Journal of Biotechnology 66 (1998) 101 – 107

Review article
Mycotechnology: the role of fungi in biotechnology1

J.W. Bennett *
Department of Cell and Molecular Biology, Tulane Uni6ersity, New Orleans, LA 70118, USA

Received 24 February 1998; accepted 8 April 1998

Abstract

Fungi have been important in both ancient and modern biotechnological processes. Processes and products that
utilize fungi include baking, brewing, and the production of antibiotics, alcohols, enzymes, organic acids, and
numerous pharmaceuticals. The advent of recombinant DNA technology and large scale genomics analysis has placed
yeasts and filamentous fungi in the forefront of contemporary commercial applications. The term ‘mycotechnology’
is introduced here to describe the enormous impact of fungi on biotechnology. © 1998 Elsevier Science B.V. All rights
reserved.

Keywords: Fungi; Biotechnological processes; Recombinant DNA technology; Genomics analysis; Mycotechnology

1. Introduction and definitions back into their cells. Diverse in morphology,

physiology and ecology, many of the best known
Fungi are lower eukaryotes classified by mod- fungi have a negative impact on human welfare as
ern biologists into their own kingdom, sometimes agents of plant disease (e.g. smuts, blights, wilts,
called ‘The Fifth Kingdom’ based on their ab- rusts), biodeterioration (rots and mildews) or as
animal pathogens (e.g. as producers of toxins and
sorptive mode of nutrition. Fungi secrete a wide
mycoses). Fungi range from microscopic molds
range of powerful enzymes into the environment
and yeasts to macroscopic mushrooms and
and then absorb these ‘pre-digested’ foodstuffs
truffles. Several species of macrofungi are consid-
ered delicacies and are gathered or cultivated for
* Corresponding author. Fax: +1 504 8656785; e-mail: the human food supply. However, it is the micro-
jbennett@mailhost.tcs.tulane.edu
1
Based on a lecture held at the symposium, ‘Progress in US
scopic species, which include genera such as As-
Biotechnology’, at the 8th European Congress on Biotechnol- pergillus, Penicillium and Saccharomyces, which
ogy (ECB8) in Budapest, Hungary, August 1997. are best known for their positive impact on hu-

0168-1656/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved.
PII S0168-1656(98)00133-3
102 J.W. Bennett / Journal of Biotechnology 66 (1998) 101–107

man affairs. They have been harnessed for their placed direct representation; in architecture func-
metabolic activities, either as producers of degra- tionalism replaced ornamentation; in literature,
dative enzymes or synthesizers of useful metabo- new stylistic forms replaced traditional narrative.
lites. These fungi form an important cornerstone In everyday life, the extensive application of the
of modern biotechnology. discoveries of basic science revolutionized the way
Biotechnology has been defined in many ways. people lived. The adjectives ‘premodern’, ‘mod-
The Spinks Commission in the United Kingdom ern’, and ‘postmodern’ are used here as a descrip-
was one the first groups to adopt a formal defini- tive device to survey the enormous number of
tion: ‘‘Biotechnology is the application of biologi- process and products encompassed under the ru-
cal organisms, systems, or processes to bric of fungal biotechnology.
manufacturing and service industries’’. The Eu- Beer, wine, bread, koji, and other fermented
ropean Federation of Biotechnology uses a similar foods and beverages have been part of the human
but somewhat broader definition: ‘‘…the inte- diet for millenia. Their origins are lost in antiquity
grated use of biochemistry, microbiology and en- (Table 1). It is clear from the historical record,
gineering sciences in order to achieve however, that people indirectly knew of microor-
technological (industrial) application of the capa- ganisms such as molds and yeasts by their activi-
bilities of micro-organisms, cultured tissue cells, ties. Scientific study of these activities, aided by
and parts thereof’’. The definition used by both the microscope, led to the disciplines now called
the US National Institutes of Health and Food microbiology and biochemistry. For example, af-
and Drug Administration is wordier still: ‘‘Bio- ter Pasteur observed that living organisms could
technology is the application of biological systems always be seen under the microscope during sugar
and organisms to technical and industrial pro- fermentations, the organisms producing them—
cesses. The technologies employed in this area mostly yeasts—were called ‘organized ferments’.
include: classic genetic selection and/or breeding The changes which occurred in solutions without
for the direct in vitro modification of genetic
any detectable microorganisms were called ‘unor-
material, e.g. recombinant DNA, or gene splicing,
ganized ferments’. Later, when it became appar-
and other novel techniques for modifying genetic
ent that the ‘unorganized ferments’ were generally
material of living organisms, e.g. cell fusion, and
the metabolic products of ‘organized ferments’,
hybridoma technology, etc.’’ For a compilation of
Kuhne suggested the new word: ‘enzyme’ (en= in,
these and other formal definitions of biotechnol-
ogy see Bennett et al. (1997). Table 1
To recapitulate, most definitions of biotechnol- ‘Premodern’ examples of mycotechnology
ogy are comprehensive and encompass fermenta-
tion processes from wine to penicillin, as well as a Process Organism
broad spectrum of contemporary methodologies
Asian food fermentations
that grow out of recombinant DNA technology. I Ang-kak Monascus purpurea
use the coined term ‘mycotechnology’ to include Miso Aspergillus oryzae
the many biotechnological processes, both old Ontjam Neurospora crassa
and new, that rely on fungal products and pro- Soy sauce Aspergillus oryzae, A. sojae
Tempeh Rhizopus ni6eus
cesses. This survey will emphasize filamentous
Brewing and bak- Saccharomyces cere6iseae, S. carlber-
fungi. ing gensis
Mold-ripened Penicillium roqueforti, P. camembetii
cheeses
2. ‘Pre-modern’ mycotechnology Mushroom culti- Agaricus bisporus, Auricularia sp.,
vation Flammulina 6elutipes, Lentinus edodes,
Pleurotus sp., Vol6ariella 6ol6acea
Modernism is a general term used to describe
20th century attempts to break with the traditions After Gray (1970), Chang and Hayes (1978), and Hesseltine
of the 19th century. In painting, abstraction re- (1983).
 J.W. Bennett / Journal of Biotechnology 66 (1998) 101–107 103

