. It is now known to contain
a specific protease enzyme called chymosin (rennin)
which breaks protein casein in milk, causing casein to
clump into a solid gel. Protests by animal rights
activists that it is inhumane to kill newly born calves
and shortages of obtaining calf rennet led to search
for substitutes. The Japanese microbiologists perceived
that since microorganisms secrete a variety of digestive
enzymes, some microorganism-derived enzyme could
be used as a substitute. From the isolation and
screening of several hundred soil microorganisms, a
thermophilic fungus, Mucor pusillus, was obtained
which had high milk-clotting activity30. Genetic
engineering was used to transfer the gene to a
historically safe fungus. Since koji mold (Fig. 4) mostly A. oryzae and A. sojae but may include A.
awamori and A. kawachii (black koji-mold) -has
long been used in Japan for production of fermented
food and beverages, and information on its cultivation
methods is available, it is presently the most widely
used filamentous fungus. A recombinant A. oryzae
strain was constructed in which the chymosin gene
was placed under the control of promoter of
glucoamylase, a well-secreted protein. The
recombinant host strain produced heterologous Mucor
(Rhizomucor) miehei protease in excess of 3 g/liter31.
This yield compares favourably with that of
recombinant proteins in milk produced by transgenic
animals (2-10 g/liter) (http://www.gtc-bio.com/science/
questions.html). Chymosin identical to calf rennet is
being produced commercially by yeast or filamentous
fungi by transformation with a plasmid containing an
artificially synthesized chymosin gene. This pure form
of “vegetarian cheese” or Chy-Max®, was the first
product of recombinant DNA technology in the U.S.
food supply. This illustrates that chance observations
often form the basis of exploitation of fungi in
industries. For example, the antibiotic industry is based
on chance discovery of penicillin produced by
Penicillium notatum in contaminated plate of bacterial
culture. Recently, the fungus Aspergillus awamori
Fig. 4. Koji mold, Aspergillus oryzae. Left, mycelium growing on a grain of steamed rice. Photo
courtesy of John Gauntner. Right, colonies growing on agar medium in a Petri dish. The colonies are
sporulating at the center. Photo courtesy of Novozymes Inc.
314 Indian J Microbiol, December 2006
has been used to produce thaumatin - a protein in the
katemfe fruit (Thaumatococcus daniellii) growing
in West Africa. This protein which on weight basis is
3000 times sweeter than sucrose has been expressed
in Pichia pastoris with a yield of 5-7 mg/liter –
higher than in the transgenic plants32.
Lipase and protease
The original idea of using enzymes in detergents
was described in 1913 by the chemist Otto Rohm,
who along with Otto Haas, founded Rohm and Haas
-one of the world’s largest specialty chemical
producing company. Rohm patented the use of crude
pancreatic extracts in laundry pre-soak composition
to improve the removal of greasy food stains
(www.novozymes.com/cgi-bin/bvisapi.dll/biotimes/
one_article.jsp?id=11394&lang=en - 41k -). In 1994,
Novozyme Inc. launched the first recombinant lipase,
LipolaseTM, obtained by cloning lipase gene from a
thermophilic fungus Thermomyces lanuginosus into
Aspergillus oryzae wherein it produced 1000-fold
more enzyme protein. It was used in formulation of
detergents for hot-water machine wash for garments
and dishes and is highly effective in removing oil
stain. Several companies manufacture lipase fortifiedhousehold detergents. Similarly alkaline proteases are
increasingly used as additives in detergents for removal
of blood stains. The search is now for psychrophilic
fungi as sources of lipases and proteases for use in
detergent formulations for cold water wash.
Lactoferrin
Lactoferrin, an iron-binding glycoprotein present
in human milk plays protective role against microbial
and viral infection. Expression of human lactoferrin
(hLF), a 78 kD glycoprotein, was achieved by placing
the cDNA under the control of the A. oryzae áamylase promoter33. Using this system, hLF is
expressed and secreted into the growth medium at
levels up to 25 mg/l. Subsequently a modification of
this production system combined with a classical strain
improvement program enabled production of
recombinant hLF in excess of 2 g/l in Aspergillus
awamori as a glucoamylase fusion polypeptide which
was secreted into the growth medium and processed
to mature hLF by an endogenous KEX-2 peptidase
34
. The recombinant lactoferrin was indistinguishable
from human milk lactoferrin with respect to its size,
immunoreactivity, and iron-binding capacity. It retained
full biological activity in terms of its ability to bind
iron and human enterocyte receptors. The recombinant
protein functioned as a potent broad spectrum
antimicrobial protein. Lactoferrin is the largest
heterologous protein and the first mammalian
glycoprotein expressed in the Aspergillus system.
