(2011) Production of Ant Proteins by Filamentous Fungi

JBA-06496; No of Pages 21
Biotechnology Advances xxx (2011) xxxxxx
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Biotechnology Advances
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Production of recombinant proteins by lamentous fungi

Owen P. Ward
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L3G1
a r t i c l e
Available online xxxx Keywords: Aspergillus Trichoderma Penicillium Recombinant protein Heterologous Protease Genome Filamentous Fungi Pathogenesis
i n f o
a b s t r a c t
The initial focus of recombinant protein production by lamentous fungi related to exploiting the extraordinary extracellular enzyme synthesis and secretion machinery of industrial strains, including Aspergillus, Trichoderma, Penicillium and Rhizopus species, was to produce single recombinant protein products. An early recognized disadvantage of lamentous fungi as hosts of recombinant proteins was their common ability to produce homologous proteases which could degrade the heterologous protein product and strategies to prevent proteolysis have met with some limited success. It was also recognized that the protein glycosylation patterns in lamentous fungi and in mammals were quite different, such that lamentous fungi are likely not to be the most suitable microbial hosts for production of recombinant human glycoproteins for therapeutic use. By combining the experience gained from production of single recombinant proteins with new scientic information being generated through genomics and proteomics research, biotechnologists are now poised to extend the biomanufacturing capabilities of recombinant lamentous fungi by enabling them to express genes encoding multiple proteins, including, for example, new biosynthetic pathways for production of new primary or secondary metabolites. It is recognized that lamentous fungi, most species of which have not yet been isolated, represent an enormously diverse source of novel biosynthetic pathways, and that the natural fungal host harboring a valuable biosynthesis pathway may often not be the most suitable organism for biomanufacture purposes. Hence it is expected that substantial effort will be directed to transforming other fungal hosts, non-fungal microbial hosts and indeed non microbial hosts to express some of these novel biosynthetic pathways. But future applications of recombinant expression of proteins will not be conned to biomanufacturing. Opportunities to exploit recombinant technology to unravel the causes of the deleterious impacts of fungi, for example as human, mammalian and plant pathogens, and then to bring forward solutions, is expected to represent a very important future focus of fungal recombinant protein technology. 2011 Published by Elsevier Inc.
1. Introduction Filamentous fungi are extraordinary organisms which impact widely on so many aspects of our lives. These organisms are characterized as having branched lamentous structures or hyphae having typical diameters of 218 um, with (higher fungi) or without (lower fungi) cross-walls or septae. Higher fungi include Aspergillus, Penicillium, Trichoderma and Fusarium species. Lower fungi include Rhizopus and Mucor species. Filamentous fungi are chemo-organotrophs meaning they obtain their energy and carbon by oxidation of organic compounds. In traditional fermentation technology lamentous fungi are dominant producers of a range of primary metabolites, including organic acids, such as citric, gluconic, fumaric, kojic, itaconic acid and fatty acids. They also produce important secondary metabolites, especially as human therapeutics, for example, penicillin, cephalosporin, ergot alkaloids, griseofulvin, lovastatin, taxol and zeranol. Some are
Tel.: + 1 519 888 4567x32427; fax: + 1 519 746 0614. E-mail address: opward@uwaterloo.ca. 0734-9750/$ see front matter 2011 Published by Elsevier Inc. doi:10.1016/j.biotechadv.2011.09.012
producers of polysaccharides and biosurfactants. Some lamentous fungi are food materials in their own right, such as mushrooms, single cell protein/biomass (single cell protein, SCP) or indeed lipid-rich biomass (single cell oil, SCO), whereas other fungi are components in fermented foods. Some are producers of an array of fungal enzymes, for example amylases, amyloglucosidases, cellulases, pectinases, laccases/ligninases, phytase, proteases, microbial rennets, lipases and glucose oxidase. Many intracellular fungal enzymes are also exploited as biocatalysts in enzyme biotransformations in bioorganic synthesis reactions while others use biodegradative processes in soil bioremediation. Some strains are well known pathogens of humans, animals and plants (Cutler et al., 2007; Maor and Shirasu, 2005; Segal and Walsh, 2006) while others, for example mycorhizal fungi, have benecial associations with plants and/or participate in nutrient recycling in soil. Some fungi are responsible for food spoilage, wood decay and infestation of damp buildings (Bennett, 2006) and many fungal spores are known allergens (Meyer, 2004). The known high productivity chartacteristics of lamentous fungi are in part related to their inherent abilities to grow at high rates and to high biomass densities supported by low cost substrates in relatively simple fermenters. For industrial fermentations, lamentous
Please cite this article as: Ward OP, Production of recombinant proteins by lamentous fungi, Biotechnol Adv (2011), doi:10.1016/ j.biotechadv.2011.09.012
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx
fungi may be cultivated using traditional surface culture methods, where oxygen uptake involves passive exposure of the culture to the atmosphere or in a semi-solid culture where a non-homogeneous culture may be aerated through various forced air and/or mixing strategies. They may also be cultured in intensively mixed stirred tank reactors where the objective is to achieve conditions within the reactor which approach homogeneity, thereby facilitating more sophisticated process control. In addition to the benecial characteristics described above, fungi are especially interesting targets for production of recombinant proteins because of their demonstrated capacities to hyperproduce and secrete enzyme proteins, for example glucoamylase production by Aspergillus with impressive titers of greater than 25 g/L. Processes for production of mold-modied foods have been implemented for many thousands of years. At least the initial stages of these fermented food processes were promoted by surface culture suggesting a likely requirement for air, as a source of oxygen, to support preferential growth of molds on the medium surfaces. Since the raw materials were derived from plants including soybeans, wheat and rice, common logic led early scientists to conclude that the molds had the capacity to at least partially degrade the principal constituents of these plant materials, namely the associated carbohydrates (including starches, pectins, celluloses and hemicelluloses), proteins and lipids. Other molds have long been known to participate in processes including pathogenesis of plants, spoilage of fruits and vegetables and rotting of wood, likewise with the presumed involvement of mold products which could mediate the biodegradation of major structural constituents of these plant materials. Since the principal structural components of plants, being subjected to biodegradative processes during tradititional food fermentations, plant pathogenesis, fruit and vegetable spoilage, wood rotting, and related processes, are polymeric substances, early scientists soon postulated that cellular assimilation of breakdown products of these polymeric substances might require that the substrates be degraded in the extracellular environment. Indeed simple methods for surface cultivation of the molds involved, on agar-like media containing individual substrates, often insoluble, demonstrated zones of hydrolysis around the mold colony, illustrating that depolymerization reactions had been facilitated or catalyzed. It was soon concluded that these biodegradations were mediated by hydrolytic and other depolymerizing enzymes which were generally secreted by the molds into the extracellular medium or sometimes were located/attached on the extracellular surfaces of the biodegradative fungal organism. Jokichi Takamine, a Japanese immigrant to the United States, was rst to commercialize an isolated microbial enzyme. In 1894, he patented a process for preparation of diastatic enzymes from molds which was marketed as Takadiastase. The method involved growth of the fungus on the surface of solid substrates, such as wheat or bran, clearly based on the traditional processes for preparation of oriental fermented foods (Ward, 1989). The enzyme and producing organism were later characterized as fungal alpha-amylase and Aspergillus oryzae, respectively (Gwynne and Devchand, 1992). The development of recombinant technology harnesses the power of many of these lamentous fungi as hosts in the production of specic recombinant proteins as nal products with applications in the agricultural, food and nutrition, biomedical and pharmaceutical, and energy and industrial sectors (Schuster et al., 2002). This focus has represented the principal effort in applied genetic engineering over the past 25 years or so and is discussed in Sections 24 of this review. Further advancements of the core transformation technologies combined with progress in the elds of genomics and proteomics is leading to a more complex level of host engineering, whereby recombinant expression of multiple proteins and enzymes is facilitating engineering of blocks of new physiological or metabolic machinery into recombinant hosts. An example of these developments relates to our ability to engineer metabolic pathways so as to enhance production of
primary or secondary metabolites, or indeed to facilitate production of novel compounds through introduction of new biosynthetic pathways. The tremendous level of metabolic diversity exhibited by currently known lamentous fungi, together with the knowledge that only a small number of the estimated 1.5 million species (Hawksworth, 2001), which are thought to exist, means lamentous fungi will continue to supply the biosynthetic tools for synthesis of a myriad of novel products for countless years to come. This research is still at an early stage and some example roles of recombinant proteins in metabolic engineering of fungi are addressed in Section 5. Of course, the enormous diversity of fungal organisms does not imply that a newly isolated natural host capable of biosynthesizing novel new benecial compounds will be a suitable host for large scale manufacture. Indeed many novel organisms that will be identied in the future are likely to be the ones that are hard to cultivate, even unculturable and will require better hosts be they other lamentous fungal organisms or non-fungal organisms. Efforts are already being directed to exploiting the unique nature of some of the enzymes or metabolic pathways or systems of lamentous fungi by transferring these capabilities to other organisms, and some preliminary examples of expression of fungal proteins in other hosts are discussed in Section 6. Filamentous fungi also interact with other organisms using a variety of extremely complicated mechanisms, many of which are as yet poorly understood and their interactions often have overall benecial and perhaps more frequently negative societal outcomes. Thus some of the rst lamentous fungal organisms to be sequenced were human or agricultural plant pathogens and, as we go forward, genomic and proteonomic research will provide new insights into the molecular mechanisms involved in pathogenesis. These studies will include cloning and expression of recombinant proteins in fungi as prospective candidate causative proteins in pathogenesis followed by application of strategies to disrupt these proteins with a view to relating these manipulations to pathogenicity and virulence of the pathogen. While this area of research is in its infancy, some indications of its potential are included in Section 7. Some recent reviews on aspects of this review topic are listed in Table 1, including reference to some informative tables from these review papers. 2. Filamentous fungi as hosts for production of recombinant proteins Many lamentous fungi are natural excellent producers of extracellular enzymes and hence are exceptional candidate hosts for the production of recombinant proteins (Iwashita, 2002; Wang et al., 2005). Organisms such as Aspergillus and Trichoderma species are notable in their abilities to produce and secrete very high levels of proteins, with Aspergillus niger being capable of producing 2530 g/L of glucoamylase and Trichoderma reesei reported to be capable of producing 100 g/L of extracellular protein (Demain and Vaishnav, 2009). Meyer (2008) discussed four common strategies for implementation of transformations of lamentous fungi: The protoplast-mediated method involves use of cell wall-degrading enzymes for protoplast preparation with subsequent uptake of foreign DNA promoted by addition of polyethylene glycol (PEG) and calcium chloride. Noted disadvantages of this method are that transformations may vary with batch variations in the lytic enzyme, that a regeneration procedure is needed and that the high copy number of insertions of DNA may result in a less controlled transformation. In the Agrobacterium tumefaciens transformation, the A. tumefaciens carries a binary vector containing the target DNA between a 24-base pair repeating unit and a virulence region required for DNA transfer. The gene-carrier organism is cocultured with the lamentous fungus and the transformation is advantageous in that the low copy number of DNA insertions facilitates a more targeted integration. Beijersbergen et al. (2001) patented a method for Agrobacterium-mediated transformation of mold species
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx Table 1 Some prior reviews related to the topic of production of recombinant proteins by lamentous fungi. Reviewshort title Genetic engineering of lamentous fungi Rening heterologous protein production in lamentous fungi Filamentous fungi as cell factories for heterologous protein production Aspergillus as a host for heterologous expression Reference Meyer (2008) Sharma et al. (2009) Punt et al. (2002) Lubertozzi and Keasling (2009) Aro et al. (2005) Kim et al. (2007) Gerngross (2004) Ward et al. (2006) 1 List of fungal proteomics papers Table 1 2 3 1 4 1 2 3 Contents Industrially important compounds produced by lamentous fungi Heterologous industrial enzymes Important heterologous proteins in recombinant Aspergillus Fungal and yeast hosts for hIL-6; Systematics of biotechnologically relevant true fungi Bioactice fungal metabolites Some fungal conversions Some fungal bioremediations
Transcriptional regulation of plant cell wall degradation by lamentous fungi Proteomics of lamentous fungi Production of human therapeutic proteins by yeasts and lamentous fungi Physiology and biotechnology of Aspergillus
Aspergillus as a cell factory for protein production Recombinant protein production systems for Aspergillus Genomics of folding, secretion, glycosylation in aspergilli Biotechnology of Trichoderma Bioconversion of lignocellulose biomass Engineering of Penicillium chrysogenum Engineered biosynthesis of peptide antibiotics Engineering primary metabolic pathways of industrial micro-organisms Taxol-producing endophytic fungi
Braaksma and Punt (2008) Fleissner and Dersch (2010) Geysens et al. (2009) Schuster and Schmoll (2010) Kumar et al. (2008) Harris et al. (2009) Stachelhaus et al. (1996) Kern et al. (2007) Zhou et al. (2010)
IV V VI,VII,VIII IX 1 1,2 1
Aspergillus genes of industrial interest Aspergillus recombinant food enzymes Important heterologous proteins expressed in Aspergillus Patents on recombinant protein production by Aspergillus Effect of secreted protease activity of protease gene disruption strains Recombinant protein production by aspergilla including hosts and promoters Genes involved in protein-folding, unfolded protein response, glycosylation
Non-ribosomally synthesized antibiotics and producing hosts
1 2
Taxol-producing strains Common transformation methods for lamentous fungi
belonging the Ascomycotina, Basidiomycotina, Deuteromycotina, Mastigomycotina and Zygomycotina and illustrated example transformations for Aspergillus awamori, Aspergillus nidulans, A. niger, Colletotrichum gloeosporiodes, Fusarium solani, Fusarium graminearum, Neurospora crassa, T. reesei, Pleurotus ostreatus and Agaricus bisporus. In applying Agrobacterium-mediated transformation strategies to lamentous fungi, for example A. bisporus, Romaine (2002) observed that it was preferable to co-cultivate the bacterium with fruit body tissue rather than with spores. A third transformation method, which often requires protoplast preparation, involves electric pulse-mediated reversible membrane permeabilization to promote DNA uptake. In contrast, the fourth more specialized method, which can be implemented without cell wall removal, involves shooting DNA-coated metal particles at high speed into cells. More specic molecular transformations involve targeting of recombinant genes to a specic position in the genome which will enhance transcription of newly introduced DNA and/or deletion of genes with potential to reduce the positive effects of the desired transformation, be it production of a specic recombinant protein or insertion of multiple enzymes or proteins participating in a specic metabolic pathway or other physiological event. In addition to DNA-based methods, introduction of RNA-based methods such as antisense RNA, hammerhead ribozymes and RNA interference approaches have been found to be very useful for silencing particular genes in lamentous fungi (Fulci and Macino, 2007; Hammond and Keller, 2005; Muller et al., 2006; Yamada et al., 2007). Detailed classical physiological and biochemical knowledge is available for many of the candidate hosts and molecular techniques, including genome sequencing and annotation strategies. These are providing data to support efforts in optimizing expression and secretion of recombinant proteins in lamentous fungi. Filamentous fungi, especially well studied Aspergillus species, have also been shown to efciently implement posttranslational modications such that heterologous 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 lamentous fungal hosts for recombinant protein production than some other lamentous fungi. The Mucor rennin gene under the control of a suitable alpha-amylase promoter, introduced into A. oryzae, resulted in production yields of the heterologous protein of 3.3 g/L (Christensen et al., 1988). Some perceived or suggested disadvantages of lamentous fungi as heterologous protein hosts relate to their relatively low frequencies of transformation, potential morphological defects, and observed protein modications due to protease activity or low pH (Kinghorn and Unkles, 1994; Radzio and Kueck, 1997). It was observed that production levels of most non-fungal recombinant proteins (mammalian, bacterial, avian, plant etc.) in lamentous fungi were generally lower as compared to those of homologous proteins and with likely bottlenecks at the level of transcription and translation, secretion, with possible limitations also at the post-translational level (i.e., inefcient translocation, folding, transport, processing, or secretion) (Broekhuijsen et al., 1993; Gouka et al., 1997a; Jeenes et al., 1994). A general model for fungal protein synthesis and secretion, based on Aspergillus species has been summarized by Fleissner and Dersch (2010). During synthesis, proteins are directed into the endoplasmic reticulum where folding takes place and glycosylation is initiated. In Aspergillus species, protein disulde isomerase (Pdi) assists in the folding and maturation of secretory proteins and the ability of PdiA to catalyze the refolding of denatured and reduced RNase has been demonstrated (Ngiam et al., 2000). Improperly folded or glycosylated proteins are sent to the proteosome or vacuoles for degradation. Further modication, including glycosylation, occurs in the golgi bodies. SNARE proteins facilitate vesicle-mediated trafcking of the proteins to the hyphal tip for extracellular secretion. In an interesting experiment, Gordon et al. (2000) fused a green uorescent protein sGFP (S65T) to truncated A. niger Gla (Gla:499) which was successfully integrated into the A. niger genome. Confocal uorescence microscopy conrmed that GFP was partially localized within the hyphal cell
wall and that protein secretion occurred at the apical or subapical hyphal regions. Limitations at the transcriptional level can be due to low steadystate mRNA levels resulting from a low transcription initiation rate or more likely from a reduced mRNA stability. It has been suggested that at least ve structural components may inuence mRNA stability. In the case of hil6 and aglA transcripts in Aspergillus species, glaA fusions appeared to stabilize mRNA levels (Gouka et al., 1997b). Jeenes et al. (1994) reported similar results for fusions of egg-white lysozyme with glucoamylase. In many cases, low levels of production of recombinant proteins are due to post-translational secretion bottlenecks rather than transcription (Conesa et al., 2001; van den Hombergh et al., 1997). After secretion, a major known problem for heterologous proteins is their degradation by high extracellular enzyme producing lametous fungi, perhaps most notably Aspergillus species which secrete a diversity of extracellular proteases (van den Hombergh et al., 1997). Proteases have been shown to be responsible for degradation of many recombinant proteins (Broekhuijsen et al., 1993; Roberts et al., 1992). Traditional fermentations for production of extracellular enzymes by lamentous fungi were based on fermented food processes. These processes which involved surface or semi-solid media are nonhomogeneous, making ne process control impossible. This motivated desires to produce fungal extracellular enzymes in submerged culture in stirred tank reactors. Some early challenges with respect to growing lamentous fungi in submerged culture related to the high viscosities which developed in the media caused by the increased concentrations of lamentous biomass, making mass transfer and especially aeration of these oxygen-requiring organisms more challenging and generally leading to early cessation of growth and limitation of desired protein product yields. These problems were addressed in various ways at the engineering, physiological and molecular levels. Improvements in fermenter design were directed towards increasing aeration while controlling mycelial shearing effects. High growth and product formation rates were achieved by manipulation of fungal morphology, generally to reduce mycelial strand length and promote formation of highly branched mycelia. An example of a manipulation at the molecular level was provided by Akin et al. (2003) who maximized heterologous protein production by transforming the cells with cotA-encoding nucleic acids controlled by a regulatable promoter. Some of these strategies are discussed in more detail elsewhere in this paper.
