Biotechnology Advances 30 (2012) 1119–1139

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Biotechnology Advances
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Production of recombinant proteins by filamentous fungi

Owen P. Ward ⁎
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L3G1

a r t i c l e i n f o a b s t r a c t

Available online 24 September 2011 The initial focus of recombinant protein production by filamentous fungi related to exploiting the extraordi-
nary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Tri-
Keywords: choderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early
Aspergillus recognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common ability
Trichoderma to produce homologous proteases which could degrade the heterologous protein product and strategies to
Penicillium
prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation
Recombinant protein
Heterologous
patterns in filamentous fungi and in mammals were quite different, such that filamentous fungi are likely not
Protease to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic
Genome use. By combining the experience gained from production of single recombinant proteins with new scientific
Filamentous information being generated through genomics and proteomics research, biotechnologists are now poised to
Fungi extend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to express
Pathogenesis genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of
new primary or secondary metabolites. It is recognized that filamentous fungi, most species of which have
not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the
natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism
for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming
other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these
novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be con-
fined to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the del-
eterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward
solutions, is expected to represent a very important future focus of fungal recombinant protein technology.
Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.

1. Introduction producers of polysaccharides and biosurfactants. Some filamentous

Filamentous fungi are extraordinary organisms which impact
widely on so many aspects of our lives. These organisms are charac-
terized as having branched filamentous structures or hyphae having
typical diameters of 2–18 um, with (higher fungi) or without (lower
fungi) cross-walls or septae. Higher fungi include Aspergillus,
Penicillium, Trichoderma and Fusarium species. Lower fungi include
Rhizopus and Mucor species. Filamentous fungi are chemo-organotrophs
meaning they obtain their energy and carbon by oxidation of organic
compounds.
In traditional fermentation technology filamentous fungi are dom-
inant producers of a range of primary metabolites, including organic
acids, such as citric, gluconic, fumaric, kojic, itaconic acid and fatty
acids. They also produce important secondary metabolites, especially
as human therapeutics, for example, penicillin, cephalosporin, ergot
alkaloids, griseofulvin, lovastatin, taxol and zeranol. Some are
⁎ Tel.: + 1 519 888 4567x32427; fax: + 1 519 746 0614.
E-mail address: opward@uwaterloo.ca.
fungi are food materials in their own right, such as mushrooms, single
cell protein/biomass (single cell protein, SCP) or indeed lipid-rich bio-
mass (single cell oil, SCO), whereas other fungi are components in
fermented foods. Some are producers of an array of fungal enzymes,
for example amylases, amyloglucosidases, cellulases, pectinases,
laccases/ligninases, phytase, proteases, microbial rennets, lipases and
glucose oxidase. Many intracellular fungal enzymes are also exploited
as biocatalysts in enzyme biotransformations in bioorganic synthesis
reactions while others use biodegradative processes in soil bioremedia-
tion. Some strains are well known pathogens of humans, animals and
plants (Cutler et al., 2007; Maor and Shirasu, 2005; Segal and Walsh,
2006) while others, for example mycorhizal fungi, have beneficial asso-
ciations with plants and/or participate in nutrient recycling in soil.
Some fungi are responsible for food spoilage, wood decay and infesta-
tion of damp buildings (Bennett, 2006) and many fungal spores are
known allergens (Meyer, 2004).
The known high productivity chartacteristics of filamentous fungi
are in part related to their inherent abilities to grow at high rates and
to high biomass densities supported by low cost substrates in rela-
tively simple fermenters. For industrial fermentations, filamentous

0734-9750/$ – see front matter. Crown Copyright © 2011 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2011.09.012
1120 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
fungi may be cultivated using traditional surface culture methods,
where oxygen uptake involves passive exposure of the culture to
the atmosphere or in a semi-solid culture where a non-homogeneous
culture may be aerated through various forced air and/or mixing
strategies. They may also be cultured in intensively mixed stirred
tank reactors where the objective is to achieve conditions within
the reactor which approach homogeneity, thereby facilitating more
sophisticated process control. In addition to the beneficial character-
istics described above, fungi are especially interesting targets for pro-
duction of recombinant proteins because of their demonstrated
capacities to hyperproduce and secrete enzyme proteins, for example
glucoamylase production by Aspergillus with impressive titers of
greater than 25 g/L.
Processes for production of mold-modified foods have been
implemented for many thousands of years. At least the initial stages
of these fermented food processes were promoted by surface culture
suggesting a likely requirement for air, as a source of oxygen, to sup-
port preferential growth of molds on the medium surfaces. Since the
raw materials were derived from plants including soybeans, wheat
and rice, common logic led early scientists to conclude that the
molds had the capacity to at least partially degrade the principal con-
stituents of these plant materials, namely the associated carbohy-
drates (including starches, pectins, celluloses and hemicelluloses),
proteins and lipids. Other molds have long been known to participate
in processes including pathogenesis of plants, spoilage of fruits and
vegetables and rotting of wood, likewise with the presumed involve-
ment of mold products which could mediate the biodegradation of
major structural constituents of these plant materials.
Since the principal structural components of plants, being subjected
to biodegradative processes during tradititional food fermentations,
plant pathogenesis, fruit and vegetable spoilage, wood rotting, and re-
lated processes, are polymeric substances, early scientists soon postu-
lated that cellular assimilation of breakdown products of these
polymeric substances might require that the substrates be degraded
in the extracellular environment. Indeed simple methods for surface
cultivation of the molds involved, on agar-like media containing indi-
vidual substrates, often insoluble, demonstrated zones of hydrolysis
around the mold colony, illustrating that depolymerization reactions
had been facilitated or catalyzed. It was soon concluded that these bio-
degradations were mediated by hydrolytic and other depolymerizing
enzymes which were generally secreted by the molds into the extracel-
lular medium or sometimes were located/attached on the extracellular
surfaces of the biodegradative fungal organism. Jokichi Takamine, a
Japanese immigrant to the United States, was first to commercialize
an isolated microbial enzyme. In 1894, he patented a process for prep-
aration of diastatic enzymes from molds which was marketed as Taka-
diastase. The method involved growth of the fungus on the surface of
solid substrates, such as wheat or bran, clearly based on the traditional
processes for preparation of oriental fermented foods (Ward, 1989).
The enzyme and producing organism were later characterized as fun-
gal alpha-amylase and Aspergillus oryzae, respectively (Gwynne and
Devchand, 1992).
The development of recombinant technology harnesses the power
of many of these filamentous fungi as hosts in the production of spe-
cific recombinant proteins as final products with applications in the
agricultural, food and nutrition, biomedical and pharmaceutical, and
energy and industrial sectors (Schuster et al., 2002). This focus has
represented the principal effort in applied genetic engineering over
the past 25 years or so and is discussed in Sections 2–4 of this review.
Further advancements of the core transformation technologies
combined with progress in the fields of genomics and proteomics is
leading to a more complex level of host engineering, whereby recom-
binant expression of multiple proteins and enzymes is facilitating en-
gineering of blocks of new physiological or metabolic machinery into
recombinant hosts. An example of these developments relates to our
ability to engineer metabolic pathways so as to enhance production of
primary or secondary metabolites, or indeed to facilitate production
of novel compounds through introduction of new biosynthetic path-
ways. The tremendous level of metabolic diversity exhibited by cur-
rently known filamentous fungi, together with the knowledge
that only a small number of the estimated 1.5 million species
(Hawksworth, 2001), which are thought to exist, means filamentous
fungi will continue to supply the biosynthetic tools for synthesis of
a myriad of novel products for countless years to come. This research
is still at an early stage and some example roles of recombinant pro-
teins in metabolic engineering of fungi are addressed in Section 5.
Of course, the enormous diversity of fungal organisms does not
imply that a newly isolated natural host capable of biosynthesizing
novel new beneficial compounds will be a suitable host for large
scale manufacture. Indeed many novel organisms that will be identi-
fied in the future are likely to be the ones that are hard to cultivate,
even unculturable and will require better hosts be they other filamen-
tous fungal organisms or non-fungal organisms. Efforts are already
being directed to exploiting the unique nature of some of the en-
zymes or metabolic pathways or systems of filamentous fungi by
transferring these capabilities to other organisms, and some prelimi-
nary examples of expression of fungal proteins in other hosts are dis-
cussed in Section 6.
Filamentous fungi also interact with other organisms using a vari-
ety of extremely complicated mechanisms, many of which are as yet
poorly understood and their interactions often have overall beneficial
and perhaps more frequently negative societal outcomes. Thus some
of the first filamentous fungal organisms to be sequenced were
human or agricultural plant pathogens and, as we go forward, geno-
mic and proteonomic research will provide new insights into the mo-
lecular mechanisms involved in pathogenesis. These studies will
include cloning and expression of recombinant proteins in fungi as
prospective candidate causative proteins in pathogenesis followed
by application of strategies to disrupt these proteins with a view to
relating these manipulations to pathogenicity and virulence of the
pathogen. While this area of research is in its infancy, some indica-
tions of its potential are included in Section 7.
Some recent reviews on aspects of this review topic are listed in
Table 1, including reference to some informative tables from these re-
view papers.
2. Filamentous fungi as hosts for production of recombinant proteins
Many filamentous fungi are natural excellent producers of extra-
cellular enzymes and hence are exceptional candidate hosts for the
production of recombinant proteins (Iwashita, 2002; Wang et al.,
2005). Organisms such as Aspergillus and Trichoderma species are no-
table in their abilities to produce and secrete very high levels of pro-
teins, with Aspergillus niger being capable of producing 25–30 g/L of
glucoamylase and Trichoderma reesei reported to be capable of pro-
ducing 100 g/L of extracellular protein (Demain and Vaishnav, 2009).
Meyer (2008) discussed four common strategies for implementa-
tion of transformations of filamentous fungi: The protoplast-mediated
method involves use of cell wall-degrading enzymes for protoplast
preparation with subsequent uptake of foreign DNA promoted by addi-
tion of polyethylene glycol (PEG) and calcium chloride. Noted disad-
vantages of this method are that transformations may vary with
batch variations in the lytic enzyme, that a regeneration procedure is
needed and that the high copy number of insertions of DNA may result
in a less controlled transformation. In the Agrobacterium tumefaciens
transformation, the A. tumefaciens carries a binary vector containing
the target DNA between a 24-base pair repeating unit and a virulence
region required for DNA transfer. The gene-carrier organism is
co-cultured with the filamentous fungus and the transformation is ad-
vantageous in that the low copy number of DNA insertions facilitates a
more targeted integration. Beijersbergen et al. (2001) patented a meth-
od for Agrobacterium-mediated transformation of mold species
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
Table 1
Some prior reviews related to the topic of production of recombinant proteins by filamentous fungi.
Review—short title Reference
Genetic engineering of filamentous fungi Meyer (2008)
Refining heterologous protein production in filamentous Sharma et al. (2009)
fungi
Filamentous fungi as cell factories for heterologous protein Punt et al. (2002)
production
Aspergillus as a host for heterologous expression Lubertozzi and Keasling
(2009)
Transcriptional regulation of plant cell wall degradation by Aro et al. (2005)
filamentous fungi
Proteomics of filamentous fungi Kim et al. (2007)
Production of human therapeutic proteins by yeasts and Gerngross (2004)
filamentous fungi
Physiology and biotechnology of Aspergillus Ward et al. (2006)
Aspergillus as a cell factory for protein production Braaksma and Punt (2008)
Recombinant protein production systems for Aspergillus Fleissner and Dersch (2010)
Genomics of folding, secretion, glycosylation in aspergilli Geysens et al. (2009)
Biotechnology of Trichoderma Schuster and Schmoll
(2010)
Bioconversion of lignocellulose biomass Kumar et al. (2008)
Engineering of Penicillium chrysogenum Harris et al. (2009)
Engineered biosynthesis of peptide antibiotics Stachelhaus et al. (1996)
Engineering primary metabolic pathways of industrial Kern et al. (2007)
micro-organisms
Taxol-producing endophytic fungi Zhou et al. (2010)
belonging the Ascomycotina, Basidiomycotina, Deuteromycotina, Mas-
tigomycotina and Zygomycotina and illustrated example transforma-
tions for Aspergillus awamori, Aspergillus nidulans, A. niger,
Colletotrichum gloeosporiodes, Fusarium solani, Fusarium graminearum,
Neurospora crassa, T. reesei, Pleurotus ostreatus and Agaricus bisporus.
In applying Agrobacterium-mediated transformation strategies to fila-
mentous fungi, for example A. bisporus, Romaine (2002) observed
that it was preferable to co-cultivate the bacterium with fruit body tis-
sue rather than with spores. A third transformation method, which
often requires protoplast preparation, involves electric pulse-mediated
reversible membrane permeabilization to promote DNA uptake. In con-
trast, the fourth more specialized method, which can be implemented
without cell wall removal, involves shooting DNA-coated metal parti-
cles at high speed into cells. More specific molecular transformations
involve targeting of recombinant genes to a specific position in the ge-
nome which will enhance transcription of newly introduced DNA
and/or deletion of genes with potential to reduce the positive effects
of the desired transformation, be it production of a specific recombi-
nant protein or insertion of multiple enzymes or proteins participating
in a specific metabolic pathway or other physiological event.
In addition to DNA-based methods, introduction of RNA-based
methods such as antisense RNA, hammerhead ribozymes and RNA in-
terference approaches have been found to be very useful for silencing
particular genes in filamentous fungi (Fulci and Macino, 2007;
Hammond and Keller, 2005; Muller et al., 2006; Yamada et al., 2007).
