(2011) Production of ant Proteins by Filamentous Fungi

Biotechnology Advances xxx (2011) xxxndashxxx

JBA-06496 No of Pages 21

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Production of recombinant proteins by filamentous fungi

Owen P WardDepartment of Biology University of Waterloo Waterloo Ontario Canada N2L3G1

Tel +1 519 888 4567x32427 fax +1 519 746 06E-mail address opwarduwaterlooca

0734-9750$ ndash see front matter copy 2011 Published by Eldoi101016jbiotechadv201109012

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KeywordsAspergillusTrichodermaPenicilliumRecombinant proteinHeterologousProteaseGenomeFilamentousFungiPathogenesis

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-choderma Penicillium and Rhizopus species was to produce single recombinant protein products An earlyrecognized disadvantage of filamentous fungi as hosts of recombinant proteins was their common abilityto produce homologous proteases which could degrade the heterologous protein product and strategies toprevent proteolysis have met with some limited success It was also recognized that the protein glycosylationpatterns in filamentous fungi and in mammals were quite different such that filamentous fungi are likely notto be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeuticuse By combining the experience gained from production of single recombinant proteins with new scientificinformation being generated through genomics and proteomics research biotechnologists are now poised toextend the biomanufacturing capabilities of recombinant filamentous fungi by enabling them to expressgenes encoding multiple proteins including for example new biosynthetic pathways for production ofnew primary or secondary metabolites It is recognized that filamentous fungi most species of which havenot yet been isolated represent an enormously diverse source of novel biosynthetic pathways and that thenatural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organismfor biomanufacture purposes Hence it is expected that substantial effort will be directed to transformingother fungal hosts non-fungal microbial hosts and indeed non microbial hosts to express some of thesenovel 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 forwardsolutions is expected to represent a very important future focus of fungal recombinant protein technology

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1 Introduction

Filamentous fungi are extraordinary organisms which impactwidely on so many aspects of our lives These organisms are charac-terized as having branched filamentous structures or hyphae havingtypical diameters of 2ndash18 um with (higher fungi) or without (lowerfungi) cross-walls or septae Higher fungi include AspergillusPenicillium Trichoderma and Fusarium species Lower fungi includeRhizopus andMucor species Filamentous fungi are chemo-organotrophsmeaning they obtain their energy and carbon by oxidation of organiccompounds

In traditional fermentation technology filamentous fungi are dom-inant producers of a range of primary metabolites including organicacids such as citric gluconic fumaric kojic itaconic acid and fattyacids They also produce important secondary metabolites especiallyas human therapeutics for example penicillin cephalosporin ergotalkaloids griseofulvin lovastatin taxol and zeranol Some are

producers of polysaccharides and biosurfactants Some filamentousfungi are food materials in their own right such as mushrooms singlecell proteinbiomass (single cell protein SCP) or indeed lipid-rich bio-mass (single cell oil SCO) whereas other fungi are components infermented foods Some are producers of an array of fungal enzymesfor example amylases amyloglucosidases cellulases pectinaseslaccasesligninases phytase proteases microbial rennets lipases andglucose oxidase Many intracellular fungal enzymes are also exploitedas biocatalysts in enzyme biotransformations in bioorganic synthesisreactions while others use biodegradative processes in soil bioremedia-tion Some strains are well known pathogens of humans animals andplants (Cutler et al 2007 Maor and Shirasu 2005 Segal and Walsh2006) while others for example mycorhizal fungi have beneficial asso-ciations with plants andor participate in nutrient recycling in soilSome fungi are responsible for food spoilage wood decay and infesta-tion of damp buildings (Bennett 2006) and many fungal spores areknown allergens (Meyer 2004)

The known high productivity chartacteristics of filamentous fungiare in part related to their inherent abilities to grow at high rates andto high biomass densities supported by low cost substrates in rela-tively simple fermenters For industrial fermentations filamentous

tous fungi Biotechnol Adv (2011) doi101016

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fungi may be cultivated using traditional surface culture methodswhere oxygen uptake involves passive exposure of the culture tothe atmosphere or in a semi-solid culture where a non-homogeneousculture may be aerated through various forced air andor mixingstrategies They may also be cultured in intensively mixed stirredtank reactors where the objective is to achieve conditions withinthe reactor which approach homogeneity thereby facilitating moresophisticated 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 demonstratedcapacities to hyperproduce and secrete enzyme proteins for exampleglucoamylase production by Aspergillus with impressive titers ofgreater than 25 gL

Processes for production of mold-modified foods have beenimplemented for many thousands of years At least the initial stagesof these fermented food processes were promoted by surface culturesuggesting a likely requirement for air as a source of oxygen to sup-port preferential growth of molds on the medium surfaces Since theraw materials were derived from plants including soybeans wheatand rice common logic led early scientists to conclude that themolds 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 participatein processes including pathogenesis of plants spoilage of fruits andvegetables and rotting of wood likewise with the presumed involve-ment of mold products which could mediate the biodegradation ofmajor structural constituents of these plant materials

Since the principal structural components of plants being subjectedto biodegradative processes during tradititional food fermentationsplant 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 thesepolymeric substances might require that the substrates be degradedin the extracellular environment Indeed simple methods for surfacecultivation of the molds involved on agar-like media containing indi-vidual substrates often insoluble demonstrated zones of hydrolysisaround the mold colony illustrating that depolymerization reactionshad been facilitated or catalyzed It was soon concluded that these bio-degradations were mediated by hydrolytic and other depolymerizingenzymes whichwere generally secreted by themolds into the extracel-lular medium or sometimes were locatedattached on the extracellularsurfaces of the biodegradative fungal organism Jokichi Takamine aJapanese immigrant to the United States was first to commercializean 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 ofsolid substrates such as wheat or bran clearly based on the traditionalprocesses 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 andDevchand 1992)

The development of recombinant technology harnesses the powerof many of these filamentous fungi as hosts in the production of spe-cific recombinant proteins as final products with applications in theagricultural food and nutrition biomedical and pharmaceutical andenergy and industrial sectors (Schuster et al 2002) This focus hasrepresented the principal effort in applied genetic engineering overthe past 25 years or so and is discussed in Sections 2ndash4 of this review

Further advancements of the core transformation technologiescombined with progress in the fields of genomics and proteomics isleading 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 intorecombinant hosts An example of these developments relates to ourability to engineer metabolic pathways so as to enhance production of

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primary or secondary metabolites or indeed to facilitate productionof 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 knowledgethat only a small number of the estimated 15 million species(Hawksworth 2001) which are thought to exist means filamentousfungi will continue to supply the biosynthetic tools for synthesis ofa myriad of novel products for countless years to come This researchis 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 notimply that a newly isolated natural host capable of biosynthesizingnovel new beneficial compounds will be a suitable host for largescale manufacture Indeed many novel organisms that will be identi-fied in the future are likely to be the ones that are hard to cultivateeven unculturable and will require better hosts be they other filamen-tous fungal organisms or non-fungal organisms Efforts are alreadybeing directed to exploiting the unique nature of some of the en-zymes or metabolic pathways or systems of filamentous fungi bytransferring 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 yetpoorly understood and their interactions often have overall beneficialand perhaps more frequently negative societal outcomes Thus someof the first filamentous fungal organisms to be sequenced werehuman 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 willinclude cloning and expression of recombinant proteins in fungi asprospective candidate causative proteins in pathogenesis followedby application of strategies to disrupt these proteins with a view torelating these manipulations to pathogenicity and virulence of thepathogen 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 inTable 1 including reference to some informative tables from these re-view papers

2 Filamentous fungi as hosts for productionof recombinant proteins

Many filamentous fungi are natural excellent producers of extra-cellular enzymes and hence are exceptional candidate hosts for theproduction of recombinant proteins (Iwashita 2002 Wang et al2005) 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 25ndash30 gL ofglucoamylase and Trichoderma reesei reported to be capable of pro-ducing 100 gL of extracellular protein (Demain and Vaishnav 2009)

Meyer (2008) discussed four common strategies for implementa-tion of transformations of filamentous fungi The protoplast-mediatedmethod involves use of cell wall-degrading enzymes for protoplastpreparation 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 withbatch variations in the lytic enzyme that a regeneration procedure isneeded and that the high copy number of insertions of DNA may resultin a less controlled transformation In the Agrobacterium tumefacienstransformation the A tumefaciens carries a binary vector containingthe target DNA between a 24-base pair repeating unit and a virulenceregion required for DNA transfer The gene-carrier organism is co-cultured with the filamentous fungus and the transformation is advan-tageous in that the low copy number of DNA insertions facilitates amore targeted integration Beijersbergen et al (2001) patented ameth-od for Agrobacterium-mediated transformation of mold species

teins by filamentous fungi Biotechnol Adv (2011) doi101016

Table 1Some prior reviews related to the topic of production of recombinant proteins by filamentous fungi

Reviewmdashshort title Reference Table Contents

Genetic engineering of filamentous fungi Meyer (2008) 1 Industrially important compounds produced by filamentous fungiRefining heterologous protein production in filamentousfungi

Sharma et al (2009) 23

Heterologous industrial enzymesImportant heterologous proteins in recombinant Aspergillus

Filamentous fungi as cell factories for heterologous proteinproduction

Punt et al (2002) 14

Fungal and yeast hosts for hIL-6Systematics of biotechnologically relevant true fungi

Aspergillus as a host for heterologous expression Lubertozzi and Keasling(2009)

Bioactice fungal metabolitesSome fungal conversionsSome fungal bioremediations

Transcriptional regulation of plant cell wall degradation byfilamentous fungi

Aro et al (2005)

Proteomics of filamentous fungi Kim et al (2007) 1 List of fungal proteomics papersProduction of human therapeutic proteins by yeasts andfilamentous fungi

Gerngross (2004)

Physiology and biotechnology of Aspergillus Ward et al (2006) IVVVIVIIVIIIIX

Aspergillus genes of industrial interestAspergillus recombinant food enzymesImportant heterologous proteins expressed in AspergillusPatents on recombinant protein production by Aspergillus

Aspergillus as a cell factory for protein production Braaksma and Punt (2008) 1 Effect of secreted protease activity of protease gene disruption strainsRecombinant protein production systems for Aspergillus Fleissner and Dersch (2010) 12 Recombinant protein production by aspergilla including hosts and

promotersGenomics of folding secretion glycosylation in aspergilli Geysens et al (2009) 1 Genes involved in protein-folding unfolded protein response

glycosylationBiotechnology 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) 1 Non-ribosomally synthesized antibiotics and producing hostsEngineering primary metabolic pathways of industrialmicro-organisms

Kern et al (2007)

Taxol-producing endophytic fungi Zhou et al (2010) 12

Taxol-producing strainsCommon transformation methods for filamentous fungi

3OP Ward Biotechnology Advances xxx (2011) xxxndashxxx

belonging the Ascomycotina Basidiomycotina Deuteromycotina Mas-tigomycotina and Zygomycotina and illustrated example transforma-tions for Aspergillus awamori Aspergillus nidulans A nigerColletotrichum gloeosporiodes Fusarium solani Fusarium graminearumNeurospora crassa T reesei Pleurotus ostreatus and Agaricus bisporusIn applying Agrobacterium-mediated transformation strategies to fila-mentous fungi for example A bisporus Romaine (2002) observedthat it was preferable to co-cultivate the bacterium with fruit body tis-sue rather than with spores A third transformation method whichoften requires protoplast preparation involves electric pulse-mediatedreversible membrane permeabilization to promote DNA uptake In con-trast the fourth more specialized method which can be implementedwithout cell wall removal involves shooting DNA-coated metal parti-cles at high speed into cells More specific molecular transformationsinvolve targeting of recombinant genes to a specific position in the ge-nome which will enhance transcription of newly introduced DNAandor deletion of genes with potential to reduce the positive effectsof the desired transformation be it production of a specific recombi-nant protein or insertion of multiple enzymes or proteins participatingin a specific metabolic pathway or other physiological event

In addition to DNA-based methods introduction of RNA-basedmethods such as antisense RNA hammerhead ribozymes and RNA in-terference approaches have been found to be very useful for silencingparticular genes in filamentous fungi (Fulci and Macino 2007Hammond and Keller 2005 Muller et al 2006 Yamada et al 2007)

Detailed classical physiological and biochemical knowledge isavailable for many of the candidate hosts and molecular techniquesincluding genome sequencing and annotation strategies These areproviding data to support efforts in optimizing expression and secre-tion of recombinant proteins in filamentous fungi Filamentous fungiespecially well studied Aspergillus species have also been shown toefficiently 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

A awamori and A oryzae appeared to be better filamentous fungalhosts for recombinant protein production than some other filamen-tous fungi The Mucor rennin gene under the control of a suitablealpha-amylase promoter introduced into A oryzae resulted in pro-duction yields of the heterologous protein of 33 gL (Christensen etal 1988)

Some perceived or suggested disadvantages of filamentous fungi asheterologous protein hosts relate to their relatively low frequencies oftransformation potential morphological defects and observed proteinmodifications due to protease activity or low pH (Kinghorn and Unkles1994 Radzio and Kueck 1997) It was observed that production levelsof most non-fungal recombinant proteins (mammalian bacterialavian plant etc) in filamentous fungi were generally lower as com-pared to those of homologous proteins and with likely bottlenecks atthe level of transcription and translation secretion with possible limi-tations also at the post-translational level (ie inefficient translocationfolding transport processing or secretion) (Broekhuijsen et al 1993Gouka et al 1997a Jeenes et al 1994)

A general model for fungal protein synthesis and secretion basedon Aspergillus species has been summarized by Fleissner and Dersch(2010) During synthesis proteins are directed into the endoplasmicreticulum where folding takes place and glycosylation is initiated InAspergillus species protein disulfide isomerase (Pdi) assists in thefolding and maturation of secretory proteins and the ability of PdiAto catalyze the refolding of denatured and reduced RNase has beendemonstrated (Ngiam et al 2000) Improperly folded or glycosylatedproteins are sent to the proteosome or vacuoles for degradation Fur-ther modification including glycosylation occurs in the golgi bodiesSNARE proteins facilitate vesicle-mediated trafficking of the proteinsto 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 (Gla499) which was successfully in-tegrated into the A niger genome Confocal fluorescence microscopyconfirmed that GFP was partially localized within the hyphal cell

Table 2Examples of GRAS notices filed since 1998 relating to filamentous fungi

Substance

8 Pectin esterase derived from Aspergillus oryzae carrying a gene encodingpectin esterase from Aspergillus aculeatus

10 Exopeptidase derived from Aspergillus oryzae carrying a gene encoding aleucine aminopeptidase from Aspergillus sojae

32 Pectin lyase derived from Trichoderma reesei carrying a gene encodingpectin lyase from Aspergillus niger

34 Aspartic proteinase derived from Aspergillus oryzae carrying a geneencoding aspartic proteinase from Rhizomucor miehei