Table 2 was probably the first to realize the technical

‘Modern’ examples of mycotechnology
possibilities of enzymes from molds and to intro-
Process or Organisms duce these fungal enzymes to industry. Takamine
product used the Japanese koji mold, Aspergillus oryzae,
to produce diastase. Takamine inoculated mold
Antibiotics and other pharmaceuticals spores on steamed rice or wheat bran and grew
Penicillins Penicillium chrysogenum
his cultures in thin layers. With a series of patents
Cephalosporin Cephalosporium acremonium
Cyclosporin Tolypocladium inflatum starting in 1894, he protected his processes and
(immunosup- suggested new uses for partially purified fungal
pressive) diastase as a substitute for malting enzyme, and
Ergot alkaloids Cla6iceps purpurea as a digestive aid for the treatment of dyspepsia
Griseofulvin Penicillium griseoful6in
(Takamine, 1894; Underkofler, 1954). During the
Mevalonin Aspergillus terreus
Enzymes early years of the Twentieth Century, similar pro-
a-Amylases A. niger, A. oryzae cesses were developed for numerous other en-
Cellulase Humicola insolens, Penicillium funiculo- zymes. By 1983, there were approximately 30
sum, Trichoderma 6iride different classes of enzyme in common commer-
Glucoamylases Aspergillus phoenicis, Rhizopus delemar,
cial use, of which approximately half were of
R. ni6eus
Glucose oxidase A. niger fungal origin. The number of commercial enzymes
Invertase A. niger, A. oryzae is growing at a rapid rate; an excellent summary
Laccase Coriolus 6ersicolor of industrial enzymology has been compiled by
Pectinase A. niger, A. oryzae, Humicola insolens Godfrey and West (1996).
Proteinases A. oryzae, Aspergillus melleus, R. dele-
Another filamentous fungal product central to
mar
Rennin (micro- Mucor miehei, M. pusillus the development of modern biotechnology is citric
bial) acid. Originally isolated from citrus fruits, since
Organic acids the end of the nineteenth century it was known
Citric acid A. niger that citric acid was also made by filamentous
Itaconic acid A. terreus fungi. The development of a commercial process
After Turner (1975), Bigelis (1985), and Godfrey and West by Pfizer, in Brooklyn, New York, USA, soon led
(1996). to widespread use of the compound in the food
and beverage industry. Additional uses included
zyme = yeast). Ultimately, ‘enzyme’ came to mean tablets, cosmetics, detergents, antifoaming, textile
all ‘unorganized ferments’, not just those pro- treatment, and as a preservative for stored blood.
duced by yeasts (Lutman, 1929). Many aspects of modern fermentation technology
were developed in the context of improving citric
acid yields by manipulating culture conditions, by
developing submerged processes, and improving
3. ‘Modern’ mycotechnology product recovery (Crueger and Crueger, 1982).
Without a doubt, however, it was the discovery
The enzymes, alcohols, organic acids, and phar- of penicillin, and the subsequent development of
maceuticals from filamentous fungi are central to penicillin into a ‘wonder drug’ that was the turn-
the development of modern biotechnology. Some ing point in the development of modern industrial
examples of major industrial fermentation prod- microbiology (Wainwright, 1990). Penicillin trig-
ucts and their producing species are given in gered searches for other secondary metabolites
Table 2. with antibacterial activity, as well as stimulating
One of the best studied of the early enzymes research on fungal physiology, fermentation tech-
was diastase (amylase), traditionally isolated from nology, and industrial strain development. So
germinating barley and used for the malting step many new antibiotics were discovered during the
in beer manufacture. Jokichi Takamine, in 1894, 1940s and 1950s that it has been called The
104 J.W. Bennett / Journal of Biotechnology 66 (1998) 101–107