Detailed classical physiological and biochemical knowledge is
available for many of the candidate hosts and molecular techniques,
including genome sequencing and annotation strategies. These are
providing data to support efforts in optimizing expression and secre-
tion of recombinant proteins in filamentous fungi. Filamentous fungi,
especially well studied Aspergillus species, have also been shown to
efficiently implement posttranslational modifications such that heter-
ologous eukaryotic proteins are expressed in a correctly folded form
(Kinghorn and Unkles, 1994). Aspergillus species, especially A. niger,
1121
Table Contents
1 Industrially important compounds produced by filamentous fungi
2 Heterologous industrial enzymes
3 Important heterologous proteins in recombinant Aspergillus
1 Fungal and yeast hosts for hIL-6;
4 Systematics of biotechnologically relevant true fungi
1 Bioactice fungal metabolites
2 Some fungal conversions
3 Some fungal bioremediations
1 List of fungal proteomics papers
IV Aspergillus genes of industrial interest
V Aspergillus recombinant food enzymes
VI,VII,VIII Important heterologous proteins expressed in Aspergillus
IX Patents on recombinant protein production by Aspergillus
1 Effect of secreted protease activity of protease gene disruption strains
1,2 Recombinant protein production by aspergilla including hosts and
promoters
1 Genes involved in protein-folding, unfolded protein response,
glycosylation
1 Non-ribosomally synthesized antibiotics and producing hosts
1 Taxol-producing strains
2 Common transformation methods for filamentous fungi
A. awamori and A. oryzae, appeared to be better filamentous fungal
hosts for recombinant protein production than some other filamen-
tous fungi. The Mucor rennin gene under the control of a suitable
alpha-amylase promoter, introduced into A. oryzae, resulted in pro-
duction yields of the heterologous protein of 3.3 g/L (Christensen et
al., 1988).
Some perceived or suggested disadvantages of filamentous fungi as
heterologous protein hosts relate to their relatively low frequencies of
transformation, potential morphological defects, and observed protein
modifications due to protease activity or low pH (Kinghorn and Unkles,
1994; Radzio and Kueck, 1997). It was observed that production levels
of most non-fungal recombinant proteins (mammalian, bacterial,
avian, plant etc.) in filamentous fungi were generally lower as com-
pared to those of homologous proteins and with likely bottlenecks at
the level of transcription and translation, secretion, with possible limi-
tations also at the post-translational level (i.e., inefficient translocation,
folding, transport, processing, or secretion) (Broekhuijsen et al., 1993;
Gouka et al., 1997a; Jeenes et al., 1994).
A general model for fungal protein synthesis and secretion, based
on Aspergillus species has been summarized by Fleissner and Dersch
(2010). During synthesis, proteins are directed into the endoplasmic
reticulum where folding takes place and glycosylation is initiated. In
Aspergillus species, protein disulfide isomerase (Pdi) assists in the
folding and maturation of secretory proteins and the ability of PdiA
to catalyze the refolding of denatured and reduced RNase has been
demonstrated (Ngiam et al., 2000). Improperly folded or glycosylated
proteins are sent to the proteosome or vacuoles for degradation. Fur-
ther modification, including glycosylation, occurs in the golgi bodies.
SNARE proteins facilitate vesicle-mediated trafficking of the proteins
to the hyphal tip for extracellular secretion. In an interesting experi-
ment, Gordon et al. (2000) fused a green fluorescent protein sGFP
(S65T) to truncated A. niger Gla (Gla:499) which was successfully in-
tegrated into the A. niger genome. Confocal fluorescence microscopy
confirmed that GFP was partially localized within the hyphal cell
1122 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
wall and that protein secretion occurred at the apical or subapical hy-
phal regions.
Limitations at the transcriptional level can be due to low steady-
state mRNA levels resulting from a low transcription initiation rate
or more likely from a reduced mRNA stability. It has been suggested
that at least five structural components may influence mRNA stability.
In the case of hil6 and aglA transcripts in Aspergillus species, glaA
fusions appeared to stabilize mRNA levels (Gouka et al., 1997b).
Jeenes et al. (1994) reported similar results for fusions of egg-white
lysozyme with glucoamylase. In many cases, low levels of production
of recombinant proteins are due to post-translational secretion
bottlenecks rather than transcription (Conesa et al., 2001; van den
Hombergh et al., 1997).
After secretion, a major known problem for heterologous proteins
is their degradation by high extracellular enzyme producing filame-
tous fungi, perhaps most notably Aspergillus species which secrete a
diversity of extracellular proteases (van den Hombergh et al., 1997).
Proteases have been shown to be responsible for degradation of
many recombinant proteins (Broekhuijsen et al., 1993; Roberts et
al., 1992).
Traditional fermentations for production of extracellular enzymes
by filamentous fungi were based on fermented food processes. These
processes which involved surface or semi-solid media are
non-homogeneous, making fine process control impossible. This mo-
tivated desires to produce fungal extracellular enzymes in submerged
culture in stirred tank reactors. Some early challenges with respect to
growing filamentous fungi in submerged culture related to the high
viscosities which developed in the media caused by the increased
concentrations of filamentous biomass, making mass transfer and es-
pecially aeration of these oxygen-requiring organisms more challeng-
ing and generally leading to early cessation of growth and limitation
of desired protein product yields. These problems were addressed in
various ways at the engineering, physiological and molecular levels.
Improvements in fermenter design were directed towards increasing
aeration while controlling mycelial shearing effects. High growth and
product formation rates were achieved by manipulation of fungal
morphology, generally to reduce mycelial strand length and promote
formation of highly branched mycelia. An example of a manipulation
at the molecular level was provided by Akin et al. (2003) who maxi-
mized heterologous protein production by transforming the cells
with cotA-encoding nucleic acids controlled by a regulatable promot-
er. Some of these strategies are discussed in more detail elsewhere in
this paper.
3. Survey of principal players
Some of the principal organisms involved in food fermentation
processes were Aspergillus and Rhizopus species. For example, the ini-
tial stage of production of soy sauce involves predominant growth of
A. oryzae strains on a mixture of soybeans and wheat, while produc-
tion of tempeh involves cultivation of Rhizopus oligosporus on cooked
soybean mash. Not surprisingly, these organisms have also been
prime candidate hosts for production of recombinant proteins. A
combination of our historical knowledge and experience of the per-
formance of GRAS (Generally Regarded As Safe) strains with new ge-
nomic information has been used to facilitate the design of a new
generation of genetically modified strains capable of efficient produc-
tion of beneficial recombinant proteins (van Dijck et al., 2003).
The GRAS food additives list of the United States Food and Drug
Administration includes enzyme products from A. niger and A. oryzae,
Endothia parasitica, Mucor miehei, Mucor pusillus and others. Since
1998, the FDA has published an inventory of notifications it has re-
ceived regarding applications for GRAS recognition/exemption. The
majority of these notices relating to products of filamentous fungi in-
volved recombinant proteins. Examples of these listings are included
in Table 2. For further information, go to the GRAS website at http://
www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=grasListing.
The availability of genomic data, combined with other methods in-
cluding proteomics (deOliveira and deGraaf, 2011) and metabolo-
mics, is and will continue to support strain development strategies
for production of recombinant proteins through use of molecular
methods for industrial fermentations. For example, comparative ge-
nomic studies among Aspergillus species suggest that A. oryzae, is
enriched with genes which participate in the degradation of biomass
and in primary and secondary metabolism (Kobayashi et al., 2007).
Also in A. oryzae when cDNA microarrays and expressed sequence
Table 2
Examples of GRAS notices filed since 1998 relating to filamentous fungi.
GRN Substance
no.
8 Pectin esterase derived from Aspergillus oryzae carrying a gene encoding
pectin esterase from Aspergillus aculeatus
10 Exopeptidase derived from Aspergillus oryzae carrying a gene encoding a
leucine aminopeptidase from Aspergillus sojae
32 Pectin lyase derived from Trichoderma reesei carrying a gene encoding
pectin lyase from Aspergillus niger
34 Aspartic proteinase derived from Aspergillus oryzae carrying a gene
encoding aspartic proteinase from Rhizomucor miehei
43 Lipase derived from Aspergillus oryzae carrying a gene encoding lipase
from Thermomyces lanuginosus
54 Xylanase derived from Fusarium venenatum carrying a gene encoding
xylanase from Thermomyces lanuginosus
75 Lipase derived from Aspergillus oryzae carrying a gene encoding lipase
from Fusarium oxysporum
89 Five enzyme preparations from Aspergillus niger: Carbohydrase enzyme
preparation, catalase enzyme preparation, glucose oxidase enzyme
preparation, pectinase enzyme preparation, and protease enzyme
preparation
90 Carbohydrase enzyme preparation from Aspergillus oryzae, protease
enzyme preparation from Aspergillus oryzae, and carbohydrase enzyme
preparation from Rhizopus oryzae
103 Lipase enzyme preparation from Aspergillus oryzae carrying a gene
constructed from a modified Thermomyces lanuginosus lipase gene and a
portion of the Fusarium oxysporum lipase gene
106 Glucose oxidase enzyme preparation from Aspergillus oryzae carrying a
gene encoding a glucose oxidase from Aspergillus niger
111 Lipase enzyme preparation from Aspergillus niger
113 Lipase enzyme preparation from Aspergillus oryzae
122 Laccase enzyme preparation produced by Aspergillus oryzae expressing
the gene encoding a laccase from Myceliophthora thermophila
132 Lactase enzyme preparation from Aspergillus niger
142 Phospholipase enzyme preparation from Aspergillus oryzae expressing the
gene encoding a phospholipase A1 from Fusarium venenatum
149 Beta-glucanase enzyme preparation from Trichoderma harzianum
150 Glucosamine hydrochloride prepared from chitin obtained from
Aspergillus niger
158 Lipase preparation from Aspergillus niger expressing a gene encoding a
lipase from Candida antartica
183 Phospholipase A2 enzyme preparation from Aspergillus niger expressing a
gene encoding a porcine phospholipase A2
195 Mixed beta-glucanase and xylanase enzyme preparation from Humicola
insolens
201 Asparaginase enzyme preparation from Aspergillus oryzae expressing the
asparaginase gene from A. oryzae
214 Asparaginase enzyme preparation from Aspergillus niger expressing the
asparaginase gene from A. niger
230 Chymosin enzyme preparation from Trichoderma reesei expressing the
bovine prochymosin B gene
238 Lipase enzyme preparation derived from Hansenula polymorpha
expressing a gene encoding a lipase from Fusarium heterosporum
296 Lipase enzyme preparation from a genetically modified strain of
Aspergillus niger
315 Transglucosidase enzyme preparation from Trichoderma reesei expressing
the gene encoding transglucosidase from Aspergillus niger
345 Carboxypeptidase enzyme preparation from modified Aspergillus niger
333 Acid fungal protease enzyme preparation from Trichoderma reesei
expressing the gene encoding acid fungal protease from T. reesei
372 Glucoamylase (GA) enzyme preparation from Trichoderma reesei
expressing the gene encoding the GA from T. reesei
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
tags were used to characterize transcriptional activity associated with
energy catabolism and hydrolytic enzyme production, transcription
levels of most catabolic genes of the EM and TCA pathways were ob-
served to be higher in glucose-rich conditions as compared with
glucose-depleted conditions (Maeda et al., 2004). As will be discussed
later, interesting studies have also been implemented in traditional in-
dustrial solid-phase media. For example, rich gene-expression profiles
for hydrolytic enzymes were observed in wheat-bran media, which
exhibited lowest expression of catabolic genes. This suggested the latter
poor expression may have released catabolite repression of hydrolytic
enzyme synthesis. Gene arrays, gene deletion and insertion strategies
and other emerging molecular techniques will undoubtedly receive
widespread application as a means to better grasp and exploit the
mechanisms of industrial product formation, regulation, and secretion
by Aspergillus species and other filamentous fungi (Akao et al., 2002;
Bautista et al., 2000; Moralejo et al., 2002; Ngiam et al., 2000; Sims et
al., 2004; Zarrin et al., 2005).
Because of the resource intensive nature of genome sequencing
and functional analysis, in the case of filamentous fungi, priority at-
tention has focussed on the most important strains based on industri-
al and/or agricultural productivity considerations or on strains which
represent important human, animal and plant pathogens. In addition
some starting species were selected for genomic characterization, for
example A. nidulans, where substantial beneficial prior physiological
and genetic knowledge had already been established. Consequently,
pioneering genomic research was implemented on industrial and
pathogenic Aspergillus species. More recent interest in developing
more efficient systems for bioconversion of biomass to energy provid-
ed the impetus to characterize the genome of the high cellulose and
hemicellulase producer, T. reesei. In the case of other industrial extra-
cellular enzyme producers which may also be excellent candidate fil-
amentous fungal hosts for production of recombinant proteins, for
example certain Penicillium, Rhizopus, Fusarium and Mucor strains, as
well as some thermophilic fungi, there was insufficient interest
and/or resources to substantially characterize these strains genetical-
ly with respect to their enzyme production, secretion function and
potential recombinant protein production potential. In some
of the latter cases, for example in the case of Penicillium and
Mucor/Fusarium, detailed sequencing and functional genomic studies
have been directed at the highest profile application of these organ-
isms, namely to penicillin production by Penicillium chrysogenum
and to the plant pathogenic properties of F. graminearum. Mention
is made of this research below, in case some of the genomic and
functional findings from these studies become relevant and applica-
ble to extracellular enzyme-producing strains as potential candidate
recombinant protein-producing hosts. In addition, molecular biolo-
gists are applying recombinant technologies to investigate the unique
metabolic properties of these organisms with the expectation that a
fuller understanding will lead to beneficial societal outcomes.
A discussion follows highlighting progress in genomics research as
it pertains to some of the more important industrial filamentous fun-
gal strains. Most filamentous fungi have estimated genomic sizes of
30–40 Mb, encoding 9000–13,000 genes (Machida, 2002).