43 Lipase derived from Aspergillus oryzae carrying a gene encoding lipasefrom Thermomyces lanuginosus

54 Xylanase derived from Fusarium venenatum carrying a gene encodingxylanase from Thermomyces lanuginosus

75 Lipase derived from Aspergillus oryzae carrying a gene encoding lipasefrom Fusarium oxysporum

89 Five enzyme preparations from Aspergillus niger Carbohydrase enzymepreparation catalase enzyme preparation glucose oxidase enzymepreparation pectinase enzyme preparation and protease enzymepreparation

90 Carbohydrase enzyme preparation from Aspergillus oryzae proteaseenzyme preparation from Aspergillus oryzae and carbohydrase enzymepreparation from Rhizopus oryzae

103 Lipase enzyme preparation from Aspergillus oryzae carrying a geneconstructed from a modified Thermomyces lanuginosus lipase gene and aportion of the Fusarium oxysporum lipase gene

106 Glucose oxidase enzyme preparation from Aspergillus oryzae carrying agene encoding a glucose oxidase from Aspergillus niger

111 Lipase enzyme preparation from Aspergillus niger113 Lipase enzyme preparation from Aspergillus oryzae122 Laccase enzyme preparation produced by Aspergillus oryzae expressing

the gene encoding a laccase from Myceliophthora thermophila132 Lactase enzyme preparation from Aspergillus niger142 Phospholipase enzyme preparation from Aspergillus oryzae expressing the

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 rateor more likely from a reduced mRNA stability It has been suggestedthat at least five structural components may influence mRNA stabilityIn the case of hil6 and aglA transcripts in Aspergillus species glaAfusions appeared to stabilize mRNA levels (Gouka et al 1997b)Jeenes et al (1994) reported similar results for fusions of egg-whitelysozyme with glucoamylase In many cases low levels of productionof recombinant proteins are due to post-translational secretionbottlenecks rather than transcription (Conesa et al 2001 van denHombergh et al 1997)

After secretion a major known problem for heterologous proteinsis their degradation by high extracellular enzyme producing filame-tous fungi perhaps most notably Aspergillus species which secrete adiversity of extracellular proteases (van den Hombergh et al 1997)Proteases have been shown to be responsible for degradation ofmany recombinant proteins (Broekhuijsen et al 1993 Roberts etal 1992)

Traditional fermentations for production of extracellular enzymesby filamentous fungi were based on fermented food processes Theseprocesses which involved surface or semi-solid media are non-homogeneous making fine process control impossible This motivat-ed desires to produce fungal extracellular enzymes in submerged cul-ture in stirred tank reactors Some early challenges with respect togrowing filamentous fungi in submerged culture related to the highviscosities which developed in the media caused by the increasedconcentrations 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 limitationof desired protein product yields These problems were addressed invarious ways at the engineering physiological and molecular levelsImprovements in fermenter design were directed towards increasingaeration while controlling mycelial shearing effects High growth andproduct formation rates were achieved by manipulation of fungalmorphology generally to reduce mycelial strand length and promoteformation of highly branched mycelia An example of a manipulationat the molecular level was provided by Akin et al (2003) who maxi-mized heterologous protein production by transforming the cellswith cotA-encoding nucleic acids controlled by a regulatable promot-er Some of these strategies are discussed in more detail elsewhere inthis paper

gene encoding a phospholipase A1 from Fusarium venenatum149 Beta-glucanase enzyme preparation from Trichoderma harzianum150 Glucosamine hydrochloride prepared from chitin obtained from

Aspergillus niger158 Lipase preparation from Aspergillus niger expressing a gene encoding a

lipase from Candida antartica183 Phospholipase A2 enzyme preparation from Aspergillus niger expressing a

gene encoding a porcine phospholipase A2195 Mixed beta-glucanase and xylanase enzyme preparation from Humicola

insolens201 Asparaginase enzyme preparation from Aspergillus oryzae expressing the

asparaginase gene from A oryzae214 Asparaginase enzyme preparation from Aspergillus niger expressing the

asparaginase gene from A niger230 Chymosin enzyme preparation from Trichoderma reesei expressing the

bovine prochymosin B gene238 Lipase enzyme preparation derived from Hansenula polymorpha

expressing a gene encoding a lipase from Fusarium heterosporum296 Lipase enzyme preparation from a genetically modified strain of

Aspergillus niger315 Transglucosidase enzyme preparation from Trichoderma reesei expressing

the gene encoding transglucosidase from Aspergillus niger345 Carboxypeptidase enzyme preparation from modified Aspergillus niger333 Acid fungal protease enzyme preparation from Trichoderma reesei

expressing the gene encoding acid fungal protease from T reesei372 Glucoamylase (GA) enzyme preparation from Trichoderma reesei

expressing the gene encoding the GA from T reesei

3 Survey of principal players

Some of the principal organisms involved in food fermentationprocesses were Aspergillus and Rhizopus species For example the ini-tial stage of production of soy sauce involves predominant growth ofA oryzae strains on a mixture of soybeans and wheat while produc-tion of tempeh involves cultivation of Rhizopus oligosporus on cookedsoybean mash Not surprisingly these organisms have also beenprime candidate hosts for production of recombinant proteins Acombination 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 newgeneration 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 DrugAdministration includes enzyme products from A niger and A oryzaeEndothia parasitica Mucor miehei Mucor pusillus and others Since1998 the FDA has published an inventory of notifications it has re-ceived regarding applications for GRAS recognitionexemption Themajority 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 httpwwwaccessdatafdagovscriptsfcnfcnNavigationcfmrpt=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 strategiesfor production of recombinant proteins through use of molecularmethods for industrial fermentations For example comparative ge-nomic studies among Aspergillus species suggest that A oryzae isenriched with genes which participate in the degradation of biomassand in primary and secondary metabolism (Kobayashi et al 2007)Also in A oryzae when cDNA microarrays and expressed sequence

tags were used to characterize transcriptional activity associated withenergy catabolism and hydrolytic enzyme production transcriptionlevels of most catabolic genes of the EM and TCA pathways were ob-served to be higher in glucose-rich conditions as compared withglucose-depleted conditions (Maeda et al 2004) As will be discussedlater interesting studies have also been implemented in traditional in-dustrial solid-phase media For example rich gene-expression profilesfor hydrolytic enzymes were observed in wheat-bran media whichexhibited lowest expression of catabolic genes This suggested the latterpoor expression may have released catabolite repression of hydrolyticenzyme synthesis Gene arrays gene deletion and insertion strategiesand other emerging molecular techniques will undoubtedly receivewidespread application as a means to better grasp and exploit themechanisms of industrial product formation regulation and secretionby Aspergillus species and other filamentous fungi (Akao et al 2002Bautista et al 2000 Moralejo et al 2002 Ngiam et al 2000 Sims etal 2004 Zarrin et al 2005)

Because of the resource intensive nature of genome sequencingand functional analysis in the case of filamentous fungi priority at-tention has focussed on the most important strains based on industri-al andor agricultural productivity considerations or on strains whichrepresent important human animal and plant pathogens In additionsome starting species were selected for genomic characterization forexample A nidulans where substantial beneficial prior physiologicaland genetic knowledge had already been established Consequentlypioneering genomic research was implemented on industrial andpathogenic Aspergillus species More recent interest in developingmore efficient systems for bioconversion of biomass to energy provid-ed the impetus to characterize the genome of the high cellulose andhemicellulase 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 forexample certain Penicillium Rhizopus Fusarium and Mucor strains aswell as some thermophilic fungi there was insufficient interestandor resources to substantially characterize these strains genetical-ly with respect to their enzyme production secretion function andpotential recombinant protein production potential In someof the latter cases for example in the case of Penicillium andMucorFusarium detailed sequencing and functional genomic studieshave been directed at the highest profile application of these organ-isms namely to penicillin production by Penicillium chrysogenumand to the plant pathogenic properties of F graminearum Mentionis made of this research below in case some of the genomic andfunctional findings from these studies become relevant and applica-ble to extracellular enzyme-producing strains as potential candidaterecombinant protein-producing hosts In addition molecular biolo-gists are applying recombinant technologies to investigate the uniquemetabolic properties of these organisms with the expectation that afuller understanding will lead to beneficial societal outcomes

A discussion follows highlighting progress in genomics research asit pertains to some of the more important industrial filamentous fun-gal strains Most filamentous fungi have estimated genomic sizes of30ndash40 Mb encoding 9000ndash13000 genes (Machida 2002)

31 Aspergillus

The genus Aspergillus consists of more than 180 officially recog-nized species most of which degrade plant polysaccharides (deVries 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) Aoryzae 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 relevantto recombinant protein production deal with A nidulans A oryzae A

niger and its awamori variant Detailed genetic analyses have alsobeen carried out on the human pathogen Aspergillus fumigatus butbecause of the severe toxigenic nature of this organism as a producerof the highly toxic aflatoxins it is not relevant as a host for biotech-nology production processes A nidulans is of particular interest as amodel filamentous fungal organism for studies of cell biology andgene regulation It is also related to A niger and A oryzae which arethe best natural filamentous fungal production hosts While Anidulans is the best genetically characterized Aspergillus species it isalso deemed to be unsuitable as a recombinant host for biotechnologyprocesses because it produces sterigmatocystin which is also a toxinalbeit much less severe than aflatoxins Aniger and A oryzae are alsorecognized potential broad based recombinant hosts for the biophar-maceutical industry for production of recombinant proteins

311 A nidulansConventional matings lead to the identification of greater than 900

genes in A nidulans (Brody et al 1991) The whole genome has a sizeof 301 Mb with eight well marked chromosomes containing approx-imately 10000 genes Physical and chromosomal linkage maps andgenome sequence have been described in detail (Archer and Dyer2004 Clutterbuck 1997 Galagan et al 2005 Lubertozzi andKeasling 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

312 A nigerSequencing of a derivative of the enzyme-producing strain A niger

NRRL 3122 (ATCC 22343 CBS 115989) indicated a genome size is359 Mb containing 14097 predicted genes (Archer and Dyer2004) The sequence data for A niger ATCC strain 9029 is held at thePacific Northwest National Laboratory (PNL) and is available to re-searchers upon request Genencor has access to the A niger genomesequence data of Integrated Genomics (Machida 2002) The JointGenome Institute (JGI) initiated a sequencing program for the citrate-producing A niger ATCC 1015 in 2004 as part of the United StatesDOE Genome Programwith participation of PNL and Oakridge NationalLaboratory Sequencing information on this strainwasmade available athttpwwwjgidoegovaspergillus The genome sequence and analysisof the ancestor of a current A niger enzyme production strains A nigerCBS 51388 indicates a genome size of 339 Mb (Pel et al 2007)Among the 14165 open reading frames identified strong functionswere predicted for 6505 of them This paper and supplementary infor-mation referenced on line includes detailed functional genomic analysesof protein secretion carbohydrases andproteases aswell 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 Aniger with genomic predictions of signal-peptide containing proteinsto confirm that the presumed secreted proteins were in fact secretedand not the result of cell autolysis Combining gene expression andproteomic data for A niger overproducing strains of lipid proteinand carbohydrate-degrading enzymes facilitated identification of898 proteins and demonstrated that the strains exhibited upregula-tion of proteins participating in carbon- and N-metabolism as wellas protein folding and protein degradation The data enabled re-searchers to manipulate the system incuding overexpression of aputative protein glycosylation gene and to increase secretion of aspecified enzyme (Jacobs et al 2009)

313 A oryzaeThe Japanese National Institute of Technology and Evaluation

completed sequencing of the A oryzae genome which consists ofeight chromosomes ranging from 28 to 70 Mb (Kitamoto et al1994 Machida 2002) The total genome size was estimated to be

368 Mbwith the number of genes being about 12074 (Machida et al2005)

Fleissner and Dersch (2010) recently reviewed the range of re-combinant protein products produced by Aspergillus species Theprincipal host species identified were A niger A awamori A oryzaeA nidulans and A terreus The predominant promoters used for re-combinant protein production were adhA alcA alcC aldA amdSamdS amyA amyB aphA exlA gdhA glaA glaA1 gpdA oliC pkiAsodM sucA tef1 and tpiA Recombinant products from humans includ-ed alpha1-proteinase inhibitor antigen-binding (Fabprime) fragmentcorticosteroid binding globulin epithelial growth factor granulocytemacrophage colony stimulating factor growth hormone humanizedIgG1(kappa) antibodies interferon-alpha-2 interleukin-6 lactoferrinlysozyme mucus proteinase inhibitor parathyroid hormone single-chain variable region fragment (scFv) anti lysozyme construct super-oxide dismutase and tissue plasminogen activator Recombinantproducts originating from other animals included porcine pancreaticphospholipase A2 and prochymosin bovine chymosin prochymosinand prochymosin B hen egg-white lysozyme and llama antibodiesRecombinant plant proteins expressed in Aspergillus includeThaumatococcus daniellii thaumatin and Cyamosis tetragonolobaalpha-galactosidase Recombinant bacterial proteins expressed inAspergillus species included Cellulomonas fimi endoglucanaseClostridium thermocellum dockerin Eschericia coli enterotoxin subunitB beta-galactosidase beta-glucuronidase and Thermobifida fusca hy-drolase Recombinant proteins from other fungal genera expressedin Aspergillus included Agaricus meleagris pyranose dehydrogenaseM miehei triglyceride lipase and aspartyl protease Phanerochaetechrysosporium lignin peroxidase H8 and manganese peroxidase H4Pleurotus eryngii peroxidase Pycnoporus cinnabarinus laccaseThermomyces lanuginosus lipase and Trametes versicolor laccaseMany recombinant proteins from one species of Aspergillus have alsobeen expressed in another Aspergillus species The very interestingswollenin-like protein from A fumigatus which like swollenin fromT reesei disrupts cellulosic materials and has similarities to the plantproteins (expansins) which have a cell wall loosening effect was pro-duced as a recombinant protein in A oryzae (Chen et al 2010) Whilethe protein exhibits no apparent enzyme activity in the presence ofcellulases it promoted efficient saccharification of crystallinecellulose

32 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 genomesof two biocontrol species Trichoderma altroviride and Trichodermavireus are proceeding T reesei is a soft-rot ascomycete filamentousfungus with a long and safe track record as a producer of commercialcellulases initially with applications in food processing (Nevalainenet al 1994) Studies aimed at understanding and optimizing factorsaffecting productivity and catalytic efficiency of cellulases are funda-mental to overcoming the major biomass pre-treatment obstacle tocommercialization of processes for production of bioenergy from lig-nocellulose biomass Applications of its cellulase and hemicellulasecompliment in the pulp and paper and textile industries are also im-portant (Buchert et al 1998 Galanti et al 1998) T reesei representsa principal target cellulase host in the quest to replace gasoline withcellulose-derived ethanol