Miscellaneous
During 1970s, Phillips Petroleum developed
Pichia pastoris as a source of single cell protein as
it can be grown economically in large scale in a
completely defined growth medium containing
methanol as a sole carbon source. P. pastoris has
emerged as an alternative expression host for
production of vaccines against bacterial toxins35. This
yeast ferments glucose and related sugars even in
the presence of air (absence of Crabtree effect) and
hence it is termed as non-conventional yeast. It has a
strong, methanol-induced alcohol oxidase promoter
(AOX1). The alcohol oxidase -the enzyme which
catalyzes the fist step in the metabolism of methanolcan constitute as much as 35% of the soluble protein
in the cell. The promoter region of the gene encoding
this enzyme is being used in gene-fusion approach to
express foreign genes with yields from milligram
to gram quantity of protein 35,36,37 . The posttranslational glycosylation is similar to that in
mammalian-proteins. It is being advocated for
heterologous protein secretion35. The production of
high (14.8 g/liter) amount of animal protein (gelatin)
has led many companies to adopt the Pichia
expression system for production of heterologous
proteins2.
Seeds constitute the main diet of poultry and pigs.
There is therefore much interest in improvement of
animal feed using phytase. Supplementation of feed
with phytase increases availability of phosphorus in
the seed which store it chiefly as phytic acid (myoinositol hexakisphosphate). The E. coli phytase gene
is reportedly ‘highly’ expressed in Pichia pastoris
under the control of AOX1 promoter. Replacement
of culture medium with fresh medium to remove
repressing glycerol and metabolic wastes prior to
methanol induction gave the highest level phytase
expression38.
Fungal cell factory 315
The most recently developed method of
manufacturing human insulin uses recombinant DNA
technology with baker’s yeast as the host cell, offering
potentially limitless supplies of insulin structurally
identical to that made by the human pancreas. The
above examples show that not only gene products
from fungi but also from quite unrelated organisms
have been successfully produced in fungi. However,
yields of heterologous proteins by fungi (Table 1) are,
for unknown reasons, several orders of magnitude
lower than of homologous proteins even when the
same expression signals were used.
Improving yield
Increase in copy number of genes
Several approaches are being tried to obtain
reproducible higher yields. The results of inserting
extra copies of the desired gene have been
disappointing. For example, Mellon and Casselton52
(1988) found that as many as eight copies of the
gene encoding isocitrate lyase gave only 25% activity
compared to the wild type Coprinus cinereus.
Analysis of Aspergillus niger transformed with
glaA gene also showed no correlation between the
Table 1. Yields of some proteins and metabolites from fungi.
Product/Source
Production (host) fungus
Yield
(per liter)
Reference
no
Aspergillus niger
1.3 g
39
Trichoderma reesei
30-40 g
12
Glucoamylase (Ho)
Aspergillus niger
20 g
15
Lipase (He)
Thermomyces lanuginosus
Aspergillus oryzae
NA
www.novozymes.com
Aspergillus oryzae
NA
40
Acremonium
chrysogenum
4g
41
Aspergillus oryzae
20 mg
42
Aspergillus oryzae
5 mg
43
Aspergillus niger
100 mg
44
Lactoferrin (He)
Human
Aspergillus niger
25 mg
45
Interleukin-6 (He)
Human
Aspergillus niger
150 mg
46
Aspergillus nidulans
4.8 mg
47
Pichia pastoris
~ 3 mg
48
Human Insulin
Pichia pastoris
1.5 g
49
Citric acid
Aspergillus niger
130-150 g
50
Penicillin
Penicillium chrysogenum
40-50 g
51
Acremonium chrysogenum
20-25 g
51
Calf chymosin (He)
Cellulase (Ho)
Rhizomucor miehei
Protease (alkaline) (He)
Fusarium
Protease (acid) (He)
Mucor pusillus
Mn Peroxidase (He)
Phanerochaete chrysosporium
Aspergillus niger (He)
Human Chorionic
Gonadotropin (He)
Cephalosporin
Abbreviation: Ho, homologous production; He, heterologous production, NA, data not available.