3.1. Aspergillus
The genus Aspergillus consists of more than 180 officially recog-
nized species, most of which degrade plant polysaccharides (de
Vries, 2003), and they are particularly important industrial filamen-
tous fungi for the large-scale production of both homologous and het-
erologous enzymes (Fawole and Odunfa, 2003; Wang et al., 2003). A.
oryzae and A. niger are on the Generally Recognized as Safe (GRAS)
list of the Food and Drug Administration (FDA) in the United States
(Tailor and Richardson, 1979).
Molecular and genetic studies of Aspergillus species most relevant
to recombinant protein production deal with A. nidulans, A. oryzae, A.
1123
niger and its awamori variant. Detailed genetic analyses have also
been carried out on the human pathogen, Aspergillus fumigatus, but
because of the severe toxigenic nature of this organism as a producer
of the highly toxic aflatoxins, it is not relevant as a host for biotech-
nology production processes. A. nidulans is of particular interest as a
model filamentous fungal organism for studies of cell biology and
gene regulation. It is also related to A. niger and A. oryzae, which are
the best natural filamentous fungal production hosts. While A.
nidulans is the best genetically characterized Aspergillus species, it is
also deemed to be unsuitable as a recombinant host for biotechnology
processes, because it produces sterigmatocystin, which is also a toxin,
albeit much less severe than aflatoxins. A.niger and A. oryzae are also
recognized potential broad based recombinant hosts for the biophar-
maceutical industry for production of recombinant proteins.
3.1.1. A. nidulans
Conventional matings lead to the identification of greater than 900
genes in A. nidulans (Brody et al., 1991). The whole genome has a size
of 30.1 Mb, with eight well marked chromosomes containing approx-
imately 10,000 genes. Physical and chromosomal linkage maps, and
genome sequence have been described in detail (Archer and Dyer,
2004; Clutterbuck, 1997; Galagan et al., 2005; Lubertozzi and
Keasling, 2009; Monsanto, 2001; Sims et al., 2004). Research is con-
tinuing with the ultimate objective of describing and relating expres-
sion patterns, cellular roles and functions of all genes.
3.1.2. A. niger
Sequencing of a derivative of the enzyme-producing strain A. niger
NRRL 3122 (ATCC 22343, CBS 115989) indicated a genome size is
35.9 Mb, containing 14,097 predicted genes (Archer and Dyer,
2004). The sequence data for A. niger ATCC strain 9029 is held at the
Pacific Northwest National Laboratory (PNL) and is available to re-
searchers upon request. Genencor has access to the A. niger genome
sequence data of Integrated Genomics (Machida, 2002). The Joint
Genome Institute (JGI) initiated a sequencing program for the citrate-
producing A. niger ATCC 1015, in 2004 as part of the United States
DOE Genome Program, with participation of PNL and Oakridge National
Laboratory. Sequencing information on this strain was made available at
http://www.jgi.doe.gov/aspergillus. The genome sequence and analysis
of the ancestor of a current A. niger enzyme production strains, A. niger
CBS 513.88, indicates a genome size of 33.9 Mb (Pel et al., 2007).
Among the 14,165 open reading frames identified, strong functions
were predicted for 6505 of them. This paper and supplementary infor-
mation referenced on line includes detailed functional genomic analyses
of protein secretion, carbohydrases and proteases as well as correspond-
ing comparative analyses among the different Aspergillus species
(A. niger, A. nidulans, A. oryzae and A. fumigatus).
Tsang et al. (2009) combined analysis of proteins secreted by A.
niger with genomic predictions of signal-peptide containing proteins
to confirm that the presumed secreted proteins were in fact secreted
and not the result of cell autolysis. Combining gene expression and
proteomic data for A. niger overproducing strains of lipid, protein
and carbohydrate-degrading enzymes, facilitated identification of
898 proteins and demonstrated that the strains exhibited upregula-
tion of proteins participating in carbon- and N-metabolism, as well
as protein folding and protein degradation. The data enabled re-
searchers to manipulate the system incuding overexpression of a
putative protein glycosylation gene and to increase secretion of a
specified enzyme (Jacobs et al., 2009).
3.1.3. A. oryzae
The Japanese National Institute of Technology and Evaluation
completed sequencing of the A. oryzae genome which consists of
eight chromosomes ranging from 2.8 to 7.0 Mb (Kitamoto et al.,
1994; Machida, 2002). The total genome size was estimated to be
1124 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
36.8 Mb with the number of genes being about 12,074 (Machida et al.,
2005).
Fleissner and Dersch (2010) recently reviewed the range of re-
combinant protein products produced by Aspergillus species. The
principal host species identified were: A. niger, A. awamori, A. oryzae,
A. nidulans and A. terreus. The predominant promoters used for re-
combinant protein production were: adhA, alcA, alcC, aldA, amdS,
amdS, amyA, amyB, aphA, exlA, gdhA, glaA, glaA1, gpdA, oliC,
pkiA, sodM, sucA, tef1 and tpiA. Recombinant products from humans
included: alpha1-proteinase inhibitor, antigen-binding (Fab′) frag-
ment, corticosteroid binding globulin, epithelial growth factor, granu-
locyte macrophage colony stimulating factor, growth hormone,
humanized IgG1(kappa) antibodies, interferon-alpha-2, interleukin-6,
lactoferrin, lysozyme, mucus proteinase inhibitor, parathyroid hor-
mone, single-chain variable region fragment (scFv) anti lysozyme con-
struct, superoxide dismutase and tissue plasminogen activator.
Recombinant products originating from other animals included: por-
cine pancreatic phospholipase A2 and prochymosin; bovine chymosin,
prochymosin and prochymosin B; hen egg-white lysozyme and llama
antibodies. Recombinant plant proteins expressed in Aspergillus
include Thaumatococcus daniellii thaumatin and Cyamosis tetragonoloba
alpha-galactosidase. Recombinant bacterial proteins expressed in
Aspergillus species included Cellulomonas fimi endoglucanase, Clostridium
thermocellum dockerin, Eschericia coli enterotoxin subunit B, beta-
galactosidase, beta-glucuronidase and Thermobifida fusca hydrolase. Re-
combinant proteins from other fungal genera expressed in Aspergillus in-
cluded: Agaricus meleagris pyranose dehydrogenase, M. miehei
triglyceride lipase and aspartyl protease, Phanerochaete chrysospor-
ium lignin peroxidase H8 and manganese peroxidase H4, Pleurotus
eryngii peroxidase, Pycnoporus cinnabarinus laccase, Thermomyces
lanuginosus lipase and Trametes versicolor laccase. Many recombi-
nant proteins from one species of Aspergillus have also been
expressed in another Aspergillus species. The very interesting
swollenin-like protein from A. fumigatus which, like swollenin
from T. reesei, disrupts cellulosic materials and has similarities to the
plant proteins (expansins) which have a cell wall loosening effect,
was produced as a recombinant protein in A. oryzae (Chen et al.,
2010). While the protein exhibits no apparent enzyme activity, in
the presence of cellulases, it promoted efficient saccharification of
crystalline cellulose.
3.2. Trichoderma
The genome sequence of the commercially important high pro-
ducer of cellulases and hemicellulases, T. reesei, has been published
(Martinez et al., 2008) while analysis and annotation of the genomes
of two biocontrol species, Trichoderma altroviride and Trichoderma
vireus are proceeding. T. reesei is a soft-rot ascomycete filamentous
fungus with a long and safe track record as a producer of commercial
cellulases, initially with applications in food processing (Nevalainen
et al., 1994). Studies aimed at understanding and optimizing factors
affecting productivity and catalytic efficiency of cellulases are funda-
mental to overcoming the major biomass pre-treatment obstacle to
commercialization of processes for production of bioenergy from lig-
nocellulose biomass. Applications of its cellulase and hemicellulase
compliment in the pulp and paper and textile industries are also im-
portant (Buchert et al., 1998; Galanti et al., 1998). T. reesei represents
a principal target cellulase host in the quest to replace gasoline with
cellulose-derived ethanol.
3.2.1. T. reesei
T. reesei has a genome size of 33 Mb and seven chromosomes
(http://genome.jgf-psf..org/Trire2/Trire2.home.html). The predicted
number of genes in the genome was 9129 (Martinez et al., 2008).
T. reesei has an extraordinary ability to secrete proteins. Cherry and
Fidantsef (2003) reported that some industrial strains following
aggressive mutation programs, could produce as much as 100 g/L ex-
tracellular protein, with up to 60% as the major cellulase Cel7a (CBHI)
and 20% of Cel 6a (CBHII).
The complete pattern of proteins related to expression of cellulase
and hemicellulase genes in T. reesei was characterized by Ouyang et
al. (2006). Cultivation of T. reesei on cellulose, xylan, a mixture of
plant polysaccharides or indeed lactose promotes high levels of ex-
pression of cellulase and hemicellulase genes (Mach and Zeilinger,
2003; Seiboth et al., 2007). Sophorose is thought to be the natural cel-
lulase inducer (Sternberg and Mandels, 1979; Vaheri et al., 1979).
That notwithstanding, genomic analysis casts little mechanistic light
on its enormous protein secretion capacity. Despite its effectiveness
in degrading plant polysaccharides, suggesting it should contain ex-
pansions of genes encoding enzymes capable of digesting plant cell
walls, T. reesei contains fewer genes encoding glycoside hydrolases
(total 200) than other phytopathogens such as F. graminearum
(total 243) and Magnaporthe grisea (total 231), A. oryzae (total 285)
or A nidulans (247). It was also noted that while plant polysaccharases
often contain a carbohydrate-binding molecule (CBM) within its re-
lated group of fungal genomes (N. crassa, F. graminearum, M. grisea
and T. reesei), they had the smallest number of CBM-containing
proteins.
First efforts to produce heterologous proteins in T. reesei focussed
on calf chymosin (Harkki et al., 1989; Uusitalo et al., 1991) after
which Nyyssonen et al. (1993) reported use of this host to produce
antibody fragments. It was observed that higher production of recom-
binant proteins was generally observed when the original source of
the gene encoding the protein was taxonomically related to the re-
combinant host. Cellulase gene promoters are most often incorporat-
ed into cassettes for production of recombinant proteins by
Trichoderma (Penttila, 1998; Schmoll and Kubicek, 2003), most fre-
quently the signal peptide of Cel 7a (CBHI) which mediates efficient
recombinant protein secretion. This topic was reviewed by Schuster
and Schmoll (2010).
Three recombinant endoxylanases from Chaetomium thermophilum
were expressed in T. reesei with a view to facilitating their production
for application in biobleaching of kraft pulp (Mantyla et al., 2007).
The expression cassettes utilized the strong T. reesei cel 7A promoter.
The host was a low protease producer where deletions in the endoglu-
canase I, endoglucanase II and cellobiohydrolase I genes rendered it the
desired low cellulase producer for applications in kraft pulp treatment.
It was demonstrated that a commercially viable recombinant thermo-
stable xylanase can be produced by T. reesei. Recently, the industrially
interesting biocatalyst cinnanoyl esterase from an unsuitable host, the
anaerobic fungus Piromyces equi, was successfully expressed in recom-
binant T. reesei as a more suitable producing host (Poidevin et al.,
2009).
Substantial effort has focussed on transforming fuel ethanol yeast
strains with cellulolytic genes from Trichoderma species to facilitate
their ability to ferment cellulose to ethanol. In a recent example,
Huang et al. (2010) described cloning and expression of the endoglu-
canase gene egVIII from Trichoderma viride into Saccharomyces
cerevisiae.
3.3. Penicillium
Limited genomic sequencing information appears to be available
on potential recombinant protein-producing filamentous fungi other
than Aspergillus and Trichoderma species. As selected Penicillium spe-
cies, for example Penicillium purpurogenum, Penicillium funiculosum
and Penicillium (Talaromyces) emersonii are high producers of cellu-
lases, hemicellulases and pectinases, they may have considerable po-
tential as recombinant protein-producing hosts. Chavez et al. (2010)
carried out transformation studies and demonstrated high transfor-
mation frequencies in two cheese ripening fungi, Penicillium
camemberti and Penicillium roqueforti, which exhibit low protease
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
activity. They concluded that these species had all of the right strain
characteristics as suitable hosts for production of recombinant pro-
teins. Gonzalez-Vogel et al. (2011) recently identified a number of
protein complexes containing enzymes including arabinofuranosi-
dases, beta-glucosidase, xylanases, acetyl esterases and ferulyl ester-
ases, in the soft rot fungus P. purpurogenum using a proteomics
strategy. Guais et al. (2010) prepared a partial DNA library for P.
funiculosum and sequenced genes encoding four GH54 α-L-
arabinofuranosidases. This organism has been used to produce com-
mercial mixtures of enzymes degrading complex agricultural residues
containing cellulose, hemicellulose, arabinoxylan, arabinogalactan,
proteases etc., with applications as a feed additive to enhance feed di-
gestibility. The enzyme mixture contained more than fifty separate
proteins (Guasi et al., 2008). The strong gpdA promoter from A.
nidulans was used to promote overexpression of pectin lyase by Pen-
icillium griseoroseum in submerged fermentation production systems
(Cardoso et al., 2010). Penicillium canescens was transformed with a
vector encoding the laccase of Trametes hirsute under control of an ef-
ficient promoter of the bgaS gene of P. canescens and efficiently
expressed and secreted the recombinant protein (Abianova et al.,
2010).
As might be expected, the predominant effort related to sequenc-
ing and annotation of Penicillium has been directed at the principal
producer of penicillins, P. chrysogenum. While antibiotic-producing
strains are generally not considered as suitable hosts for production
of natural or recombinant enzymes or other proteins for use in
foods or pharmaceuticals, some of the P. chrysogenum genomic infor-
mation may be applicable to development of non-antibiotic-
producing Penicillium strains as recombinant protein-producing
hosts. Promoters of the genes encoding glutamate dehydrogenase,
β-acetylhexosaminidase and gamma-actin from P. chrysogenum may
be used to construct potent vectors for expression and secretion of
homologous and heterologous proteins in these strains and also in
other hosts (Barredo Fuente et al., 2001).