321 T reeseiT reesei has a genome size of 33 Mb and seven chromosomes

(httpgenomejgf-psforgTrire2Trire2homehtml) The predictednumber of genes in the genome was 9129 (Martinez et al 2008)T reesei has an extraordinary ability to secrete proteins Cherry andFidantsef (2003) reported that some industrial strains following

aggressive mutation programs could produce as much as 100 gL 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 cellulaseand hemicellulase genes in T reesei was characterized by Ouyang etal (2006) Cultivation of T reesei on cellulose xylan a mixture ofplant polysaccharides or indeed lactose promotes high levels of ex-pression of cellulase and hemicellulase genes (Mach and Zeilinger2003 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 lighton its enormous protein secretion capacity Despite its effectivenessin degrading plant polysaccharides suggesting it should contain ex-pansions of genes encoding enzymes capable of digesting plant cellwalls 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 polysaccharasesoften contain a carbohydrate-binding molecule (CBM) within its re-lated group of fungal genomes (N crassa F graminearum M griseaand T reesei) they had the smallest number of CBM-containingproteins

First efforts to produce heterologous proteins in T reesei focussedon calf chymosin (Harkki et al 1989 Uusitalo et al 1991) afterwhich Nyyssonen et al (1993) reported use of this host to produceantibody fragments It was observed that higher production of recom-binant proteins was generally observed when the original source ofthe 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 byTrichoderma (Penttila 1998 Schmoll and Kubicek 2003) most fre-quently the signal peptide of Cel 7a (CBHI) which mediates efficientrecombinant protein secretion This topic was reviewed by Schusterand Schmoll (2010)

Three recombinant endoxylanases from Chaetomium thermophilumwere expressed in T reesei with a view to facilitating their productionfor application in biobleaching of kraft pulp (Mantyla et al 2007)The expression cassettes utilized the strong T reesei cel 7A promoterThe host was a low protease producer where deletions in the endoglu-canase I endoglucanase II and cellobiohydrolase I genes rendered it thedesired low cellulase producer for applications in kraft pulp treatmentIt was demonstrated that a commercially viable recombinant thermo-stable xylanase can be produced by T reesei Recently the industriallyinteresting biocatalyst cinnanoyl esterase from an unsuitable host theanaerobic fungus Piromyces equi was successfully expressed in recom-binant T reesei as a more suitable producing host (Poidevin et al2009)

Substantial effort has focussed on transforming fuel ethanol yeaststrains with cellulolytic genes from Trichoderma species to facilitatetheir ability to ferment cellulose to ethanol In a recent exampleHuang et al (2010) described cloning and expression of the endoglu-canase gene egVIII from Trichoderma viride into Saccharomycescerevisiae

33 Penicillium

Limited genomic sequencing information appears to be availableon potential recombinant protein-producing filamentous fungi otherthan Aspergillus and Trichoderma species As selected Penicillium spe-cies for example Penicillium purpurogenum Penicillium funiculosumand 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 Penicilliumcamemberti and Penicillium roqueforti which exhibit low protease

activity They concluded that these species had all of the right straincharacteristics as suitable hosts for production of recombinant pro-teins Gonzalez-Vogel et al (2011) recently identified a number ofprotein complexes containing enzymes including arabinofuranosi-dases beta-glucosidase xylanases acetyl esterases and ferulyl ester-ases in the soft rot fungus P purpurogenum using a proteomicsstrategy Guais et al (2010) prepared a partial DNA library for Pfuniculosum and sequenced genes encoding four GH54 α-L-arabinofuranosidases This organism has been used to produce com-mercial mixtures of enzymes degrading complex agricultural residuescontaining cellulose hemicellulose arabinoxylan arabinogalactanproteases etc with applications as a feed additive to enhance feed di-gestibility The enzyme mixture contained more than fifty separateproteins (Guasi et al 2008) The strong gpdA promoter from Anidulans 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 avector encoding the laccase of Trametes hirsute under control of an ef-ficient promoter of the bgaS gene of P canescens and efficientlyexpressed and secreted the recombinant protein (Abianova et al2010)

As might be expected the predominant effort related to sequenc-ing and annotation of Penicillium has been directed at the principalproducer of penicillins P chrysogenum While antibiotic-producingstrains are generally not considered as suitable hosts for productionof natural or recombinant enzymes or other proteins for use infoods or pharmaceuticals some of the P chrysogenum genomic infor-mation may be applicable to development of non-antibiotic-producing Penicillium strains as recombinant protein-producinghosts Promoters of the genes encoding glutamate dehydrogenaseβ-acetylhexosaminidase and gamma-actin from P chrysogenum maybe used to construct potent vectors for expression and secretion ofhomologous and heterologous proteins in these strains and also inother hosts (Barredo Fuente et al 2001)

331 P chrysogenumThe complete genome sequence of the penicillin producer P

chtysogenum Wisconsin 54ndash1255 strain (ATCC 28089 see Elander1983) was published in 2008 Genome size was 3219 Mb compara-ble with that of other filamentous fungi and the total gene numberwas 12943 (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 informationmay provide more general direction for manipulationengineeringof 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 sequencedstrain and a high penicillin-producing strain were compared andas might have been expected many of the genes involved in synthe-sis of the penicillin precursors valine cysteine and α-aminoadipicacid were observed to be increased in the high penicillin-producingstrain Some genes were identified which control β-lactam outputand genes with predicted roles as transporters appeared to be upre-gulated under penicillin-producing conditions Culmination of thiswork clearly represents a milestone for future metabolic engineer-ing strategies which of course may involve participation or use ofrecombinant proteins

34 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 Rhizomucorpusillus and digestive lipases proteases and amylases are producedby Rhizopus arrhizus However the major fungal genomics resourcesrelated to this group of filamentous fungi have been directed to

pathogenic strains and indeed the first of the zygomycetes to befully sequenced was Rhizopus oryzae which is the primary cause ofthe potentially lethal angioinvasive mucormycosis infection (Ibrahimet al 2003 Kwon-Chung and Bennett 1992) RelatedMortierella spe-cies are of great interest in the area of lipid production and moleculartransformations involving these species are being investigated (Mac-Kenzie et al 2000) Nevertheless as is indicated below some of thegenomic information is directly relevant to the long established abil-ities of this and related strains to produce hydrolytic enzymes

341 Rhizopus oryzaeThe genome sequence of R oryzae strain 99ndash880 isolated from a

fatal infection of mucormycosis has recently been published (Ma etal 2009) Total length of the R oryzae genome was found to be4526 Mb while total number of protein-encoding genes was 17467Evidence was provided that there is whole-genome duplication inthis 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-wallsynthesis enzymes required for fungal cell wall metabolism butwhich are not present in mammalian hosts and hence which maybe targeted by novel future drugs Of interest both to therapy aswell as to use of R oryzae and related species as hosts for recombinantprotein 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 thepathogenic infection process as these enzymes have previouslybeen thought to be associated with virulence of pathogenic Rhizopusspecies (Schoen et al 2002 Spreer et al 2006) In this case theseproteases may mediate penetration of hyphae through decaying or-ganic matter (Ma et al 2009)

35 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 cellulosemicrofibrils of plant cell walls especially woody plants The whiterot fungi are particularly important because they degrade the ligninwhile not attacking the cellulose These filamentous fungi are theonly microbes capable of efficient depolymerization and mineraliza-tion of lignin P chrysosporium has been the most intensively studiedwhite rot fungus White rot fungi secrete an array of peroxidases andoxidases that attack lignin non-specifically by producing lignin-freeradicals 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 proteomicscoupled to LC-MSMS was used to monitor enzyme expression andchemical products present during the process of degradation of aro-matic substrates by P chrysosporium as a means of gaining a betterinsight into the process of lignin degradation (Matsuzaki et al2008) Not surprisingly the first basidiomycete genome to be se-quenced was the white rot fungus P chrysosporium

351 P chrysosporiumIts thirty million base-pair genome was sequenced using a whole

genome shotgun method The genome length was 299 Mb similarin size to most of the other sequenced filamentous fungi genomesThe genome contains 11777 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 areknown 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 humancytokine associated with some inflammatory and autoimmune

diseases was successfully introduced and expressed in the ediblemushroom P eryngii via an A tumefaciens transformation (Chung etal 2011) There is continuing interest in expressing the diversedegrading enzymes from basidiomycetes in more conventional indus-trial work-horse hosts For example ligninolytic basidiomycetes con-tain a sugar oxidoreductase (pyranose dehydrogenase) that has verybroad substrate specificity towards breakdown constituents of ligno-cellulose In order to extend the biodegradative capability of moreconventional industrial strains this enzyme from A meleagris washeterologously expressed in A nidulans and A niger (Pisanelli et al2010) The white rot fungus T versicolor produces two groups of lac-cases with several isoforms Two of these laccases were expressed asrecombinant enzymes in A oryzae and the recombinant enzymesexhibited 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 largeapplications in delignification basidiomycetes are in general not asversatile or suitable as industrial fermentation producers as com-pared to ascomycetes and consequently much effort has focussed ontransforming the more suitable fermentation hosts to produce recom-binant basidiomycetes laccase However there have been problems inachieving production of recombinant laccases in good fermentationhosts primarily due to glycosylation deficiencies and these challengesare currently being addressed with a view to mass producing effectivelaccases

Sakaki and Munetsuna (2010) have surveyed the various enzymeswhich could co-operate to degrade complex pollutants such as poly-chlorinated dibenzo-dioxins and furans including angular dioxygenasecytochrome P450 (CYP) lignin peroxidase manganese-dependent per-oxidase and dehalogenase and concluded that combinations of distinctenzymes could have significant application in these biodegradationsGiven that white rot fungi already produce lignin and Mn-dependentperoxidases and CYPs it was concluded that supplementing this hostby adding additional recombinant capability wouldmake this organisma very powerful bioremediation strain While the risks associated withreleasing genetically engineered organisms to the environment wererecognized it was suggested this could be addressed by creating suicid-al engineered strains (Paul et al 2005)

36 Fusarium

While a high profile Fusarium species F graminearum is the caus-ative agent of some important plant diseases other Fusarium strainsare used in fermentations processes including production of singlecell protein approved for human consumption and some of thesestrains may have potential for production of recombinant proteinsNevertheless the predominant scientific research to date has fo-cussed on F graminearum which causes plant diseases of substantialeconomic importance including Fusarium ear root of maize andhead blight of cereals In addition F graminearum produces myco-toxins in infected plants which if they find their way into food andfeed products constitute a health risk

361 F graminearumThe sequencing and annotation of F graminearumwas reported by

Cuomo et al (2007) and gene annotation information was revisitedby Wong et al (2011) Updated resource information may beassessed at httpmipsgsfdegenreprojFGDB

The Cuomo et al paper indicates a genome size of 361 Mb includ-ing 32 genes being predicted plant cell-degrading enzymes includingxylanases 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 recentannotated information indicated a set of 13718 protein coding genes

37 N crassa

While N crassa is not recognized as an important industrial host itis included in this discussion as a powerful model filamentous fungalsystem which has been characterized biochemically and geneticallyThis host can be grown at high growth rates in simple definedmedia and can produce high amounts of recombinant proteins Theapproximate genome size is 40 Mb and it contains about 10000 pro-tein-coding genes andmanyof the genes involved in interesting aspectsof Neurospora biology including its secondary metabolism have beenannotated (Colot et al 2006 Galagan et al 2003) Up-to-date informa-tion may be obtained online from httpwwwfgscnet

Tian et al (2009) applied microarray and shotgun proteomicsanalysis on strains of a cellulolytic N crassa fungus grown in differentmedia in order to combine data fromgene expression and the proteome-secretome in an attempt to better understand the cellulose-degradingsystem 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 theone hand but also enables mixtures of different antigens to be co-expressed together thereby facilitating tailoring of a vaccine directedat a particular pathogen target or variant

38 Selected key genomic resources

A variety of institutional and online resources are available to re-searchers with interests in genomic aspects of filamentous fungi andare clearly relevant to the topic of recombinant protein productionby these hosts Reference is made to some of these below

httpwwwaspgdorg ldquois 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 CandidaGenome Database and is funded by the National Institute of Allergyand Infectious Diseases at the US National Institutes of Healthrdquo Subsitesdeal with the annotated Aspergillus genomes of strains of A fumigatusA clavatus A nidulans A niger A oryzae and Aspergillus terreus

The aim of the JGI Fungal Genomics Program is ldquoto scale up sequenc-ing and analysis of fungal genomes to explore the diversity of fungi inDOE mission areas and to develop the Genomic Encyclopedia of Fungiin the areas of Plant feedstock health (mycorrhizal symbiosis plantpathogenicity biocontrol) Biorefinery (lignocellulose degradationsugar fermentation industrial organisms) and Fungal diversityrdquo (httpjgi-psforgprogramsfungiabout-programsjsf) Subsites deal with im-portant filamentous hosts including Aspergillus carbonarius P chrysos-porium Sporotrichum thermophile Thielavia terrestris T versicolor andT reesei

The Fungal Genome Initiative (FGI) of the Broad Institute of MITand Harvard ldquoproduces and analyzes sequence data from fungal or-ganisms that are important to medicine agriculture and industryOver 50 fungi have been sequenced or are being sequenced includinghuman and plant pathogens as well as fungi that serve as basicmodels for molecular and cellular biology In partnership with thewider fungal research community organisms are selected for se-quencing as part of a cohesive strategy that considers not only thevalue of data from each organism given their role in basic researchhealth agriculture and industry but also their value in comparativegenomicsrdquo It includes databases on R oryzae and on the FusariumComparative project (httpbroadinstituteorgscientific-communityscienceprojectsfungal-genome-initiativefungal-genome-initiative)

The Fungal Genetics Stock Center (httpwwwfgscnet) ldquois a re-source available to the Fungal Genetics research community and toeducational and research organizations in general The FGSC is fundedlargely by a grant from the National Science Foundation (Award

Number 0235887) of the United States of America and to a lesser extentby the payments made by researchers who use our services Most fungalstrains in the FGSC collection are listed in the online searches Specificgroups of materials are listed by category include NeurosporaAspergillus Fusarium and Magnaporthe Ustilago Cryptococcus otherFungirdquo The FGSC together with other organizations is a major sponsorof the Fungal Genetics Conference (httpwwwfgscnet26thFGCindexhtm)

The above websites in turn provide links to other resources

4 Improving recombinant protein expression in filamentous fungi

41 Molecular strategies

A primary practical motivation for studying gene expression in fil-amentous fungal hosts is to understand the molecular mechanisms oftranscription regulation in these organisms and to improve recombi-nant protein expression especially by the study of DNA sequencesparticipating in transcription initiation andor regulation and selec-tion of strong promoters Transcriptional regulation of extracellularplant cell wall-degrading enzymes produced by filamentous fungihas been reviewed by Aro et al (2005)