Golden Age of Antibiotics. The research group is also under development (Rasmussen-Wilson et
associated with Selman Waksman at Rutgers Uni- al., 1997). Originally, researchers hoped to pro-
versity and Merck Corporation, in New Jersey, duce high levels of mammalian proteins in
was particularly successful in screening for an- filamentous fungi. For bovine prochymosin (ren-
tibacterial metabolites. Many of the new antibi- nin) this was achieved for several species (Davies,
otics discovered by the Waksman group came 1991). However, the levels of expression and se-
from soil bacteria, especially actinomycetes. The cretion for most mammalian gene products have
filamentous nature of both molds and actino- been lower than hoped. The molecular steps of
mycetes challenged chemical engineers to develop the fungal secretion pathway, post-translational
industrial-scale sterile fermentation procedures. modification of metabolites, and the release of
Both batch and continuous methods were per- proteins from hyphae into their environment are
fected (Smith and Berry, 1975; Demain, 1981). understood only in a descriptive sense (Wosten et
After the recombinant DNA revolution, these al., 1991). There is a need for continued research
processes required only minor modification for on the molecular biology of filamentous fungal
the production of genetically engineered microbial gene expression and secretion.
products. Secondary metabolic pathways are also
A development growing out of the Gold Age of amenable to molecular analyses. Predictably, the
Antibiotics was the systematic search for drugs penicillin family of antibiotics was the first group
with activities other than anti-infective. Laborato- of compounds to benefit from the new method-
ries in Japan under the leadership of Umezawa ologies. After cloning the gene for isopenicillin
were particularly innovative in establishing new N-synthase, it was found that many of the genes
screens to look for anti-tumor, anti-hypertensive, for the pathway were clustered; these were iso-
immunostimulant, anti-diarrheal, anti-mutagenic lated in rapid order (Skatrud, 1991). It was dis-
and other such biological activities (Umezawa, covered that certain high-yielding strains
1982). Of these, the immunosuppressant cy- contained multiple copies of the genes for key
enzymes in the penicillin pathway. In some cases
closporins and the anti-hypertensive mevalonins
it has been possible to engineer portions of the
are two of the most important pharmaceuticals to
pathway using unusual precursors and production
be discovered from filamentous fungi (Von Wart-
hosts, thereby embellishing nature’s chemical di-
burg and Trabor, 1986; Monghan and Tkacz,
versity (Skatrud, 1992).
1990).
Table 3
‘Postmodern’ examples of mycotechnology.
4. ‘Postmodern’ mycotechnology
Development Examples
Recombinant DNA and its cognate technolo-
Expression of heterologous Fungal, plant, mammalian
gies have revolutionized biology. As used here, genes
the term ‘postmodern’ mycotechnology refers to Amplification of Antibiotic pathways, enzymes
the new biotechnological developments spawned homologous genes
by marrying the techniques of gene splicing and Manipulation of secondary New semi-synthetic
pathways antibiotics; hybrid antibiotics
other post-recombinant DNA methodologies to
Large scale genomics Aspergillus nidulans;
those of conventional industrial mycology. Some Neurospora crassa;
examples of the developments coming out of this Magnaporthe grisea;
hybrid mix are listed in Table 3. Phytophthora infestans
The expression of heterologous proteins in DNA chips High-density DNA arrays for
screening gene expression
filamentous fungi has received considerable atten-
‘Mining’ fungal biodiversity Sampling environmental
tion. Currently, the major production hosts are for new pharmaceuticals DNA genomics-based
Aspergillus nidulans, A. niger, A. oryzae, and Tri- screening
choderma reseii (Davies, 1991). Neurospora crassa
 J.W. Bennett / Journal of Biotechnology 66 (1998) 101–107 105