316 Indian J Microbiol, December 2006
copy number of transforming gene and its level of
glucoamylase 53. Since journals are reluctant to
publish negative results, one must suppose that
failures to increase yields by increasing gene dosage
have outnumbered successes. In view of the
discovery of gene-silencing processes in fungi such
as “RIP, repeat-induced point mutation” 54 and
“quelling”55 it is now understood why overexpression
of a gene product by increasing copy number has
failed, as in the plants.
Cultural manipulation of morphology
The few cases where this has been studied show
that morphology can vary from one product to another.
The gross colony morphology of filamentous fungi in
submerged cultures is broadly of three types: freely
dispersed hyphae, pellets of densely interwoven
hyphae, or clumped mycelia, with zero concentration
of oxygen in center 56, 57. A fungus can have all three
different morphologies but the form most suitable
imposed by cultural conditions for secretion of a
particular protein is seldom reported. The filamentous
form of A. niger was better for pectic enzyme
synthesis, whereas the pellet form was optimum for
citric acid production58. A deficiency of manganese
leads to loss of growth polarity and formation of
bulbous hyphae and increased citric acid production.
Pellet form was also required for penicillin production
by Penicillium chrysogenum. The mode of aeration
can significantly affect morphology and the yield. In
Aspergillus terreus, large fluffy pellet form obtained
with supply of oxygen-enriched gas, but not with air,
produced higher titers of lovastatin, a cholesterol
lowering drug, than freely dispersed form59. A cross
section of a 2 mm pellet of Penicillium chrysogenum
showed differentiation of hyphae with cytoplasm-rich
outer layer, partially lysed middle layers and
disintegrated hyphae in the center60, indicating that
the region of synthesis and release of product may
be quite different. One of the more important
parameters influencing culture morphology is aeration.
For example, Trichophyton rubrum (a wooddegrading fungus) had different morphologies
depending on its cultivation in baffled or unbaffled
flasks. The pellet size in baffled flask was small and
yield of MnP ligninase higher61. In Phanerochaete
chrysosporium the ligninolytic activity (manganese
peroxidase) was produced during differentiation of
spore62. These cells disappeared, coinciding with the
time of enzyme secretion, suggesting that the
differentiated cells acted as enzyme reservoir releasing
their contents by autolysis process. Development of
spores requires the presence of new enzymes for a
limited period of time. Optimal productivity of
secondary metabolites is dependent on a specific
morphology, for example Penicillium urticae produced
the antibiotics patulin and griseofulvin following
conidiation63. Physical and chemical conditions of
culture affect the expression of regulatory genes,
affecting gross and microscopic morphology and the
yield of product.
Hyperbranching mutants
Surprisingly, though filamentous fungi are known for
their protein secreting abilities, the region of the hypha
that secretes proteins is contentious. Wösten et al.64
found that in A. niger, protein synthesis occurs
throughout growing hypha but glucoamylase is secreted
only from the growing apical region of the hypha
where nascent wall is laid down and is therefore
porous. However, apical secretion may not be because
the hyphal tip walls are relatively porous, but due to
polarized nature of the hypha directing secretion
vectorially at the tip. Nonetheless, this implies increased
apical surface and continuous growth due to enhanced
hyphal branching may increase productivity of strains.
The mcb mutant of Neurospora crassa shows loss
of growth polarity at 37 ºC with swollen hyphal tips,
i.e. large increase in growing-surface area65. The
effect is due to mutation in the gene encoding
regulatory subunit of protein kinase. The mutant
secreted 3-5-fold more extracellular proteins and a
20-fold increased level of carboxymethylcellulase
relative to wild type. Another approach of obtaining
higher protein yield may be through mutants with
increased branching intensity. Hyperbranching mutants
of Aspergillus oryzae produced higher amylase and
protease on solid substrates66. The above results
suggest that yields of extracellular protein by a
filamentous fungus can be significantly increased by
selecting strains with desirable morphologies for
fermentation67.