The promoter regions of the Aspergillus amylase genes consist offour 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 withregion IIIa (Minetoki et al 1998) A sequence of CCAAT present inthe 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 Anidulans genes (Papagiannopoulos et al 1996) One of the moststrongly 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 tobe effective as a mediator of strong synthesis of recombinant proteinsin Aspergillus hosts (Ishida et al 2001) The A oryzae TAKA-amylasepromoter preceded by its upstream activating sequences was foundto 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 3prime terminus of the DNAsequence encoding the heterologous protein and which may includepromoter 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 hydrophobinfrom A nidulans in T reesei When the class II hydrophobin-encodingpromoter from T reesei hfb2 was used with lactose as carbon sourcethe majority of the recombinant protein was secreted into the medi-um by T reesei In contrast when the T reesei cel7A promoter wasused the recombinant protein was not secreted into the mediumbut remained cell wall-bound High expression of the fumR genewhich encodes fumarase in a high fumaric acid producing strain ofR 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 metabolicengineering in Rhizopus species Gene expression was primarily regu-lated at the level of transcription

411 Gene-fusions strategiesSome early recombinant research on filamenous fungal sought to

produce recombinant proteins by lsquocoat-tailingrsquo a hyper-producedand secreted homologous protein with subsequent cleavage of result-ing fused proteins Thus techniques involving fusing the target geneto the 3prime end of a homologous gene encoding glucoamylase improvedproduction 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 nigerA awamori promoted production of high

levels of a variety of secreted recombinant proteins including bovineprochymosin (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 etal 2003)In the case of chymosin and lactoferrin production gramto multigram quantities of recombinant product were produced perliter 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 ofglucoamylase rather than the complete enzyme (Gouka et al1997b) To facilitate subsequent cleavage of the two protein ele-ments a linker proteolytic processing site is incorporated betweenthe carrier moiety component and the protein of interest The linkerregion 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 therecombinant protein into the ER as well as its folding is mediatedby the N-terminal fungal protein Subsequently in most cases the fu-sion protein is cleaved facilitating secretion of the separate proteinsby a KEX2-like endopeptidase at a KEX2 recognition site introducedspecifically 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 genefusion strategies are employed When a part of the fungal glucoamy-lase protein (GAM) linked via a KEX2 processing site was also usedin a gene-fusion strategy in A niger to produce extracellular bovinepancreatic 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 proteinor indeed involved another endoprotease (MacKenzie et al 1998)For example while the desired recombinant protein is normallylinked to the glucoamylase via a Lys-Arg KEX2-like cleavage site inA niger the fidelity of cleavage to release mature protein is not al-ways observed to be consistent and appears to be also influenced bysequences immediately downstream and upstream of the KEX2 site(Spencer et al 1998)

The protein neoculin (NCL) naturally produced in the fruits of thetropical plant Curculigo latifolia is about 500 times sweeter thansugar 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 usingseparate NAS and NBS constructs each fused to the A oryzae α-amylase via KEX2 cleavage sites (Nakajima et al 2006) The NAScomponent 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 expressedproteins containing a tag that may facilitate product extraction duringdownstream processing By way of example Collen et al(2001) ge-netically engineered endoglucanase (Cel7B) from T viridewith a pep-tide extension containing non-polar tryptophan-proline residueswhich facilitated preferential partitioning of the protein into the lesspolar phase of an aqueous two phase model system

412 Overproduction of foldases and chaperonesFoldases catalyze the isomerizations and disulfide bond forma-

tions and molecular chaperones which are non-catalytic mediatefolding of the nascent polypeptides into functional proteins and pre-vent non-productive proteinndashprotein interactions (Conesa et al2000) 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 degradationof defective proteins

It has been postulated that hyper-production of recombinant pro-teins into the ER has the potential to overload the folding assemblyand secretion machinery of filamentous fungi Therefore the effects

of overexpression of genes for several ER chaperones and foldases infilamentous fungi including bipA (from a family of binding proteinsBiP) pdiA (from a family of protein disulfide isomerase) and a familyof calnexins on overproduction of recombinant proteases have beenevaluated (Conesa et al 2001 Jeenes et al 1997 Ngiam et al2000 van Gemeren et al 1997) It was found that filamentousfungi overproducing specific proteins both homologous and heterol-ogous exhibited increased levels of bipA transcription whereas directinterventions to overexpress bipA overexpression appeared not to af-fect yields of secreted proteins (Punt et al 1998) Overproduction offungal proteins generally increased bipA mRNA levels in A niger Inthe case of two transformed A niger strains which produced HEWLa twofold induction in bipA mRNA levels was observed (Ngiam etal 2000) BiP overexpression did not increase secreted levels of hIL-6 in Aspergillus (Gouka et al 1997a) and pdiA overexpression didnot increase secreted yields of HEWL in A niger (Ngiam et al2000) Disruption of a vacuolar protein sorting receptor gene in Aoryzae which targets aberrant and recombinant proteins for vacuolardegradation enhanced production and secretion of the bovine chy-mosin and human lysozyme heterologous proteins (Yoon et al2010)

413 GlycosylationGlycosylation 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 mainglycosylation processes common to eukaryotes involve N- and O-glycosylation whereby oligosaccharides attach to the beta-amidemoiety of asparagine residues and mainly to serine and threonineβ-hydroxy groups N-glycosylation involves transfer of pre-assembled glycosyl precursors to specific asparagine residues of thenascent polypeptide chain after which glycosidase- and glycosyltransferase-mediated modifications of the oligosaccharide occurresulting in production of a common trimannosyl-chitobiose corewith branched N-acetylglucosamine residues generating the highmannose N-glycans characteristic of filamentous fungi and yeastsO-glycosylation in fungi starts in the endoplasmic reticulum and in-volves O-mannosylations resulting in the sequential build up of theO-glucosyl structure Geysens et al (2009) has recently used analysisof the genome sequences to review folding secretion and glycosyla-tion especially the N-glycosylation processes while Goto (2007) hasdescribed the O-glycosylation process both in Aspergillus

Filamentous fungi have two distinct alpha-12-mannosidases oneof which is similar to the mammalian Golgi alpha-12-mannosidasesthat trim 3 mannose moieties off Man8GlcNAc2 to form Man5GlcNAc2as substrate for GlcNAc transferase 1 and another distinct fungalalpha-12-mannosidase (Ichishima et al 1999 Yoshida et al 2000)However the mammalian-like enzyme is neither well expressed norsecreted such that very little of the lower mannosylated moiety getstransferred (Maras et al 1997) N-glycans from fungi also differfrom mammalian N-glycans in having terminal altered substituentssuch as glucose galactose or phosphoesters (De Pourcq et al 2010)Maras et al (1997) employed recombinant mammalian beta-14-galactosyl transferase and alpha-26-sialyltransferase to make Treesei cellobiohydrolase 1 more mammalian-like with respect to itsglycosylation pattern Recombinant human β-12-GlcNAc transferasewas subsequently overexpressed in Trichoderma thereby enhancingits GlcNAc transfer capability (Maras et al 1999a 1999b) and similartransformations with the corresponding rat GlcNAc transferase wereimplemented in A nidulans (Kasajima et al 2006) Kainz et al(2008) has carried out other molecular stratefies to successfully pro-duce lower mannosylated Man3GlcNAc2 N-glycans in recombinantAspergillus strains

For production of therapeutic proteins glycoform is very impor-tant as incorrectly glycosylated proteins for example recombinanthuman 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 ofhigh mannose-containing glycoproteins has been found to be verycomplex (Gerngross 2004)

Antitrypsin the human a1-proteinase inhibitor (a1-PI) is themost abundant inhibitor of serine proteases in plasma (Brantly etal 1991) Progressive emphysema develops in antitrypsin-deficientpatients ultimately leading to death (Crystal 1996) Conventionalantitrypsin-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 ofproduction altered glycosylation patterns or complete absence of gly-cosylation in the recombinant product reduced in vitro stability of theinhibitor and resulted in its rapid removal from the circulation system(Karnaukhova et al 2006)

Amature and biologically active glycosylated recombinant a1-PI pro-duced by A niger exhibited improved stability over a non-glycosylatedrecombinant 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 wascleaved in vivo by a KEX2-type protease Implementation of strategiesfor increasing glycosylation in Aspergillus resulted in increased pro-duction of the recombinant protein chymosin (van den Brink et al2006) 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 theN-glycosylation site was located away from the native chymosin at-tached via a linker a substantial increase in recombinant proteinwas observed

414 Other molecular strategiesThe following are miscellaneous examples of molecular strategies

used to enhance production of recombinant proteins by filamentousfungi

ndash Hastrup et al (1997) proposed production of a proenzyme incases where the enzyme was unstable or harmful to the producinghost which could be proteolytically activated after secretion

ndash An activator protein binding site containing the CCAAT sequencewas identified within the cis regulatory region of the A nigerglaA gene Insertion of multiple copies of this binding site intothe promoter of transformed recombinant plasmid sequence en-hanced promoter production of the heterologous protein (Liu etal 2003)

ndash Berka et al (2002 2003) disclosed constructed novel vectorswhich encoded the desired heterologous polypeptide and a secre-tory sequence functional in the filamentous fungus secretorysystem

ndash A oryzae produces two predominant proteases serine-type car-boxypeptidase (CPase) and aspartic endopeptidase under acidicconditions (Takuchi and Ichishima 1986)A typical antisense control strategy whereby vectors are createdto express a high level of the antisense RNA complementary tothe RNA transcript of a target gene used to inhibit fungal gene ex-pression was used to isolate an low CPase-producing A oryzaemutant expressing high and stable levels of lysozyme (Zheng etal 1998)

ndash Researchers had limited success in striving for overproduction ofmanganese peroxidase in its natural host P chrysogenum (Cullen1997) However a combination of strategies including use of astrong glucoamylase promoter a protease-deficient A niger hostculture 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 2000Punt et al 2002 Stewart et al 1996)

ndash Promoters of the genes encoding glutamate dehydrogenase beta-acetylhexosaminidase and gamma-actin from P chrysogenummaybe used to block expression of undesired genes through anti-senseconstruction (Barredo Fuente et al 2001)

42 Protease-deficient strategies

Production properties and classification of microbial proteaseshave recently been reviewed (Ward 2011 Ward et al 2009) Inaddition to the observed variabilities in processing of fusion proteinsby KEX-like endoproteases in Aspergillus discussed above recombi-nant protein-degrading fungal proteases have long been known tobe problematic (Ward et al 2006) Braaksma and Punt (2008)reviewed various strategies for controlling protease activity as ameans of supporting recombinant protein production Methods in-cluded classical selection of protease mutants molecular geneticmethods to construct protease mutants targeted to protease genesand protease regulators manipulation of fermentation conditionsspecifically pH control of metabolitescatabolites such as carbon nitro-gen sulfur and phosphorus induction of proteases and physiologicaland morphological effects

With enzyme-overproducing industrial strains one approach wasto 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 andmethods for transforming strains to produce the protease and per-haps more importantly for development of Aspergillusmutants defec-tive in the production of aspartic protease Given that filamentousfungi can contain as many as 80 proteolytic genes of varying knownand unknown function researchers are cautioned against trying todevelop mutants deficient in multiple proteases (Machida 2002) Im-pacts on recombinant protein production of constructing stable Aniger recombinants containing up to three disrupted protease geneswere characterized (Van den Hombergh et al 1997) Specific mu-tants of A nidulans deficient in the aspartic protease gene exhibitedthe ability to produce chymosin as well as other recombinant proteins(Berka et al 2003) When the alkaline protease gene of a strain of Aoryzae 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 particulargenes including proteases in recombinant hosts PEPB protein re-cently characterized as a member of the glutamic proteases wasthought to be the causative agent in degradation of recombinantthaumatin in A awamori containing a disrupted pepA gene producinginactive PEPA Thaumatin production was improved by expression ofpepB antisense RNA but results indicated antisense mRNA had onlypartially silenced pepB gene expression A substantial further increasein 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 insubstantially reducing protease activity in Aspergillus species For ex-ample disruption of the prtT gene which is a regulatory gene whichencodes a member of the Zn-binuclear cluster family appears to elim-inate two Aspergillus proteases from the medium including PEPA andreduces total protease activity by 80 (Punt et al 2008) Yoon et al(2011) reported on experiments which demonstrated how successivedisruption 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 foractivity and implementation of cultivation strategies which preventrelease of intracellular proteases via mycelial cell lysis have been shownto reduce proteolysis of secreted recombinant proteases (Denison2000 ODonnell et al 2001 Wang et al 2005) Use of peptide-rich

media typically induces protease production by A niger (Ahamed etal 2005) and productivity of secreted egg lysozyme by a recombinantstrain of A niger was reduced in such rich media (Archer et al 1990)Double disruption of the two protease genes in A oryzae tppA andpepE facilitated an increase of 63 in the production level of human ly-sozyme (Jin et al 2007) Combination strategies of using non-proteaseinducing medium and use of the aspartyl protease inhibitor pepstatinrepresent an alternative strategy tominimizing the impacts of proteaseson fungal recombinant protein activity (Ahamed et al 2005) In two re-cent proteomic studies involving A niger it was observed that underconditions of culture starvation resulting from depletion of carbonsource proteases were found to predominate in the secretome andhence these conditions should be avoided to minimize protease secre-tion during production of recombinant proteins (Adav et al 2010Braaksma et al 2010)

43 Manipulations of morphology

Vegetative growth involves hyphal extension and occurs at thehyphal 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 theprincipal locus for biological process-related recombinant proteinproduction Further back from the tip protoplasmic compartmentsbecomemore vacuolated It follows that a greater degree of branchingwill 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 includinginoculum size and type (vegetative spores etc) On the surface ofsolid media filamentous fungi grow as mycelial mats In submergedcultures fungi may attach to suspended particles if present or growas diffuse filamentous mycelia or as dense pellets which may developto different sizes Morphological form influences rate of growth andproduct formation Predominant growth and metabolism of fungi inpelleted form occurs at the pellet surface where there is maximumaccess to nutrients and oxygen Inside the pellet inward diffusion ofnutrients and outward diffusion of product become limiting andvacuolization and lysis are frequently observed Recently Driouch etal (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 infermenters which result in rheology and viscosity problems leadingto 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 strainsincluding Acremonium Corynascus ThielaviaMyceliophthora Thermo-ascus and Chaetomium species were grown in batch fermentationsunder the same conditions used to culture A oryzae medium viscos-ities observed were much lower

Impact of morphology changes as they effect recombinant proteinproduction may be at least partially related to protease production orrelease Growth of the A nigermycelium as large pellets was associat-ed with lower specific protease activities and increased specific glu-coamylase activities were found when A niger was cultured inmedia which generated large pellets (Papagianni and Young 2002)In general fungal pelleted growth mediates greater lysis in fungifor example in Aspergillus species and this results in the presenceof higher levels of proteolytic activity in filtrates of pelleted culturesas compared to filamentous growth (Ahamed et al 2005) While thegreater proteolytic activity in pellet cultures is likely to be partly dueto intrapellet cell lysis differential expression may also be a factorDai 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 mutantformation and recombinant glucoamylase production in A niger