in Table 2. For further information, go to the GRAS website at http:// www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=grasListing. The availability of genomic data, combined with other methods including proteomics (deOliveira and deGraaf, 2011) and metabolomics, is and will continue to support strain development strategies for production of recombinant proteins through use of molecular methods for industrial fermentations. For example, comparative genomic studies among Aspergillus species suggest that A. oryzae, is enriched with genes which participate in the degradation of biomass and in primary and secondary metabolism (Kobayashi et al., 2007). Also in A. oryzae when cDNA microarrays and expressed sequence
Table 2 Examples of GRAS notices led since 1998 relating to lamentous fungi. GRN no. 8 10 32 34 43 54 75 89 Substance Pectin esterase derived from Aspergillus oryzae carrying a gene encoding pectin esterase from Aspergillus aculeatus Exopeptidase derived from Aspergillus oryzae carrying a gene encoding a leucine aminopeptidase from Aspergillus sojae Pectin lyase derived from Trichoderma reesei carrying a gene encoding pectin lyase from Aspergillus niger Aspartic proteinase derived from Aspergillus oryzae carrying a gene encoding aspartic proteinase from Rhizomucor miehei Lipase derived from Aspergillus oryzae carrying a gene encoding lipase from Thermomyces lanuginosus Xylanase derived from Fusarium venenatum carrying a gene encoding xylanase from Thermomyces lanuginosus Lipase derived from Aspergillus oryzae carrying a gene encoding lipase from Fusarium oxysporum Five enzyme preparations from Aspergillus niger: Carbohydrase enzyme preparation, catalase enzyme preparation, glucose oxidase enzyme preparation, pectinase enzyme preparation, and protease enzyme preparation Carbohydrase enzyme preparation from Aspergillus oryzae, protease enzyme preparation from Aspergillus oryzae, and carbohydrase enzyme preparation from Rhizopus oryzae Lipase enzyme preparation from Aspergillus oryzae carrying a gene constructed from a modied Thermomyces lanuginosus lipase gene and a portion of the Fusarium oxysporum lipase gene Glucose oxidase enzyme preparation from Aspergillus oryzae carrying a gene encoding a glucose oxidase from Aspergillus niger Lipase enzyme preparation from Aspergillus niger Lipase enzyme preparation from Aspergillus oryzae Laccase enzyme preparation produced by Aspergillus oryzae expressing the gene encoding a laccase from Myceliophthora thermophila Lactase enzyme preparation from Aspergillus niger Phospholipase enzyme preparation from Aspergillus oryzae expressing the gene encoding a phospholipase A1 from Fusarium venenatum Beta-glucanase enzyme preparation from Trichoderma harzianum Glucosamine hydrochloride prepared from chitin obtained from Aspergillus niger Lipase preparation from Aspergillus niger expressing a gene encoding a lipase from Candida antartica Phospholipase A2 enzyme preparation from Aspergillus niger expressing a gene encoding a porcine phospholipase A2 Mixed beta-glucanase and xylanase enzyme preparation from Humicola insolens Asparaginase enzyme preparation from Aspergillus oryzae expressing the asparaginase gene from A. oryzae Asparaginase enzyme preparation from Aspergillus niger expressing the asparaginase gene from A. niger Chymosin enzyme preparation from Trichoderma reesei expressing the bovine prochymosin B gene Lipase enzyme preparation derived from Hansenula polymorpha expressing a gene encoding a lipase from Fusarium heterosporum Lipase enzyme preparation from a genetically modied strain of Aspergillus niger Transglucosidase enzyme preparation from Trichoderma reesei expressing the gene encoding transglucosidase from Aspergillus niger Carboxypeptidase enzyme preparation from modied Aspergillus niger Acid fungal protease enzyme preparation from Trichoderma reesei expressing the gene encoding acid fungal protease from T. reesei Glucoamylase (GA) enzyme preparation from Trichoderma reesei expressing the gene encoding the GA from T. reesei
90
103
106 111 113 122 132 142 149 150 158 183 195 201 214 230 238 296 315 345 333 372
3. Survey of principal players Some of the principal organisms involved in food fermentation processes were Aspergillus and Rhizopus species. For example, the initial stage of production of soy sauce involves predominant growth of A. oryzae strains on a mixture of soybeans and wheat, while production of tempeh involves cultivation of Rhizopus oligosporus on cooked soybean mash. Not surprisingly, these organisms have also been prime candidate hosts for production of recombinant proteins. A combination of our historical knowledge and experience of the performance of GRAS (Generally Regarded As Safe) strains with new genomic information has been used to facilitate the design of a new generation of genetically modied strains capable of efcient production of benecial recombinant proteins (van Dijck et al., 2003). The GRAS food additives list of the United States Food and Drug Administration includes enzyme products from A. niger and A. oryzae, Endothia parasitica, Mucor miehei, Mucor pusillus and others. Since 1998, the FDA has published an inventory of notications it has received regarding applications for GRAS recognition/exemption. The majority of these notices relating to products of lamentous fungi involved recombinant proteins. Examples of these listings are included
tags were used to characterize transcriptional activity associated with energy catabolism and hydrolytic enzyme production, transcription levels of most catabolic genes of the EM and TCA pathways were observed to be higher in glucose-rich conditions as compared with glucose-depleted conditions (Maeda et al., 2004). As will be discussed later, interesting studies have also been implemented in traditional industrial solid-phase media. For example, rich gene-expression proles for hydrolytic enzymes were observed in wheat-bran media, which exhibited lowest expression of catabolic genes. This suggested the latter poor expression may have released catabolite repression of hydrolytic enzyme synthesis. Gene arrays, gene deletion and insertion strategies and other emerging molecular techniques will undoubtedly receive widespread application as a means to better grasp and exploit the mechanisms of industrial product formation, regulation, and secretion by Aspergillus species and other lamentous fungi (Akao et al., 2002; Bautista et al., 2000; Moralejo et al., 2002; Ngiam et al., 2000; Sims et al., 2004; Zarrin et al., 2005). Because of the resource intensive nature of genome sequencing and functional analysis, in the case of lamentous fungi, priority attention has focussed on the most important strains based on industrial and/or agricultural productivity considerations or on strains which represent important human, animal and plant pathogens. In addition some starting species were selected for genomic characterization, for example A. nidulans, where substantial benecial prior physiological and genetic knowledge had already been established. Consequently, pioneering genomic research was implemented on industrial and pathogenic Aspergillus species. More recent interest in developing more efcient systems for bioconversion of biomass to energy provided the impetus to characterize the genome of the high cellulose and hemicellulase producer, T. reesei. In the case of other industrial extracellular enzyme producers which may also be excellent candidate lamentous fungal hosts for production of recombinant proteins, for example certain Penicillium, Rhizopus, Fusarium and Mucor strains, as well as some thermophilic fungi, there was insufcient interest and/or resources to substantially characterize these strains genetically with respect to their enzyme production, secretion function and potential recombinant protein production potential. In some of the latter cases, for example in the case of Penicillium and Mucor/Fusarium, detailed sequencing and functional genomic studies have been directed at the highest prole application of these organisms, namely to penicillin production by Penicillium chrysogenum and to the plant pathogenic properties of F. graminearum. Mention is made of this research below, in case some of the genomic and functional ndings from these studies become relevant and applicable to extracellular enzyme-producing strains as potential candidate recombinant protein-producing hosts. In addition, molecular biologists are applying recombinant technologies to investigate the unique metabolic properties of these organisms with the expectation that a fuller understanding will lead to benecial societal outcomes. A discussion follows highlighting progress in genomics research as it pertains to some of the more important industrial lamentous fungal strains. Most lamentous fungi have estimated genomic sizes of 3040 Mb, encoding 900013,000 genes (Machida, 2002). 3.1. Aspergillus The genus Aspergillus consists of more than 180 ofcially recognized species, most of which degrade plant polysaccharides (de Vries, 2003), and they are particularly important industrial lamentous fungi for the large-scale production of both homologous and heterologous enzymes (Fawole and Odunfa, 2003; Wang et al., 2003). A. oryzae and A. niger are on the Generally Recognized as Safe (GRAS) list of the Food and Drug Administration (FDA) in the United States (Tailor and Richardson, 1979). Molecular and genetic studies of Aspergillus species most relevant to recombinant protein production deal with A. nidulans, A. oryzae, A.
niger and its awamori variant. Detailed genetic analyses have also been carried out on the human pathogen, Aspergillus fumigatus, but because of the severe toxigenic nature of this organism as a producer of the highly toxic aatoxins, it is not relevant as a host for biotechnology production processes. A. nidulans is of particular interest as a model lamentous fungal organism for studies of cell biology and gene regulation. It is also related to A. niger and A. oryzae, which are the best natural lamentous fungal production hosts. While A. nidulans is the best genetically characterized Aspergillus species, it is also deemed to be unsuitable as a recombinant host for biotechnology processes, because it produces sterigmatocystin, which is also a toxin, albeit much less severe than aatoxins. A.niger and A. oryzae are also recognized potential broad based recombinant hosts for the biopharmaceutical industry for production of recombinant proteins. 3.1.1. A. nidulans Conventional matings lead to the identication of greater than 900 genes in A. nidulans (Brody et al., 1991). The whole genome has a size of 30.1 Mb, with eight well marked chromosomes containing approximately 10,000 genes. Physical and chromosomal linkage maps, and genome sequence have been described in detail (Archer and Dyer, 2004; Clutterbuck, 1997; Galagan et al., 2005; Lubertozzi and Keasling, 2009; Monsanto, 2001; Sims et al., 2004). Research is continuing with the ultimate objective of describing and relating expression patterns, cellular roles and functions of all genes. 3.1.2. A. niger Sequencing of a derivative of the enzyme-producing strain A. niger NRRL 3122 (ATCC 22343, CBS 115989) indicated a genome size is 35.9 Mb, containing 14,097 predicted genes (Archer and Dyer, 2004). The sequence data for A. niger ATCC strain 9029 is held at the Pacic Northwest National Laboratory (PNL) and is available to researchers upon request. Genencor has access to the A. niger genome sequence data of Integrated Genomics (Machida, 2002). The Joint Genome Institute (JGI) initiated a sequencing program for the citrateproducing A. niger ATCC 1015, in 2004 as part of the United States DOE Genome Program, with participation of PNL and Oakridge National Laboratory. Sequencing information on this strain was made available at http://www.jgi.doe.gov/aspergillus. The genome sequence and analysis of the ancestor of a current A. niger enzyme production strains, A. niger CBS 513.88, indicates a genome size of 33.9 Mb (Pel et al., 2007). Among the 14,165 open reading frames identied, strong functions were predicted for 6505 of them. This paper and supplementary information referenced on line includes detailed functional genomic analyses of protein secretion, carbohydrases and proteases as well as corresponding comparative analyses among the different Aspergillus species (A. niger, A. nidulans, A. oryzae and A. fumigatus). Tsang et al. (2009) combined analysis of proteins secreted by A. niger with genomic predictions of signal-peptide containing proteins to conrm that the presumed secreted proteins were in fact secreted and not the result of cell autolysis. Combining gene expression and proteomic data for A. niger overproducing strains of lipid, protein and carbohydrate-degrading enzymes, facilitated identication of 898 proteins and demonstrated that the strains exhibited upregulation of proteins participating in carbon- and N-metabolism, as well as protein folding and protein degradation. The data enabled researchers to manipulate the system incuding overexpression of a putative protein glycosylation gene and to increase secretion of a specied enzyme (Jacobs et al., 2009). 3.1.3. A. oryzae The Japanese National Institute of Technology and Evaluation completed sequencing of the A. oryzae genome which consists of eight chromosomes ranging from 2.8 to 7.0 Mb (Kitamoto et al., 1994; Machida, 2002). The total genome size was estimated to be
36.8 Mb with the number of genes being about 12,074 (Machida et al., 2005). Fleissner and Dersch (2010) recently reviewed the range of recombinant protein products produced by Aspergillus species. The principal host species identied were: A. niger, A. awamori, A. oryzae, A. nidulans and A. terreus. The predominant promoters used for recombinant protein production were: adhA, alcA, alcC, aldA, amdS, amdS, amyA, amyB, aphA, exlA, gdhA, glaA, glaA1, gpdA, oliC, pkiA, sodM, sucA, tef1 and tpiA. Recombinant products from humans included: alpha1-proteinase inhibitor, antigen-binding (Fab) fragment, corticosteroid binding globulin, epithelial growth factor, granulocyte macrophage colony stimulating factor, growth hormone, humanized IgG1(kappa) antibodies, interferon-alpha-2, interleukin-6, lactoferrin, lysozyme, mucus proteinase inhibitor, parathyroid hormone, singlechain variable region fragment (scFv) anti lysozyme construct, superoxide dismutase and tissue plasminogen activator. Recombinant products originating from other animals included: porcine pancreatic phospholipase A2 and prochymosin; bovine chymosin, prochymosin and prochymosin B; hen egg-white lysozyme and llama antibodies. Recombinant plant proteins expressed in Aspergillus include Thaumatococcus daniellii thaumatin and Cyamosis tetragonoloba alpha-galactosidase. Recombinant bacterial proteins expressed in Aspergillus species included Cellulomonas mi endoglucanase, Clostridium thermocellum dockerin, Eschericia coli enterotoxin subunit B, beta-galactosidase, beta-glucuronidase and Thermobida fusca hydrolase. Recombinant proteins from other fungal genera expressed in Aspergillus included: Agaricus meleagris pyranose dehydrogenase, M. miehei triglyceride lipase and aspartyl protease, Phanerochaete chrysosporium lignin peroxidase H8 and manganese peroxidase H4, Pleurotus eryngii peroxidase, Pycnoporus cinnabarinus laccase, Thermomyces lanuginosus lipase and Trametes versicolor laccase. Many recombinant proteins from one species of Aspergillus have also been expressed in another Aspergillus species. The very interesting swollenin-like protein from A. fumigatus which, like swollenin from T. reesei, disrupts cellulosic materials and has similarities to the plant proteins (expansins) which have a cell wall loosening effect, was produced as a recombinant protein in A. oryzae (Chen et al., 2010). While the protein exhibits no apparent enzyme activity, in the presence of cellulases, it promoted efcient saccharication of crystalline cellulose. 3.2. Trichoderma The genome sequence of the commercially important high producer of cellulases and hemicellulases, T. reesei, has been published (Martinez et al., 2008) while analysis and annotation of the genomes of two biocontrol species, Trichoderma altroviride and Trichoderma vireus are proceeding. T. reesei is a soft-rot ascomycete lamentous fungus with a long and safe track record as a producer of commercial cellulases, initially with applications in food processing (Nevalainen et al., 1994). Studies aimed at understanding and optimizing factors affecting productivity and catalytic efciency of cellulases are fundamental to overcoming the major biomass pre-treatment obstacle to commercialization of processes for production of bioenergy from lignocellulose biomass. Applications of its cellulase and hemicellulase compliment in the pulp and paper and textile industries are also important (Buchert et al., 1998; Galanti et al., 1998). T. reesei represents a principal target cellulase host in the quest to replace gasoline with cellulose-derived ethanol. 3.2.1. T. reesei T. reesei has a genome size of 33 Mb and seven chromosomes (http://genome.jgf-psf..org/Trire2/Trire2.home.html). The predicted number of genes in the genome was 9129 (Martinez et al., 2008). T. reesei has an extraordinary ability to secrete proteins. Cherry and Fidantsef (2003) reported that some industrial strains following