3.3.1. P. chrysogenum
The complete genome sequence of the penicillin producer P.
chtysogenum Wisconsin 54–1255 strain (ATCC 28089, see Elander,
1983) was published in 2008. Genome size was 32.19 Mb, compara-
ble with that of other filamentous fungi, and the total gene number
was 12,943 (van den Berg et al., 2008). In addition to cellular func-
tional characterization of the P. chrysogenum genes, particular atten-
tion was paid to the penicillin biosynthetic genes. This information
may provide more general direction for manipulation/engineering
of metabolic pathways to increase production of natural target me-
tabolites or indeed to facilitate production of wholly novel metabo-
lites in filamentous fungi. The transcriptomes of the sequenced
strain and a high penicillin-producing strain were compared and
as might have been expected, many of the genes involved in synthe-
sis of the penicillin precursors, valine, cysteine and α-aminoadipic
acid were observed to be increased in the high penicillin-producing
strain. Some genes were identified which control β-lactam output
and genes with predicted roles as transporters appeared to be upre-
gulated under penicillin-producing conditions. Culmination of this
work clearly represents a milestone for future metabolic engineer-
ing strategies, which of course may involve participation or use of
recombinant proteins.
3.4. Rhizopus
A number of important extracellular industrial and medical en-
zymes are produced by the zygomycetes, including the important mi-
crobial rennets produced by Rhizomucor miehei and Rhizomucor
pusillus and digestive lipases, proteases and amylases are produced
by Rhizopus arrhizus. However, the major fungal genomics resources
related to this group of filamentous fungi have been directed to
1125
pathogenic strains and indeed the first of the zygomycetes to be
fully sequenced was Rhizopus oryzae which is the primary cause of
the potentially lethal angioinvasive mucormycosis infection (Ibrahim
et al., 2003; Kwon-Chung and Bennett, 1992). Related Mortierella spe-
cies are of great interest in the area of lipid production and molecular
transformations involving these species are being investigated (Mac-
Kenzie et al., 2000). Nevertheless, as is indicated below, some of the
genomic information is directly relevant to the long established abil-
ities of this and related strains to produce hydrolytic enzymes.
3.4.1. Rhizopus oryzae
The genome sequence of R. oryzae strain 99–880, isolated from a
fatal infection of mucormycosis, has recently been published (Ma et
al., 2009). Total length of the R. oryzae genome was found to be
45.26 Mb while total number of protein-encoding genes was 17,467.
Evidence was provided that there is whole-genome duplication in
this strain mainly attributed to an ancestral duplication event. Of spe-
cific interest to diagnostic and therapeutic treatment of mucormyco-
sis is the genomic characterization of expanded families of cell-wall
synthesis enzymes required for fungal cell wall metabolism but
which are not present in mammalian hosts and hence which may
be targeted by novel future drugs. Of interest both to therapy as
well as to use of R. oryzae and related species as hosts for recombinant
protein production are the annotated expanded gene families of se-
creted proteases characterized, especially aspartic proteases and sub-
tilases. It was suggested that these proteases may mediate the
pathogenic infection process, as these enzymes have previously
been thought to be associated with virulence of pathogenic Rhizopus
species (Schoen et al., 2002; Spreer et al., 2006). In this case, these
proteases may mediate penetration of hyphae through decaying or-
ganic matter (Ma et al., 2009).
3.5. White rot fungi
White rot fungi are basidiomycetes that are of great interest as en-
zyme producers as they produce unique extracellular oxidative en-
zymes that degrade lignin which surrounds and protects cellulose
microfibrils of plant cell walls, especially woody plants. The white
rot fungi are particularly important because they degrade the lignin
while not attacking the cellulose. These filamentous fungi are the
only microbes capable of efficient depolymerization and mineraliza-
tion of lignin. P. chrysosporium has been the most intensively studied
white rot fungus. White rot fungi secrete an array of peroxidases and
oxidases that attack lignin non-specifically by producing lignin-free
radicals, which subsequently facilitate spontaneous cleavage reac-
tions (Kirk and Farrel, 1987). These enzymes also participate in deg-
radation of organic pollutants in bioremediation. Recently,
high-resolution two dimensional electrophoresis-based proteomics
coupled to LC-MS/MS was used to monitor enzyme expression and
chemical products present during the process of degradation of aro-
matic substrates by P. chrysosporium, as a means of gaining a better
insight into the process of lignin degradation (Matsuzaki et al.,
2008). Not surprisingly, the first basidiomycete genome to be se-
quenced was the white rot fungus P. chrysosporium.
3.5.1. P. chrysosporium
Its thirty million base-pair genome was sequenced using a whole
genome shotgun method. The genome length was 29.9 Mb, similar
in size to most of the other sequenced filamentous fungi genomes.
The genome contains 11,777 protein coding genes. Analysis of the ge-
nome indicates an array of genes which encode secreted enzymes, in-
cluding oxidases, peroxidases and hydrolytic enzymes which are
known to co-operatively cause wood decay (Martinez et al., 2004).
Recombinant proteins have been expressed in a variety of basidio-
mycetes. For example, a vector encoding interleukin-32, the human
cytokine associated with some inflammatory and autoimmune
1126 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
diseases, was successfully introduced and expressed in the edible
mushroom P. eryngii, via an A. tumefaciens transformation (Chung et
al., 2011). There is continuing interest in expressing the diverse
degrading enzymes from basidiomycetes in more conventional indus-
trial work-horse hosts. For example ligninolytic basidiomycetes con-
tain a sugar oxidoreductase (pyranose dehydrogenase) that has very
broad substrate specificity towards breakdown constituents of ligno-
cellulose. In order to extend the biodegradative capability of more
conventional industrial strains, this enzyme from A. meleagris was
heterologously expressed in A. nidulans and A. niger (Pisanelli et al.,
2010). The white rot fungus, T. versicolor produces two groups of lac-
cases with several isoforms. Two of these laccases were expressed as
recombinant enzymes in A. oryzae and the recombinant enzymes
exhibited catabolic degradative activity against hydroxylated PCBs
(Fujihiro et al., 2009). Rodgers et al. (2010) noted that while basidio-
mycetes are the predominant sources of laccases with potential large
applications in delignification, basidiomycetes are in general not as
versatile or suitable as industrial fermentation producers as com-
pared to ascomycetes and consequently much effort has focussed on
transforming the more suitable fermentation hosts to produce recom-
binant basidiomycetes laccase. However, there have been problems in
achieving production of recombinant laccases in good fermentation
hosts primarily due to glycosylation deficiencies and these challenges
are currently being addressed with a view to mass producing effective
laccases.
Sakaki and Munetsuna (2010) have surveyed the various enzymes
which could co-operate to degrade complex pollutants such as poly-
chlorinated dibenzo-dioxins and furans, including angular dioxygenase,
cytochrome P450 (CYP), lignin peroxidase, manganese-dependent per-
oxidase and dehalogenase and concluded that combinations of distinct
enzymes could have significant application in these biodegradations.
Given that white rot fungi already produce lignin and Mn-dependent
peroxidases and CYPs it was concluded that supplementing this host
by adding additional recombinant capability would make this organism
a very powerful bioremediation strain. While the risks associated with
releasing genetically engineered organisms to the environment were
recognized, it was suggested this could be addressed by creating suicid-
al engineered strains (Paul et al., 2005).
3.6. Fusarium
While a high profile Fusarium species, F. graminearum is the caus-
ative agent of some important plant diseases, other Fusarium strains
are used in fermentations processes, including production of single
cell protein approved for human consumption and some of these
strains may have potential for production of recombinant proteins.
Nevertheless, the predominant scientific research to date has fo-
cussed on F. graminearum which causes plant diseases of substantial
economic importance including Fusarium ear root of maize and
head blight of cereals. In addition F. graminearum produces myco-
toxins in infected plants which, if they find their way into food and
feed products, constitute a health risk.
3.6.1. F. graminearum
The sequencing and annotation of F. graminearum was reported by
Cuomo et al. (2007) and gene annotation information was revisited
by Wong et al. (2011). Updated resource information may be
assessed at http://mips.gsf.de/genre/proj/FGDB/.
The Cuomo et al. paper indicates a genome size of 36.1 Mb includ-
ing 32 genes being predicted plant cell-degrading enzymes, including
xylanases, pectate lyase and cutinases which were postulated to func-
tion in pathogenesis by facilitating plant tissue penetration and mac-
eration and nutrient provision for the invading organism. The recent
annotated information indicated a set of 13,718 protein coding genes.
3.7. N. crassa
While N. crassa is not recognized as an important industrial host, it
is included in this discussion as a powerful model filamentous fungal
system which has been characterized biochemically and genetically.
This host can be grown at high growth rates in simple defined
media and can produce high amounts of recombinant proteins. The
approximate genome size is 40 Mb, and it contains about 10,000 pro-
tein-coding genes and many of the genes involved in interesting aspects
of Neurospora biology, including its secondary metabolism have been
annotated (Colot et al., 2006; Galagan et al., 2003). Up-to-date informa-
tion may be obtained online from http://www.fgsc.net.
Tian et al. (2009) applied microarray and shotgun proteomics
analysis on strains of a cellulolytic N. crassa fungus grown in different
media in order to combine data from gene expression and the proteome/
secretome in an attempt to better understand the cellulose-degrading
system and the principal genes involved.
Recently, N. crassa has been used as a host for production or re-
combinant subunit vaccines including influenza hemagglutinin (HA)
and neuraminidase antigens (NA) (Allgaier et al., 2009). High molec-
ular weight particles containing NA could be generated in a hetero-
karyon expression system facilitating downstream processing on the
one hand but also enables mixtures of different antigens to be co-
expressed together, thereby facilitating tailoring of a vaccine directed
at a particular pathogen target or variant.
3.8. Selected key genomic resources
A variety of institutional and online resources are available to re-
searchers with interests in genomic aspects of filamentous fungi and
are clearly relevant to the topic of recombinant protein production
by these hosts. Reference is made to some of these below:
http://www.aspgd.org/ “is the home of the Aspergillus Genome Da-
tabase, a resource for genomic sequence data and gene and protein in-
formation for Aspergillus species. AspGD is based on the Candida
Genome Database and is funded by the National Institute of Allergy
and Infectious Diseases at the US National Institutes of Health”. Subsites
deal with the annotated Aspergillus genomes of strains of A. fumigatus,
A. clavatus, A. nidulans, A. niger, A. oryzae and Aspergillus terreus.
The aim of the JGI Fungal Genomics Program is “to scale up sequenc-
ing and analysis of fungal genomes to explore the diversity of fungi in
DOE mission areas and to develop the Genomic Encyclopedia of Fungi
in the areas of: Plant feedstock health (mycorrhizal symbiosis, plant
pathogenicity, biocontrol); Biorefinery (lignocellulose degradation,
sugar fermentation, industrial organisms) and Fungal diversity” (http://
jgi-psf.org/programs/fungi/about-programs.jsf). Subsites deal with im-
portant filamentous hosts including Aspergillus carbonarius, P. chrysos-
porium, Sporotrichum thermophile, Thielavia terrestris, T. versicolor and
T. reesei.
The Fungal Genome Initiative (FGI) of the Broad Institute of MIT
and Harvard “produces and analyzes sequence data from fungal or-
ganisms that are important to medicine, agriculture and industry.
Over 50 fungi have been sequenced or are being sequenced, including
human and plant pathogens as well as fungi that serve as basic
models for molecular and cellular biology. In partnership with the
wider fungal research community, organisms are selected for se-
quencing as part of a cohesive strategy that considers not only the
value of data from each organism given their role in basic research,
health, agriculture, and industry, but also their value in comparative
genomics”. It includes databases on R. oryzae and on the Fusarium
Comparative project. (http://broadinstitute.org/scientific-community/
science/projects/fungal-genome-initiative/fungal-genome-initiative).
The Fungal Genetics Stock Center (http://www.fgsc.net) “is a re-
source available to the Fungal Genetics research community and to
educational and research organizations in general. The FGSC is funded
largely by a grant from the National Science Foundation (Award
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
Number 0235887) of the United States of America and to a lesser extent
by the payments made by researchers who use our services. Most fungal
strains in the FGSC collection are listed in the online searches. Specific
groups of materials are listed by category include: Neurospora,
Aspergillus, Fusarium and Magnaporthe, Ustilago, Cryptococcus, other
Fungi”. The FGSC together with other organizations is a major sponsor
of the Fungal Genetics Conference (http://www.fgsc.net/26thFGC/
index.htm).
The above websites in turn provide links to other resources.
4. Improving recombinant protein expression in filamentous fungi
4.1. Molecular strategies
A primary practical motivation for studying gene expression in fil-
amentous fungal hosts is to understand the molecular mechanisms of
transcription regulation in these organisms and to improve recombi-
nant protein expression, especially by the study of DNA sequences
participating in transcription initiation and/or regulation and selec-
tion of strong promoters. Transcriptional regulation of extracellular
plant cell wall-degrading enzymes produced by filamentous fungi
has been reviewed by Aro et al. (2005).