(Swift et al 1998) In studies of an A niger strain containing a genefor the Gla-GFP fusion protein protease activity in pelleted growthwas only one-third of that observed with filamentous growth (Xu etal 2000)

Morphology of the filamentous fungus Chrysosporium lucknowensehas been manipulated genetically to produce a non-filamentous formthereby reducing viscosity of culture systems The non-filamentousform also exhibits low protease activity and is capable of producingvery high yields of heterologous proteins (Verdoes et al 2007)

General aspects of the relationships between morphology andproductivity in filamentous fungi have been reviewed by Grimm etal (2005)

44 Solid-state fermentation approaches

While the mechanisms have not been fully elucidated variationsbetween solid-state and submerged culture fermentation conditionscan 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 offilamentous growth mediate differential levels of gene expressionand product formation (Masai et al 2005) In some cases the con-trasting activities reflect whether the activity is cell wall-associatedor excreted into the culture medium in a case in point in submergedcultures some enzyme activities were associated with the cell wallwhereas in solid-state cultures they were secreted into the medium(Wang et al 2005) In solid state culture wheat bran media causedlowest expression of catabolic genes which were thought to have re-leased catabolite repression of synthesis of hydrolytic enzymesthereby mediating rich hydrolytic enzyme gene expression Oda etal (2006) concluded that certain enzymes were selectively secretedunder conditions of solid-state culture or in submerged culture inde-pendent of the composition of the medium In a different but a partlyanalogous situation proteome differences were observed when Fgraminearum was cultured in vitro and in planta with some proteinsbeing expressed in only one of the two conditions (Paper et al 2007)

Imanaka et al (2010) compared cultivation of A oryzae usingthree different culture methods ie shake-flasks agar-plate andmembrane surface liquid culture and observed differences in growthsecretion of proteases and alpha-amylase secreted protein level andgene transcriptional profile by DNA microarray analysis Protease ac-tivities especially oryzin (alkaline protease) and alpha-amylase weremuch higher in agar-plate and membrane surface liquid culture thanin shake flasks Transcriptional gene profiles from the agar-plate andmembrane 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 morehomogeneous glycosylated recombinant product Submerged cul-tures by A oryzae transformed to produce the important heterologousantigenic protein PrendashS2 of human hepatitis B virus resulted in pro-duction of a partially degraded heterologously glycosylated proteinwhereas in solid-state culture a homogeneous glycosylated form ofthe whole fusion protein was obtained (Maruyama et al 2000) Useof wheat bran (2) in solid state culture mediated a 500-fold increasein production of recombinant chymosin as compared with submergedculture (Tsuchiya et al 1994) te Biesebeke et al (2002) found thatpelleted growth in submerged fermentations and semi-solid fermen-tations showed different patterns of gene expression and proteinsecretion

45 Concluding comments

Because of their extraordinarily high productivities and simplegrowth requirements filamentous fungi can compete or outcompetemost other recombinant hosts in processes for production of non-

glycosylated recombinant proteins assuming for a particular productthat problems related to recombinant protein degradation by proteasesof the host producing strain have been resolved It is also clear thatwhere the interest relates to recombinant glycosylated proteins espe-cially recombinant proteins for human therapeutic use the quite differ-ent native protein glycosylation patterns observed in fungi renderthem unsuitable hosts for recombinant human glycoprotein produc-tion and some other hosts for example Pichia species may be moresuitable microbial hosts to be engineered for mammalian glycoproteinproduction Nevertheless research is continuing to be aimed at engi-neering the appropriate human glycosylation machinery into filamen-tous fungi

5 Recombinant proteins in metabolic engineering of fungi

In recombinant protein production the initial priority targetswere individual proteins and glycoproteins for beneficial industrialagricultural food or pharmaceutical use However the developmentof genomic and proteomic capabilities combined with moleculartools provides the tools to manipulate hosts with respect to morecomplex combinations of proteins with diverse functions be theycatalytic regulatory or with other physiological roles As otherorganisms the filamentous fungi offer potential opportunities for im-proving desired metabolite product yields reducing or eliminatingundesired metabolite bi-products extending substrate ranges and in-troducing new enzymes or pathways leading to production of newproducts (Kern et al 2007) For example hosts may be transformedto produce the multiple enzymes associated as part or all of a newmetabolite biosynthesis sequence Alternatively an existing pathwaymay 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 invariablyinvolve genetic modifications including expression of recombinantenzymes and manipulation of host transcription andor translationmachinery 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 fungalspecies With an estimated 15 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 10000 genes per spe-cies the resource is almost unlimited Some of these concepts asthey apply to primary and secondary metabolites are briefly dis-cussed in this section through use of selected examples

51 Primary metabolites

511 Citric acidWith 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 citrateproduction (Ruijter et al 2002) Seven or more enzymes were select-ed as principal targets and two genes encoding regulatory enzymes inthe pathway were overexpressed (Ruijter et al 1997 Torres et al1996)

512 BiodieselBiodiesel originating from some form of biological host arguably

represents part of the ldquobiosolutionrdquo to the worlds energy problems(Demain 2009) The products of fungi and algae are currently beingused as beneficial food and feed additives and nutritional productsWhile it is still unclear if fungal and algal strains are appropriate forproduction of microbial oils for biodiesel production they do containthe machinery for bio-oil biosynthesis and the tools for transformingtheir biosynthetic enzymes to other hosts which may be deemed

more suitable for energy production are well on the way to beingestablished

Several filamentous fungi accumulate very high amounts of micro-bial lipids especially highly unsaturated lipids (Ward and Singh2005) The concept of using filamentous fungi as producers as lipidswas introduced during World War 1 in Germany and processesusing a variety of strains including Mortierella isabellina Mortierellaalpina andMucor vinacea have been scaled up to produce commercialquantities of lipids (Ratledge 1978) While the primary emphasis wason the nutritional benefit of unsaturated fatty acids scientists in theSoviet Union considered the Mucorales as potential candidate hostsfor production of lipids for use in biofuels (Feofilova et al 2010) Ifmomentum in this area continues strategies will be needed for opti-mization of processes which will include detailed examination of thelipid synthetic pathways in order to determine metabolic bottle-necks In addition sequencing and annotation of the genes encodingthe participating enzymes will provide opportunities for transforma-tion of hosts to produce recombinant enzymes These biochemicaland molecular approaches may be combined with fermentation opti-misation strategies both to optimize production rates and yields andto produce products of varying lipid compositions

513 Higher unsaturated fatty acids (HUFAs)M alpina produces the C-20 highly unsaturated fatty acids dihomo-

gamma-linolenic acid arachidonic acid and eicosapentaenoic and hasbeen used for industrial production of arachidonic acid (Shimizu etal 1997) In mammals γ-linolenic acid (GLA) is an intermediate ofthe (nminus6) pathway conversion of linolenic acid to arachidonic acidand a direct precursor of dihomo-gamma-linolenic acid Certain condi-tions (aging stress some infections diabetes eczema) may be mani-fested as causing a deficiency of Δ6-desaturation and of GLA andhence there is considerable interest in investigating production of re-combinant forms of Δ6-desaturase and the efficient enzyme from Malpina as a target of interest GLA is further metabolized to produceanti-inflammatory eicosanoids such as prostaglandins and leukotri-enes and GLA and its metabolites also regulate expression levels of var-ious gene products As a first step this enzyme was cloned from Malpina and expressed in A oryzae under the control of the amyB pro-moter While GLA was not produced by the control A oryzae strainGLA constituted 252 of total fatty acids in the recombinant hosttransformed to expressΔ6-desaturase (Sakuradani et al 1999) Similarenzymes have now been cloned from a range of filamentous fungi in-cluding Cunninghamella echinulata R arrhizus Rhizopus nigricans Rhi-zopus stolonifer Mucor rouxii and Pythium irregulare The geneencoding the enzyme from R stoloniferwas recently expressed in Pichiapastoris enabling the transformed Pichia strain to produce about 224of its fatty acid content as GLA (Wan et al 2011)

52 Secondary metabolites

Some of the recent exciting developments in fungal secondarymetabolite production relate to the continuing developments in ourknowledge 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 sixPKSs (synthesizing monodictyphenone asperfuranone orsellinicacidF9775 asperthicin napthopyrone sterigmatocystin) and fourNRPSs responsible for the biosynthesis of terrequinone aspyridonesemericellamide and penicillin are reported to have been character-ized in A nidulans (Balibar et al 2007 Bergmann et al 2007 Chianget al 2010 Evans et al 2011 Sanchez et al 2009 Szewczyk et al2008 von Dohren 2009)

521 Engineered biosynthesis of novel peptidesIn addition to the predominant and ubiquitous ribosomal machin-

ery for synthesis of peptides and proteins certain organisms

including filamentous fungi contain complex peptide synthetasetemplates which mediate synthesis of a variety of bioactive peptidesmore than 300 of which are known without ribosomal involvement(Stachelhaus et al 1996)The bioactive products include antibioticstoxins enzyme inhibitors antiviral agents antitumor agents and im-munosuppressants These non-ribosomal large protein templatescontain protein domains which catalyze synthesis of a wide range oflow molecular weight linear branched and cyclic peptides whichmay be further transformed by acylation glycosylation and other re-actions (Marahiel 1992) Research is ongoing related to manipulationof 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 synthesisof novel peptides including novel antibiotics and other therapeutics(Kleinkauf and von Doumlhren 1990 Stachelhaus and Marahiel 1995)

522 AntibioticsBiochemistry 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 Pchrysogenum and A nidulans and production of cyclosporin A by Toly-pocladium niveum (Weber et al 1994 Aharonowitz et al 1993Smith et al 1990 Gutierrez et al 1991)

Numerous genes involved in biosynthesis of antibiotics and othersecondary metabolites have been cloned including genes involved inthe synthesis of β-lactam antibiotics by the filamentous fungi Pchrysogenum Cephalosporium acremonium and A nidulans (Martiacuten andLiras 1989) As with other antibiotics genes involved in penicillincephalosporin and cephamycin biosynthesis are organized in clustersand pathway-specific regulatory genes positively or negatively modu-late genes expression in these clusters and hence regulate antibioticbiosynthesis (Loder and Abraham 1971 Martiacuten and Liras 1989Nuesch et al 1987) Positive regulatory elements also impact the inter-related processes of secondary metabolism and differentiation in theseorganisms Clustering of biosynthetic enzymes is facilitating researchinto the molecular mechanisms which mediate expression of genes in-volved in antibiotic synthesis which will enable gene expression to bepositively regulated and optimized to systematically maximize antibiot-ic formation in industrial fermentation processes (Martin 1992)Routes to production of penicillin G cephalosporins and cephamycinsshare a common early pathway to synthesis of the isopenicillin-N inter-mediate The lsquonaturalrsquo cephalosporins produced by A chrysogenum haverelatively low clinical efficacy and are produced semi-synthetically fromintermediate building blocks 7-aminodeacetoxycephalosporanic acid(7-ADCA) and 7-aminocephalosporanic acid (7-ACA) While Pchrysogenum cannot produce cephalosporins and cephamycins it is anattractive potential recombinant host because of its hyper capacity toproduce penicillin via the common precursor isopenicillin N and in-deed the host was successfully engineered to synthesize cephalosporinsand 7-ADCA (Crawford et al 1995 Robin et al 2001 2003ab van deSandt and de Vroom 2000) The principal transformations of Pchrysogenum involved introduction of the gene cefE from Streptomycesclavuligerus encoding production of the expandase enzyme deacetoxy-cephalosporin-C synthase and the genes cefEF and cefG from Pchrysogenum encoding the dual expandasehydroxylase and acetyl-transferas respectively Other recombinant proteins for example thecmcH gene from S clavuligerus encoding deacetylcephalosporin O-carbamoyltransferase have been successfully expressed in Pchrysogenum 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 strainsalready improved through classical mutation programs can lead to de-velopment of multipurpose host platforms for production of non-nativesecondary metabolites

In related work two dimensional electrophoresis-based proteo-mics was used to compare protein maps with up to 950 proteins

being identified from a three P chrysogenum strains a high and lowpenicillin 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 whilepathways for synthesis of other metabolites were reduced

Peroxisomes participate in important functional aspects of second-ary metabolite synthesis in filamentous fungi including the final stepsof penicillin synthesis mediated by isopenicillin N-acetyltransferase(Muller et al 19911992)

523 TaxolThe tetracyclic diterpene lactam taxol is a low toxicity broad

spectrum anticancer drug with beneficial application against breastuterine and other cancers (Wani et al 1971) Taxol is present natu-rally at low concentrations of 001ndash005 in the bark roots andbranches of the western yew Taxus brevifolia an endangered speciesIn the past few years significant progress has been made in our un-derstanding of the general biology and taxol biosynthetic pathwayof taxol-producing endophytic fungi with a view to producing taxolby microbial fermentation (Lin et al 2003 Zhou et al 2008 2010)Some of the 20 or so genes encoding enzymes of the pathway havebeen sequenced and some genetic transformation systems havebeen established which included development of some fungal pro-moter-containing expression cassettes ultimately directed at devel-oping efficient taxol-producing recombinant fungal hosts (Guo etal 2006 Wang et al 2007abc)

6 Expressing fungal multi-protein complexes or pathways inother hosts

While much effort has been fruitfully invested in transforminghosts to produce individual recombinant proteins and to optimizingrecombinant product formation the evolution in our understandingof these systems combined with newly acquired knowledge of geno-mics and proteomics is facilitating more advanced engineering ofhosts with whole new pathways or functions The huge diversity offungal 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 optimalexpression of the pathway or synthesis and exploitation of the de-sired metabolic products In this section a small number of fungalcomplex systems expressed in non fungal hosts will be briefly dis-cussed It is hoped that the examples point to the great potential toexploit fungal diversity in part through transfer of fungal machineryto other hosts