aggressive mutation programs, could produce as much as 100 g/L extracellular protein, with up to 60% as the major cellulase Cel7a (CBHI) and 20% of Cel 6a (CBHII). The complete pattern of proteins related to expression of cellulase and hemicellulase genes in T. reesei was characterized by Ouyang et al. (2006). Cultivation of T. reesei on cellulose, xylan, a mixture of plant polysaccharides or indeed lactose promotes high levels of expression of cellulase and hemicellulase genes (Mach and Zeilinger, 2003; Seiboth et al., 2007). Sophorose is thought to be the natural cellulase inducer (Sternberg and Mandels, 1979; Vaheri et al., 1979). That notwithstanding, genomic analysis casts little mechanistic light on its enormous protein secretion capacity. Despite its effectiveness in degrading plant polysaccharides, suggesting it should contain expansions of genes encoding enzymes capable of digesting plant cell walls, T. reesei contains fewer genes encoding glycoside hydrolases (total 200) than other phytopathogens such as F. graminearum (total 243) and Magnaporthe grisea (total 231), A. oryzae (total 285) or A nidulans (247). It was also noted that while plant polysaccharases often contain a carbohydrate-binding molecule (CBM) within its related group of fungal genomes (N. crassa, F. graminearum, M. grisea and T. reesei), they had the smallest number of CBM-containing proteins. First efforts to produce heterologous proteins in T. reesei focussed on calf chymosin (Harkki et al., 1989; Uusitalo et al., 1991) after which Nyyssonen et al. (1993) reported use of this host to produce antibody fragments. It was observed that higher production of recombinant proteins was generally observed when the original source of the gene encoding the protein was taxonomically related to the recombinant host. Cellulase gene promoters are most often incorporated into cassettes for production of recombinant proteins by Trichoderma (Penttila, 1998; Schmoll and Kubicek, 2003), most frequently the signal peptide of Cel 7a (CBHI) which mediates efcient recombinant protein secretion. This topic was reviewed by Schuster and Schmoll (2010). Three recombinant endoxylanases from Chaetomium thermophilum were expressed in T. reesei with a view to facilitating their production for application in biobleaching of kraft pulp (Mantyla et al., 2007). The expression cassettes utilized the strong T. reesei cel 7A promoter. The host was a low protease producer where deletions in the endoglucanase I, endoglucanase II and cellobiohydrolase I genes rendered it the desired low cellulase producer for applications in kraft pulp treatment. It was demonstrated that a commercially viable recombinant thermostable xylanase can be produced by T. reesei. Recently, the industrially interesting biocatalyst cinnanoyl esterase from an unsuitable host, the anaerobic fungus Piromyces equi, was successfully expressed in recombinant T. reesei as a more suitable producing host (Poidevin et al., 2009). Substantial effort has focussed on transforming fuel ethanol yeast strains with cellulolytic genes from Trichoderma species to facilitate their ability to ferment cellulose to ethanol. In a recent example, Huang et al. (2010) described cloning and expression of the endoglucanase gene egVIII from Trichoderma viride into Saccharomyces cerevisiae. 3.3. Penicillium Limited genomic sequencing information appears to be available on potential recombinant protein-producing lamentous fungi other than Aspergillus and Trichoderma species. As selected Penicillium species, for example Penicillium purpurogenum, Penicillium funiculosum and Penicillium (Talaromyces) emersonii are high producers of cellulases, hemicellulases and pectinases, they may have considerable potential as recombinant protein-producing hosts. Chavez et al. (2010) carried out transformation studies and demonstrated high transformation frequencies in two cheese ripening fungi, Penicillium camemberti and Penicillium roqueforti, which exhibit low protease
activity. They concluded that these species had all of the right strain characteristics as suitable hosts for production of recombinant proteins. Gonzalez-Vogel et al. (2011) recently identied a number of protein complexes containing enzymes including arabinofuranosidases, beta-glucosidase, xylanases, acetyl esterases and ferulyl esterases, in the soft rot fungus P. purpurogenum using a proteomics strategy. Guais et al. (2010) prepared a partial DNA library for P. funiculosum and sequenced genes encoding four GH54 -Larabinofuranosidases. This organism has been used to produce commercial mixtures of enzymes degrading complex agricultural residues containing cellulose, hemicellulose, arabinoxylan, arabinogalactan, proteases etc., with applications as a feed additive to enhance feed digestibility. The enzyme mixture contained more than fty separate proteins (Guasi et al., 2008). The strong gpdA promoter from A. nidulans was used to promote overexpression of pectin lyase by Penicillium griseoroseum in submerged fermentation production systems (Cardoso et al., 2010). Penicillium canescens was transformed with a vector encoding the laccase of Trametes hirsute under control of an efcient promoter of the bgaS gene of P. canescens and efciently expressed and secreted the recombinant protein (Abianova et al., 2010). As might be expected, the predominant effort related to sequencing and annotation of Penicillium has been directed at the principal producer of penicillins, P. chrysogenum. While antibiotic-producing strains are generally not considered as suitable hosts for production of natural or recombinant enzymes or other proteins for use in foods or pharmaceuticals, some of the P. chrysogenum genomic information may be applicable to development of non-antibioticproducing Penicillium strains as recombinant protein-producing hosts. Promoters of the genes encoding glutamate dehydrogenase, -acetylhexosaminidase and gamma-actin from P. chrysogenum may be used to construct potent vectors for expression and secretion of homologous and heterologous proteins in these strains and also in other hosts (Barredo Fuente et al., 2001). 3.3.1. P. chrysogenum The complete genome sequence of the penicillin producer P. chtysogenum Wisconsin 541255 strain (ATCC 28089, see Elander, 1983) was published in 2008. Genome size was 32.19 Mb, comparable with that of other lamentous fungi, and the total gene number was 12,943 (van den Berg et al., 2008). In addition to cellular functional characterization of the P. chrysogenum genes, particular attention was paid to the penicillin biosynthetic genes. This information may provide more general direction for manipulation/engineering of metabolic pathways to increase production of natural target metabolites or indeed to facilitate production of wholly novel metabolites in lamentous fungi. The transcriptomes of the sequenced strain and a high penicillin-producing strain were compared and as might have been expected, many of the genes involved in synthesis of the penicillin precursors, valine, cysteine and -aminoadipic acid were observed to be increased in the high penicillin-producing strain. Some genes were identied which control -lactam output and genes with predicted roles as transporters appeared to be upregulated under penicillin-producing conditions. Culmination of this work clearly represents a milestone for future metabolic engineering strategies, which of course may involve participation or use of recombinant proteins. 3.4. Rhizopus A number of important extracellular industrial and medical enzymes are produced by the zygomycetes, including the important microbial rennets produced by Rhizomucor miehei and Rhizomucor pusillus and digestive lipases, proteases and amylases are produced by Rhizopus arrhizus. However, the major fungal genomics resources related to this group of lamentous fungi have been directed to
pathogenic strains and indeed the rst of the zygomycetes to be fully sequenced was Rhizopus oryzae which is the primary cause of the potentially lethal angioinvasive mucormycosis infection (Ibrahim et al., 2003; Kwon-Chung and Bennett, 1992). Related Mortierella species are of great interest in the area of lipid production and molecular transformations involving these species are being investigated (MacKenzie et al., 2000). Nevertheless, as is indicated below, some of the genomic information is directly relevant to the long established abilities of this and related strains to produce hydrolytic enzymes. 3.4.1. Rhizopus oryzae The genome sequence of R. oryzae strain 99880, isolated from a fatal infection of mucormycosis, has recently been published (Ma et al., 2009). Total length of the R. oryzae genome was found to be 45.26 Mb while total number of protein-encoding genes was 17,467. Evidence was provided that there is whole-genome duplication in this strain mainly attributed to an ancestral duplication event. Of specic interest to diagnostic and therapeutic treatment of mucormycosis is the genomic characterization of expanded families of cell-wall synthesis enzymes required for fungal cell wall metabolism but which are not present in mammalian hosts and hence which may be targeted by novel future drugs. Of interest both to therapy as well as to use of R. oryzae and related species as hosts for recombinant protein production are the annotated expanded gene families of secreted proteases characterized, especially aspartic proteases and subtilases. It was suggested that these proteases may mediate the pathogenic infection process, as these enzymes have previously been thought to be associated with virulence of pathogenic Rhizopus species (Schoen et al., 2002; Spreer et al., 2006). In this case, these proteases may mediate penetration of hyphae through decaying organic matter (Ma et al., 2009). 3.5. White rot fungi White rot fungi are basidiomycetes that are of great interest as enzyme producers as they produce unique extracellular oxidative enzymes that degrade lignin which surrounds and protects cellulose microbrils of plant cell walls, especially woody plants. The white rot fungi are particularly important because they degrade the lignin while not attacking the cellulose. These lamentous fungi are the only microbes capable of efcient depolymerization and mineralization of lignin. P. chrysosporium has been the most intensively studied white rot fungus. White rot fungi secrete an array of peroxidases and oxidases that attack lignin non-specically by producing lignin-free radicals, which subsequently facilitate spontaneous cleavage reactions (Kirk and Farrel, 1987). These enzymes also participate in degradation of organic pollutants in bioremediation. Recently, highresolution two dimensional electrophoresis-based proteomics coupled to LC-MS/MS was used to monitor enzyme expression and chemical products present during the process of degradation of aromatic substrates by P. chrysosporium, as a means of gaining a better insight into the process of lignin degradation (Matsuzaki et al., 2008). Not surprisingly, the rst basidiomycete genome to be sequenced was the white rot fungus P. chrysosporium. 3.5.1. P. chrysosporium Its thirty million base-pair genome was sequenced using a whole genome shotgun method. The genome length was 29.9 Mb, similar in size to most of the other sequenced lamentous fungi genomes. The genome contains 11,777 protein coding genes. Analysis of the genome indicates an array of genes which encode secreted enzymes, including oxidases, peroxidases and hydrolytic enzymes which are known to co-operatively cause wood decay (Martinez et al., 2004). Recombinant proteins have been expressed in a variety of basidiomycetes. For example, a vector encoding interleukin-32, the human cytokine associated with some inammatory and autoimmune
diseases, was successfully introduced and expressed in the edible mushroom P. eryngii, via an A. tumefaciens transformation (Chung et al., 2011). There is continuing interest in expressing the diverse degrading enzymes from basidiomycetes in more conventional industrial work-horse hosts. For example ligninolytic basidiomycetes contain a sugar oxidoreductase (pyranose dehydrogenase) that has very broad substrate specicity towards breakdown constituents of lignocellulose. In order to extend the biodegradative capability of more conventional industrial strains, this enzyme from A. meleagris was heterologously expressed in A. nidulans and A. niger (Pisanelli et al., 2010). The white rot fungus, T. versicolor produces two groups of laccases with several isoforms. Two of these laccases were expressed as recombinant enzymes in A. oryzae and the recombinant enzymes exhibited catabolic degradative activity against hydroxylated PCBs (Fujihiro et al., 2009). Rodgers et al. (2010) noted that while basidiomycetes are the predominant sources of laccases with potential large applications in delignication, basidiomycetes are in general not as versatile or suitable as industrial fermentation producers as compared to ascomycetes and consequently much effort has focussed on transforming the more suitable fermentation hosts to produce recombinant basidiomycetes laccase. However, there have been problems in achieving production of recombinant laccases in good fermentation hosts primarily due to glycosylation deciencies and these challenges are currently being addressed with a view to mass producing effective laccases. Sakaki and Munetsuna (2010) have surveyed the various enzymes which could co-operate to degrade complex pollutants such as polychlorinated dibenzo-dioxins and furans, including angular dioxygenase, cytochrome P450 (CYP), lignin peroxidase, manganese-dependent peroxidase and dehalogenase and concluded that combinations of distinct enzymes could have signicant application in these biodegradations. Given that white rot fungi already produce lignin and Mn-dependent peroxidases and CYPs it was concluded that supplementing this host by adding additional recombinant capability would make this organism a very powerful bioremediation strain. While the risks associated with releasing genetically engineered organisms to the environment were recognized, it was suggested this could be addressed by creating suicidal engineered strains (Paul et al., 2005).
3.7. N. crassa While N. crassa is not recognized as an important industrial host, it is included in this discussion as a powerful model lamentous fungal system which has been characterized biochemically and genetically. This host can be grown at high growth rates in simple dened media and can produce high amounts of recombinant proteins. The approximate genome size is 40 Mb, and it contains about 10,000 protein-coding genes and many of the genes involved in interesting aspects of Neurospora biology, including its secondary metabolism have been annotated (Colot et al., 2006; Galagan et al., 2003). Up-to-date information may be obtained online from http://www.fgsc.net. Tian et al. (2009) applied microarray and shotgun proteomics analysis on strains of a cellulolytic N. crassa fungus grown in different media in order to combine data from gene expression and the proteome/secretome in an attempt to better understand the cellulose-degrading system and the principal genes involved. Recently, N. crassa has been used as a host for production or recombinant subunit vaccines including inuenza hemagglutinin (HA) and neuraminidase antigens (NA) (Allgaier et al., 2009). High molecular weight particles containing NA could be generated in a heterokaryon expression system facilitating downstream processing on the one hand but also enables mixtures of different antigens to be coexpressed together, thereby facilitating tailoring of a vaccine directed at a particular pathogen target or variant. 3.8. Selected key genomic resources A variety of institutional and online resources are available to researchers with interests in genomic aspects of lamentous fungi and are clearly relevant to the topic of recombinant protein production by these hosts. Reference is made to some of these below: http://www.aspgd.org/ is the home of the Aspergillus Genome Database, a resource for genomic sequence data and gene and protein information for Aspergillus species. AspGD is based on the Candida Genome Database and is funded by the National Institute of Allergy and Infectious Diseases at the US National Institutes of Health. Subsites deal with the annotated Aspergillus genomes of strains of A. fumigatus, A. clavatus, A. nidulans, A. niger, A. oryzae and Aspergillus terreus. The aim of the JGI Fungal Genomics Program is to scale up sequencing and analysis of fungal genomes to explore the diversity of fungi in DOE mission areas and to develop the Genomic Encyclopedia of Fungi in the areas of: Plant feedstock health (mycorrhizal symbiosis, plant pathogenicity, biocontrol); Biorenery (lignocellulose degradation, sugar fermentation, industrial organisms) and Fungal diversity (http:// jgi-psf.org/programs/fungi/about-programs.jsf). Subsites deal with important lamentous hosts including Aspergillus carbonarius, P. chrysosporium, Sporotrichum thermophile, Thielavia terrestris, T. versicolor and T. reesei. The Fungal Genome Initiative (FGI) of the Broad Institute of MIT and Harvard produces and analyzes sequence data from fungal organisms that are important to medicine, agriculture and industry. Over 50 fungi have been sequenced or are being sequenced, including human and plant pathogens as well as fungi that serve as basic models for molecular and cellular biology. In partnership with the wider fungal research community, organisms are selected for sequencing as part of a cohesive strategy that considers not only the value of data from each organism given their role in basic research, health, agriculture, and industry, but also their value in comparative genomics. It includes databases on R. oryzae and on the Fusarium Comparative project. (http://broadinstitute.org/scientic-community/ science/projects/fungal-genome-initiative/fungal-genome-initiative). The Fungal Genetics Stock Center (http://www.fgsc.net) is a resource available to the Fungal Genetics research community and to educational and research organizations in general. The FGSC is funded largely by a grant from the National Science Foundation (Award
3.6. Fusarium While a high prole Fusarium species, F. graminearum is the causative agent of some important plant diseases, other Fusarium strains are used in fermentations processes, including production of single cell protein approved for human consumption and some of these strains may have potential for production of recombinant proteins. Nevertheless, the predominant scientic research to date has focussed on F. graminearum which causes plant diseases of substantial economic importance including Fusarium ear root of maize and head blight of cereals. In addition F. graminearum produces mycotoxins in infected plants which, if they nd their way into food and feed products, constitute a health risk.