Modification of cell wall
Why some proteins are secreted better than
others, and why some strains secrete a protein better
Fungal cell factory 317
than others? Trevithick and Metzenberg68 found
that if the quaternary structure of invertase of
Neurospora is of a size that it can pass through the
pores (40-70 Å) in the multi-layered network of cell
wall, it was secreted externally as a light form. The
large form of enzyme remained in the periplasm or
was bound to cell wall. This observation is of unusual
significance for it suggests that structural features of
the protein and/or the cell wall are important
determinants of level of secreted protein. Cellular
regulatory mechanisms avoid wasteful synthesis of a
protein that is unable to exit because of unfavourable
pore size of the cell wall. Structural changes in mycelia
with altered physical and chemical properties can
occur in response to carbon sources69 affecting
secretion. One example is α-glucosidase in the
thermophilic, cellulolytic fungus Sporotrichum
thermophile. Culture morphology of this fungus was
strikingly different when grown with cellulose (filter
paper) or its depolymerized form (cellobiose) as carbon
source. In cellulose-medium the mycelium autolysed
releasing the cell-wall-bound β-glucosidase whereas
in cellobiose-medium the hyphae remained healthy in
agglomerated state and β-glucosidase was not
released into the medium70. That hyphal morphology
is important in secretion is also emphasized by
observations in a temperature-controlled morphological
mutant of N. crassa. Increase in number of hyphal
tips per hypha in secreted more cellulase relative to a
wild-type strain65. The amount of protein secreted
was seven-fold more when initial growth at 18 ºC
was followed by growth at 37 ºC. In this mutant the
site of secretion was not limited to the tip. Besides
genetic factors, chemical environment of growth
greatly affects thickness, chemical composition and
structure of wall resulting in phenotypic change in the
colony morphology71, 72. The results emphasize that
structure of hyphal cell-wall is one of the important
factors determining the amount of protein secreted.
New expression hosts
The fungi used currently do not fulfill all of the
requirements, i.e., the production of enzymes stable
at broad pH and temperature range, culture
morphology giving non-viscous growth thereby
reducing energy cost in operation in large-scale
fermentation, free of undesirable pigment or protease
enzymes. From the screening of more than 100 fungi,
Novozymes Inc. (www.novozymes.com) selected a
filamentous fungus Fusarium venenatum as a new
expression host which has the advantages of low
secreted protease levels, low total spectrum of
secreted protein, high level of heterologous expression,
‘favourable’ fermentation morphology and is
‘Generally Regarded As Safe’ (GRAS, i.e., no skin
or respiratory allergy). Using F. venenatum the first
microbe-produced recombinant alternative to animal
trypsin was commercialized in November 2002. It
has better stability than animal-derived trypsin. Dyadic
International, Inc. has patented a novel-gene
expression system based on a filamentous fungus
Chrysosporium lucknowense that was isolated from
alkaline soils in far-east Russia. This fungus grows at
a broader range of pH from 4.5 to 9.0, compatible
with stability of secreted proteins73. UV and N-methylN’-nitro-N-nitrosoguanidine treatment yielded
protease-deficient mutants which formed dispersed
fragmented mycelia and produced 200-fold more
neutral cellulase with application in softening denim
used in manufacture of jeans
Molecular manipulations
As proteins are significant constituents of all
organic matter, fungi secrete proteases to utilize it.
Not surprisingly, proteolytic destruction of enzyme is
the single most important factor in low enzyme levels
in the culture medium. By comparing the mRNA
levels of a number of heterologous genes integrated
in a single copy at a single site with the secreted
levels of proteins, it was found that higher protein
yields may be obtained through development of
protease-deficient strains74, 75. In efforts to understand
reasons of low yield, the authors compared protein
levels of a number of heterologous genes integrated
in a single copy at a defined locus, controlled by the
expression signals of the host Aspergillus awamori
endoxylanase gene. The results of mRNA analyses
showed that mRNA stability is partly a reason for
the low or undetectable protein levels. Other strategies
are improvement of the mRNA stability by fusion
with highly expressed genes 76; improvement in
translation efficiency by construction of a synthetic
gene with codon usage optimized for species;
overproduction of protein disulphide isomerase77, 78,
of chaperone79; and the use of protease-deficient
strain so that the protein yield is not reduced 80, 81, 82.
318 Indian J Microbiol, December 2006
However, despite this knowledge, success has been
limited. For example, lignin peroxidase of
Phanerochaete chrysosporium could not be detected
when fused to cellobiohydrolase-encoding cbh1 gene
promoter of Trichoderma reesei 83; neither was
ligninolytic activity (MnP) overproduced when cDNA
encoding it was fused to the well-secreted
glucoamylase in a protease-deficient strain of
Aspergillus niger 44 . The results suggest that
secretion of fusion protein is a complex process.