61 Expression of fungal systems in plants

There is considerable interest in metabolic engineering of fattyacids into plants to produce new oil seed crops Since filamentousfungi are among the best producers of highly unsaturated fatty acidsand since higher plants do not produce these fatty acid there is inter-est in transforming plants with some of these key highly unsaturatedfatty acids (HUFA) biosynthetic enzymes with M alpina being recog-nized as one of the most useful gene sources (Drexler et al 2003Huang 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 stepsinvolved seed-specific co-expression of an oleate delta12 desaturaseand a Δ6 desaturase both from Mortierella species in canola (Liu etal 2001) The fifth generation of field trials contained 44 γ-linolenicacid (GLA) not present in rapeseed Similar high proportions of GLAwere accumulated when a Δ6 desaturase from P irregulare wasexpressed in Brassica juncea (Hong et al 2002) Some other fungallong 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 Fusariummoniliforme co-expressed in both soybean and yeast (Yersinialipolytica) with the primary LC-PUFA biosynthetic enzymes enhancedlevels of eicosapentaenoic acid (EPA) production (Damude et al2006) Enzymes with similar biofunctional properties and potentialare produced by Claviceps purpurea (Meesapyodsuk et al 2007) andCoprinus cinereus (Zhang et al 2007) A recent review on metabolicengineering of oil-seed crops to produce omega-3 long chained fattyacids 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 differentpivotal amino acid linked processes for ammonium assimilation Infilamentous 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 gdhAfrom A niger was expressed in the cytoplasm of rice plants positivechanges 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 filamentousfungi have significant potential applications in genetically-engineered plants and hydrophobin-fusion technology is predictedto become a very useful tool in plant biotechnology By way of exam-ple when the hydrophobin HFBI sequence from T reesei was fused togreen fluorescent protein (GFP) and transiently expressed inNicotiana benthamiana plants mediated by A tumefaciens the fusionenhanced GFP accumulation (Joensuu et al 2010) The fusion proteinconstituted more than 50 of total soluble protein and delayed leafnecrosis

62 Expression of fungal systems in bacteria and yeast

621 Polyketide biosynthetic machineryPolyketides result from biosyntheses involving polymerization of

acetyl and propionyl subunits in a manner analogous to fatty acidsynthesis Polyketide products generated by different PKS types in-clude erythromycin tacrolimus epothilone tetracycline tetraceno-mycin daunorubicin 6-methyl salicylic acid aflatoxin B1 andlovastatin (Crawford et al 2006 Fujii 2010 Kennedy et al 1999Moriguchi et al 2010 Shen 2003 Watanabe et al 1996) Fungalpolyketide synthases (PKSs) utilize a variety of acyl-CoA substratesto catalyze carbon to carbon bond forming reactions to synthesizethe well known complex fungal polyketides which exhibit a rangeof bioactivities Many gene clusters thought to encode the enzymesresponsible for synthesizing polyketides such as bikaverin zearale-none and hypothemycin including genes encoding PKSs have beencharacterized Recent reports indicate that these recombinant fungalenzymes have been expressed in E coli rendering the bacterial hostcapable of production of the corresponding secondary metabolite(Saruwatari et al 2011) The aromatic polyketide 6-methylsalicylicacid is synthesized by ATX encoding gene atX present in A terreusand Penicillium patulum and this gene has successfully beenexpressed in E coli S cerevisiae and Streptomyces species and an en-hanced production of 6-methylsalicylic acid was noted in the case ofS cerevisiae (Bedford et al 1995 Kealey et al 1998) The huge fungalgene responsible for bikaverin (aromatic polyketide anticancer drug)biosynthesis PKS4 originating in Giberella fujikuroi was successfullyexpressed in E coli with production of the polyketide (Ma et al2007) Ma et al (2009) also reported that the lovastatin nonaketidesynthase (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 contributingsubstantially to determining the complex set of functions and struc-tures of the polyketide synthases Mixing of domains of differentPKS genes including domains originating from fungi and bacteriagenerated different combinations of biosynthetic enzymes and

demonstrated the potential to vary the synthezing machinery to pro-duce wholly novel metabolites not produced naturally by the nativestrains (Saruwatari et al 2011 Zhang et al 2008)

622 Penicillin biosynthetic machineryWhile 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 forexample yeasts for production of new beta lactams and perhapsother bioactive compounds such as peptides An industrial precedentexists for changing from a filamentous fungal host to a yeast host inthe case of citric acid production when the Pfizer Company modifiedtheir process by introducing a Candida guilliermondii yeast strain inplace of the conventional Aspergillus producer thereby increasing cit-rate production rate (Ward 1989 1991) In P chrysogenum productionof penicillin is compartmentalized in peroxisomes and the cytosol Theyeast Hansenula polymorpha has a number of features that make it po-tentially an attractive host as an alternative producer of beta-lactamsincluding 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 peroxisomesof H polymorpha may be hyperinduced Gidijala et al(2009) success-fully engineering H polymorpha to produce and efficiently secretepenicillin a) by introducing the P chrysogenum gene encodingthe non-ribosomal peptide synthase (NRPS) δ-(L-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) into H polymorpha resulting inproduction of active ACVS enzyme b) co-expression of the B subtilisstp gene encoding the enzyme phosphopantetheinyl transferasewhich activates ACVS and c) co-expression of P chrysogenum genesencoding cytosolic isopenicillin N synthase and the peroxisomal en-zymes isopenicillin N acyl transferase (IAT) and phenylacetyl CoA li-gase (PCL) Recombinant fungal ACVS has also been expressed inbakerrsquos yeast (Siewers et al 2009)

7 Engineering of pathogenic fungi

A very large number of filamentous fungi are pathogenic to otherorganisms including animals and humans plants and insects and inthe case of animals and plants the resulting diseases have vast nega-tive economic and social impacts Molecular techniques includingstrategies involving production of recombinant proteins may beused to facilitate elucidation of the causes of pathogenesis and to pro-vide direction on solutions on pathogenesis problems The followingbrief examples are offered to illustrate potential opportunities

71 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 asherbicides insecticides or biological inhibitors against undesired tar-gets Recently published examples include a recombinant funguswhich kills human malaria parasites in mosquitoes mediated by se-creted recombinant toxic peptides and enhancement of infectivity ofa recombinant entomopathogenic Beauveria bassiana towards larvaeof 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 ofpyrethroid-resistantmosquitoes Several fungi includingMetarhiziumanisopliae are pathogenic to adult mosquitoes but the infective pro-cess through contact with the cuticle is slow (12ndash14 days) Recombi-nant strains of M anisopliae expressing the antimicrobial toxinscorpine or a salivary gland and mid-gut peptide 1 (SM1) reducedentry andor development of Plasmodium falciparum the parasiticcausative agent of malaria into the Anopheles gut where it gets

transformed from gametocytes into infective sporozoites Sporozoitecount reductions mediated by the expressed SM1 and scorpine were71 and 90 respectively The SMI peptide binds to the surface of thesalivary gland of Anopheles gambiae the principal malaria vectorand blocks entry of the parasite into themosquito vector The scorpionantimicrobial scorpine is a hybrid of two toxins a cecropin and adefensin which is two orders of magnitudemore toxic against Plasmo-dium A recombinant M anisopliae expressing an [SM1]sub8scorpinefusion protein reduced sporozite counts by 98 (Fang et al 2011)

The infective action of the filamentous fungus B bassiana towardsinsects via their cuticle is generally a slow process However lepi-dopteran pests are rapidly killed by ingestion of the Vip3A insecticidalproteins produced by Bacillus thuriengensis A recombinant strain of Bbassiana which highly expressed one of the Vip3A toxins Vip3Aa1 inits conidial cytoplasm when sprayed on cabbage leaves was shown todramatically reduce larval numbers feeding on the cabbage leaves (by262 fold) (Qin et al 2010) The mid gut of the infected larvae con-tained a digested form of the toxin and the larvae exhibited theexpected symptoms of toxin action namely shrinkage and palsyMethods have also been established whereby larvae of organismssuch as the silk worm have been directly transformed to produce re-combinant fungal proteins For example the 1320-bp endoglucanase1 gene from T viride was cloned and expressed in silkworm larvaeand in a silkworm cell line using a mutant baculovirus expression sys-tem (Li et al 2010)

72 Plant pathogens

Molecular methods are also being used to develop a better under-standing of the mechanisms related to fungal pathogenicity of plantsIn 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 Colletotrichumorbiculare and the rice blast pathogen Magnaporthe oryzae (Bhambraet al 2006 Wang et al 2007d) and in production of host selectiveAK-protein toxins by Alternaria alternata which mediate its pathoge-nicity to multiple plant species including Japanese pear (Imanaka etal 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 recom-binant A alternata strains with AK-toxin disruptions to confirm therole of AK-toxins in pathogenicity

It was noted earlier (341) that the genome of the human Roryzae pathogen contained expanded gene families of encoding se-creted proteases and that these proteases are involved in the infectiveprocess and are characteristic of the virulence of Rhizopus pathogens(Ma et al 2009) It was also noted in the same section that R mieheiwhich causes fungal infections in humans and animals can resisthigher concentrations of statins than is observed in Mucorcircinelloides where statins inhibit HMG-CoA and ultimately lead toMucor cell death A recombinant M circinelloides strain expressingthe gene hmgR encoding the HMG-CoA of R miehei was renderedmore tolerant to statins Clearly our knowledge of fungal pathogene-sis will greatly expand over the next few years as the genomes ofmore plant pathogens are sequenced and their gene functions areannotated

8 Engineering of biocontrol fungi

Filamentous fungi can also have beneficial roles in plant diseasemanagement as will be illustrated here by focusing on Trichodermaspecies Many strains of Trichoderma are highly competent in coloniz-ing plant root surfaces and thrive and proliferate in association withhealthy root surfaces (Sriram and Ray 2005) Many other fungi lackthis ability and Trichoderma species have evolved a variety of mecha-nisms to attack and parasitize these other fungi and benefit from

nutrients from these other fungi as they support and enhance plantgrowth as biocontrol agents (Harman et al 2004) Biocontrol agentsutilize several antagonistic strategies against pathogens and all ofthese mechanisms are exploited by Trichoderma (Dennis andWebster 1971abc) These mechanisms include competition by lim-iting access to the pathogens nutritional needs production ofinhibitory 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 antibioticand production of hydrolytic enzymes had a synergistic effect on thepathogens (Schirmboumlck et al 1994) Enzymes thought beneficial toTrichoderma species in attacking pathogens such as Fusarium oxy-sporum and Botrytis cinerea include the lytic enzymes chitinasebeta-13-glucanases and protease (Cherif and Benhamou 1990Elad and Kapat 1999 Geremia et al 1993 Tronsmo et al 1993)When a recombinant chitinase from a soil bacterium Serratiamarcescens used to control Sclerotium rolfsii was introduced into Tharzianum under control of a constitutive promoter the constitutiveproduction of chitinase by the Trichoderma biocontrol agent enabledit to overgrow the S rolfsii pathogen (Haran et al 1993) An alkalineprotease 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 fungalplant pathogens is to directly arm the plants with the requisite biode-fense machinery and indeed several genes encoding the Trichodermaenzymes mediating Trichodermas parasitic role have been expressedin 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 proteinresearch 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 recombinantprotein 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 levelsof homologous enzymes are widely recognized In exploiting theseadvantages for production of recombinant proteins researchershave generally used the most effective regulatory expression and se-cretion machinery identified in these organisms and hooked it up tothe genes encoding the desired recombinant protein an approachwhich has met with considerable commercial success

Filamentous fungi also have several disadvantages as hosts forheterologous protein production perhaps the most serious beingtheir abilities also to produce and secrete homologous proteaseswhich may degrade the desired recombinant product In addition tothe secreted proteases filamentous fungi may be subject to differentdegrees of lysis during the fermentation process the extent ofwhich will vary with other aspects of fungal physiology includingmorphology such that intracellular proteases may be releasedwhich 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 isbeing invested in modifying the glycosylation machinery in fungi touse them as hosts for production hosts of more human-like glycopro-teins it is widely accepted that yeasts such as Pichia may be bettercandidate hosts for production of recombinant human therapeuticglycoproteins

The vast amounts of scientific information being generatedthrough advances in genomics and proteomics are expanding the op-portunities for new applications for recombinant filamentous fungi Itis facilitating a shift from simply inserting a gene encoding a singleprotein product to inserting machinery encoding multiple genes forexample 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 earlystage it will clearly gain momentum and will become a more impor-tant pursuit in the field of industrial microbiology in the years aheadIt has also been recognized that fungi represent an enormouslydiverse range of organisms and hence a huge resource for novel bio-synthetic systems especially since the vast majority of fungal specieson the planet have yet to be cultured identified and characterized Itis clear that many of the fungi naturally harboring novel biosyntheticmachinery will likely not be the best hosts for large-scale exploitationof these capabilities It is expected that in many cases the target bio-synthetic abilities will be better utilized by transformation into otherfungal hosts perhaps also to other microbial hosts and in some casesto non-microbial hosts for example to plants

One of the most exciting new aspects of the study of recombinantproteins expression in fungi is the realization that the future applica-tions will not be confined to the biomanufacturing capacities of theseorganisms Some of the natural beneficial impacts and especially thedeleterious 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 areknown to cause diseases in humans (Monk and Goffeau 2008) andmany of these organisms exhibit multidrug resistance mediated bymultidrug efflux pumps (Balzi et al 1994) A myriad of fungal patho-gens attack plants and a quantum shift is currently taking place in theadvancement of our knowledge of the plantndashmicrobe interactions in-volved in these diseases (Boller and He 2009) The underlying causa-tive mechanisms involved in these attacks are now beingcharacterized using modern genomic and molecular strategies Putsimply the economic and social costs associated with the impacts offungal pathogenesis are enormous The opportunities to exploit re-combinant technology to unravel the causes and then bring forwardsolutions will represent a very important future focus of recombinantprotein technology as it relates to fungi

References

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Imanaka H Tanaka S Feng B Imamura K Nakanishi K Cultivation characteristicsand gene expression profiles of Aspergillus oryzae by membrane-surface liquidculture shaking-flask culture and agar-plate culture J Biosci Bioeng2010109267ndash73

Imazaka A Tanaka A Harimoto Y Yamamoto M Akimitsu K Park P et al Contributionof peroxisomes to secondary metabolism and pathogenicity in the fungal plantpathogen Alternaria alternata Eukaryot Cell 20109682ndash94

Ishida H Matsumura K Hata Y Kawato A Suginami K Abe Y et al Establishment of ahyper-protein production system in submerged Aspergillus oryzae culture undertyrosinase-encoding gene (melO) promoter control Appl Microbiol Biotechnol200157131ndash7

Iwashita K Recent studies of protein secretion by filamentous fungimdashreview J BiosciBioeng 200294530ndash5

Jacobs DI Olsthoorn MMA Maillet I Akeroyd M Breestraat S Donkers S et al Effectivelead saelection for improved porotein production in Aspergillus niger based on in-tegrated genomics Fungal Genet Biol 200946S141ndash52

Jami MS Barreiro C Garcia Estradfa C Martin JF Proteome analysis of the penicillinproducer Penicillium chrysogenum characterization of protein changes during in-dustrial strain improvement Mol Cell Proteomics 201091182ndash98

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Jeenes DJ Marczinke B MacKenzie DA Archer DB A truncated glucoamylase gene fu-sion for heterologous protein secretion from A niger FEMS Microbiol Lett1993107267ndash71

Jeenes DJ Pfaller R Archer DB Isolation and characterization of a novel stress induciblePDI family gene from A niger Gene 1997194151ndash6