3.6.1. F. graminearum The sequencing and annotation of F. graminearum was reported by Cuomo et al. (2007) and gene annotation information was revisited by Wong et al. (2011). Updated resource information may be assessed at http://mips.gsf.de/genre/proj/FGDB/. The Cuomo et al. paper indicates a genome size of 36.1 Mb including 32 genes being predicted plant cell-degrading enzymes, including xylanases, pectate lyase and cutinases which were postulated to function in pathogenesis by facilitating plant tissue penetration and maceration and nutrient provision for the invading organism. The recent annotated information indicated a set of 13,718 protein coding genes.
Number 0235887) of the United States of America and to a lesser extent by the payments made by researchers who use our services. Most fungal strains in the FGSC collection are listed in the online searches. Specic groups of materials are listed by category include: Neurospora, Aspergillus, Fusarium and Magnaporthe, Ustilago, Cryptococcus, other Fungi. The FGSC together with other organizations is a major sponsor of the Fungal Genetics Conference (http://www.fgsc.net/26thFGC/ index.htm). The above websites in turn provide links to other resources. 4. Improving recombinant protein expression in lamentous fungi 4.1. Molecular strategies A primary practical motivation for studying gene expression in lamentous fungal hosts is to understand the molecular mechanisms of transcription regulation in these organisms and to improve recombinant protein expression, especially by the study of DNA sequences participating in transcription initiation and/or regulation and selection of strong promoters. Transcriptional regulation of extracellular plant cell wall-degrading enzymes produced by lamentous fungi has been reviewed by Aro et al. (2005). The promoter regions of the Aspergillus amylase genes consist of four highly conserved sequences, one of which (region IIIa) is essential for high-level expression and another of which (Region IIIb) contains sequences thought to enhance expression in combination with region IIIa (Minetoki et al., 1998). A sequence of CCAAT present in the promoter region of the A. nidulans amdS (encoding acetamidase) is required for high-level expression of amdS, and related CCAAT sequences are present in the promoter regions of a number of other A. nidulans genes (Papagiannopoulos et al., 1996). One of the most strongly expressed genes in A. oryzae, the enolase gene (enoA), contains a15-bp element with a sequence essential for transcription regulation of the gene (Toida et al., 2000). The melO promoter appears to be effective as a mediator of strong synthesis of recombinant proteins in Aspergillus hosts (Ishida et al., 2001). The A. oryzae TAKA-amylase promoter preceded by its upstream activating sequences, was found to be suitable for expression of protein products in Aspergillus species (Boel et al., 1996). Berka et al. (2002) patented novel vectors containing polyadenylation sequences linked to the 3 terminus of the DNA sequence encoding the heterologous protein and which may include promoter and signal sequences for promotion of expression and secretion of heterologous proteins in lamentous fungi. Schmoll et al. (2010) described the construct used to produce class 1 hydrophobin from A nidulans in T. reesei. When the class II hydrophobin-encoding promoter from T. reesei, hfb2, was used with lactose as carbon source, the majority of the recombinant protein was secreted into the medium by T. reesei. In contrast when the T. reesei cel7A promoter was used, the recombinant protein was not secreted into the medium, but remained cell wall-bound. High expression of the fumR gene, which encodes fumarase in a high fumaric acid producing strain of R. oryzae, was observed under good fumaric acid-producing conditions (high sugar, low N) and the regulation of this gene may be of interest for production of recombinant proteins and metabolic engineering in Rhizopus species. Gene expression was primarily regulated at the level of transcription. 4.1.1. Gene-fusions strategies Some early recombinant research on lamenous fungal sought to produce recombinant proteins by coat-tailing a hyper-produced and secreted homologous protein with subsequent cleavage of resulting fused proteins. Thus, techniques involving fusing the target gene to the 3 end of a homologous gene encoding glucoamylase improved production of recombinant proteins, for example, of mammalian proteins, by lamentous fungi (Gouka et al., 1997a, b). Fusions to the glucoamylase gene of A. niger/A. awamori promoted production of high
levels of a variety of secreted recombinant proteins including bovine prochymosin (Ward et al., 1990), hIL-6 (Broekhuijsen et al., 1993), hen egg-white lysozyme (Jeenes et al., 1993), human lactoferrin (Ward et al., 1992, 1995), and phytases from A. awamori (Martin et al., 2003).In the case of chymosin and lactoferrin production, gram to multigram quantities of recombinant product were produced per liter when the high-level-production strains were put through a mutation program (Dunn-Coleman et al., 1991; Ward et al., 1995). Enhancements to this approach involved use of the catalytic domain of glucoamylase rather than the complete enzyme (Gouka et al., 1997b). To facilitate subsequent cleavage of the two protein elements, a linker proteolytic processing site is incorporated between the carrier moiety component and the protein of interest. The linker region is designed to allow the catalytic domain and the rest of the fusion protein to fold independently. The N-terminal fungal protein appears to serve as a carrier, improving translocation of the recombinant protein into the ER, as well as its folding, is mediated by the N-terminal fungal protein. Subsequently in most cases the fusion protein is cleaved facilitating secretion of the separate proteins by a KEX2-like endopeptidase at a KEX2 recognition site introduced specically 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, aberrant forms of the recombinant product are observed when gene fusion strategies are employed. When a part of the fungal glucoamylase protein (GAM), linked via a KEX2 processing site, was also used in a gene-fusion strategy in A. niger to produce extracellular bovine pancreatic trypsin inhibitor (BPTI), aberrant forms of the recombinant protein were attributed to possible variations in A. niger KEX2-like endoprotease point of attack of the GAM-BPTI fusion protein or indeed involved another endoprotease (MacKenzie et al., 1998). For example, while the desired recombinant protein is normally linked to the glucoamylase via a Lys-Arg KEX2-like cleavage site in A. niger, the delity of cleavage to release mature protein is not always observed to be consistent and appears to be also inuenced by sequences immediately downstream and upstream of the KEX2 site (Spencer et al., 1998). The protein, neoculin (NCL), naturally produced in the fruits of the tropical plant Curculigo latifolia, is about 500 times sweeter than sugar. It is a heterodimer consisting of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS) linked by disulphide bonds. Recombinant neoculin (rNCL) was produced in A. oryzae by using separate NAS and NBS constructs, each fused to the A. oryzae amylase via KEX2 cleavage sites (Nakajima et al., 2006). The NAS component was properly N-glycosylated and the sweetness properties of the rNCL were comparable with the native NCL. Gene fusion strategies are also exploited to produce expressed proteins containing a tag that may facilitate product extraction during downstream processing. By way of example, Collen et al.(2001) genetically engineered endoglucanase (Cel7B) from T. viride with a peptide extension containing non-polar tryptophan-proline residues which facilitated preferential partitioning of the protein into the less polar phase of an aqueous two phase model system. 4.1.2. Overproduction of foldases and chaperones Foldases catalyze the isomerizations and disulde bond formations and molecular chaperones, which are non-catalytic, mediate folding of the nascent polypeptides into functional proteins and prevent non-productive proteinprotein interactions (Conesa et al., 2000). Chaperones may act in diverse ways such as identifying defective proteins in the ER, inducing synthesis of folding enzymes or indeed ER-associated protein degradation responses for degradation of defective proteins. It has been postulated that hyper-production of recombinant proteins into the ER has the potential to overload the folding, assembly and secretion machinery of lamentous fungi. Therefore, the effects
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of overexpression of genes for several ER chaperones and foldases in lamentous fungi, including bipA (from a family of binding proteins, BiP), pdiA (from a family of protein disulde isomerase) and a family of calnexins on overproduction of recombinant proteases have been evaluated (Conesa et al., 2001; Jeenes et al., 1997; Ngiam et al., 2000; van Gemeren et al., 1997). It was found that lamentous fungi overproducing specic proteins, both homologous and heterologous, exhibited increased levels of bipA transcription, whereas direct interventions to overexpress bipA overexpression appeared not to affect yields of secreted proteins (Punt et al., 1998). Overproduction of fungal proteins generally increased bipA mRNA levels in A. niger. In the case of two transformed A. niger strains which produced HEWL, a twofold induction in bipA mRNA levels was observed (Ngiam et al., 2000). BiP overexpression did not increase secreted levels of hIL6 in Aspergillus (Gouka et al., 1997a) and pdiA overexpression did not increase secreted yields of HEWL in A. niger (Ngiam et al., 2000). Disruption of a vacuolar protein sorting receptor gene in A. oryzae, which targets aberrant and recombinant proteins for vacuolar degradation, enhanced production and secretion of the bovine chymosin and human lysozyme heterologous proteins (Yoon et al., 2010). 4.1.3. Glycosylation Glycosylation patterns from lamentous fungi are more similar to those of mammals than the patterns observed in common yeast hosts (Maras et al., 1999a, 1999b; Nevalainen et al., 2005). The two main glycosylation processes common to eukaryotes involve N- and Oglycosylation, whereby oligosaccharides attach to the beta-amide moiety of asparagine residues and mainly to serine and threonine -hydroxy groups. N-glycosylation involves transfer of preassembled glycosyl precursors to specic asparagine residues of the nascent polypeptide chain after which glycosidase- and glycosyl transferase-mediated modications of the oligosaccharide occur resulting in production of a common trimannosyl-chitobiose core with branched N-acetylglucosamine residues generating the high mannose N-glycans characteristic of lamentous fungi and yeasts. O-glycosylation in fungi starts in the endoplasmic reticulum, and involves O-mannosylations resulting in the sequential build up of the O-glucosyl structure. Geysens et al. (2009) has recently used analysis of the genome sequences to review folding, secretion and glycosylation, especially the N-glycosylation processes while Goto (2007) has described the O-glycosylation process, both in Aspergillus. Filamentous fungi have two distinct alpha-1,2-mannosidases, one of which is similar to the mammalian Golgi alpha-1,2-mannosidases that trim 3 mannose moieties off Man8GlcNAc2 to form Man5GlcNAc2 as substrate for GlcNAc transferase 1, and another distinct fungal alpha-1,2-mannosidase (Ichishima et al., 1999; Yoshida et al., 2000). However, the mammalian-like enzyme is neither well expressed nor secreted such that very little of the lower mannosylated moiety gets transferred (Maras et al., 1997). N-glycans from fungi also differ from mammalian N-glycans in having terminal altered substituents such as glucose, galactose or phosphoesters (De Pourcq et al., 2010). Maras et al. (1997) employed recombinant mammalian beta-1,4galactosyl transferase and alpha-2,6-sialyltransferase to make T. reesei cellobiohydrolase 1 more mammalian-like with respect to its glycosylation pattern. Recombinant human -1,2-GlcNAc transferase was subsequently overexpressed in Trichoderma, thereby enhancing its GlcNAc transfer capability (Maras et al., 1999a, 1999b) and similar transformations with the corresponding rat GlcNAc transferase were implemented in A. nidulans (Kasajima et al., 2006). Kainz et al. (2008) has carried out other molecular stratees to successfully produce lower mannosylated Man3GlcNAc2 N-glycans in recombinant Aspergillus strains. For production of therapeutic proteins, glycoform is very important as incorrectly glycosylated proteins, for example recombinant human therapeutic glycoproteins produced by lamentous fungi,
may induce an immune response in the patient being treated, reducing treatment efcacy. Engineering humanized glycosylation pathways into lamentous fungi, including trimming the branches of high mannose-containing glycoproteins, has been found to be very complex (Gerngross, 2004). Antitrypsin, the human a1-proteinase inhibitor (a1-PI), is the most abundant inhibitor of serine proteases in plasma (Brantly et al., 1991). Progressive emphysema develops in antitrypsin-decient patients ultimately leading to death (Crystal, 1996). Conventional antitrypsin-inhibitor replacement therapy uses a limited plasma derived source which has created momentum for production of the recombinant form. While several hosts have been tested for efcacy of production, altered glycosylation patterns or complete absence of glycosylation in the recombinant product reduced in vitro stability of the inhibitor and resulted in its rapid removal from the circulation system (Karnaukhova et al., 2006). A mature and biologically active glycosylated recombinant a1-PI produced by A. niger exhibited improved stability over a non-glycosylated recombinant product produced by E. coli (Karnaukhova et al., 2007). The recombinant protein was fused to a well secreted native fungal protein with a KEX2 recognition site at the fusion junction which was cleaved in vivo by a KEX2-type protease. Implementation of strategies for increasing glycosylation in Aspergillus resulted in increased production of the recombinant protein chymosin (van den Brink et al., 2006). In one case, a poorly used glycosylation site within the chymosin molecule was improved resulting in much more efcient production of the glycosylated chymosin. In the second case, when the N-glycosylation site was located away from the native chymosin attached via a linker, a substantial increase in recombinant protein was observed. 4.1.4. Other molecular strategies The following are miscellaneous examples of molecular strategies used to enhance production of recombinant proteins by lamentous fungi: Hastrup et al. (1997) proposed production of a proenzyme, in cases where the enzyme was unstable or harmful to the producing host, which could be proteolytically activated after secretion. An activator protein binding site containing the CCAAT sequence was identied within the cis regulatory region of the A. niger glaA gene. Insertion of multiple copies of this binding site into the promoter of transformed recombinant plasmid sequence enhanced promoter production of the heterologous protein (Liu et al., 2003). Berka et al. (2002, 2003) disclosed constructed novel vectors, which encoded the desired heterologous polypeptide and a secretory sequence functional in the lamentous fungus secretory system. A. oryzae produces two predominant proteases, serine-type carboxypeptidase (CPase) and aspartic endopeptidase under acidic conditions (Takuchi and Ichishima, 1986). A typical antisense control strategy, whereby vectors are created to express a high level of the antisense RNA complementary to the RNA transcript of a target gene, used to inhibit fungal gene expression, was used to isolate an low CPase-producing A. oryzae mutant expressing high and stable levels of lysozyme (Zheng et al., 1998). Researchers had limited success in striving for overproduction of manganese peroxidase in its natural host, P. chrysogenum (Cullen, 1997). However, a combination of strategies including use of a strong glucoamylase promoter, a protease-decient A. niger host, culture pH manipulation and incorporation of hemin into the culture medium facilitated strong recombinant enzyme production (Broekhuijsen et al., 1993; Conesa., 2001; Conesa et al., 2000; Punt et al., 2002; Stewart et al., 1996).