The promoter regions of the Aspergillus amylase genes consist of
four highly conserved sequences, one of which (region IIIa) is essen-
tial for high-level expression and another of which (Region IIIb) con-
tains sequences thought to enhance expression in combination with
region IIIa (Minetoki et al., 1998). A sequence of CCAAT present in
the promoter region of the A. nidulans amdS (encoding acetamidase)
is required for high-level expression of amdS, and related CCAAT se-
quences are present in the promoter regions of a number of other A.
nidulans genes (Papagiannopoulos et al., 1996). One of the most
strongly expressed genes in A. oryzae, the enolase gene (enoA), con-
tains a15-bp element with a sequence essential for transcription reg-
ulation of the gene (Toida et al., 2000). The melO promoter appears to
be effective as a mediator of strong synthesis of recombinant proteins
in Aspergillus hosts (Ishida et al., 2001). The A. oryzae TAKA-amylase
promoter preceded by its upstream activating sequences, was found
to be suitable for expression of protein products in Aspergillus species
(Boel et al., 1996). Berka et al. (2002) patented novel vectors contain-
ing polyadenylation sequences linked to the 3′ terminus of the DNA
sequence encoding the heterologous protein and which may include
promoter and signal sequences for promotion of expression and se-
cretion of heterologous proteins in filamentous fungi. Schmoll et al.
(2010) described the construct used to produce class 1 hydrophobin
from A nidulans in T. reesei. When the class II hydrophobin-encoding
promoter from T. reesei, hfb2, was used with lactose as carbon source,
the majority of the recombinant protein was secreted into the medi-
um by T. reesei. In contrast when the T. reesei cel7A promoter was
used, the recombinant protein was not secreted into the medium,
but remained cell wall-bound. High expression of the fumR gene,
which encodes fumarase in a high fumaric acid producing strain of
R. oryzae, was observed under good fumaric acid-producing condi-
tions (high sugar, low N) and the regulation of this gene may be of in-
terest for production of recombinant proteins and metabolic
engineering in Rhizopus species. Gene expression was primarily regu-
lated at the level of transcription.
4.1.1. Gene-fusions strategies
Some early recombinant research on filamenous fungal sought to
produce recombinant proteins by ‘coat-tailing’ a hyper-produced
and secreted homologous protein with subsequent cleavage of result-
ing fused proteins. Thus, techniques involving fusing the target gene
to the 3′ end of a homologous gene encoding glucoamylase improved
production of recombinant proteins, for example, of mammalian pro-
teins, by filamentous fungi (Gouka et al., 1997a, b). Fusions to the glu-
coamylase gene of A. niger/A. awamori promoted production of high
1127
levels of a variety of secreted recombinant proteins including bovine
prochymosin (Ward et al., 1990), hIL-6 (Broekhuijsen et al., 1993),
hen egg-white lysozyme (Jeenes et al., 1993), human lactoferrin
(Ward et al., 1992, 1995), and phytases from A. awamori (Martin et
al., 2003).In the case of chymosin and lactoferrin production, gram
to multigram quantities of recombinant product were produced per
liter when the high-level-production strains were put through a mu-
tation program (Dunn-Coleman et al., 1991; Ward et al., 1995). En-
hancements to this approach involved use of the catalytic domain of
glucoamylase rather than the complete enzyme (Gouka et al.,
1997b). To facilitate subsequent cleavage of the two protein ele-
ments, a linker proteolytic processing site is incorporated between
the carrier moiety component and the protein of interest. The linker
region is designed to allow the catalytic domain and the rest of the fu-
sion protein to fold independently. The N-terminal fungal protein ap-
pears to serve as a carrier, improving translocation of the
recombinant protein into the ER, as well as its folding, is mediated
by the N-terminal fungal protein. Subsequently in most cases the fu-
sion protein is cleaved facilitating secretion of the separate proteins
by a KEX2-like endopeptidase at a KEX2 recognition site introduced
specifically into the fusion protein as a linker, as indicated above
(Broekhuijsen et al., 1993; Punt et al., 2002; Ward et al., 1990, 1995).
Fidelity of cleavage of the KEX2 processing site: Sometimes, aber-
rant forms of the recombinant product are observed when gene
fusion strategies are employed. When a part of the fungal glucoamy-
lase protein (GAM), linked via a KEX2 processing site, was also used
in a gene-fusion strategy in A. niger to produce extracellular bovine
pancreatic trypsin inhibitor (BPTI), aberrant forms of the recombi-
nant protein were attributed to possible variations in A. niger
KEX-2-like endoprotease point of attack of the GAM-BPTI fusion pro-
tein or indeed involved another endoprotease (MacKenzie et al.,
1998). For example, while the desired recombinant protein is normal-
ly linked to the glucoamylase via a Lys-Arg KEX2-like cleavage site in
A. niger, the fidelity of cleavage to release mature protein is not al-
ways observed to be consistent and appears to be also influenced by
sequences immediately downstream and upstream of the KEX2 site
(Spencer et al., 1998).
The protein, neoculin (NCL), naturally produced in the fruits of the
tropical plant Curculigo latifolia, is about 500 times sweeter than
sugar. It is a heterodimer consisting of an N-glycosylated acidic subu-
nit (NAS) and a basic subunit (NBS) linked by disulphide bonds. Re-
combinant neoculin (rNCL) was produced in A. oryzae by using
separate NAS and NBS constructs, each fused to the A. oryzae
α-amylase via KEX2 cleavage sites (Nakajima et al., 2006). The NAS
component was properly N-glycosylated and the sweetness proper-
ties of the rNCL were comparable with the native NCL.
Gene fusion strategies are also exploited to produce expressed
proteins containing a tag that may facilitate product extraction during
downstream processing. By way of example, Collen et al.(2001) ge-
netically engineered endoglucanase (Cel7B) from T. viride with a pep-
tide extension containing non-polar tryptophan-proline residues
which facilitated preferential partitioning of the protein into the less
polar phase of an aqueous two phase model system.
4.1.2. Overproduction of foldases and chaperones
Foldases catalyze the isomerizations and disulfide bond forma-
tions and molecular chaperones, which are non-catalytic, mediate
folding of the nascent polypeptides into functional proteins and pre-
vent non-productive protein–protein interactions (Conesa et al.,
2000). Chaperones may act in diverse ways such as identifying defec-
tive proteins in the ER, inducing synthesis of folding enzymes or in-
deed ER-associated protein degradation responses for degradation
of defective proteins.
It has been postulated that hyper-production of recombinant pro-
teins into the ER has the potential to overload the folding, assembly
and secretion machinery of filamentous fungi. Therefore, the effects
1128 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
of overexpression of genes for several ER chaperones and foldases in
filamentous fungi, including bipA (from a family of binding proteins,
BiP), pdiA (from a family of protein disulfide isomerase) and a family
of calnexins on overproduction of recombinant proteases have been
evaluated (Conesa et al., 2001; Jeenes et al., 1997; Ngiam et al.,
2000; van Gemeren et al., 1997). It was found that filamentous
fungi overproducing specific proteins, both homologous and heterol-
ogous, exhibited increased levels of bipA transcription, whereas direct
interventions to overexpress bipA overexpression appeared not to af-
fect yields of secreted proteins (Punt et al., 1998). Overproduction of
fungal proteins generally increased bipA mRNA levels in A. niger. In
the case of two transformed A. niger strains which produced HEWL,
a twofold induction in bipA mRNA levels was observed (Ngiam et
al., 2000). BiP overexpression did not increase secreted levels of
hIL-6 in Aspergillus (Gouka et al., 1997a) and pdiA overexpression
did not increase secreted yields of HEWL in A. niger (Ngiam et al.,
2000). Disruption of a vacuolar protein sorting receptor gene in A.
oryzae, which targets aberrant and recombinant proteins for vacuolar
degradation, enhanced production and secretion of the bovine chy-
mosin and human lysozyme heterologous proteins (Yoon et al.,
2010).
4.1.3. Glycosylation
Glycosylation patterns from filamentous fungi are more similar to
those of mammals than the patterns observed in common yeast hosts
(Maras et al., 1999a, 1999b; Nevalainen et al., 2005). The two main
glycosylation processes common to eukaryotes involve N- and
O-glycosylation, whereby oligosaccharides attach to the beta-amide
moiety of asparagine residues and mainly to serine and threonine
β-hydroxy groups. N-glycosylation involves transfer of pre-
assembled glycosyl precursors to specific asparagine residues of the
nascent polypeptide chain after which glycosidase- and glycosyl
transferase-mediated modifications of the oligosaccharide occur
resulting in production of a common trimannosyl-chitobiose core
with branched N-acetylglucosamine residues generating the high
mannose N-glycans characteristic of filamentous fungi and yeasts.
O-glycosylation in fungi starts in the endoplasmic reticulum, and in-
volves O-mannosylations resulting in the sequential build up of the
O-glucosyl structure. Geysens et al. (2009) has recently used analysis
of the genome sequences to review folding, secretion and glycosyla-
tion, especially the N-glycosylation processes while Goto (2007) has
described the O-glycosylation process, both in Aspergillus.
Filamentous fungi have two distinct alpha-1,2-mannosidases, one
of which is similar to the mammalian Golgi alpha-1,2-mannosidases
that trim 3 mannose moieties off Man8GlcNAc2 to form Man5GlcNAc2
as substrate for GlcNAc transferase 1, and another distinct fungal
alpha-1,2-mannosidase (Ichishima et al., 1999; Yoshida et al., 2000).
However, the mammalian-like enzyme is neither well expressed nor
secreted such that very little of the lower mannosylated moiety gets
transferred (Maras et al., 1997). N-glycans from fungi also differ
from mammalian N-glycans in having terminal altered substituents
such as glucose, galactose or phosphoesters (De Pourcq et al., 2010).
Maras et al. (1997) employed recombinant mammalian
beta-1,4-galactosyl transferase and alpha-2,6-sialyltransferase to
make T. reesei cellobiohydrolase 1 more mammalian-like with respect
to its glycosylation pattern. Recombinant human β-1,2-GlcNAc trans-
ferase was subsequently overexpressed in Trichoderma, thereby en-
hancing its GlcNAc transfer capability (Maras et al., 1999a, 1999b)
and similar transformations with the corresponding rat GlcNAc trans-
ferase were implemented in A. nidulans (Kasajima et al., 2006). Kainz
et al. (2008) has carried out other molecular stratefies to successfully
produce lower mannosylated Man3GlcNAc2 N-glycans in recombi-
nant Aspergillus strains.
For production of therapeutic proteins, glycoform is very impor-
tant as incorrectly glycosylated proteins, for example recombinant
human therapeutic glycoproteins produced by filamentous fungi,
may induce an immune response in the patient being treated, reduc-
ing treatment efficacy. Engineering humanized glycosylation path-
ways into filamentous fungi, including trimming the branches of
high mannose-containing glycoproteins, has been found to be very
complex (Gerngross, 2004).
Antitrypsin, the human a1-proteinase inhibitor (a1-PI), is the
most abundant inhibitor of serine proteases in plasma (Brantly et
al., 1991). Progressive emphysema develops in antitrypsin-deficient
patients ultimately leading to death (Crystal, 1996). Conventional
antitrypsin-inhibitor replacement therapy uses a limited plasma de-
rived source which has created momentum for production of the re-
combinant form. While several hosts have been tested for efficacy of
production, altered glycosylation patterns or complete absence of gly-
cosylation in the recombinant product reduced in vitro stability of the
inhibitor and resulted in its rapid removal from the circulation system
(Karnaukhova et al., 2006).
A mature and biologically active glycosylated recombinant a1-PI pro-
duced by A. niger exhibited improved stability over a non-glycosylated
recombinant product produced by E. coli (Karnaukhova et al., 2007).
The recombinant protein was fused to a well secreted native fungal pro-
tein with a KEX2 recognition site at the fusion junction which was
cleaved in vivo by a KEX2-type protease. Implementation of strategies
for increasing glycosylation in Aspergillus resulted in increased pro-
duction of the recombinant protein chymosin (van den Brink et al.,
2006). In one case, a poorly used glycosylation site within the chy-
mosin molecule was improved resulting in much more efficient pro-
duction of the glycosylated chymosin. In the second case, when the
N-glycosylation site was located away from the native chymosin at-
tached via a linker, a substantial increase in recombinant protein
was observed.
4.1.4. Other molecular strategies
The following are miscellaneous examples of molecular strategies
used to enhance production of recombinant proteins by filamentous
fungi:
– Hastrup et al. (1997) proposed production of a proenzyme, in
cases where the enzyme was unstable or harmful to the producing
host, which could be proteolytically activated after secretion.
– An activator protein binding site containing the CCAAT sequence
was identified within the cis regulatory region of the A. niger
glaA gene. Insertion of multiple copies of this binding site into
the promoter of transformed recombinant plasmid sequence en-
hanced promoter production of the heterologous protein (Liu et
al., 2003).
– Berka et al. (2002, 2003) disclosed constructed novel vectors,
which encoded the desired heterologous polypeptide and a secre-
tory sequence functional in the filamentous fungus secretory
system.
– A. oryzae produces two predominant proteases, serine-type car-
boxypeptidase (CPase) and aspartic endopeptidase under acidic
conditions (Takuchi and Ichishima, 1986).
A typical antisense control strategy, whereby vectors are created
to express a high level of the antisense RNA complementary to
the RNA transcript of a target gene, used to inhibit fungal gene ex-
pression, was used to isolate an low CPase-producing A. oryzae
mutant expressing high and stable levels of lysozyme (Zheng et
al., 1998).
– Researchers had limited success in striving for overproduction of
manganese peroxidase in its natural host, P. chrysogenum (Cullen,
1997). However, a combination of strategies including use of a
strong glucoamylase promoter, a protease-deficient A. niger host,
culture pH manipulation and incorporation of hemin into the cul-
ture medium facilitated strong recombinant enzyme production
(Broekhuijsen et al., 1993; Conesa., 2001; Conesa et al., 2000;
Punt et al., 2002; Stewart et al., 1996).
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
– Promoters of the genes encoding glutamate dehydrogenase,
beta-acetylhexosaminidase and gamma-actin from P. chryso-
genum may be used to block expression of undesired genes
through anti-sense construction (Barredo Fuente et al., 2001).