Since the ability to secrete protein hydrolyzing an
available substrate is essential for growth, use was
made of the yeast temperature-sensitive mutants to
identify steps in secretion. In these mutants a
secretory step is normal at lower temperature but
blocked only at a higher temperature. Using the
temperature-sensitive mutants of yeast and invertase
as a model secretory proteins the pathway (Fig. 5)
indicated was84,85,: ER→ Berkeley bodies→Golgi
→Secretory vesicles→Cell surface, i.e., after being
translated in the ribosome, the protein is transported
into the endoplasmic reticulum, where its subunits are
assembled by correct disulphide bonds and the protein
molecule correctly folded with the help of folding
enzymes and chaperones85, and other modifications
such as disulphide bond formation and
glycosylation86,87, take place catalyzed by an array of
proteins. Following quality control checks the protein
is transported into the Golgi and finally in carrier
vesicles to the plasma membrane for fusion with
plasma membrane and release into the periplasm
between the plasma membrane and cell wall, the
latter acting as a sieve allowing proteins of a particular
size to pass through depending on its porosity. The
low protein secretion can be due to defect in any of
the steps. At least 23 genes with roles in secretory
pathway have been identified in the yeast and a similar
pathway is assumed in filamentous hypha. From the
yeast genome sequence, about 20% of genes control
functions that are related to cell wall biogenesis88,
indicating that remodeling cell wall structure for
enhancing protein secretion will be not be easy; it
might even make the organism vulnerable to osmotic
bursting due to lack or altered cross-linking of wall
polymers89.
Interacting factors
Besides genetic factors, morphology of the fungus
depends on the concentration of inoculum, composition
of medium, the design of cultivation vessel (fermentor),
Fig. 5. Diagram of main events in the secretory path of protein. Abbreviations: N, nucleus; ER, endoplasmic reticulum;
VAC, vacuole; G, Golgi ; SV, secretory vesicle. Fungi do not have stacked Golgi cisternae. Adapted from Conesa et al.
(2001).
Fungal cell factory 319
although carried out by a different type of mycelium
formed late in culture. A new finding is that of mycelial
differentiation — of exploring hyphae penetrating into
the substrate and of branching hyphae growing on
surface — opening a new field of control of hyphal
type in industrial fermentations92 (Vinck et al. 2005).
When interacting factors in growth and differentiation
are better understood, a system can be set up for
best productivity, perhaps by slow feeding of nutrients,
overlapping trophophase and idiophase.
Human antibody production by fungi
Fig. 6. Growth forms of steroid- transforming mold Rhizopus
nigricans. Fungus was grown in 100 ml medium/500 ml flask
under different submerged cultivation conditions. Up, left: T=23
C, N=225 rpm, Inoculum= 103 spores/liter. Up, right; T= 19 C,
N=150 rpm, Inoculum = 8 X 104 spores/liter; Bottom, left: T=23
C, N=100 rpm, Inoculum= 103 spores/liter; Bottom, right: T= 23
C, N=225 rpm, Inoculum = 107 spores/liter. Photo courtesy of
Dr. Polona Znidarsic-Plazl (Food Technol. Biotechnol. 39: 237252, 2001)
addition of surfactants, mode of aeration (shearing
forces), etc. In Rhizopus nigricans (Fig. 6) low
inoculum resulted in small pellets which gave higher
steroid transformation activity (progesterone 11α
hydroxylation)57. In small pellets the ratio of growing
hyphal length and inactive/dead hyphae is much higher.
In concluding this section, we note that secretion of
proteins is correlated with the period active growth
of fungus (trophophase) or its release from autolysing
cells (idiophase)57. Earlier, it had been thought that
ligninolytic enzyme production occurs after primary
growth has ceased in liquid-grown cultures due to
nutrient limitation90 (Jeffries et al. 1981). However,
Moukha et al. (1993) obtained different results with
agar-surface-grown fungus sandwiched between two
perforated membranes91. Though the radial growth
of the fungal colony had stopped, new short branches
were initiated at the colony centre which secreted
Mn2+- dependent lignin peroxidase, suggesting that
ligninase is produced by a specialized type of hyphae
that develop after much of the assimilable carbon
source has been consumed. In surface-grown cultures,
this period coincided with accumulation of RNA
transcripts and secretion of ligninase. Therefore lignin
degradation is hyphal growth-associated process
Currently mammalian cell cultures are used for
production of therapeutic proteins for inactivating or
sequestering specific host proteins. These require
expensive media of undefined composition for
cultivation. Moreover, the average yield of antibody
from hybridoma cells is about 100 milligram /liter.
The major limitation in the therapeutic use of antibodies
is producing a useful antibody in quantities required
for clinical trials and use 93 (Nyssönen et al. 1993).