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Promoters of the genes encoding glutamate dehydrogenase, betaacetylhexosaminidase and gamma-actin from P. chrysogenum may be used to block expression of undesired genes through anti-sense construction (Barredo Fuente et al., 2001). 4.2. Protease-decient strategies Production, properties and classication of microbial proteases have recently been reviewed (Ward., 2011; Ward et al., 2009). In addition to the observed variabilities in processing of fusion proteins by KEX-like endoproteases in Aspergillus discussed above recombinant protein-degrading fungal proteases have long been known to be problematic (Ward et al., 2006). Braaksma and Punt (2008) reviewed various strategies for controlling protease activity as a means of supporting recombinant protein production. Methods included classical selection of protease mutants, molecular genetic methods to construct protease mutants, targeted to protease genes and protease regulators, manipulation of fermentation conditions, specically pH, control of metabolites/catabolites such as carbon, nitrogen, sulfur and phosphorus, induction of proteases and physiological and morphological effects. With enzyme-overproducing industrial strains, one approach was to partially inactivate some of the more prominent extracellular proteases, for example the alkaline proteases and the metallo-proteases (Christensen and Lehmbeck, 2000). Buxton and Gabor (1997) patented a sequence encoding the vacuolar PEPA aspartic protease and methods for transforming strains to produce the protease and perhaps more importantly for development of Aspergillus mutants defective in the production of aspartic protease. Given that lamentous fungi can contain as many as 80 proteolytic genes of varying known and unknown function, researchers are cautioned against trying to develop mutants decient in multiple proteases (Machida, 2002). Impacts on recombinant protein production of constructing stable A. niger recombinants containing up to three disrupted protease genes were characterized (Van den Hombergh et al., 1997). Specic mutants of A. nidulans, decient in the aspartic protease gene, exhibited the ability to produce chymosin as well as other recombinant proteins (Berka et al., 2003). When the alkaline protease gene of a strain of A. oryzae was transformed to produce heterologous endoglucanase, enhanced production and stability of the recombinant protein was observed in shake ask cultures (Lehmbeck., 2001). Antisense RNA may be used to reduce expression of particular genes, including proteases in recombinant hosts. PEPB protein, recently characterized as a member of the glutamic proteases, was thought to be the causative agent in degradation of recombinant thaumatin in A. awamori containing a disrupted pepA gene producing inactive PEPA. Thaumatin production was improved by expression of pepB antisense RNA, but results indicated antisense mRNA had only partially silenced pepB gene expression. A substantial further increase in thaumatin production was achieved by disruption of the pepB gene (Fujinaga et al., 2004; Moralejo et al., 2002). Disruption of some protease regulator genes has been effective in substantially reducing protease activity in Aspergillus species. For example, disruption of the prtT gene which is a regulatory gene which encodes a member of the Zn-binuclear cluster family appears to eliminate two Aspergillus proteases from the medium including PEPA and reduces total protease activity by 80% (Punt et al., 2008). Yoon et al. (2011) reported on experiments which demonstrated how successive disruption of ten protease genes in A. oryzae was effective in enhancing heterologous production of human lysozyme and bovine chymosin production. Manipulation of fungal culture pH away from the optimal pH for activity and implementation of cultivation strategies which prevent release of intracellular proteases via mycelial cell lysis have been shown to reduce proteolysis of secreted recombinant proteases (Denison, 2000; O'Donnell et al., 2001; Wang et al., 2005). Use of peptide-rich
media, typically induces protease production by A. niger (Ahamed et al., 2005) and productivity of secreted egg lysozyme by a recombinant strain of A. niger was reduced in such rich media (Archer et al., 1990). Double disruption of the two protease genes in A. oryzae, tppA and pepE, facilitated an increase of 63% in the production level of human lysozyme (Jin et al., 2007). Combination strategies of using non-protease inducing medium and use of the aspartyl protease inhibitor pepstatin represent an alternative strategy to minimizing the impacts of proteases on fungal recombinant protein activity (Ahamed et al., 2005). In two recent proteomic studies involving A. niger, it was observed that under conditions of culture starvation, resulting from depletion of carbon source, proteases were found to predominate in the secretome and hence these conditions should be avoided to minimize protease secretion during production of recombinant proteins (Adav et al., 2010; Braaksma et al., 2010). 4.3. Manipulations of morphology Vegetative growth involves hyphal extension and occurs at the hyphal tip. Branching leads to new hyphal extension units. The hyphal tip is the principal region of protoplasmic activity, protein production and extracellular protein secretion and hence this is the principal locus for biological process-related recombinant protein production. Further back from the tip, protoplasmic compartments become more vacuolated. It follows that a greater degree of branching will increase rates of fungal growth, protein synthesis and extracellular protein secretion. Morphology of the mycelium is strongly inuenced by the surrounding environment and other factors including inoculum size and type (vegetative, spores, etc.). On the surface of solid media, lamentous fungi grow as mycelial mats. In submerged cultures, fungi may attach to suspended particles if present or grow as diffuse lamentous mycelia or as dense pellets, which may develop to different sizes. Morphological form inuences rate of growth and product formation. Predominant growth and metabolism of fungi in pelleted form occurs at the pellet surface where there is maximum access to nutrients and oxygen. Inside the pellet, inward diffusion of nutrients and outward diffusion of product become limiting and vacuolization and lysis are frequently observed. Recently, Driouch et al. (2010) described a novel approach, involving use of silicate microparticles, to engineering different morphology states in A. niger to improve enzyme production. Because of morphological problems noted for Aspergillus species in fermenters which result in rheology and viscosity problems leading to mass transfer limitations, Jensen (1997) proposed use of alternative thermophilic fungal hosts for production of recombinant proteins. It was observed that when thermophilic fungal strains including Acremonium, Corynascus, Thielavia, Myceliophthora, Thermoascus and Chaetomium species, were grown in batch fermentations under the same conditions used to culture A. oryzae, medium viscosities observed were much lower. Impact of morphology changes, as they effect recombinant protein production, may be at least partially related to protease production or release. Growth of the A. niger mycelium as large pellets was associated with lower specic protease activities and increased specic glucoamylase activities were found when A. niger was cultured in media which generated large pellets (Papagianni and Young, 2002). In general, fungal pelleted growth mediates greater lysis in fungi, for example, in Aspergillus species, and this results in the presence of higher levels of proteolytic activity in ltrates of pelleted cultures, as compared to lamentous growth (Ahamed et al., 2005). While the greater proteolytic activity in pellet cultures is likely to be partly due to intrapellet cell lysis, differential expression may also be a factor. Dai et al. (2004) has reported that one of seven genes that were differentially expressed in A. niger pellets encoded a pepsin-type protease. pH could be manipulated to cause morphological mutant formation and recombinant glucoamylase production in A niger
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(Swift et al., 1998). In studies of an A. niger strain containing a gene for the Gla-GFP fusion protein, protease activity in pelleted growth was only one-third of that observed with lamentous growth (Xu et al., 2000). Morphology of the lamentous fungus, Chrysosporium lucknowense, has been manipulated genetically to produce a non-lamentous form, thereby reducing viscosity of culture systems. The non-lamentous form also exhibits low protease activity and is capable of producing very high yields of heterologous proteins (Verdoes et al., 2007). General aspects of the relationships between morphology and productivity in lamentous fungi have been reviewed by Grimm et al. (2005). 4.4. Solid-state fermentation approaches While the mechanisms have not been fully elucidated, variations between solid-state and submerged culture fermentation conditions can alter the expression of genes and rates of enzyme production (Iwashita, 2002). During growth of A. oryzae on solid substrates, regions of differentiation of lamentous mycelia exist, from aerial mycelium to medium-associated mycelium, and the different regions of lamentous growth mediate differential levels of gene expression and product formation (Masai et al., 2005). In some cases, the contrasting activities reect whether the activity is cell wall-associated or excreted into the culture medium: in a case in point in submerged cultures, some enzyme activities were associated with the cell wall, whereas in solid-state cultures, they were secreted into the medium (Wang et al., 2005). In solid state culture, wheat bran media caused lowest expression of catabolic genes, which were thought to have released catabolite repression of synthesis of hydrolytic enzymes, thereby mediating rich hydrolytic enzyme gene expression. Oda et al. (2006) concluded that certain enzymes were selectively secreted under conditions of solid-state culture or in submerged culture, independent of the composition of the medium. In a different but a partly analogous situation, proteome differences were observed when F. graminearum was cultured in vitro and in planta, with some proteins being expressed in only one of the two conditions (Paper et al., 2007). Imanaka et al. (2010) compared cultivation of A. oryzae, using three different culture methods, i.e., shake-asks, agar-plate and membrane surface liquid culture, and observed differences in growth, secretion of proteases and alpha-amylase, secreted protein level and gene transcriptional prole by DNA microarray analysis. Protease activities, especially oryzin (alkaline protease) and alpha-amylase, were much higher in agar-plate and membrane surface liquid culture than in shake asks. Transcriptional gene proles from the agar-plate and membrane surface liquid culture manifested somewhat similar patterns but were quite different from the shake ask proles. In some cases, solid state culture promoted production of a more homogeneous glycosylated recombinant product. Submerged cultures by A. oryzae transformed to produce the important heterologous antigenic protein PreS2 of human hepatitis B virus, resulted in production of a partially degraded heterologously glycosylated protein, whereas in solid-state culture a homogeneous glycosylated form of the whole fusion protein was obtained (Maruyama et al., 2000). Use of wheat bran (2%) in solid state culture mediated a 500-fold increase in production of recombinant chymosin as compared with submerged culture (Tsuchiya et al., 1994). te Biesebeke et al. (2002) found that pelleted growth in submerged fermentations and semi-solid fermentations showed different patterns of gene expression and protein secretion. 4.5. Concluding comments Because of their extraordinarily high productivities and simple growth requirements, lamentous fungi can compete or outcompete most other recombinant hosts in processes for production of non-
glycosylated recombinant proteins, assuming for a particular product that problems related to recombinant protein degradation by proteases of the host producing strain have been resolved. It is also clear that where the interest relates to recombinant glycosylated proteins, especially recombinant proteins for human therapeutic use, the quite different native protein glycosylation patterns observed in fungi render them unsuitable hosts for recombinant human glycoprotein production, and some other hosts, for example Pichia species, may be more suitable microbial hosts to be engineered for mammalian glycoprotein production. Nevertheless, research is continuing to be aimed at engineering the appropriate human glycosylation machinery into lamentous fungi. 5. Recombinant proteins in metabolic engineering of fungi In recombinant protein production, the initial priority targets were individual proteins and glycoproteins for benecial industrial, agricultural, food or pharmaceutical use. However, the development of genomic and proteomic capabilities combined with molecular tools provides the tools to manipulate hosts with respect to more complex combinations of proteins, with diverse functions, be they catalytic, regulatory or with other physiological roles. As other organisms, the lamentous fungi offer potential opportunities for improving desired metabolite product yields, reducing or eliminating undesired metabolite bi-products, extending substrate ranges and introducing new enzymes or pathways leading to production of new products (Kern et al., 2007). For example, hosts may be transformed to produce the multiple enzymes associated as part or all of a new metabolite biosynthesis sequence. Alternatively, an existing pathway may be engineered to address bottlenecks and maximize rate of metabolite biosynthesis or indeed to alter specicity of one or more reactions to facilitate expansion of product range. Approaches invariably involve genetic modications, including expression of recombinant enzymes and manipulation of host transcription and/or translation machinery. The opportunities to exploit lamentous fungi for production of a vast range of new products, most of which are not yet conceived, is enormous and derives from the diverse nature of fungal species. With an estimated 1.5 million species (Hawksworth, 2001), only a small number of which has been sampled by genomic and proteomic methods, and with a likely average of 10,000 genes per species, the resource is almost unlimited. Some of these concepts, as they apply to primary and secondary metabolites, are briey discussed in this section through use of selected examples. 5.1. Primary metabolites 5.1.1. Citric acid With respect to primary metabolites, A niger has been investigated as a target model for metabolic engineering of lamentous fungi, especially with respect to citric acid production with a view to increasing metabolic uxes through the glycolytic pathway leading to citrate production (Ruijter et al., 2002). Seven or more enzymes were selected as principal targets and two genes encoding regulatory enzymes in the pathway were overexpressed (Ruijter et al., 1997; Torres et al., 1996). 5.1.2. Biodiesel Biodiesel, originating from some form of biological host, arguably represents part of the biosolution to the world's energy problems (Demain, 2009). The products of fungi and algae are currently being used as benecial food and feed additives and nutritional products. While it is still unclear if fungal and algal strains are appropriate for production of microbial oils for biodiesel production, they do contain the machinery for bio-oil biosynthesis and the tools for transforming their biosynthetic enzymes to other hosts, which may be deemed
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more suitable for energy production, are well on the way to being established. Several lamentous fungi accumulate very high amounts of microbial lipids, especially highly unsaturated lipids (Ward and Singh, 2005). The concept of using lamentous fungi as producers as lipids was introduced during World War 1 in Germany and processes using a variety of strains including Mortierella isabellina, Mortierella alpina and Mucor vinacea have been scaled up to produce commercial quantities of lipids (Ratledge, 1978). While the primary emphasis was on the nutritional benet of unsaturated fatty acids, scientists in the Soviet Union considered the Mucorales as potential candidate hosts for production of lipids for use in biofuels (Feolova et al., 2010). If momentum in this area continues, strategies will be needed for optimization of processes which will include detailed examination of the lipid synthetic pathways, in order to determine metabolic bottlenecks. In addition, sequencing and annotation of the genes encoding the participating enzymes will provide opportunities for transformation of hosts to produce recombinant enzymes. These biochemical and molecular approaches may be combined with fermentation optimisation strategies, both to optimize production rates and yields and to produce products of varying lipid compositions. 5.1.3. Higher unsaturated fatty acids (HUFAs) M. alpina produces the C-20 highly unsaturated fatty acids dihomogamma-linolenic acid, arachidonic acid and eicosapentaenoic and has been used for industrial production of arachidonic acid (Shimizu et al., 1997). In mammals, -linolenic acid (GLA) is an intermediate of the (n6) pathway conversion of linolenic acid to arachidonic acid and a direct precursor of dihomo-gamma-linolenic acid. Certain conditions (aging, stress, some infections, diabetes, eczema) may be manifested as causing a deciency of 6-desaturation and of GLA, and hence there is considerable interest in investigating production of recombinant forms of 6-desaturase and the efcient enzyme from M. alpina as a target of interest. GLA is further metabolized to produce anti-inammatory eicosanoids, such as prostaglandins and leukotrienes, and GLA and its metabolites also regulate expression levels of various gene products. As a rst step, this enzyme was cloned from M. alpina and expressed in A. oryzae under the control of the amyB promoter. While GLA was not produced by the control A. oryzae strain, GLA constituted 25.2% of total fatty acids in the recombinant host transformed to express 6-desaturase (Sakuradani et al., 1999). Similar enzymes have now been cloned from a range of lamentous fungi including Cunninghamella echinulata, R. arrhizus, Rhizopus nigricans, Rhizopus stolonifer, Mucor rouxii and Pythium irregulare. The gene encoding the enzyme from R. stolonifer was recently expressed in Pichia pastoris, enabling the transformed Pichia strain to produce about 22.4% of its fatty acid content as GLA (Wan et al., 2011). 5.2. Secondary metabolites Some of the recent exciting developments in fungal secondary metabolite production relate to the continuing developments in our knowledge of mechanisms for production of non-ribosomal peptides (NRPs) and polyketides (PKSs). While the A. nidulans genome indicates the presence of 26 PKSs and twenty four NRPSs, only six PKSs (synthesizing monodictyphenone, asperfuranone, orsellinic acid/F9775, asperthicin, napthopyrone, sterigmatocystin) and four NRPSs responsible for the biosynthesis of terrequinone, aspyridones, emericellamide and penicillin, are reported to have been characterized in A. nidulans (Balibar et al., 2007; Bergmann et al., 2007; Chiang et al., 2010; Evans et al., 2011; Sanchez et al., 2009; Szewczyk et al., 2008; von Dohren, 2009). 5.2.1. Engineered biosynthesis of novel peptides In addition to the predominant and ubiquitous ribosomal machinery for synthesis of peptides and proteins, certain organisms,