Jensen EB Boominathan KC Thermophilic fungal expression system United States Pat-ent 5695985 1997

Jin FJ Watanabe T Juvvadi PR Maruyama J-I Arioka M Kitamoto K Double dis-ruption of the proteinase genes tppA and pepE increase the productionlevel of human lysozyme by Aspergillus oryzae Appl Gen Mol Biotechnol2007761059ndash68

Joensuu JJ Conley AJ Lienemann M Brandle JE Linder MB Menassa R Hydrophobin fu-sions for high-level transient protein expression and purification in Nicotianabenthamiana Plant Physiol 2010152622ndash33

Kainz E Gallmetzer A Hatzl C Nett JH Li H Schinko T et al N-glycan modification inAspergillus species Appl Environ Microbiol 2008741076ndash86

Karnaukhova E Ophir Y Golding B Recombinant human alpha-1-proteinase inhibitortowards therapeutic use Amino Acids 200630317ndash32

Karnaukhova E Ophir Y Trinh L Dalal N Punt P Golding B et al Expression of humanα(I)-proteinase inhibitor in Aspergillus niger Microb Cell Factories 20076(34)1-10

Kasajima T Yamaguichi M Hirai N Ohmachi T Yoshida T In vivo expression of UDP-N-acetylglucosamine α-3-D-mannoside β-12-N-acetylglucosyl-transferase 1 (GnT-1) in Aspergillus oryzae and effects on the sugar chain of alpha-amylase Biosci Bio-technol Biochem 2006702662ndash8

Kealey JT Liu L Santi DV Betlach MC Barr PJ Production of a polyketide natural prod-uct in nonpolyketide-producing prokaryotic and eukaryotic hosts Proc Natl AcadSci USA 199895499ndash505

Kennedy J Auclair K Kendrew SG Park C Vederas JC Hutchinson CR Modulation ofpolyketide synthase activity by accessory proteins during lovastatin biosynthesisScience 19992841368ndash72

Kern A Tilley E Hunter IS Legisa M Glieder A Engineering primary metabolic path-ways of industrial micro-organisms J Biotechnol 20071296-29

Kim Y Nandakumar MP Marten MR Proteomics of filamentous fungi Trends Biotech-nol 200725395ndash400

Kinghorn JR Unkles SE Aspergillus New York Plenum Press 1994Kirk TK Farrel RL Enzymatic ldquocombustionrdquo the microbial degradation of lignin Ann

Rev Microbiol 198741465ndash505Kitamoto K Kimura K Gomi K Kumagai C Electrophoretic karyotype and gene assign-

ment to chromosomes of Aspergillus oryzae Biosci Biotechnol Biochem 1994581467ndash70

Kleinkauf H von Doumlhren H Non-ribosomal biosynthesis of peptide antibiotics Eur JBiochem 19901921-15

Knutzon DS Thurmond JM Huang Y-S Chaudhary S Bobik EG Chan GM et al Identi-fication of Δ5-desaturase from Mortierella alpina by heterologous expression inbakers yeast and canola J Biol Chem 199827329360ndash6

Kobayashi T Abe K Asai K Gomi K Juwadi PR Kitamoto K et al Genomics of Aspergil-lus oryzae Biosci Biotechnol Biochem 200771646ndash70

Kumar R Singh S Singh OV Bioconversion of lignocellulosic biomass biochemical andmolecular perspectives J Indust Microbiol Biotechnol 200835377ndash91

Kwon-Chung KJ Bennett JE Mucormycosis Medical mycology Philadelphia Lea andFebiger 1992 p 524ndash59

Lehmbeck J Alkaline protease deficient filamentous fungi United States Patent 6291209 2001

Li X Wang D Zhou F Yang H Bhaskar R Hu J et al Cloning and expression of a cellu-lose gene in the silkworm Bombyx mori by improved Bac-to-BacBmNPV baculo-virus expression system Mol Biol Rep 201083721ndash8

Lin FC Liu XH Wang HK Zhang CL Recent research and prospect on taxol and its pro-ducing fungi Acta Microbiol Sin 200343534ndash8

Liu J-W Huang Y-S DeMichele S Bergana M Bobik Jr E Hastilow C et al Evaluation ofthe seed oils from a canola plant genetically transformed to produce high levels ofγ-linolenic acid In Hunag Y-S Ziboh VA editors γ-Linolenic acid recent ad-vances in biotechnology and clinical applications Champaign IL AOCS Press2001 p 61ndash71

Liu L Liu J Qiu RX Zhu XG Dong ZY Tang GM Improving heterologous gene ex-pression in Aspergillus niger by introducing multiple copies of protein-bindingsequence containing CCAAT to the promoter Lett Appl Microbiol 200336358ndash61

Loder PB Abraham EB Isolation and nature of intracellular peptides from a cephalo-sporin C producing Cephalosporium sp Biochem J 1971123471ndash6

Lorito MWoo SL Fernandez IG Colucci G Harman GE Pintor-Toro JA et al Genes frommycoparasitic fungi as a source for improving plant resistance to fungal pathogensProc Natl Acad Sci USA 1998957860ndash5

Lubertozzi D Keasling JD Developing Aspergillus as a host for heterologous expressionBiotechnol Adv 20092753ndash75

Ma LJ Ibrahim AS Skory C Grabherr MG Burger G Butler M et al Genomic analysis ofthe basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication au-thor(s) source PLoS Genet 20095(7) Article No e1000549

Ma SM et al Enzymatic synthesis of aromatic polyketides using PKS4 from Gibberellafujikuroi J Am Chem Soc 200712910642ndash3

Mach RL Zeilinger S Regulation of gene expression in industrial fungi TrichodermaAppl Microbiol Biotechnol 200360515ndash22

Machida M Progress of Aspergillus oryzae genomics Adv Appl Microbiol 20025181-106

Machida M Asai K Sano M Tanaka T Kumagai T Terai G et al Genome sequencing andanalysis of Aspergillus oryzae Nature 20054381157ndash61

MacKenzie DA Wongwathanarat P Carter AT Archer DB Isolation and use of a homol-ogous histone H4 promoter and a ribosomal DNA region in a transformation vectorfor the oil-producing fungus Mortierella alpina Appl Environ Microbiol 2000664655ndash61

MacKenzie DA Kraunsoe JAE Chesshyre JA Lowe G Komiyama T Fuller RS et al Aber-rant processing of wild-type and mutant bovine pancreatic trypsin inhibitor se-creted by Aspergillus niger J Biotechnol 199863137ndash46

Maeda H Sano M Maruyama Y Tanno T Akao T Totsuka Y et al Transcriptional anal-ysis of genes for energy catabolism and hydrolytic enzymes in the filamentous fun-gus Aspergillus oryzae using cDNA microarrays and expressed sequence tags ApplMicrobiol Biotechnol 20046574ndash83

Mantyla A Paloheimo M Hakola S Lindberg E Leskinen S Kallio J et al Production inTrichoderma reesei of three xylanases from Chaetomium thermophilum a recombi-nant thermoxylanase for biobleaching of kraft pulp Appl Microbiol Biotechnol200776377ndash86

Maor R Shirasu K The arms race continues battle strategies between plants and fungalpathogens Curr Opin Microbiol 20058399ndash404

Marahiel MA Multidomain enzymes involved in peptide synthesis FEBS Lett199230740ndash3

Maras M De Bruyn A Vervecken W Uusitalo J Penttila M Busson R et al In vivosynthesis of complex N-glycans by expression of human N-acetylglucosamyl-transferase 1 in the filamentous fungus Trichoderma reesei FEBS Lett1999a452365ndash70

Maras M Saelens X Laroy W Piens K Claeyssens M Fiers W et al In vitro conversionof the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosac-charidesmdashevidence for N-acetylglucosaminetransferase-1-accepting glycans fromTrichoderma reesei Eur J Biochem 1997249701ndash7

Maras M van Die I Contreras R van den Hondel CAMJJ Filamentous fungi as productionorganisms for glycoproteins of bio-medical interest Glycoconj J 1999b1699-107

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Martin JF Clusters of genes for the biosynthesis of antibioticsmdashregulatory genes andoverproduction of pharmaceuticals J Ind Microbiol Biotechnol 1992973ndash90

Martiacuten JF Liras P Organization and expression of genes involved in the biosynthesis ofantibiotics and other secondary metabolites Annu Rev Microbiol 198943173ndash206

Martinez D Berka RM Henrissat B Saloheimo M Arvas M Baker SE et al Genome se-quencing and analysis of the biomass-degrading fungus Trichoderma reesei (synHypocrea jecorina) Nature Biotechnol 200826553ndash60

Martinez D Larrondo LF Putnam N Gelpke MD Huang K Chapman J et al Genome se-quence of the lignocellulose degrading fungus Phanerochaete chrysosporium strainRP78 Nature Biotechnol 200422695ndash700

Maruyama J Ohnuma H Yoshikawa A Kadokura H Nakajima H Kitamoto K Produc-tion and product quality assessment of human hepatitis B virus-preS2 antigen insubmerged and solid state cultures of A oryzae J Biosci Bioeng 200090118ndash20

Masai K Maruyama J Nakajima H Sakamoto K Akita O Kitamoto K Analysis of differ-ential gene expression across regions of the Aspergillus oryzae mycelium FungalGenet Newslett 200552161

Matsuzaki F Shimizu M Wariishi H Proteomic and metabolomic analyses of thewhite-rot fungus Phanerochaete chrysogenum exposed to exogenous benzoic acidJ Proteome Res 200872342ndash50

Meesapyodsuk D Reed DW Covello PS Qiu X Primary structure regioselectivity andevolution of the membrane-bound fatty acid desaturases of Claviceps purpurea JBiol Chem 200728220191ndash9

Meyer HW Molds in floor dust and building-related symptoms in adolescent schoolchildren Indoor Air 20041465ndash72

Meyer V Genetic engineering of filamentous fungi-progress obstacles and futuretrends Biotechnol Adv 200826177ndash85

Michaelson LV Lazarus CM Griffiths G Napier JA Isolation of a Δ5-fatty acid desaturasegene from Mortierella alpina J Biol Chem 199827319055ndash9

Minetoki T Kumagai C Gomi K Kitamoto K Takahashi K Improvement of promoter ac-tivity by the introduction of multiple copies of the conserved region III sequenceinvolved in the efficient expression of Aspergillus oryzae amylase-encoding genesAppl Microbiol Biotechnol 199850459ndash67

Monk BC Goffeau A Outwitting multidrug resistance to antifungals Science 2008321367ndash9

Monsanto Microbial sequence database httpmicrobialcereoncom2001Moralejo FJ Cardoza RE Gutierrez S Lombrana M Fierro F Martin JF Silencing of the

Aspergillopepsin B (pepB) gene of Aspergillus awamori by antisense RNA expres-sion or protease removal by gene disruption results in a large increase in thauma-tin production Appl Environ Microbiol 2002683550ndash9

Moriguchi T Kezuka Y Nonaka T Ebizuka Y Fujii I Hidden function of catalytic domainin 6-methylsalicylic acid synthase for product release J Biol Chem 201028515637ndash43

Muller D Stahl U Meyer V Application of hammerhead ribozymes in filamentousfungi J Microbiol Meth 200665585ndash95

Muller WH Bovenberg RA Groothuis MH Kattevilder F Smaal EB van der Voort LHet al Involvement of microbodies in penicillin biosynthesis Biochim BiophysActa 19921116210ndash3

Muller WH van der Krift TP Krouwer AJJ Wosten HAB van der Voort L Smaal EB et allocalization of the pathway of the penicillin biosynthesis in Penicillium chryso-genum EMBO 199110489ndash95

Nakajima K Asakura T Maruyama J Morita Y Oike H Shimizu-Ibuka A et al Extracel-lular production of neoculin a sweet-tasting heterodimeric protein with taste-modifying activity by Aspergillus oryzae Appl Environ Microbiol 2006723716ndash23

Nevalainen H Souminen P Taimisto K On the safety of Trichoderma reesei J Biotechnol199437193ndash200

Nevalainen KMH Teo VS Bergquist PL Heterologous protein expression in filamen-tous fungi Trends Biotechnol 200523468ndash74

Ngiam C Jeenes DA Punt PJ van den Hondel CAMJJ Archer DA Characterization of afoldase protein disulfide isomerase A in the protein secretory pathway of Aspergil-lus niger Appl Environ Microbiol 200066775ndash82

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Nyyssonen E Penttila M Harkki M Saloheimo A Knowles JK Keranen S Efficient pro-duction of antibody fragments by the filamentous fungus Trichoderma reesei Bio-technology 199311591ndash5

ODonnell D Wang L Xu J Ridgway D Gu T Moo-Young M Enhanced heterologousprotein production in Aspergillus niger through pH control of extracellular proteaseactivity Biochem Eng 20018187ndash93

Oda K Kakizono D Yamada O Lefuji H Akita O Iwashita K Proteomic analysis of extra-cellular proteins of Aspergillus oryzae grown under submerged and solid-state cul-ture conditions Appl Environ Microbiol 2006723448ndash57

Ouyang J Yan M Kong D Xu L A complete protein pattern of cellulose and hemicellu-lase genes in the filamentous fungus Trichoderma reesei Biotechnol J 200611266ndash74

Papagianni M Young MM Protease secretion in glucoamylase producer Aspergillusniger cultures fungal morphology and inoculum effects Proc Biochem 2002l371271ndash8

Papagiannopoulos P Andrianopoulos A Sharp JA Davis MA Hynes MJ The hapC geneof Aspergillus nidulans is involved in the expression of CCAAT-containing pro-moters Mol Gen Genet 1996251412ndash21

Paper JM Scott-Craig JS Adhikari ND Cuomo CA Walton JD Comparative proteomicsof extracellular proteins in vitro and in planta from the pathogenic fungus Fusari-um graminearum Proteomics 200773171ndash83

Paul D Pandey G Jain RK Suicidal genetically engineered microorganisms for bioreme-diation need and perspectives Bioessays 2005563ndash73

Pel HJ de Winde JH Archer DB Dyer PS Hofmann G Schaap PJ et al Genome sequenc-ing and analysis of the versatile cell factory Aspergillus niger CBS 51388 NatureBiotechnol 200725221ndash31

Penttila M Heterologous protein production in Trichoderma In Harman GE Kubicek CPeditors Trichoderma and Gliocladium London Taylor and Francis 1998 p 365ndash82

Pisanelli I Kujawa M Gschnitzer D Spaduit O Seiboth B Peterbauer C Heterologousexpression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in As-pergillus spp and characterization of the recombinant enzyme Appl Microbiol Bio-technol 201086599ndash606

Poidevin L Levasseur A Paes G Navarro D Heiss-Blanquet S Asther M et al Heterol-ogous production of the Piromyces equi cinnamoyl esterase in Trichoderma reeseifor biotechnological applications Letts Appl Microbiol 200949673ndash8