Novozymes (USA), Genencor (USA) and DSM (The
Netherlands) are using the yeast expression systems
for producing large quantities of human-like antibody
with addition of human-like N-glycan structures for
therapeutic use is by a blending of fungal genetic
techniques, recombinant DNA, and monoclonal
antibody 94. Single chain antibody fragments have
been produced in Aspergillus and Trichoderma as
proteins fused to Trichoderma cellulase cbh1
promoter 95. Although fungi possess cell wall, hyphae
of two related strains can fuse through short lateral
branches to form a multinucleate mycelium containing
a mixture of nuclei in a common cytoplasm
(heterokaryon) without involving nuclear fusion. An
exciting idea, based on fundamental knowledge being
pursued by Neugenesis Corporation 96, 97 is to
construct two separate vectors of light and heavy
antibody chains as fusion proteins with a well-secreted
protein (such as glucoamylase or cellobiohydrolase).
A specific amino acid sequence is engineered between
the secretory enzyme and the antibody chain in order
that the secretory enzyme is clipped off by a host
protease during the secretory process in the Golgi.
These vectors are subsequently used to transform
auxotrophic strains of N. crassa. The two transformed
strains, i.e., one producing the light chain and the
other producing the heavy chain (Fig 7) are fused to
320 Indian J Microbiol, December 2006
Heavy
chain
Light
chain
Heterokaryon
formation
Auxotroph A
Auxotroph B
Fusion
Fermentation
Heterokaryotic mycelium
Whole antibody
Fig. 7. A scheme for production of humanized antibody by heterokaryotic mycelium. Germinating spores of two auxotrophic strains
(A and B) fuse to produce a heterokaryotic mycelium which can grow on a minimal medium lacking supplements because of
complementation of non-allelic mutant genes. The mycelium secretes the heterologous whole antibody molecule. Based on a figure
kindly provided by Dr. W. Dorsey Stuart (Novozyme-Neugenesis, Davis).
form a heterokaryon which produces both the antibody
subunits and processes them into the intact monoclonal
antibody molecules in synthetic medium of defined
composition. As the information is classified, details
are not available,
Future researches
“Each species has evolved to become a unique
chemical factory producing substances in an
unforgiving world.” This quote from E. O. Wilson98
is also true for fungi. However, the list of useful
fungal products is small, and the list of exploited fungi
even smaller - out of approximately 70,000
documented species of fungi, only a mere handful is
exploited in industry despite the demonstration that
fungi are sources of metabolites with antimicrobial,
antidiabetic or anticancer properties99. One of the
reasons is that isolation of fungi, their axenic culturing
and investigations of their physiology and biochemistry
is not fashionable science anymore. An alternative
proposed in lieu of pure cultures of microorganisms is
the direct extraction and cloning of DNA, obtained
from unidentified mixtures (consortia) of microbial
communities taken from soil, seawater, insect guts,
etc. 100, forming a metagenomic library, and finally its
expression in E. coli 101. However, it is premature to
infer general applicability of this approach to fungi. A
coherent strategy of white biotechnology (i.e., the
high-tech technology of using microorganisms by
white-coated scientists and technicians) should
combine traditional methods of new fungal hosts for
microbial factories, and the development of suitable
transformation protocols to accommodate the large
gene clusters that are involved in secondary metabolite
biosynthesis for a new species. Obtaining significant
levels (greater than 10-15 percent of total protein) of
heterologous protein requires fundamental researches
— a comprehensive understanding of the signals for
gene expression, strategies for adding, modifying or
deleting regulatory genes; of the structure of gene
product and its intracellular localization and
interactions. Ultimately, since secretory proteins must
exit through the cell wall, investigations of composition
and three-dimensional structure of cell wall 102 and
methods of manipulating its dynamic structure are
important for improving the fungus’ secretion potential.
Fungal cell factory 321
Acknowledgments
15. van Brunt J (1986) Fungi: the perfect hosts? Biotechnology
4: 1057-1063.
I thank the following for granting permission to reproduce
illustrations: John Gauntner (Sake World, Inc), W. Dorsey Stuart
(Neugenesis Corporation), Polona Znidarsic-Plazl (University of
Ljublanca), Bioengineering AG (Switzerland) and Novozymes
Biotech Inc. (Denmark). Masayuki Machida (Ibaraki, Japan)
advised on scientific nomenclature of koji mold, Manjuli
Maheshwari edited the text and P. V. Balasubramanyam helped
in graphics.
16. Foster JW (1947) Some introspections on mold metabolism.
Bact Rev 11: 167-188.
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