including lamentous fungi, contain complex peptide synthetase templates which mediate synthesis of a variety of bioactive peptides, more than 300 of which are known, without ribosomal involvement (Stachelhaus et al., 1996).The bioactive products include antibiotics, toxins, enzyme inhibitors, antiviral agents, antitumor agents and immunosuppressants. These non-ribosomal large protein templates contain protein domains which catalyze synthesis of a wide range of low molecular weight linear, branched and cyclic peptides which may be further transformed by acylation, glycosylation and other reactions (Marahiel, 1992). Research is ongoing related to manipulation of the participating genes encoding synthetases with altered substrate specicities, using gene disruption and reconstitution strategies, with a view to determining the potential to engineer synthesis of novel peptides, including novel antibiotics and other therapeutics (Kleinkauf and von Dhren, 1990; Stachelhaus and Marahiel, 1995). 5.2.2. Antibiotics Biochemistry of non-ribosomal peptide synthesis has been investigated for many years as it relates, for example, to production of peptide antibiotics, including beta-lactams by the lamentous fungi P. chrysogenum and A. nidulans and production of cyclosporin A by Tolypocladium niveum (Weber et al., 1994; Aharonowitz et al., 1993; Smith et al., 1990; Gutierrez et al., 1991). Numerous genes involved in biosynthesis of antibiotics and other secondary metabolites have been cloned, including genes involved in the synthesis of -lactam antibiotics by the lamentous fungi, P. chrysogenum, Cephalosporium acremonium and A. nidulans (Martn and Liras, 1989). As with other antibiotics, genes involved in penicillin, cephalosporin and cephamycin biosynthesis are organized in clusters and pathway-specic regulatory genes positively or negatively modulate genes expression in these clusters and hence regulate antibiotic biosynthesis (Loder and Abraham, 1971; Martn and Liras, 1989; Nuesch et al., 1987). Positive regulatory elements also impact the interrelated processes of secondary metabolism and differentiation in these organisms. Clustering of biosynthetic enzymes is facilitating research into the molecular mechanisms which mediate expression of genes involved in antibiotic synthesis, which will enable gene expression to be positively regulated and optimized to systematically maximize antibiotic formation in industrial fermentation processes (Martin, 1992). Routes to production of penicillin G, cephalosporins and cephamycins share a common early pathway to synthesis of the isopenicillin-N intermediate. The natural cephalosporins produced by A. chrysogenum have relatively low clinical efcacy and are produced semi-synthetically from intermediate building blocks, 7-aminodeacetoxycephalosporanic acid (7-ADCA) and 7-aminocephalosporanic acid (7-ACA). While P. chrysogenum cannot produce cephalosporins and cephamycins, it is an attractive potential recombinant host because of its hyper capacity to produce penicillin, via the common precursor isopenicillin N and indeed the host was successfully engineered to synthesize cephalosporins and 7-ADCA (Crawford et al., 1995; Robin et al., 2001; 2003a,b; van de Sandt and de Vroom, 2000). The principal transformations of P. chrysogenum involved introduction of the gene cefE from Streptomyces clavuligerus, encoding production of the expandase enzyme deacetoxycephalosporin-C synthase, and the genes cefEF and cefG from P. chrysogenum, encoding the dual expandase/hydroxylase and acetyltransferas, respectively. Other recombinant proteins, for example the cmcH gene from S. clavuligerus, encoding deacetylcephalosporin Ocarbamoyltransferase, have been successfully expressed in P. chrysogenum with a view to producing other novel cephem precursors (Harris et al., 2009). This work demonstrates how metabolic engineering strategies involving production of recombinant enzymes into strains already improved through classical mutation programs can lead to development of multipurpose host platforms for production of non-native secondary metabolites. In related work, two dimensional electrophoresis-based proteomics was used to compare protein maps, with up to 950 proteins
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being identied, from a three P. chrysogenum strains, a high and low penicillin producer and a wild-type strain (Jami et al., 2010). The cysteine biosynthesis, pentose phosphate and some stress response proteins predominated in the high penicillin producing strain while pathways for synthesis of other metabolites were reduced. Peroxisomes participate in important functional aspects of secondary metabolite synthesis in lamentous fungi, including the nal steps of penicillin synthesis, mediated by isopenicillin N-acetyltransferase (Muller et al., 1991;1992). 5.2.3. Taxol The tetracyclic diterpene lactam, taxol, is a low toxicity broad spectrum anticancer drug with benecial application against breast, uterine and other cancers (Wani et al., 1971). Taxol is present naturally at low concentrations of 0.010.05% in the bark, roots and branches of the western yew, Taxus brevifolia, an endangered species. In the past few years, signicant progress has been made in our understanding of the general biology and taxol biosynthetic pathway of taxol-producing endophytic fungi, with a view to producing taxol by microbial fermentation (Lin et al., 2003; Zhou et al., 2008, 2010). Some of the 20 or so genes encoding enzymes of the pathway have been sequenced and some genetic transformation systems have been established which included development of some fungal promoter-containing expression cassettes, ultimately directed at developing efcient taxol-producing recombinant fungal hosts (Guo et al., 2006; Wang et al., 2007a,b,c). 6. Expressing fungal multi-protein complexes or pathways in other hosts While much effort has been fruitfully invested in transforming hosts to produce individual recombinant proteins and to optimizing recombinant product formation, the evolution in our understanding of these systems combined with newly acquired knowledge of genomics and proteomics is facilitating more advanced engineering of hosts with whole new pathways or functions. The huge diversity of fungal species makes fungi a rich reservoir for mining novel biosynthetic capabilities and clearly the native fungal hosts of new pathways, deemed to be of value, may not be suitable hosts for optimal expression of the pathway, or synthesis and exploitation of the desired metabolic products. In this section, a small number of fungal complex systems expressed in non fungal hosts will be briey discussed. It is hoped that the examples point to the great potential to exploit fungal diversity in part through transfer of fungal machinery to other hosts. 6.1. Expression of fungal systems in plants There is considerable interest in metabolic engineering of fatty acids into plants to produce new oil seed crops. Since lamentous fungi are among the best producers of highly unsaturated fatty acids and since higher plants do not produce these fatty acid, there is interest in transforming plants with some of these key highly unsaturated fatty acids (HUFA) biosynthetic enzymes, with M. alpina being recognized as one of the most useful gene sources (Drexler et al., 2003; Huang et al., 1999; Knutzon et al., 1998; Michaelson et al., 1998). Initial strategies involve enhancing the very long chain fatty acid content of current plant oils. Some of the early promising steps involved seed-specic co-expression of an oleate delta12 desaturase and a 6 desaturase, both from Mortierella species, in canola (Liu et al., 2001). The fth generation of eld trials contained 44% -linolenic acid (GLA) not present in rapeseed. Similar high proportions of GLA were accumulated when a 6 desaturase from P. irregulare was expressed in Brassica juncea (Hong et al., 2002). Some other fungal long chain polyunsaturated fatty acid (LC-PUFA)-synthesizing enzymes are also being used as part of the strategy to produce HUFA
oils in plants. A bifunctional 12- and 15-desaturase from Fusarium moniliforme, co-expressed in both soybean and yeast (Yersinia lipolytica) with the primary LC-PUFA biosynthetic enzymes, enhanced levels of eicosapentaenoic acid (EPA) production (Damude et al., 2006). Enzymes with similar biofunctional properties and potential are produced by Claviceps purpurea (Meesapyodsuk et al., 2007) and Coprinus cinereus (Zhang et al., 2007). A recent review on metabolic engineering of oil-seed crops to produce omega-3 long chained fatty acids shows where recombinant HUFA biosynthetic enzymes from lamentous fungi play their part (Venegas-Caleron et al., 2010). Plants and lamentous fungi such as Ascomycota, have different pivotal amino acid linked processes for ammonium assimilation. In lamentous fungi, NADP(H)-dependant glutamate dehydrogenase (GDH) and glutamine synthetase participate in ammonium assimilation whereas in plants glutamine synthetase alone is primarily responsible for assimilation. When the gene encoding GDH, gdhA, from A. niger was expressed in the cytoplasm of rice plants, positive changes in metabolism were observed which lead to enhanced ammonium assimilation and better plant growth (Abiko et al., 2010). Hydrophobins, surface-active proteins obtained from lamentous fungi, have signicant potential applications in geneticallyengineered plants and hydrophobin-fusion technology is predicted to become a very useful tool in plant biotechnology. By way of example, when the hydrophobin HFBI sequence from T. reesei was fused to green uorescent protein (GFP) and transiently expressed in Nicotiana benthamiana plants mediated by A. tumefaciens, the fusion enhanced GFP accumulation (Joensuu et al., 2010). The fusion protein constituted more than 50% of total soluble protein and delayed leaf necrosis. 6.2. Expression of fungal systems in bacteria and yeast 6.2.1. Polyketide biosynthetic machinery Polyketides result from biosyntheses involving polymerization of acetyl and propionyl subunits in a manner analogous to fatty acid synthesis. Polyketide products, generated by different PKS types, include erythromycin, tacrolimus, epothilone, tetracycline, tetracenomycin, daunorubicin, 6-methyl salicylic acid, aatoxin B1 and lovastatin (Crawford et al., 2006; Fujii, 2010; Kennedy et al., 1999; Moriguchi et al., 2010; Shen, 2003; Watanabe et al., 1996). Fungal polyketide synthases (PKSs) utilize a variety of acyl-CoA substrates to catalyze carbon to carbon bond forming reactions to synthesize the well known complex fungal polyketides which exhibit a range of bioactivities. Many gene clusters, thought to encode the enzymes responsible for synthesizing polyketides such as bikaverin, zearalenone and hypothemycin, including genes encoding PKSs, have been characterized. Recent reports indicate that these recombinant fungal enzymes have been expressed in E. coli rendering the bacterial host capable of production of the corresponding secondary metabolite (Saruwatari et al., 2011). The aromatic polyketide, 6-methylsalicylic acid, is synthesized by ATX, encoding gene atX, present in A. terreus and Penicillium patulum, and this gene has successfully been expressed in E. coli, S. cerevisiae and Streptomyces species and an enhanced production of 6-methylsalicylic acid was noted in the case of S. cerevisiae (Bedford et al., 1995; Kealey et al., 1998). The huge fungal gene responsible for bikaverin (aromatic polyketide anticancer drug) biosynthesis, PKS4 originating in Giberella fujikuroi, was successfully expressed in E. coli with production of the polyketide (Ma et al., 2007). Ma et al. (2009) also reported that the lovastatin nonaketide synthase (LovB) from A. terreus, involved in synthesis of the cholesterol lowering drug, lovastatin, could be efciently expressed by a recombinant S. cerevisiae host, research work which is contributing substantially to determining the complex set of functions and structures of the polyketide synthases. Mixing of domains of different PKS genes, including domains originating from fungi and bacteria, generated different combinations of biosynthetic enzymes and
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demonstrated the potential to vary the synthezing machinery to produce wholly novel metabolites not produced naturally by the native strains (Saruwatari et al., 2011; Zhang et al., 2008). 6.2.2. Penicillin biosynthetic machinery While current industrial production of penicillin is mediated by P. chrysogenum, the intrinsic growth limitations of fungi in large fermenters are recognized and there is interest in developing new hosts, for example yeasts, for production of new beta lactams and perhaps other bioactive compounds such as peptides. An industrial precedent exists for changing from a lamentous fungal host to a yeast host in the case of citric acid production when the Pzer Company modied their process by introducing a Candida guilliermondii yeast strain in place of the conventional Aspergillus producer, thereby increasing citrate production rate (Ward, 1989; 1991). In P. chrysogenum, production of penicillin is compartmentalized in peroxisomes and the cytosol. The yeast Hansenula polymorpha has a number of features that make it potentially an attractive host as an alternative producer of beta-lactams, including the availability of some very strong and regulatable promoters. Furthermore, penicillin production in Penicillium is signicantly mediated by peroxisomal enzymes and it is known that peroxisomes of H. polymorpha may be hyperinduced. Gidijala et al.(2009) successfully engineering H. polymorpha to produce and efciently secrete penicillin: a) by introducing the P. chrysogenum gene encoding the non-ribosomal peptide synthase (NRPS) -(L-aminoadipyl)-Lcysteinyl-D-valine synthetase (ACVS) into H. polymorpha resulting in production of active ACVS enzyme; b) co-expression of the B. subtilis stp gene encoding the enzyme phosphopantetheinyl transferase which activates ACVS; and c) co-expression of P. chrysogenum genes encoding cytosolic isopenicillin N synthase and the peroxisomal enzymes, isopenicillin N acyl transferase (IAT) and phenylacetyl CoA ligase (PCL). Recombinant fungal ACVS has also been expressed in bakers yeast (Siewers et al., 2009). 7. Engineering of pathogenic fungi A very large number of lamentous fungi are pathogenic to other organisms, including animals and humans, plants and insects and, in the case of animals and plants, the resulting diseases have vast negative economic and social impacts. Molecular techniques, including strategies involving production of recombinant proteins may be used to facilitate elucidation of the causes of pathogenesis and to provide direction on solutions on pathogenesis problems. The following brief examples are offered to illustrate potential opportunities. 7.1. Entomopathogens Recombinant fungal technology offers potential to create recombinant hosts which interact with other organisms. Examples would include transformed fungi which produce molecular species such as herbicides, insecticides or biological inhibitors against undesired targets. Recently published examples include a recombinant fungus which kills human malaria parasites in mosquitoes, mediated by secreted recombinant toxic peptides and enhancement of infectivity of a recombinant entomopathogenic Beauveria bassiana towards larvae of the oriental leafworm moth. While there have been effective chemical pesticides against malaria and its mosquito carrier, especially pyrethroids, the progress towards eradication has been hampered by the development of pyrethroid-resistant mosquitoes. Several fungi, including Metarhizium anisopliae, are pathogenic to adult mosquitoes but the infective process through contact with the cuticle is slow (1214 days). Recombinant strains of M. anisopliae expressing the antimicrobial toxin, scorpine, or a salivary gland and mid-gut peptide 1 (SM1) reduced entry and/or development of Plasmodium falciparum, the parasitic causative agent of malaria, into the Anopheles gut where it gets