4.2. Protease-deficient strategies
Production, properties and classification of microbial proteases
have recently been reviewed (Ward., 2011; Ward et al., 2009). In
addition to the observed variabilities in processing of fusion proteins
by KEX-like endoproteases in Aspergillus discussed above recombi-
nant protein-degrading fungal proteases have long been known to
be problematic (Ward et al., 2006). Braaksma and Punt (2008)
reviewed various strategies for controlling protease activity as a
means of supporting recombinant protein production. Methods in-
cluded classical selection of protease mutants, molecular genetic
methods to construct protease mutants, targeted to protease genes
and protease regulators, manipulation of fermentation conditions,
specifically pH, control of metabolites/catabolites such as carbon, nitro-
gen, sulfur and phosphorus, induction of proteases and physiological
and morphological effects.
With enzyme-overproducing industrial strains, one approach was
to partially inactivate some of the more prominent extracellular pro-
teases, for example the alkaline proteases and the metallo-proteases
(Christensen and Lehmbeck, 2000). Buxton and Gabor (1997) patent-
ed a sequence encoding the vacuolar PEPA aspartic protease and
methods for transforming strains to produce the protease and per-
haps more importantly for development of Aspergillus mutants defec-
tive in the production of aspartic protease. Given that filamentous
fungi can contain as many as 80 proteolytic genes of varying known
and unknown function, researchers are cautioned against trying to
develop mutants deficient in multiple proteases (Machida, 2002). Im-
pacts on recombinant protein production of constructing stable A.
niger recombinants containing up to three disrupted protease genes
were characterized (Van den Hombergh et al., 1997). Specific mu-
tants of A. nidulans, deficient in the aspartic protease gene, exhibited
the ability to produce chymosin as well as other recombinant proteins
(Berka et al., 2003). When the alkaline protease gene of a strain of A.
oryzae was transformed to produce heterologous endoglucanase, en-
hanced production and stability of the recombinant protein was ob-
served in shake flask cultures (Lehmbeck., 2001).
Antisense RNA may be used to reduce expression of particular
genes, including proteases in recombinant hosts. PEPB protein, re-
cently characterized as a member of the glutamic proteases, was
thought to be the causative agent in degradation of recombinant
thaumatin in A. awamori containing a disrupted pepA gene producing
inactive PEPA. Thaumatin production was improved by expression of
pepB antisense RNA, but results indicated antisense mRNA had only
partially silenced pepB gene expression. A substantial further increase
in thaumatin production was achieved by disruption of the pepB gene
(Fujinaga et al., 2004; Moralejo et al., 2002).
Disruption of some protease regulator genes has been effective in
substantially reducing protease activity in Aspergillus species. For ex-
ample, disruption of the prtT gene which is a regulatory gene which
encodes a member of the Zn-binuclear cluster family appears to elim-
inate two Aspergillus proteases from the medium including PEPA and
reduces total protease activity by 80% (Punt et al., 2008). Yoon et al.
(2011) reported on experiments which demonstrated how successive
disruption of ten protease genes in A. oryzae was effective in enhanc-
ing heterologous production of human lysozyme and bovine chymo-
sin production.
Manipulation of fungal culture pH away from the optimal pH for
activity and implementation of cultivation strategies which prevent
release of intracellular proteases via mycelial cell lysis have been shown
to reduce proteolysis of secreted recombinant proteases (Denison,
2000; O'Donnell et al., 2001; Wang et al., 2005). Use of peptide-rich
1129
media, typically induces protease production by A. niger (Ahamed et
al., 2005) and productivity of secreted egg lysozyme by a recombinant
strain of A. niger was reduced in such rich media (Archer et al., 1990).
Double disruption of the two protease genes in A. oryzae, tppA and
pepE, facilitated an increase of 63% in the production level of human ly-
sozyme (Jin et al., 2007). Combination strategies of using non-protease
inducing medium and use of the aspartyl protease inhibitor pepstatin
represent an alternative strategy to minimizing the impacts of proteases
on fungal recombinant protein activity (Ahamed et al., 2005). In two re-
cent proteomic studies involving A. niger, it was observed that under
conditions of culture starvation, resulting from depletion of carbon
source, proteases were found to predominate in the secretome and
hence these conditions should be avoided to minimize protease secre-
tion during production of recombinant proteins (Adav et al., 2010;
Braaksma et al., 2010).
4.3. Manipulations of morphology
Vegetative growth involves hyphal extension and occurs at the
hyphal tip. Branching leads to new hyphal extension units. The hy-
phal tip is the principal region of protoplasmic activity, protein pro-
duction and extracellular protein secretion and hence this is the
principal locus for biological process-related recombinant protein
production. Further back from the tip, protoplasmic compartments
become more vacuolated. It follows that a greater degree of branching
will increase rates of fungal growth, protein synthesis and extracellu-
lar protein secretion. Morphology of the mycelium is strongly influ-
enced by the surrounding environment and other factors including
inoculum size and type (vegetative, spores, etc.). On the surface of
solid media, filamentous fungi grow as mycelial mats. In submerged
cultures, fungi may attach to suspended particles if present or grow
as diffuse filamentous mycelia or as dense pellets, which may develop
to different sizes. Morphological form influences rate of growth and
product formation. Predominant growth and metabolism of fungi in
pelleted form occurs at the pellet surface where there is maximum
access to nutrients and oxygen. Inside the pellet, inward diffusion of
nutrients and outward diffusion of product become limiting and
vacuolization and lysis are frequently observed. Recently, Driouch et
al. (2010) described a novel approach, involving use of silicate micro-
particles, to engineering different morphology states in A. niger to im-
prove enzyme production.
Because of morphological problems noted for Aspergillus species in
fermenters which result in rheology and viscosity problems leading
to mass transfer limitations, Jensen (1997) proposed use of alterna-
tive thermophilic fungal hosts for production of recombinant pro-
teins. It was observed that when thermophilic fungal strains
including Acremonium, Corynascus, Thielavia, Myceliophthora, Thermo-
ascus and Chaetomium species, were grown in batch fermentations
under the same conditions used to culture A. oryzae, medium viscos-
ities observed were much lower.
Impact of morphology changes, as they effect recombinant protein
production, may be at least partially related to protease production or
release. Growth of the A. niger mycelium as large pellets was associat-
ed with lower specific protease activities and increased specific glu-
coamylase activities were found when A. niger was cultured in
media which generated large pellets (Papagianni and Young, 2002).
In general, fungal pelleted growth mediates greater lysis in fungi,
for example, in Aspergillus species, and this results in the presence
of higher levels of proteolytic activity in filtrates of pelleted cultures,
as compared to filamentous growth (Ahamed et al., 2005). While the
greater proteolytic activity in pellet cultures is likely to be partly due
to intrapellet cell lysis, differential expression may also be a factor.
Dai et al. (2004) has reported that one of seven genes that were dif-
ferentially expressed in A. niger pellets encoded a pepsin-type prote-
ase. pH could be manipulated to cause morphological mutant
formation and recombinant glucoamylase production in A niger
1130 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
(Swift et al., 1998). In studies of an A. niger strain containing a gene
for the Gla-GFP fusion protein, protease activity in pelleted growth
was only one-third of that observed with filamentous growth (Xu et
al., 2000).
Morphology of the filamentous fungus, Chrysosporium lucknowense,
has been manipulated genetically to produce a non-filamentous form,
thereby reducing viscosity of culture systems. The non-filamentous
form also exhibits low protease activity and is capable of producing
very high yields of heterologous proteins (Verdoes et al., 2007).
General aspects of the relationships between morphology and
productivity in filamentous fungi have been reviewed by Grimm et
al. (2005).
4.4. Solid-state fermentation approaches
While the mechanisms have not been fully elucidated, variations
between solid-state and submerged culture fermentation conditions
can alter the expression of genes and rates of enzyme production
(Iwashita, 2002). During growth of A. oryzae on solid substrates, re-
gions of differentiation of filamentous mycelia exist, from aerial my-
celium to medium-associated mycelium, and the different regions of
filamentous growth mediate differential levels of gene expression
and product formation (Masai et al., 2005). In some cases, the con-
trasting activities reflect whether the activity is cell wall-associated
or excreted into the culture medium: in a case in point in submerged
cultures, some enzyme activities were associated with the cell wall,
whereas in solid-state cultures, they were secreted into the medium
(Wang et al., 2005). In solid state culture, wheat bran media caused
lowest expression of catabolic genes, which were thought to have re-
leased catabolite repression of synthesis of hydrolytic enzymes,
thereby mediating rich hydrolytic enzyme gene expression. Oda et
al. (2006) concluded that certain enzymes were selectively secreted
under conditions of solid-state culture or in submerged culture, inde-
pendent of the composition of the medium. In a different but a partly
analogous situation, proteome differences were observed when F.
graminearum was cultured in vitro and in planta, with some proteins
being expressed in only one of the two conditions (Paper et al., 2007).
Imanaka et al. (2010) compared cultivation of A. oryzae, using
three different culture methods, i.e., shake-flasks, agar-plate and
membrane surface liquid culture, and observed differences in growth,
secretion of proteases and alpha-amylase, secreted protein level and
gene transcriptional profile by DNA microarray analysis. Protease ac-
tivities, especially oryzin (alkaline protease) and alpha-amylase, were
much higher in agar-plate and membrane surface liquid culture than
in shake flasks. Transcriptional gene profiles from the agar-plate and
membrane surface liquid culture manifested somewhat similar pat-
terns but were quite different from the shake flask profiles.
In some cases, solid state culture promoted production of a more
homogeneous glycosylated recombinant product. Submerged cul-
tures by A. oryzae transformed to produce the important heterologous
antigenic protein Pre–S2 of human hepatitis B virus, resulted in pro-
duction of a partially degraded heterologously glycosylated protein,
whereas in solid-state culture a homogeneous glycosylated form of
the whole fusion protein was obtained (Maruyama et al., 2000). Use
of wheat bran (2%) in solid state culture mediated a 500-fold increase
in production of recombinant chymosin as compared with submerged
culture (Tsuchiya et al., 1994). te Biesebeke et al. (2002) found that
pelleted growth in submerged fermentations and semi-solid fermen-
tations showed different patterns of gene expression and protein
secretion.
4.5. Concluding comments
Because of their extraordinarily high productivities and simple
growth requirements, filamentous fungi can compete or outcompete
most other recombinant hosts in processes for production of
non-glycosylated recombinant proteins, assuming for a particular
product that problems related to recombinant protein degradation by
proteases of the host producing strain have been resolved. It is also
clear that where the interest relates to recombinant glycosylated pro-
teins, especially recombinant proteins for human therapeutic use, the
quite different native protein glycosylation patterns observed in fungi
render them unsuitable hosts for recombinant human glycoprotein
production, and some other hosts, for example Pichia species, may be
more suitable microbial hosts to be engineered for mammalian glyco-
protein production. Nevertheless, research is continuing to be aimed
at engineering the appropriate human glycosylation machinery into fil-
amentous fungi.
5. Recombinant proteins in metabolic engineering of fungi
In recombinant protein production, the initial priority targets
were individual proteins and glycoproteins for beneficial industrial,
agricultural, food or pharmaceutical use. However, the development
of genomic and proteomic capabilities combined with molecular
tools provides the tools to manipulate hosts with respect to more
complex combinations of proteins, with diverse functions, be they
catalytic, regulatory or with other physiological roles. As other
organisms, the filamentous fungi offer potential opportunities for im-
proving desired metabolite product yields, reducing or eliminating
undesired metabolite bi-products, extending substrate ranges and in-
troducing new enzymes or pathways leading to production of new
products (Kern et al., 2007). For example, hosts may be transformed
to produce the multiple enzymes associated as part or all of a new
metabolite biosynthesis sequence. Alternatively, an existing pathway
may be engineered to address bottlenecks and maximize rate of me-
tabolite biosynthesis or indeed to alter specificity of one or more reac-
tions to facilitate expansion of product range. Approaches invariably
involve genetic modifications, including expression of recombinant
enzymes and manipulation of host transcription and/or translation
machinery. The opportunities to exploit filamentous fungi for produc-
tion of a vast range of new products, most of which are not yet con-
ceived, is enormous and derives from the diverse nature of fungal
species. With an estimated 1.5 million species (Hawksworth, 2001),
only a small number of which has been sampled by genomic and pro-
teomic methods, and with a likely average of 10,000 genes per spe-
cies, the resource is almost unlimited. Some of these concepts, as
they apply to primary and secondary metabolites, are briefly dis-
cussed in this section through use of selected examples.
5.1. Primary metabolites
5.1.1. Citric acid
With respect to primary metabolites, A niger has been investigated
as a target model for metabolic engineering of filamentous fungi, es-
pecially with respect to citric acid production with a view to increas-
ing metabolic fluxes through the glycolytic pathway leading to citrate
production (Ruijter et al., 2002). Seven or more enzymes were select-
ed as principal targets and two genes encoding regulatory enzymes in
the pathway were overexpressed (Ruijter et al., 1997; Torres et al.,
1996).
5.1.2. Biodiesel
Biodiesel, originating from some form of biological host, arguably
represents part of the “biosolution” to the world's energy problems
(Demain, 2009). The products of fungi and algae are currently being
used as beneficial food and feed additives and nutritional products.
While it is still unclear if fungal and algal strains are appropriate for
production of microbial oils for biodiesel production, they do contain
the machinery for bio-oil biosynthesis and the tools for transforming
their biosynthetic enzymes to other hosts, which may be deemed
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
more suitable for energy production, are well on the way to being
established.