Polyketides are another group of secondary the bacterium Haemophilus influenzae Rd (Fleis-
metabolites that have benefited from molecular chmann et al., 1995); since then seven additional
analyses. To date, most of the research on mixing- genomes have been completed and it is estimated
and-matching polyketide synthases has been con- that about 100 species are under investigation.
ducted in actinomycetes (Kao et al., 1994); The first complete eukaryotic genome was the
however, these strategies will also be useful in yeast Saccharomyces cere6isiae (Dujon, 1996). The
fungi. The permutations generated by the use of yeast genome consists of 16 chromosomes encom-
different starter units; adjustment of oxidation passing 12067266 base pairs (excluding repeats).
and stereochemistry during elongation; and the Many yeast genes have mammalian homologues
introduction of various post-polyketide modifica- and this functional compatibility has been ex-
tions lead to an almost astronomical number of ploited in studying cancers and other human dis-
theoretically possible different molecules. By using eases (Botstein et al., 1997). On the other hand,
engineered modular polyketide synthases, one can most of the open reading frames identified by the
create ‘unnatural natural products’. sequencing project had gone unnoticed, i.e. they
The development of fungal genetic transforma- had no known phenotypes.
tion systems has been used to bring the power of Genomics projects have been initiated for the
genetic analysis to species lacking sexual and yeasts Schizosaccharomyces pombe and Candida
parasexual cycles (Esser, 1997). Traditional fungal albicans as well as for several filamentous fungal
fermentations have been improved and genetic species, including the genetic models Aspergillus
engineering makes it possible to tailor fungal en- nidulans and Neurospora crassa (Bennett, 1997).
zymes for specific purposes. Both homologous The large amount of classical genetic and bio-
and heterologous genes have been introduced into chemical information available for these species
fungal production hosts. These genes can be ma- will make it easier to correlate sequence data with
nipulated in various ways in order to improve the phenotypes.
properties of the enzymes or to increase yields. Microbial genomics promises to revolutionize
Enzymes with wider substrate range, temperature not only basic biology but also to accelerate the
optima, and the like can be produced (Kinghorn drug discovery processes by guiding clinical trials.
and Lucena, 1994). Correlations between sets of genes and drug activ-
Even as traditional a field as mushroom cultiva- ities open new avenues for screening. Using pho-
tion has been stimulated by the new mycotechnol- tolithography and technology derived from the
ogy. Many delectable species cannot be cultured silica chip industry, immobilized oligonucleotides
and must therefore be collected during their spo- encompassing the entire yeast genome can be
radic fruitings in nature. Coprinus cinereus arrayed on a small surface creating a DNA mi-
(Pukkila, 1993) and Schizophyllum commune crochip. Using probes to measure hybridization,
(Raper and Horton, 1993) have been used as the chip can be used as a reagent for measuring
models to study the genetic basis of fruit body the expression of any number of genes. Genome-
formation in these species. It is predicted that based diagnostic products are also likely. In con-
improved strains of commercially cultivated junction with combinatorial chemistry and assay
mushroom can be obtained using similar breeding miniaturization, DNA chips can be used to iden-
strategies based on molecular tools. Moreover, it tify important molecular targets for drug discov-
is hoped that the isolation of genes associated ery. They also are being used to search for
with mushroom development in these laboratory bioactive products using environmental DNA
models will facilitate understanding of fruiting in samples from organisms that have been over-
exotic wild species as well. looked in the past, including difficult-to-culture
The acceleration and automation of DNA se- marine and macrofungi (Blanchard and Hood,
quencing has made large-scale genomics possible, 1996).
and genomics is changing all of biology. The first Like all of biotechnology, mycotechnology op-
complete DNA sequence of an organism was for erates at the scientific frontier. New scientific ad-
106 J.W. Bennett / Journal of Biotechnology 66 (1998) 101–107

vances drive new approaches in agriculture, indus- Demain, A.L., 1981. Industrial microbiology. Science 214,
try, and medicine. The molecular biotechnology 987 – 991.
Dujon, B., 1996. The yeast genome project; what did we learn?
revolution has spawned social and political con- Trends Genet. 12, 263 – 270.
troversy. Sometimes regulatory issues overshadow Esser, K., 1997. Fungal genetics: from fundamental research to
the scientific issues. Although economic factors biotechnology. Prog. Bot. 58, 3 – 38.
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tors have taken on certain urgency in the era of Kirkness, E.F., et al., 1995. Whole-genome random se-
quencing and assembly of Haemophilus influenzae Rd. Sci-
biotechnology start-up firms. They have also gen- ence 269, 496 – 512.
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Science 265, 509 – 512.
Kinghorn, J.R., Lucena, N. 1994. Biotechnology of filamen-
Fungi, the industrial workhorses of traditional
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