transformed from gametocytes into infective sporozoites. Sporozoite count reductions mediated by the expressed SM1 and scorpine were 71 and 90%, respectively. The SMI peptide binds to the surface of the salivary gland of Anopheles gambiae, the principal malaria vector, and blocks entry of the parasite into the mosquito vector. The scorpion antimicrobial scorpine, is a hybrid of two toxins, a cecropin and a defensin which is two orders of magnitude more toxic against Plasmodium. A recombinant M. anisopliae expressing an [SM1]sub8:scorpine fusion protein, reduced sporozite counts by 98% (Fang et al., 2011). The infective action of the lamentous fungus, B. bassiana, towards insects, via their cuticle, is generally a slow process. However, lepidopteran pests are rapidly killed by ingestion of the Vip3A insecticidal proteins produced by Bacillus thuriengensis. A recombinant strain of B. bassiana, which highly expressed one of the Vip3A toxins, Vip3Aa1 in its conidial cytoplasm, when sprayed on cabbage leaves was shown to dramatically reduce larval numbers feeding on the cabbage leaves (by 26.2 fold) (Qin et al., 2010). The mid gut of the infected larvae contained a digested form of the toxin and the larvae exhibited the expected symptoms of toxin action, namely shrinkage and palsy. Methods have also been established whereby larvae of organisms such as the silk worm have been directly transformed to produce recombinant fungal proteins. For example, the 1320-bp endoglucanase 1 gene from T. viride was cloned and expressed in silkworm larvae and in a silkworm cell line using a mutant baculovirus expression system (Li et al., 2010). 7.2. Plant pathogens Molecular methods are also being used to develop a better understanding of the mechanisms related to fungal pathogenicity of plants. In addition to having an important role in secondary metabolite production, as indicated above, peroxisomes are also involved in lamentous fungal pathogenicity, for example, of cucumber by Colletotrichum orbiculare and the rice blast pathogen, Magnaporthe oryzae (Bhambra et al., 2006; Wang et al., 2007d) and in production of host selective AK-protein toxins by Alternaria alternata which mediate its pathogenicity to multiple plant species, including Japanese pear (Imanaka et al., 2010). Imazaka et al. (2010) conrmed the peroxisome localization of AK-toxins by developing and expressing recombinant GFPtagged AK-protein toxins in A. alternata and then used mutant recombinant A. alternata strains, with AK-toxin disruptions, to conrm the role of AK-toxins in pathogenicity. It was noted earlier (3.4.1) that the genome of the human R. oryzae pathogen contained expanded gene families of encoding secreted proteases and that these proteases are involved in the infective process and are characteristic of the virulence of Rhizopus pathogens (Ma et al., 2009). It was also noted in the same section that R. miehei, which causes fungal infections in humans and animals, can resist higher concentrations of statins than is observed in Mucor circinelloides, where statins inhibit HMG-CoA and ultimately lead to Mucor cell death. A recombinant M. circinelloides strain expressing the gene hmgR encoding the HMG-CoA of R. miehei, was rendered more tolerant to statins. Clearly, our knowledge of fungal pathogenesis will greatly expand over the next few years as the genomes of more plant pathogens are sequenced and their gene functions are annotated. 8. Engineering of biocontrol fungi Filamentous fungi can also have benecial roles in plant disease management as will be illustrated here by focusing on Trichoderma species. Many strains of Trichoderma are highly competent in colonizing plant root surfaces and thrive and proliferate in association with healthy root surfaces (Sriram and Ray, 2005). Many other fungi lack this ability and Trichoderma species have evolved a variety of mechanisms to attack and parasitize these other fungi and benet from
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nutrients from these other fungi as they support and enhance plant growth as biocontrol agents (Harman et al., 2004). Biocontrol agents utilize several antagonistic strategies against pathogens and all of these mechanisms are exploited by Trichoderma (Dennis and Webster, 1971a,b,c). These mechanisms include: competition by limiting access to the pathogen's nutritional needs; production of inhibitory metabolites or other toxins including trichothecin, sesquiterpene and certain enzymes; and parasitism. In the case of T. harzianum, it was noted that combined effect of production of an antibiotic and production of hydrolytic enzymes had a synergistic effect on the pathogens (Schirmbck et al., 1994). Enzymes thought benecial to Trichoderma species in attacking pathogens such as Fusarium oxysporum and Botrytis cinerea include the lytic enzymes, chitinase, beta-1,3-glucanases, and protease (Cherif and Benhamou, 1990; Elad and Kapat, 1999; Geremia et al., 1993; Tronsmo et al., 1993). When a recombinant chitinase from a soil bacterium Serratia marcescens used to control Sclerotium rolfsii was introduced into T. harzianum under control of a constitutive promoter, the constitutive production of chitinase by the Trichoderma biocontrol agent enabled it to overgrow the S. rolfsii pathogen (Haran et al., 1993). An alkaline protease producing transformant of T. harzianum was thought to enhance its mycoparasitic activity (Sriram and Ray, 2005). Of course, another approach to protecting plants against fungal plant pathogens is to directly arm the plants with the requisite biodefense machinery and indeed several genes encoding the Trichoderma enzymes mediating Trichoderma's parasitic role have been expressed in plants, rendering them more resistant to fungal plant pathogens (Bolar et al., 2000; Lorito et al., 1998; St Leger et al., 1996). 9. Conclusions The principal focus over the past 25 years of recombinant protein research efforts as applied to lamentous fungi related to the evaluation and development of the workhorse fungi, especially the industrial extracellular enzyme-producing fungi, as hosts for recombinant protein production. The special advantages of these organisms, namely their abilities to grow at high rates and to high densities in commercial fermenters and their natural abilities to secrete high levels of homologous enzymes, are widely recognized. In exploiting these advantages for production of recombinant proteins, researchers have generally used the most effective regulatory, expression and secretion machinery identied in these organisms and hooked it up to the genes encoding the desired recombinant protein, an approach which has met with considerable commercial success. Filamentous fungi also have several disadvantages as hosts for heterologous protein production, perhaps the most serious being their abilities also to produce and secrete homologous proteases which may degrade the desired recombinant product. In addition to the secreted proteases, lamentous fungi may be subject to different degrees of lysis during the fermentation process, the extent of which will vary with other aspects of fungal physiology including morphology, such that intracellular proteases may be released which can attack the recombinant protein. In addition, there are signicant differences in the mechanisms of protein glycosylation observed in fungi and mammalian cells. While considerable research is being invested in modifying the glycosylation machinery in fungi to use them as hosts for production hosts of more human-like glycoproteins, it is widely accepted that yeasts such as Pichia, may be better candidate hosts for production of recombinant human therapeutic glycoproteins. The vast amounts of scientic information being generated through advances in genomics and proteomics are expanding the opportunities for new applications for recombinant lamentous fungi. It is facilitating a shift from simply inserting a gene encoding a single protein product to inserting machinery encoding multiple genes, for example encoding metabolic enzymes for a new or improved
biosynthetic pathway related to the production of current or new primary or secondary metabolites. While this activity is still at an early stage, it will clearly gain momentum and will become a more important pursuit in the eld of industrial microbiology in the years ahead. It has also been recognized that fungi represent an enormously diverse range of organisms and hence a huge resource for novel biosynthetic systems, especially since the vast majority of fungal species on the planet have yet to be cultured, identied and characterized. It is clear that many of the fungi naturally harboring novel biosynthetic machinery will likely not be the best hosts for large-scale exploitation of these capabilities. It is expected that in many cases, the target biosynthetic abilities will be better utilized by transformation into other fungal hosts, perhaps also to other microbial hosts, and in some cases, to non-microbial hosts, for example, to plants. One of the most exciting new aspects of the study of recombinant proteins expression in fungi is the realization that the future applications will not be conned to the biomanufacturing capacities of these organisms. Some of the natural benecial impacts and especially the deleterious impacts of fungi relate to their extremely complex interactions with other organisms, for example through mutualism, commensalisms and especially pathogenesis. About 300 fungi are known to cause diseases in humans (Monk and Goffeau, 2008) and many of these organisms exhibit multidrug resistance mediated by multidrug efux pumps (Balzi et al., 1994). A myriad of fungal pathogens attack plants and a quantum shift is currently taking place in the advancement of our knowledge of the plantmicrobe interactions involved in these diseases (Boller and He, 2009). The underlying causative mechanisms involved in these attacks are now being characterized using modern genomic and molecular strategies. Put simply, the economic and social costs associated with the impacts of fungal pathogenesis are enormous. The opportunities to exploit recombinant technology to unravel the causes and then bring forward solutions will represent a very important future focus of recombinant protein technology as it relates to fungi.
References
Abianova AR, Chulkin AM, Vavilova EA, Federova TV, Loginov DS, Koroleva OV, Benevolenskii SV. A heterologous production of the Trametes hirsuta laccase in the fungus Penicillium canescens. Prikl Biokhim Mikrobiol 2010;46:3427. Abiko T, Wakayama M, Kawakami A, Obara M, Kisaka H, Miwa T, et al. Changes in nitrogen assimilation, metabolism and growth in transgenic rice plants expressing a fungal NADP(H)-dependent glutamate dehydrogenase (gdhA). Planta 2010;232:299311. Adav SS, Li AA, Manavalan A, Punt P, Sze SK. Quantitative iTRAQ secretome analysis of Aspergillus niger reveals nivel hydrolytic enzymes. J Proteome Res 2010;9:393240. Ahamed A, Singh A, Ward OP. Culture-based strategies for minimization of protease activity in ltrates from Aspergillus niger. World J Microbiol Biotechnol 2005;21: 157783. Aharonowitz Y, Bergmeyer J, Cantoral JM, Cohen G, Demain AL, Fink U, et al. -(L -Aminoadipyl)-L-cysteinyl-D-valine synthetase, the multienzyme integrating the four primary reactions in -lactam biosynthesis, as a model peptide synthetase. Biotechnology 1993;11:80710. Akao T, Gomi K, Goto K, Okazaki N, Akita O. Subtractive cloning of cDNA from Aspergillus oryzae differentially regulated between solid-state and liquid (submerged) culture. Curr Genet 2002;41:27581. Akin AR, Bodie, EA, Burrow, S, Dunn-Coleman N, Turner G, Ward M. Regulatable growth of lamentous fungi. United States Patent Application No. 20030045697. 2003. Allgaier S, Taylor RD, Brudnaya Y, Jacobson DJ, Cambareri E, Stuart WD. Vaccine production in Neurospora crassa. Biologicals 2009;37:12832. Archer DB, Dyer PS. From genomics to post-genomics in Aspergillus. Curr Opin Microbiol 2004;7:499504. Archer DB, Jeens DJ, MacKenzie DA, Brightwell G, Lambert N, Lorne G, et al. Hen egg white lysozyme expressed and secreted from A. niger is correctly processed and folded. Biotechnology 1990;8:7415. Aro N, Pakula T, Penttila M. Transcriptional regulation of plant cell wall degradation by lamentous fungi. FEMS Microbiol Rev 2005;29:71939. Balibar CJ, Howard-Jones AR, Walsh CT. Terrequinone A biosynthesis through ltryptophan oxidation, dimerization and bisprenylation. Nat Chem Biol 2007;3: 58492. Balzi E, Wang S, Leterme S, Van Dyck L, Goffeau A. PDR5, a novel yeast multidrug conferring transporter controlled by a transcription regulator PDR1. J Biol Chem 1994;269:220614.
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx Barredo Fuente JL, Rodriguez Saiz M, Collados De La Vieja AJ, Moreno Valle MA, Salto Maldonado F, Diez Garcia B. Promoters of the genes glutamate dehydrogenase beta-N-acetylhexosaminidase and -actin and their use in lamentous fungi expression, secretion and antisense systems. United States Patent 6,300,095. 2001. Bautista LF, Aleksenko A, Hentzer M, Santerre-Henriksen A, Nielsen J. Antisense silencing of the CreA gene in Aspergillus nidulans. Appl Environ Microbiol 2000;66: 457981. Bedford DJ, Schweizer E, Hopwood DA, Khosla C. Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2). J Bacteriol 1995;177:45448. Beijersbergen AGM, Bundock P, Gouka RJ, de Groot MJA, Hooykaas PJJ. Agrobacterium mediated transformation of moulds, in particular those belonging to the genus Aspergillus. United States Patent 6255115. 2001. Bennett JW. The molds of Katrina. Update (NY Acad Sci) 2006; Jan/Feb: 69. Bergmann S, Schumann J, Scherlach K, Lange C, Brakhage AA, Hertweck C. Genomicsdriven discovery of PKSNRPS hybrid metabolites from Aspergillus nidulans. Nature Chem Biol 2007;3:2137. Berka RM, Cullen D, Gray GL, Gregory Lawrence H, Hayenga KJ, Lawlis VB. Heterologous polypeptides expressed in lamentous fungi, processes for making same, and vectors for making same. United States Patent 6,379,928. 2002. Berka RM, Cullen D, Gray GL, Hayenga KJ, and Lawlis VB. Heterologous polypeptides expressed in lamentous fungi, processes for making same, vectors for making same. United States Patent Application 20030224482. 2003. Bhambra GK, Wang Z-Y, Soanes DM, Wakley GE, Talbot NJ. Peroxisome carnithine acetyl transferase is required for elaboration of penetration hyphae during plant infection by Magnaporthe grisea. Mol Microbiol 2006;72:634554. Boel E, Christensen T, Woldike H. Process for production of protein products in Aspergillus. United States Patent 5,536,661. 1996. Bolar JP, Norelli JL, Wong KW, Hayes CK, Harman GE, Aldwinckle HS. Expression of endochitinase from Trichoderma harzianum in transgenic apple increases resistance to apple scab and reduces vigor. Phytopathology 2000;90:727. Boller T, He SY. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science 2009;324:7424. Braaksma M, Martens-Uzonova EM, Punt PJ, Schaap PJ. An inventory of the Aspergillus niger secretome by combining in silico predictions with shotgun proteomics data. BMC Genome 2010;11:58495. Braaksma M, Punt PJ. Aspergillus as a cell factory for protein production: controlling protease activity in fungal production. In: Goldman GH, Osmani SA, editors. The Aspergilli: genomics, medical aspects, biotechnology and research methods. Boca Raton: CRC Press; 2008. p. 44155. Brantly ML, Wittes JT, Vogelmeier CF, Hubbard RC, Fells GA, Crystal RG. Use of highly puried alpha 1-antitrypsin standard to establish ranges for the common normal and decient alpha 1-antitrypsin phenotypes. Chest 1991;100:7038. Brody H, Grifth J, Cuticchia AJ, Arnold J, Timberlake WE. Chromosome-specic recombinant DNA libraries from the fungus Aspergillus nidulans. Nucleic Acids Res 1991;19:31059. Broekhuijsen MP, Mattern IE, Contreras R, Kinghorn JR, van den Hondel CAMJJ. Secretion of heterologous proteins by Aspergillus niger: Production of active human interleukin-6 in a protease decient mutant by KEX2-like processing of a glucoamylase-hIL6 fusion protein. J Biotechnol 1993;31:13545. Buchert T, Oksanen T, Pere J, Siika-Aho M, Suurnakki A, Viikari L. Applications of Trichoderma reesei enzymes in the pulp and paper industry. In: Harman GE, Kubicek CP, editors. Trichoderma and Gliocladium, 2. London: Taylor and Francis; 1998. p. 34363. Buxton FJ, Gabor V, Jacob NL. Aspergillus niger vacuolar aspartyl protease. United States Patent 5674728. 1997. Cardoso PG, Teixeira JA, de Queiroz MV, de Araujo EF. Pectin lyase production by recombinant Penicillium griseoroseum. Can J Microbiol 2010;56:8317. Chavez R, Roa A, Navarrete K, Trebotich J, Espinosa Y, Vaca I. Evaluation of properties of several cheese-ripening fungi for potential biotechnological applications. Mycoscience 2010;51:847. Chen XA, Ishida N, Todaka N, Nakamura R, Maruyama JI, Takahashi H, et al. Promotion of efcient saccharication of crystalline cellulose by Aspergillus fumigatus Swo1. Appl Environ Microbiol 2010;76:255661. Cherif M, Benhamou N. Cytochemical aspects of chitin breakdown during the parasitic action of a Trichoderma sp. on Fusarium oxysporum f.sp. radicislycopersici. Phytopathology 1990;80:140614. Cherry J, Fidantsef A. Directed evolution of industrial enzymes: an update. Curr Opin Biotechnol 2003;14:43843. Chiang YM, Szewczyk E, Davidson AD, Entwistle R, Keller NP, Wang CC, et al. Characterization of the Aspergillus nidulans monodictyphenone gene cluster. Appl Environ Microbiol 2010;76:206774. Christensen T, Lehmbeck J. Fungus wherein the areA, pepC and/or pepE genes have been inactivated. United States Patent 6013452. 2000. Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L, et al. High level expression of recombinant genes in Aspergillus oryzae. Bio-Technology 1988;6:141922. Chung SJ, Kim S, Sapkota K, Choi BS, Shin C, Kim SJ. Expression of recombinant interleukin-32 in Pleurotus eryngii. Ann Microbiol 2011;61:3318. Clutterbuck AJ. The validity of the Aspergillus nidulans linkage map. Fungal Genet Biol 1997;21:26777. Collen A, Ward M, Tjerneld F, Stalbrand H. Genetic engineering of the Trichoderma reesei endoglucanase I (Cel7B) for enhanced partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers. J Biotechnol 2001;87:17991. Colot HV, Park G, Turner GE, Ringelberg C, Crew CM, Litvinkova L, et al. A highthroughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 2006;103:103527.