Several filamentous fungi accumulate very high amounts of micro-
bial lipids, especially highly unsaturated lipids (Ward and Singh,
2005). The concept of using filamentous fungi as producers as lipids
was introduced during World War 1 in Germany and processes
using a variety of strains including Mortierella isabellina, Mortierella
alpina and Mucor vinacea have been scaled up to produce commercial
quantities of lipids (Ratledge, 1978). While the primary emphasis was
on the nutritional benefit of unsaturated fatty acids, scientists in the
Soviet Union considered the Mucorales as potential candidate hosts
for production of lipids for use in biofuels (Feofilova et al., 2010). If
momentum in this area continues, strategies will be needed for opti-
mization of processes which will include detailed examination of the
lipid synthetic pathways, in order to determine metabolic bottle-
necks. In addition, sequencing and annotation of the genes encoding
the participating enzymes will provide opportunities for transforma-
tion of hosts to produce recombinant enzymes. These biochemical
and molecular approaches may be combined with fermentation opti-
misation strategies, both to optimize production rates and yields and
to produce products of varying lipid compositions.
5.1.3. Higher unsaturated fatty acids (HUFAs)
M. alpina produces the C-20 highly unsaturated fatty acids
dihomo-gamma-linolenic acid, arachidonic acid and eicosapentaenoic
and has been used for industrial production of arachidonic acid
(Shimizu et al., 1997). In mammals, γ-linolenic acid (GLA) is an inter-
mediate of the (n−6) pathway conversion of linolenic acid to arachidonic
acid and a direct precursor of dihomo-gamma-linolenic acid. Certain
condi tions (aging, stress, some infections, diabetes, eczema) may be
manifested as causing a deficiency of Δ6-desaturation and of GLA, and
hence there is considerable interest in investigating production of re-
combinant forms of Δ6-desaturase and the efficient enzyme from M.
alpina as a target of interest. GLA is further metabolized to produce
anti-inflammatory eicosanoids, such as prostaglandins and leukotrienes,
and GLA and its metabolites also regulate expression levels of various
gene products. As a first step, this enzyme was cloned from M. alpina
and expressed in A. oryzae under the control of the amyB promoter.
While GLA was not produced by the control A. oryzae strain, GLA
constituted 25.2% of total fatty acids in the recombinant host
transformed to express Δ6-desaturase (Sakuradani et al., 1999).
Similar enzymes have now been cloned from a range of filamentous
fungi including Cunninghamella echinulata, R. arrhizus, Rhizopus nigricans,
Rhizopus stolonifer, Mucor rouxii and Pythium irregulare. The gene encod-
ing the enzyme from R. stolonifer was recently expressed in Pichia
pastoris, enabling the transformed Pichia strain to produce about 22.4%
of its fatty acid content as GLA (Wan et al., 2011).
5.2. Secondary metabolites
Some of the recent exciting developments in fungal secondary
metabolite production relate to the continuing developments in our
knowledge of mechanisms for production of non-ribosomal peptides
(NRPs) and polyketides (PKSs). While the A. nidulans genome indi-
cates the presence of 26 PKSs and twenty four NRPSs, only six
PKSs (synthesizing monodictyphenone, asperfuranone, orsellinic
acid/F9775, asperthicin, napthopyrone, sterigmatocystin) and four
NRPSs responsible for the biosynthesis of terrequinone, aspyridones,
emericellamide and penicillin, are reported to have been character-
ized in A. nidulans (Balibar et al., 2007; Bergmann et al., 2007; Chiang
et al., 2010; Evans et al., 2011; Sanchez et al., 2009; Szewczyk et al.,
2008; von Dohren, 2009).
5.2.1. Engineered biosynthesis of novel peptides
In addition to the predominant and ubiquitous ribosomal machin-
ery for synthesis of peptides and proteins, certain organisms,
1131
including filamentous fungi, contain complex peptide synthetase
templates which mediate synthesis of a variety of bioactive peptides,
more than 300 of which are known, without ribosomal involvement
(Stachelhaus et al., 1996).The bioactive products include antibiotics,
toxins, enzyme inhibitors, antiviral agents, antitumor agents and im-
munosuppressants. These non-ribosomal large protein templates
contain protein domains which catalyze synthesis of a wide range of
low molecular weight linear, branched and cyclic peptides which
may be further transformed by acylation, glycosylation and other re-
actions (Marahiel, 1992). Research is ongoing related to manipulation
of the participating genes encoding synthetases with altered sub-
strate specificities, using gene disruption and reconstitution strate-
gies, with a view to determining the potential to engineer synthesis
of novel peptides, including novel antibiotics and other therapeutics
(Kleinkauf and von Döhren, 1990; Stachelhaus and Marahiel, 1995).
5.2.2. Antibiotics
Biochemistry of non-ribosomal peptide synthesis has been inves-
tigated for many years as it relates, for example, to production of pep-
tide antibiotics, including beta-lactams by the filamentous fungi P.
chrysogenum and A. nidulans and production of cyclosporin A by Toly-
pocladium niveum (Weber et al., 1994; Aharonowitz et al., 1993;
Smith et al., 1990; Gutierrez et al., 1991).
Numerous genes involved in biosynthesis of antibiotics and other
secondary metabolites have been cloned, including genes involved in
the synthesis of β-lactam antibiotics by the filamentous fungi, P.
chrysogenum, Cephalosporium acremonium and A. nidulans (Martín and
Liras, 1989). As with other antibiotics, genes involved in penicillin,
cephalosporin and cephamycin biosynthesis are organized in clusters
and pathway-specific regulatory genes positively or negatively modu-
late genes expression in these clusters and hence regulate antibiotic
biosynthesis (Loder and Abraham, 1971; Martín and Liras, 1989;
Nuesch et al., 1987). Positive regulatory elements also impact the inter-
related processes of secondary metabolism and differentiation in these
organisms. Clustering of biosynthetic enzymes is facilitating research
into the molecular mechanisms which mediate expression of genes in-
volved in antibiotic synthesis, which will enable gene expression to be
positively regulated and optimized to systematically maximize antibiot-
ic formation in industrial fermentation processes (Martin, 1992).
Routes to production of penicillin G, cephalosporins and cephamycins
share a common early pathway to synthesis of the isopenicillin-N inter-
mediate. The ‘natural’ cephalosporins produced by A. chrysogenum have
relatively low clinical efficacy and are produced semi-synthetically from
intermediate building blocks, 7-aminodeacetoxycephalosporanic acid
(7-ADCA) and 7-aminocephalosporanic acid (7-ACA). While P.
chrysogenum cannot produce cephalosporins and cephamycins, it is an
attractive potential recombinant host because of its hyper capacity to
produce penicillin, via the common precursor isopenicillin N and in-
deed the host was successfully engineered to synthesize cephalosporins
and 7-ADCA (Crawford et al., 1995; Robin et al., 2001; 2003a,b; van de
Sandt and de Vroom, 2000). The principal transformations of P.
chrysogenum involved introduction of the gene cefE from Streptomyces
clavuligerus, encoding production of the expandase enzyme deacetoxy-
cephalosporin-C synthase, and the genes cefEF and cefG from P.
chrysogenum, encoding the dual expandase/hydroxylase and acetyl-
transferas, respectively. Other recombinant proteins, for example the
cmcH gene from S. clavuligerus, encoding deacetylcephalosporin
O-carbamoyltransferase, have been successfully expressed in P.
chrysogenum with a view to producing other novel cephem precursors
(Harris et al., 2009). This work demonstrates how metabolic engineer-
ing strategies involving production of recombinant enzymes into strains
already improved through classical mutation programs can lead to de-
velopment of multipurpose host platforms for production of non-native
secondary metabolites.
In related work, two dimensional electrophoresis-based proteo-
mics was used to compare protein maps, with up to 950 proteins
1132 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
being identified, from a three P. chrysogenum strains, a high and low
penicillin producer and a wild-type strain (Jami et al., 2010). The cys-
teine biosynthesis, pentose phosphate and some stress response pro-
teins predominated in the high penicillin producing strain while
pathways for synthesis of other metabolites were reduced.
Peroxisomes participate in important functional aspects of second-
ary metabolite synthesis in filamentous fungi, including the final steps
of penicillin synthesis, mediated by isopenicillin N-acetyltransferase
(Muller et al., 1991;1992).
5.2.3. Taxol
The tetracyclic diterpene lactam, taxol, is a low toxicity broad
spectrum anticancer drug with beneficial application against breast,
uterine and other cancers (Wani et al., 1971). Taxol is present natu-
rally at low concentrations of 0.01–0.05% in the bark, roots and
branches of the western yew, Taxus brevifolia, an endangered species.
In the past few years, significant progress has been made in our un-
derstanding of the general biology and taxol biosynthetic pathway
of taxol-producing endophytic fungi, with a view to producing taxol
by microbial fermentation (Lin et al., 2003; Zhou et al., 2008, 2010).
Some of the 20 or so genes encoding enzymes of the pathway have
been sequenced and some genetic transformation systems have
been established which included development of some fungal promo-
ter-containing expression cassettes, ultimately directed at developing
efficient taxol-producing recombinant fungal hosts (Guo et al., 2006;
Wang et al., 2007a,b,c).
6. Expressing fungal multi-protein complexes or pathways in
other hosts
While much effort has been fruitfully invested in transforming
hosts to produce individual recombinant proteins and to optimizing
recombinant product formation, the evolution in our understanding
of these systems combined with newly acquired knowledge of geno-
mics and proteomics is facilitating more advanced engineering of
hosts with whole new pathways or functions. The huge diversity of
fungal species makes fungi a rich reservoir for mining novel biosyn-
thetic capabilities and clearly the native fungal hosts of new path-
ways, deemed to be of value, may not be suitable hosts for optimal
expression of the pathway, or synthesis and exploitation of the de-
sired metabolic products. In this section, a small number of fungal
complex systems expressed in non fungal hosts will be briefly dis-
cussed. It is hoped that the examples point to the great potential to
exploit fungal diversity in part through transfer of fungal machinery
to other hosts.
6.1. Expression of fungal systems in plants
There is considerable interest in metabolic engineering of fatty
acids into plants to produce new oil seed crops. Since filamentous
fungi are among the best producers of highly unsaturated fatty acids
and since higher plants do not produce these fatty acid, there is inter-
est in transforming plants with some of these key highly unsaturated
fatty acids (HUFA) biosynthetic enzymes, with M. alpina being recog-
nized as one of the most useful gene sources (Drexler et al., 2003;
Huang et al., 1999; Knutzon et al., 1998; Michaelson et al., 1998). Ini-
tial strategies involve enhancing the very long chain fatty acid con-
tent of current plant oils. Some of the early promising steps
involved seed-specific co-expression of an oleate delta12 desaturase
and a Δ6 desaturase, both from Mortierella species, in canola (Liu et
al., 2001). The fifth generation of field trials contained 44% γ-linolenic
acid (GLA) not present in rapeseed. Similar high proportions of GLA
were accumulated when a Δ6 desaturase from P. irregulare was
expressed in Brassica juncea (Hong et al., 2002). Some other fungal
long chain polyunsaturated fatty acid (LC-PUFA)-synthesizing en-
zymes are also being used as part of the strategy to produce HUFA
oils in plants. A bifunctional Δ12- and Δ15-desaturase from Fusarium
moniliforme, co-expressed in both soybean and yeast (Yersinia
lipolytica) with the primary LC-PUFA biosynthetic enzymes, enhanced
levels of eicosapentaenoic acid (EPA) production (Damude et al.,
2006). Enzymes with similar biofunctional properties and potential
are produced by Claviceps purpurea (Meesapyodsuk et al., 2007) and
Coprinus cinereus (Zhang et al., 2007). A recent review on metabolic
engineering of oil-seed crops to produce omega-3 long chained fatty
acids shows where recombinant HUFA biosynthetic enzymes from fil-
amentous fungi play their part (Venegas-Caleron et al., 2010).
Plants and filamentous fungi such as Ascomycota, have different
pivotal amino acid linked processes for ammonium assimilation. In
filamentous fungi, NADP(H)-dependant glutamate dehydrogenase
(GDH) and glutamine synthetase participate in ammonium assimila-
tion whereas in plants glutamine synthetase alone is primarily re-
sponsible for assimilation. When the gene encoding GDH, gdhA,
from A. niger was expressed in the cytoplasm of rice plants, positive
changes in metabolism were observed which lead to enhanced am-
monium assimilation and better plant growth (Abiko et al., 2010).
Hydrophobins, surface-active proteins obtained from filamentous
fungi, have significant potential applications in genetically-
engineered plants and hydrophobin-fusion technology is predicted
to become a very useful tool in plant biotechnology. By way of exam-
ple, when the hydrophobin HFBI sequence from T. reesei was fused to
green fluorescent protein (GFP) and transiently expressed in
Nicotiana benthamiana plants mediated by A. tumefaciens, the fusion
enhanced GFP accumulation (Joensuu et al., 2010). The fusion protein
constituted more than 50% of total soluble protein and delayed leaf
necrosis.