Punt PJ van Biezen N Conesa A Albers A Mangnus J van den Hondel C Filamentousfungi as cell factories for heterologous protein production Trends Biotechnol200220200ndash6

Punt PJ van Gemeren IA Drint-Kuijvenhoven A Hessing JGM van Muijlwijk-Har-teveld GM Beijersbergen A et al Analysis of the role of the gene bipA encod-ing the major endoplasmic reticulum chaperone protein in the secretion ofhomologous and heterologous proteins in black Aspergilli Appl MicrobiolBiotechnol 199850447ndash54

Punt PJ Schuren FHJ Lehmbeck J Christens J Hjort C van den Hondel C Characteriza-tion of the Aspergillus niger prtT a unique regulator of extracellular proteaseencoding genes Fungal Genet Biol 2008451591ndash9

Qin Y Ying SH Chen Y Shen ZC Feng MG Integration of insecticidal Vip3Aa1 intoBeauveria bassiana enhances fungal virulence to Spodoptera litura larvae by cuticleand per os infection Appl Environ Microbiol 2010764611ndash8

Radzio R Kueck U Synthesis of biotechnologically relevant heterologous proteins in fil-amentous fungi Proc Biochem 199732529ndash37

Ratledge C Lipids in fatty acids In Rose AH editor Primary products of metabolismLondon Academic Press 1978 p 263ndash96

Roberts IN Jeenes DJ Mackenzie DA Wilkinson AP Summer IG Archer DB Heterolo-gous gene expression in A niger A glucoamylasendashporcine pancreatic phospholi-pase A2 fusion protein is secreted and processed to yield mature enzyme Gene1992122155ndash61

Robin J Jakobsen M Beyer M Noorman H Nielsen J Physiological characterisation ofPenicillium chrysogenum strains expressing the expandase gene from Streptomycesclavuligerus during batch cultivations Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production Appl Microbiol Biotechnol200157357ndash62

Robin J Lettier G McIntyre M Noorman H Nielsen J Continuous cultivations of a Pen-icillium chrysogenum strain expressing the expandase gene from Streptomyces cla-vuligerus growth yields and morphological characterization Biotechnol Bioeng2003a83361ndash8

Robin J Bonneau S Schipper D Noorman H Nielsen J Influence of the adipate and dis-solved oxygen concentrations on the beta-lactam production during continuouscultivations of a Penicillium chrysogenum strain expressing the expandase genefrom Streptomyces clavuligerus Metab Eng 2003b542ndash8

Rodgers CJ Blanford CF Giddens SR Skamnioto P Armstrong FA Gurr SJ Designer lac-cases a vogue for high-potential fungal enzymes Trends Biotechnol 20102863ndash72

Romaine PC Chen X Methods and compositions for highly efficient transformation offilamentous fungi United States Patent Application 20020070007 2002

Ruijter GJG Kubicek CP Visser J Production of organic acids by fungi Mycota 200210213ndash20

Ruijter GJG Panneman H Visser J Overexpression of phosphofructokinase and pyru-vate kinase in citric acid producing Aspergillus niger Biochim Biophys Acta19971334317ndash26

Sakaki T Munetsuna E Enzyme systems for biodegradation of polychlorinateddibenzo-p-dioxins Appl Microbiol Biotechnol 20108823ndash30

Sakuradani E Kobayashi M Shimizu S Δ6-fatty acid desaturase from an arachidonicacid-producing Mortierella fungusmdashgene cloning and its heterologous expressionin a fungus Aspergillus Gene 1999238445ndash53

Sanchez JF Chiang YM Szewczyk E Davidson AD Ahuja M Oakley CE et al Moleculargenetic analysis of the orsellinic acidF9775 gene cluster of Aspergillus nidulansMol Biosyst 20096587ndash93

Saruwatari T Praseuth AP Sato M Torikai K Noguchi H Watanabe K A comprehensiveoverview on genomically directed assembly of aromatic polyketides and macrolidelactones using fungal megasynthases J Antibiotics 2011649-17

Schirmboumlck M Lorito M Wang YL Hayes CK Arisan-Atac I Scala F et al Parallel forma-tion and synergism of hydrolytic enzymes and peptaibol antibiotics molecularmechanisms involved in the antagonistic action of Trichoderma harzianum againstphytopathogenic fungi Appl Environ Microbiol 1994604364ndash70

Schmoll M Kubicek CP Regulation of Trichoderma cellulose formation lessons in mo-lecular biology from an industrial fungus A review Acta Microbiol Immunol Hung200350125ndash45

Schmoll M Seibel C Kotlowski C Vendt FWG Liebmann B Kubicek C Recombinantproduction of an Aspergillus bidulans class 1 hydrophobin (DewA) in Hypocreajecorina (Trichoderma reesei) is promoter dependent Appl Microbiol Biotechnol20108895-103

Schoen C Reichard U Monod M Kratzin HD Ruchel R Molecular cloning of an extra-cellular aspartic proteinase from Rhizopus microsporus and evidence for its expres-sion during infection Med Mycol 20024061ndash71

Schuster A Schmoll M Biology and biotechnology of Trichoderma Appl Microbiol Bio-technol 201087787ndash99

Schuster E Dunn-Coleman N Frisvad JC van Dijck PWM On the safety of Aspergillusnigermdasha review Appl Microbiol Biotechnol 200259426ndash35

Segal BH Walsh TJ Current approaches to diagnosis and treatment of invasive asper-gillosis Am J Respir Crit Care Med 2006173707ndash17

Seiboth B Gamauf C Pail M Hartl L Kubicek CP The D-xylose reductase of Hypocreajecorina is the major aldose reductase in pentose and D-galactose catabolism andnecessary for beta-galactosidase and cellulase induction by lactose Mol Microbiol200766890ndash900

Sharma R Katoch M Srivastava P Qazi G Approaches for refining heterologous proteinproduction in filamentous fungi World J Microbiol Biotechnol 2009252083ndash94

Shen B Polyketide biosynthesis beyond the type I II and III polyketide synthase para-digms Curr Opin Chem Biol 20037285ndash95

Shimizu S Ogawa J Kataoka M Kobayashi M Screening of novel microbial enzymes forthe production of biologically and chemically useful compounds Adv Biochem EngBiotech 19975845ndash87

Siewers V Chen X Huang L Zhang J Nielsen J Heterologous production of non-ribo-somal peptide LLD-ACV in Saccharomyces cerevisiae Metab Eng 200911391ndash7

Sims AH Robson GD Hoyle DC Oliver SG Turner G Prade RA et al Use of expressedsequence tag analysis and cDNA microarrays of the filamentous fungus Aspergillusnidulans Fungal Genet Biol 200441199ndash212

Smith DJ Earl AJ Turner G The multifunctional peptide synthetase performing the firststep of penicillin biosynthesis in Penicillium chrysogenum is a 421 073 dalton proteinsimilar to Bacillus brevis peptide antibiotic synthetases EMBO J 199092743ndash50

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Spreer A Ruchel R Reichard U Characterization of an extracellular subtilisin proteaseof Rhizopus microsporus and evidence for its expression during invasive rhinoorbi-tal mycosis Med Mycol 200644723ndash31

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Stachelhaus T Schneider A Marahiel MA Engineered biosynthesis of peptide antibi-otics Biochem Pharmacol 199652177ndash86

Sternberg D Mandels GR Induction of cellulolytic enzymes in Trichoderma reesei bysophorose J Bacteriol 1979139761ndash9

Stewart P Whitwam RE Kersten PJ Cullen D Tien M Efficient expression of a Phaner-ochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae ApplMicrobiol Biotechnol 199662860ndash4

Swift RJ Wiebe MG Robson GD Trinci APJ Recombinant glucoamylase production byAspergillus niger B1 in chemostat and pH-autostat cultures Fungal Genet Biol199825100ndash9

Szewczyk E Chiang YM Oakley CE Davidson AD Wang CC Oakley BR Identificationand characterization of the asperthecin gene cluster of Aspergillus nidulans ApplEnviron Microbiol 2008747607ndash12

Tailor MJ Richardson T Application of microbial enzymes in food systems and in bio-technology Adv Appl Microbiol 1979257-35

Takuchi M Ichishima E A 155 K acid carboxypeptidase O from Aspergillus oryzae AgricBiol Chem 198650633ndash8

te Biesebeke R Ruijter G Rahardjo YSP Hoogschagen MJ Heerikhuisen M Levin Aet al Aspergillus oryzae in solid-state and submerged fermentations FEMS YeastRes 20022245ndash8

Tian C Beeson WT Iaverone AT Sun J Marletta MA Cate JH Glass NL Systems anasly-sis of plant cell wall degradation by the model filamentous fungus Neurosporacrassa Proc Natl Acad Sci USA 200910622157ndash62

Toida J Fukuzawa M Kobayashi G Ito K Sekiguchi J Cloning and sequencing of thetriacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichiacoli FEMS Microbiol Lett 2000189159ndash64

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Venegas-Caleron M Sayanova O Napier JA An alternative to fish oils metabolic engi-neering of oil-seed crops to produce omega-3 long chain polyunsaturated fattyacids Prog Lipid Res 201049108ndash19

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von Dohren H A survey of nonribosomal peptide synthetase (NRPS) genes in Aspergil-lus nidulans Fungal Genet Biol 200946(Suppl 1)S45ndash52

Wan X Zhang YB Wang P Jiang M Molecular cloning and expression analysis of adelta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can ac-cumulate high levels of gamma-linolenic acid J Microbiol 201149151ndash4

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Wang L Darin R Tingyue G Moo-Young M Effects of process parameters on heterolo-gous protein production in Aspergillus niger fermentation J Chem Technol Biotech-nol 2003781259ndash66

Wang L Ridgway D Gu T Moo-Young M Bioprocessing strategies to improve heterol-ogous protein production in filamentous fungal fermentations Biotechnol Ad-vances 200523115ndash29

Wang SW Ma X PingWX Zhou DP Research advances on taxol production by microbefermentation Microbiology 2007b34561ndash5

Wang YC Guo BH Miao ZQ Tang KX Transformation of taxol-producing endophyticfungi by restriction enzyme-mediated integration (REMI) FEMS Microbiol Lett2007c273253ndash9

Wang Z-Y Soanes DM Kershaw MJ Talbot NJ Functional analysis of lipid metabolisminMagnaporthe grisea reveals a requirement for peroxisomal fatty acid β-oxidationduring appressorium-mediated plant infection Mol Plant Microbe Interact2007d20475ndash91

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nes Open University Press 1989Ward OP Proteases Comprehensive Biotechnology 2nd ed Moo-Young M editor Vol 3

2011 pp 571ndash582Ward PP May GS Headon DR Conneely OM An inducible expression system for the

production of human lactoferrin in A nidulans Gene 1992122219ndash23Ward PP Peddington CS Cunnighham GA Zhou X Wyatt RD Conneely OM A system

for production of commercial quantities of human lactoferrin a broad spectrumnatural antibiotic Biotechnology 199513498ndash503

Watanabe CM Wilson D Linz JE Townsend CA Demonstration of the catalytic rolesand evidence for the physical association of type I fatty acid synthases and a poly-ketide synthase in the biosynthesis of aflatoxin B1 Chem Biol 19963463ndash9

Weber G Schoumlrgendorfer K Schneider-Scherzer E Leitner E The peptide synthetasecatalyzing cyclosporin production in Tolypocladium niveum is encoded by a giant458-kilobase open reading frame Curr Genet 199426120ndash5

Wong P Walter M Lee W Mannhaupt G Munsterkotter M Mewes H-W et al FGDBrevisiting the genome annotation of the plant pathogen Fusarium graminearumNucleic Acid Res 201139D637ndash9

Xu J Wang L Ridgway D Gu T Moo-Young M Increased heterologous protein produc-tion in Aspergillus niger fermentation through extracellular proteases inhibition bypelleted growth Biotechnol Prog 200016222ndash7

Yamada O Ikeda R Ohkita Y Hayashi R Sakamoto K Akita O Gene silencing by RNAinterference in the koji mold Aspergillus oryzae Biosci Biotechnol Biochem200771138ndash44

Yoon J Aishan T Maruyama J Kitamoto K Enhanced production and secretion of het-erologous proteins by the filamentous fungus Aspergillus oryzae via disruption ofthe vacuolar protein sorting receptor gene Aovps10 Appl Environ Microbiol2010765718ndash27

Yoon J Maruyama J Kitamoto K Disruption of ten protease genes in the filamentousfungus Aspergillus oryzae highly improves production of heterologous proteinsAppl Microbiol Biotechnol 201189747ndash59

Yoshida T Kato Y Asada Y Nakajima T Filamentous fungus Aspergillus oryzae has twotypes of alpha-12-mannosidase one of which is a microsomal enzyme thatremoves a single mannose residue from Man9GlcNAc2 Glycoconj J 200017745ndash8

Zarrin M Leeder AC Turner G A rapid method for promoter exchange in Aspergillusnidulans using recombinant PCR Fungal Genet Biol 2005421ndash8

Zhang S Sakuradani E Ito K Shimizu S Identification of a novel bifunctional 1215 fattyacid desaturase from a basidiomycete Coprinus cinereus TD822ndash2 FEBS Lett2007581315ndash9

Zhang W Li Y Tang Y Engineered biosynthesis of bacterial aromatic polyketides inEscherichia coli Proc Natl Acad Sci USA 200810520683ndash8

Zheng XF Kobayashi Y Takeuchi A Construction of a low-serine-typecarboxypeptidase-producing mutant of Aspergillus oryzae by the expressionof antisense RNA and its use as a host for heterologous protein secretion ApplMicrobiol Biotechnol 19984939ndash44

Zhou X Zhu H Liu L Lin J Tang K A review recent advances and future prospects oftaxol-producing endophytic fungi Appl Microbiol Biotechnol 2010861707ndash17

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1 Introduction

2 Filamentous fungi as hosts for production of recombinant proteins


31 Aspergillus

311 A nidulans

312 A niger

313 A oryzae

32 Trichoderma

321 T reesei

33 Penicillium

331 P chrysogenum

34 Rhizopus

341 Rhizopus oryzae

35 White rot fungi

351 P chrysosporium

36 Fusarium

361 F graminearum

37 N crassa




411 Gene-fusions strategies

412 Overproduction of foldases and chaperones

413 Glycosylation

414 Other molecular strategies







511 Citric acid

512 Biodiesel

513 Higher unsaturated fatty acids (HUFAs)


521 Engineered biosynthesis of novel peptides

522 Antibiotics

523 Taxol

6 Expressing fungal multi-protein complexes or pathways in other hosts



621 Polyketide biosynthetic machinery

622 Penicillin biosynthetic machinery


71 Entomopathogens

72 Plant pathogens


9 Conclusions

References