6.2. Expression of fungal systems in bacteria and yeast
6.2.1. Polyketide biosynthetic machinery
Polyketides result from biosyntheses involving polymerization of
acetyl and propionyl subunits in a manner analogous to fatty acid
synthesis. Polyketide products, generated by different PKS types, in-
clude erythromycin, tacrolimus, epothilone, tetracycline, tetraceno-
mycin, daunorubicin, 6-methyl salicylic acid, aflatoxin B1 and
lovastatin (Crawford et al., 2006; Fujii, 2010; Kennedy et al., 1999;
Moriguchi et al., 2010; Shen, 2003; Watanabe et al., 1996). Fungal
polyketide synthases (PKSs) utilize a variety of acyl-CoA substrates
to catalyze carbon to carbon bond forming reactions to synthesize
the well known complex fungal polyketides which exhibit a range
of bioactivities. Many gene clusters, thought to encode the enzymes
responsible for synthesizing polyketides such as bikaverin, zearale-
none and hypothemycin, including genes encoding PKSs, have been
characterized. Recent reports indicate that these recombinant fungal
enzymes have been expressed in E. coli rendering the bacterial host
capable of production of the corresponding secondary metabolite
(Saruwatari et al., 2011). The aromatic polyketide, 6-methylsalicylic
acid, is synthesized by ATX, encoding gene atX, present in A. terreus
and Penicillium patulum, and this gene has successfully been
expressed in E. coli, S. cerevisiae and Streptomyces species and an en-
hanced production of 6-methylsalicylic acid was noted in the case of
S. cerevisiae (Bedford et al., 1995; Kealey et al., 1998). The huge fungal
gene responsible for bikaverin (aromatic polyketide anticancer drug)
biosynthesis, PKS4 originating in Giberella fujikuroi, was successfully
expressed in E. coli with production of the polyketide (Ma et al.,
2007). Ma et al. (2009) also reported that the lovastatin nonaketide
synthase (LovB) from A. terreus, involved in synthesis of the choles-
terol lowering drug, lovastatin, could be efficiently expressed by a re-
combinant S. cerevisiae host, research work which is contributing
substantially to determining the complex set of functions and struc-
tures of the polyketide synthases. Mixing of domains of different
PKS genes, including domains originating from fungi and bacteria,
generated different combinations of biosynthetic enzymes and
 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
demonstrated the potential to vary the synthezing machinery to pro-
duce wholly novel metabolites not produced naturally by the native
strains (Saruwatari et al., 2011; Zhang et al., 2008).
6.2.2. Penicillin biosynthetic machinery
While current industrial production of penicillin is mediated by P.
chrysogenum, the intrinsic growth limitations of fungi in large fermen-
ters are recognized and there is interest in developing new hosts, for
example yeasts, for production of new beta lactams and perhaps
other bioactive compounds such as peptides. An industrial precedent
exists for changing from a filamentous fungal host to a yeast host in
the case of citric acid production when the Pfizer Company modified
their process by introducing a Candida guilliermondii yeast strain in
place of the conventional Aspergillus producer, thereby increasing cit-
rate production rate (Ward, 1989; 1991). In P. chrysogenum, production
of penicillin is compartmentalized in peroxisomes and the cytosol. The
yeast Hansenula polymorpha has a number of features that make it po-
tentially an attractive host as an alternative producer of beta-lactams,
including the availability of some very strong and regulatable pro-
moters. Furthermore, penicillin production in Penicillium is significant-
ly mediated by peroxisomal enzymes and it is known that peroxisomes
of H. polymorpha may be hyperinduced. Gidijala et al.(2009) success-
fully engineering H. polymorpha to produce and efficiently secrete
penicillin: a) by introducing the P. chrysogenum gene encoding
the non-ribosomal peptide synthase (NRPS) δ-(L-aminoadipyl)-
L-cysteinyl-D-valine synthetase (ACVS) into H. polymorpha resulting
in production of active ACVS enzyme; b) co-expression of the B. subtilis
stp gene encoding the enzyme phosphopantetheinyl transferase which
activates ACVS; and c) co-expression of P. chrysogenum genes encoding
cytosolic isopenicillin N synthase and the peroxisomal enzymes, isope-
nicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL). Re-
combinant fungal ACVS has also been expressed in baker’s yeast
(Siewers et al., 2009).
7. Engineering of pathogenic fungi
A very large number of filamentous fungi are pathogenic to other
organisms, including animals and humans, plants and insects and, in
the case of animals and plants, the resulting diseases have vast nega-
tive economic and social impacts. Molecular techniques, including
strategies involving production of recombinant proteins may be
used to facilitate elucidation of the causes of pathogenesis and to pro-
vide direction on solutions on pathogenesis problems. The following
brief examples are offered to illustrate potential opportunities.
7.1. Entomopathogens
Recombinant fungal technology offers potential to create recombi-
nant hosts which interact with other organisms. Examples would in-
clude transformed fungi which produce molecular species such as
herbicides, insecticides or biological inhibitors against undesired tar-
gets. Recently published examples include a recombinant fungus
which kills human malaria parasites in mosquitoes, mediated by se-
creted recombinant toxic peptides and enhancement of infectivity of
a recombinant entomopathogenic Beauveria bassiana towards larvae
of the oriental leafworm moth.
While there have been effective chemical pesticides against malar-
ia and its mosquito carrier, especially pyrethroids, the progress to-
wards eradication has been hampered by the development of
pyrethroid-resistant mosquitoes. Several fungi, including Metarhizium
anisopliae, are pathogenic to adult mosquitoes but the infective pro-
cess through contact with the cuticle is slow (12–14 days). Recombi-
nant strains of M. anisopliae expressing the antimicrobial toxin,
scorpine, or a salivary gland and mid-gut peptide 1 (SM1) reduced
entry and/or development of Plasmodium falciparum, the parasitic
causative agent of malaria, into the Anopheles gut where it gets
1133
transformed from gametocytes into infective sporozoites. Sporozoite
count reductions mediated by the expressed SM1 and scorpine were
71 and 90%, respectively. The SMI peptide binds to the surface of the
salivary gland of Anopheles gambiae, the principal malaria vector,
and blocks entry of the parasite into the mosquito vector. The scorpion
antimicrobial scorpine, is a hybrid of two toxins, a cecropin and a
defensin which is two orders of magnitude more toxic against Plasmo-
dium. A recombinant M. anisopliae expressing an [SM1]sub8:scorpine
fusion protein, reduced sporozite counts by 98% (Fang et al., 2011).
The infective action of the filamentous fungus, B. bassiana, towards
insects, via their cuticle, is generally a slow process. However, lepi-
dopteran pests are rapidly killed by ingestion of the Vip3A insecticidal
proteins produced by Bacillus thuriengensis. A recombinant strain of B.
bassiana, which highly expressed one of the Vip3A toxins, Vip3Aa1 in
its conidial cytoplasm, when sprayed on cabbage leaves was shown to
dramatically reduce larval numbers feeding on the cabbage leaves (by
26.2 fold) (Qin et al., 2010). The mid gut of the infected larvae con-
tained a digested form of the toxin and the larvae exhibited the
expected symptoms of toxin action, namely shrinkage and palsy.
Methods have also been established whereby larvae of organisms
such as the silk worm have been directly transformed to produce re-
combinant fungal proteins. For example, the 1320-bp endoglucanase
1 gene from T. viride was cloned and expressed in silkworm larvae
and in a silkworm cell line using a mutant baculovirus expression sys-
tem (Li et al., 2010).
7.2. Plant pathogens
Molecular methods are also being used to develop a better under-
standing of the mechanisms related to fungal pathogenicity of plants.
In addition to having an important role in secondary metabolite pro-
duction, as indicated above, peroxisomes are also involved in filamen-
tous fungal pathogenicity, for example, of cucumber by Colletotrichum
orbiculare and the rice blast pathogen, Magnaporthe oryzae (Bhambra
et al., 2006; Wang et al., 2007d) and in production of host selective
AK-protein toxins by Alternaria alternata which mediate its pathoge-
nicity to multiple plant species, including Japanese pear (Imanaka et
al., 2010). Imazaka et al. (2010) confirmed the peroxisome localiza-
tion of AK-toxins by developing and expressing recombinant
GFP-tagged AK-protein toxins in A. alternata and then used mutant
recombinant A. alternata strains, with AK-toxin disruptions, to con-
firm the role of AK-toxins in pathogenicity.
It was noted earlier (3.4.1) that the genome of the human R.
oryzae pathogen contained expanded gene families of encoding se-
creted proteases and that these proteases are involved in the infective
process and are characteristic of the virulence of Rhizopus pathogens
(Ma et al., 2009). It was also noted in the same section that R. miehei,
which causes fungal infections in humans and animals, can resist
higher concentrations of statins than is observed in Mucor
circinelloides, where statins inhibit HMG-CoA and ultimately lead to
Mucor cell death. A recombinant M. circinelloides strain expressing
the gene hmgR encoding the HMG-CoA of R. miehei, was rendered
more tolerant to statins. Clearly, our knowledge of fungal pathogene-
sis will greatly expand over the next few years as the genomes of
more plant pathogens are sequenced and their gene functions are
annotated.
8. Engineering of biocontrol fungi
Filamentous fungi can also have beneficial roles in plant disease
management as will be illustrated here by focusing on Trichoderma
species. Many strains of Trichoderma are highly competent in coloniz-
ing plant root surfaces and thrive and proliferate in association with
healthy root surfaces (Sriram and Ray, 2005). Many other fungi lack
this ability and Trichoderma species have evolved a variety of mecha-
nisms to attack and parasitize these other fungi and benefit from
1134 O.P. Ward / Biotechnology Advances 30 (2012) 1119–1139
nutrients from these other fungi as they support and enhance plant
growth as biocontrol agents (Harman et al., 2004). Biocontrol agents
utilize several antagonistic strategies against pathogens and all of
these mechanisms are exploited by Trichoderma (Dennis and
Webster, 1971a,b,c). These mechanisms include: competition by lim-
iting access to the pathogen's nutritional needs; production of
inhibitory metabolites or other toxins including trichothecin, sesqui-
terpene and certain enzymes; and parasitism. In the case of T. harzia-
num, it was noted that combined effect of production of an antibiotic
and production of hydrolytic enzymes had a synergistic effect on the
pathogens (Schirmböck et al., 1994). Enzymes thought beneficial to
Trichoderma species in attacking pathogens such as Fusarium oxy-
sporum and Botrytis cinerea include the lytic enzymes, chitinase,
beta-1,3-glucanases, and protease (Cherif and Benhamou, 1990;
Elad and Kapat, 1999; Geremia et al., 1993; Tronsmo et al., 1993).
When a recombinant chitinase from a soil bacterium Serratia
marcescens used to control Sclerotium rolfsii was introduced into T.
harzianum under control of a constitutive promoter, the constitutive
production of chitinase by the Trichoderma biocontrol agent enabled
it to overgrow the S. rolfsii pathogen (Haran et al., 1993). An alkaline
protease producing transformant of T. harzianum was thought to en-
hance its mycoparasitic activity (Sriram and Ray, 2005).
Of course, another approach to protecting plants against fungal
plant pathogens is to directly arm the plants with the requisite biode-
fense machinery and indeed several genes encoding the Trichoderma
enzymes mediating Trichoderma's parasitic role have been expressed
in plants, rendering them more resistant to fungal plant pathogens
(Bolar et al., 2000; Lorito et al., 1998; St Leger et al., 1996).
9. Conclusions
The principal focus over the past 25 years of recombinant protein
research efforts as applied to filamentous fungi related to the evalua-
tion and development of the workhorse fungi, especially the industri-
al extracellular enzyme-producing fungi, as hosts for recombinant
protein production. The special advantages of these organisms, name-
ly their abilities to grow at high rates and to high densities in com-
mercial fermenters and their natural abilities to secrete high levels
of homologous enzymes, are widely recognized. In exploiting these
advantages for production of recombinant proteins, researchers
have generally used the most effective regulatory, expression and se-
cretion machinery identified in these organisms and hooked it up to
the genes encoding the desired recombinant protein, an approach
which has met with considerable commercial success.
Filamentous fungi also have several disadvantages as hosts for
heterologous protein production, perhaps the most serious being
their abilities also to produce and secrete homologous proteases
which may degrade the desired recombinant product. In addition to
the secreted proteases, filamentous fungi may be subject to different
degrees of lysis during the fermentation process, the extent of
which will vary with other aspects of fungal physiology including
morphology, such that intracellular proteases may be released
which can attack the recombinant protein. In addition, there are sig-
nificant differences in the mechanisms of protein glycosylation ob-
served in fungi and mammalian cells. While considerable research is
being invested in modifying the glycosylation machinery in fungi to
use them as hosts for production hosts of more human-like glycopro-
teins, it is widely accepted that yeasts such as Pichia, may be better
candidate hosts for production of recombinant human therapeutic
glycoproteins.
The vast amounts of scientific information being generated
through advances in genomics and proteomics are expanding the op-
portunities for new applications for recombinant filamentous fungi. It
is facilitating a shift from simply inserting a gene encoding a single
protein product to inserting machinery encoding multiple genes, for
example encoding metabolic enzymes for a new or improved
biosynthetic pathway related to the production of current or new pri-
mary or secondary metabolites. While this activity is still at an early
stage, it will clearly gain momentum and will become a more impor-
tant pursuit in the field of industrial microbiology in the years ahead.
It has also been recognized that fungi represent an enormously
diverse range of organisms and hence a huge resource for novel bio-
synthetic systems, especially since the vast majority of fungal species
on the planet have yet to be cultured, identified and characterized. It
is clear that many of the fungi naturally harboring novel biosynthetic
machinery will likely not be the best hosts for large-scale exploitation
of these capabilities. It is expected that in many cases, the target bio-
synthetic abilities will be better utilized by transformation into other
fungal hosts, perhaps also to other microbial hosts, and in some cases,
to non-microbial hosts, for example, to plants.
One of the most exciting new aspects of the study of recombinant
proteins expression in fungi is the realization that the future applica-
tions will not be confined to the biomanufacturing capacities of these
organisms. Some of the natural beneficial impacts and especially the
deleterious impacts of fungi relate to their extremely complex inter-
actions with other organisms, for example through mutualism, com-
mensalisms and especially pathogenesis. About 300 fungi are
known to cause diseases in humans (Monk and Goffeau, 2008) and
many of these organisms exhibit multidrug resistance mediated by
multidrug efflux pumps (Balzi et al., 1994). A myriad of fungal patho-
gens attack plants and a quantum shift is currently taking place in the
advancement of our knowledge of the plant–microbe interactions in-
volved in these diseases (Boller and He, 2009). The underlying causa-
tive mechanisms involved in these attacks are now being
characterized using modern genomic and molecular strategies. Put
simply, the economic and social costs associated with the impacts of
fungal pathogenesis are enormous. The opportunities to exploit re-
combinant technology to unravel the causes and then bring forward
solutions will represent a very important future focus of recombinant
protein technology as it relates to fungi.
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