17
Conesa A, Punt PJ, Luijk N, van den Hondel CAMJJ. The secretion pathway in lamentous fungi: a biotechnological view. Fungal Genet Biol 2001;33:15571. Conesa A, van den Hondel CAMJJ, Punt PJ. Studies on the production of fungal peroxidases in Aspergillus niger. Appl Environ Microbiol 2000;66:301623. Conesa A. Overproduction of fungal peroxidases in lamentous fungi (Thesis). University of Leiden; 2001. Crawford JM, Dancy BCR, Hill EA, Udwary DW, Townsend CA. Identication of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase. Proc Natl Acad Sci USA 2006;103:1672833. Crawford L, Stepan AM, McAda PC, Rambosek JA, Conder MJ, Vinci VA, et al. Production of cephalosporin intermediates by feeding adipic acid to recombinant Penicillium chrysogenum strains expressing ring expansion activity. Bio-Technology 1995;13: 5862. Crystal R. 1-Antitrypsin deciency. Marcel Dekker Inc; 1996. Cullen D. Recent advances on the molecular genetics of ligninolytic fungi. J Biotechnol 1997;53:27389. Cuomo CA, Guldener U, Xu J-R, Trail F, Turgeon BG, Di Pietro A, et al. The Fusarium graminearum genome reveals a link between localized polymorphism and pathogen specialization. Science 2007;317:14002. Cutler JE, Deepe GS, Klein BS. Advances in combating fungal diseases: vaccines on the threshold. Nat Rev Microbiol 2007;5:1328. Dai Z, Mao X, Magnuson JK, Lasure LL. Identication of genes associated with morphology in Aspergillus niger by using suppression subtractive hybridization. Appl Environ Microbiol 2004;70:247485. Damude HG, Zhang H, Farrall L, Ripp KG, Tomb J-F, Hollerbach D, et al. Identication of bifunctional 12/3 fatty acid desaturases for improving the ratio of 36 fatty acids in microbes and plants. Proc Natl Acad Sci USA 2006;103:944651. De Pourcq K, De Schutter K, Callewaert N. Engineering of glycosylation in yeast and other fungi: current state and perspectives. Appl Microbiol Biotechnol 2010;87: 161731. de Vries RP. Regulation of Aspergillus genes encoding plant cell wall polysaccharidedegrading enzymes; relevance for industrial production. Appl Microbiol Biotechnol 2003;61:1020. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Advances 2009;27:297306. Demain AL. Biosolutions to the energy problem. J Indust Microbiol 2009;36:31932. Denison SH. pH regulation of gene expression. Fungal Genet Biol 2000;29:6171. Dennis C, Webster J. Antagonistic properties of species-groups of Thrichoderma. 1. Production of non-volatile antibiotics. Trans Brit Mycol Soc 1971a;57:2539. Dennis C, Webster J. Antagonistic properties of species-groups of Thrichoderma. 2. Production of volatile antibiotics. Trans Brit Mycol Soc 1971b;57:418. Dennis C, Webster J. Antagonistic properties of species-groups of Thrichoderma. 3. Hyphal interaction. Trans Brit Mycol Soc 1971c;57:3639. deOliveira JMPF, deGraaf LH. Proteomics of industrial fungi: trends and insights. Appl Microbiol Biotechnol 2011;89:22537. Drexler H, Spiekermann P, Meyer A, Domergue F, Zank T, Sperling P, et al. Metabolic engineering of fatty acids for breeding of new oilseed crops: strategies, problems and rst results. J Plant Physiol 2003;160:779802. Driouch H, Sommer B, Wittmann C. Morphology engineering of Aspergillus niger for improved enzyme production. Biotech Bioeng 2010;105:105868. Dunn-Coleman NS, Bloebaum P, Barka M, Bodie E, Robinson N, Armstrong G, et al. Commercial levels of chymosin production by Aspergillus. Mol Gen Genet 1991;230:28894. Elad Y, Kapat A. The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur J Plant Pathol 1999;105:17789. Elander R. Strain improvement and preservation of beta-lactam producing microorganisms. In: Demain AL, Solomon N, editors. Antibiotics containing the -lactam structure I. New York: Springer-Verlag; 1983. p. 97-146. Evans BS, Robinson SJ, Kelleher NL. Surveys of non-ribosomal peptide and polyketide assembly lines in fungi and prospects for their analysis in vitro and in vivo. Fungal Genet Biol 2011;48:4961. Fang W, Vega-Rodriguez J, Ghosh AK, Jacobs-Lorena M, Kang A, St Leger RJ. Development of transgenic fungi that kill human malaria parasites in mosquitoes. Science 2011;331:10747. Fawole OB, Odunfa SA. Some factors affecting production of pectic enzymes by Aspergillus niger. Int Biodeter Biodegr 2003;52:2237. Feolova EP, Dergeeva YE, Inashechkin AA. Biodiesel-fuel: content, production, producers, contemporary biotechnology (review). Appl Biochem Microbiol 2010;46:36978. Fleissner A, Dersch P. Expression and export: recombinant protein production systems for Aspergillus. Appl Microbiol Biotechnol 2010;87:125570. Fujihiro S, Higuchi R, Hisamatsu S, Sonoki S. Metabolism of hydroxylated PCBs by cloned laccase isoforms. Appl Microbiol Biotechnol 2009;82:85360. Fujii I. Functional analysis of fungal polyketide biosynthesis genes. J Antibiot 2010;63: 20718. Fujinaga M, Cherney MM, Oyama H, Oda K, James MNG. The molecular structure and catalytic mechanism of a novel carboxyl peptidase from Scytalidium lignicolum. Proc Natl Acad Sci USA 2004;101:33649. Fulci V, Macino G. Quelling: post-translational gene silencing guided by small RNAs in Neurospora crassa. Curr Opin Microbiol 2007;10:199203. Galagan JE, Calvo SE, Borkovich KA, Selker EU, Read ND, Jaffe D. The genome sequence of the lamentous fungus Neurospora crassa. Nature 2003;422:85968. Galagan JE, Calvo SE, Cuomo C, Ma L-J, Wortman JR, Batzoglou S, et al. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 2005;438:110515. Galanti YM, De Conti A, Monteverdi R. Applications of Trichoderma enzymes in the textile industry. In: Harman GE, Kubicek CP, editors. Trichoderma and Gliocladium, Vol 2. London: Taylor and Francis; 1998. p. 31126.
18
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx Iwashita K. Recent studies of protein secretion by lamentous fungireview. J Biosci Bioeng 2002;94:5305. Jacobs DI, Olsthoorn MMA, Maillet I, Akeroyd M, Breestraat S, Donkers S, et al. Effective lead saelection for improved porotein production in Aspergillus niger based on integrated genomics. Fungal Genet Biol 2009;46:S14152. Jami MS, Barreiro C, Garcia Estradfa C, Martin JF. Proteome analysis of the penicillin producer Penicillium chrysogenum: characterization of protein changes during industrial strain improvement. Mol Cell Proteomics 2010;9:118298. Jeenes DJ, MacKenzie DA, Archer DB. Transcriptional and post translational events affect the production of secreted hen egg white lysozyme by A. niger. Transgen Res 1994;3:297303. Jeenes DJ, Marczinke B, MacKenzie DA, Archer DB. A truncated glucoamylase gene fusion for heterologous protein secretion from A. niger. FEMS Microbiol Lett 1993;107:26771. Jeenes DJ, Pfaller R, Archer DB. Isolation and characterization of a novel stress inducible PDI family gene from A. niger. Gene 1997;194:1516. Jensen EB, Boominathan KC. Thermophilic fungal expression system. United States Patent 5695985. 1997. Jin FJ, Watanabe T, Juvvadi PR, Maruyama J-I, Arioka M, Kitamoto K. Double disruption of the proteinase genes, tppA and pepE, increase the production level of human lysozyme by Aspergillus oryzae. Appl Gen Mol Biotechnol 2007;76:105968. Joensuu JJ, Conley AJ, Lienemann M, Brandle JE, Linder MB, Menassa R. Hydrophobin fusions for high-level transient protein expression and purication in Nicotiana benthamiana. Plant Physiol 2010;152:62233. Kainz E, Gallmetzer A, Hatzl C, Nett JH, Li H, Schinko T, et al. N-glycan modication in Aspergillus species. Appl Environ Microbiol 2008;74:107686. Karnaukhova E, Ophir Y, Golding B. Recombinant human alpha-1-proteinase inhibitor towards therapeutic use. Amino Acids 2006;30:31732. Karnaukhova E, Ophir Y, Trinh L, Dalal N, Punt P, Golding B, et al. Expression of human (I)-proteinase inhibitor in Aspergillus niger. Microb Cell Factories 2007;6(34): 1-10. Kasajima T, Yamaguichi M, Hirai N, Ohmachi T, Yoshida T. In vivo expression of UDP-Nacetylglucosamine: -3-D-mannoside -1,2-N-acetylglucosyl-transferase 1 (GnT1) in Aspergillus oryzae and effects on the sugar chain of alpha-amylase. Biosci Biotechnol Biochem 2006;70:26628. Kealey JT, Liu L, Santi DV, Betlach MC, Barr PJ. Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts. Proc Natl Acad Sci USA 1998;95:499505. Kennedy J, Auclair K, Kendrew SG, Park C, Vederas JC, Hutchinson CR. Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 1999;284:136872. Kern A, Tilley E, Hunter IS, Legisa M, Glieder A. Engineering primary metabolic pathways of industrial micro-organisms. J Biotechnol 2007;129:6-29. Kim Y, Nandakumar MP, Marten MR. Proteomics of lamentous fungi. Trends Biotechnol 2007;25:395400. Kinghorn JR, Unkles SE. Aspergillus. New York: Plenum Press; 1994. Kirk TK, Farrel RL. Enzymatic combustion: the microbial degradation of lignin. Ann Rev Microbiol 1987;41:465505. Kitamoto K, Kimura K, Gomi K, Kumagai C. Electrophoretic karyotype and gene assignment to chromosomes of Aspergillus oryzae. Biosci Biotechnol Biochem 1994;58: 146770. Kleinkauf H, von Dhren H. Non-ribosomal biosynthesis of peptide antibiotics. Eur J Biochem 1990;192:1-15. Knutzon DS, Thurmond JM, Huang Y-S, Chaudhary S, Bobik EG, Chan GM, et al. Identication of 5-desaturase from Mortierella alpina by heterologous expression in bakers' yeast and canola. J Biol Chem 1998;273:293606. Kobayashi T, Abe K, Asai K, Gomi K, Juwadi PR, Kitamoto K, et al. Genomics of Aspergillus oryzae. Biosci Biotechnol Biochem 2007;71:64670. Kumar R, Singh S, Singh OV. Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives. J Indust Microbiol Biotechnol 2008;35:37791. Kwon-Chung KJ, Bennett JE. Mucormycosis. Medical mycology. Philadelphia: Lea and Febiger; 1992. p. 52459. Lehmbeck J. Alkaline protease decient lamentous fungi. United States Patent 6, 291,209. 2001. Li X, Wang D, Zhou F, Yang H, Bhaskar R, Hu J, et al. Cloning and expression of a cellulose gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system. Mol Biol Rep 2010;8:37218. Lin FC, Liu XH, Wang HK, Zhang CL. Recent research and prospect on taxol and its producing fungi. Acta Microbiol Sin 2003;43:5348. Liu J-W, Huang Y-S, DeMichele S, Bergana M, Bobik Jr E, Hastilow C, et al. Evaluation of the seed oils from a canola plant genetically transformed to produce high levels of -linolenic acid. In: Hunag Y-S, Ziboh VA, editors. -Linolenic acid: recent advances in biotechnology and clinical applications. Champaign IL: AOCS Press; 2001. p. 6171. Liu L, Liu J, Qiu RX, Zhu XG, Dong ZY, Tang GM. Improving heterologous gene expression in Aspergillus niger by introducing multiple copies of protein-binding sequence containing CCAAT to the promoter. Lett Appl Microbiol 2003;36: 35861. Loder PB, Abraham EB. Isolation and nature of intracellular peptides from a cephalosporin C producing Cephalosporium sp. Biochem J 1971;123:4716. Lorito M, Woo SL, Fernandez IG, Colucci G, Harman GE, Pintor-Toro JA, et al. Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens. Proc Natl Acad Sci USA 1998;95:78605. Lubertozzi D, Keasling JD. Developing Aspergillus as a host for heterologous expression. Biotechnol Adv 2009;27:5375.
Geremia RA, Goldman GH, Jacobs D, Ardrtes W, Vila SB, Van Montagu M, et al. Molecular characterization of the proteinase-encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol 1993;8:60313. Gerngross TU. Advances in the production of human therapeutic proteins yeasts and lamentous fungi. Nature Biotechnol 2004;22:140914. Geysens S, Whyteside G, Archer DB. Genomics of protein folding in the endoplasmic reticulum, secretion stress and glycosylation in the aspergilli. Fungal Genet Biol 2009;46:S12140. Gidijala L, Kiel JAKW, Douma RD, Seifar RM, van Gulik WM, Bovenberg RA, et al. Amn engineered yeast efciently secreting penicillin. PLoS One 2009;4(12):e8317. Gonzalez-Vogel A, Eyzaguirre J, Oleas G, Callegari E, Navarette M. Proteomic analysis in non-denaturing condition of the secretome reveals the presence of multienzyme complexes in Penicillium purpurogenum. Appl Microbiol Biotechnol 2011;89: 14555. Gordon CL, Archer DB, Jeenes DJ, Doonan JH, Wells B, Trinci APJ, et al. A glucoamylase:: GFP gene fusion to study protein secretion by individual hyphae of Aspergillus niger. J Microbiol Meth 2000;42:3948. Goto M. Protein O-glycosylation in fungi: diverse structures and multiple functions. Biosci Biotechnol Biochem 2007:141527. Gouka RJ, Punt PJ, van den Hondel CAMJJ. Efcient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 1997a;47:1-11. Gouka RJ, Punt PJ, van den Hondel CAMJJ. Glucoamylase gene fusions alleviate limitations for protein production in Aspergillus awamori at the transcriptional and (post) translational levels. Appl Environ Microbiol 1997b;63:48897. Grimm LH, Kelly S, Krull R, Hempel DC. Morphology and productivity of lamentous fungi. Appl Microbiol Biotechnol 2005;69:37584. Guais O, Tourrasse O, Dourdoigne M, Parrou JL, Francois JM. Characterization of the family GH54 -l-arabinofuranosidases in Penicillium funiculosum, including a novel protein bearing a cellulose-binding domain. Applied Microbiol Biotechnol 2010;87:100721. Guasi O, Borderies G, Pichereaux C, Maestracci M, Neugnot V, Rossignol M, et al. Proteomics analysis of Rovabio Excel, a secreted protein cocktail from the lamentous fungus Penicillium funiculosum grown under industrial process fermentation. J Indust Microbiol Biotechnol 2008;35:165968. Guo BH, Wang YC, Zhou XW, Hu K, Tan F, Miao ZQ, Tang KX. An endophytic taxolproducing fungus BT2 isolated from Taxus chinensis var. mairei. Afr J Biotechnol 2006;5:8757. Gutierrez S, Diez B, Alvarez E, Barredo JL, Martin JF. Expression of the penDE gene of Penicillium chrysogenum encoding isopenicillin N acyltransferase in Cephalosporium acremonium: production of benzylpenicillin by the transformants. Mol Gen Genet 1991;225:5664. Gwynne DI, Devchand M. Expression of foreign proteins in the genus Aspergillus. Biotechnology 1992;23:20314. Hammond TM, Keller NP. RNA silencing in Aspergillus nidulans is independent of RNAdependent RNA polymerases. Genetics 2005;169:60717. Haran S, Schickler H, Peer S, Logemann S, Oppenheim A, Chet I. Increased constitutive chitinase activity in transformed Trichoderma harzianum. Biological Control 1993;3:1018. Harkki A, Uusitalo J, Bailey M, Penttila M, Knowles JKC. A novel fungal expression system: secretion of actice calf chymosin from the lamentous fungus Trichoderma reesei. Nature Biotechnol 1989;7:596603. Harman GE, Howell CR, Viterbo A, Chet I, Lorito M. Trichoderma speciesopportunistic, avirulent plant symbionts. Nature Revs Microbiol 2004;2(1):4356. Harris DM, Westerlaken I, Schipper D, van der Krogt ZA, Gombert AK, Sutherland J, et al. Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor. Metab Eng 2009;11:12537. Hastrup S, Branner S, Jorgensen BR, Christensen T, Jorgensen BB, Shuster JR et al. Processes for producing an enzyme. United States Patent 5,702,934. 1997. Hawksworth DL. The magnitude of fungal diversity: the 1.5 million species estimate revisited. Mycol Res 2001;105:142232. Hong H, Datla N, Reed DW, Covello PS, MacKenzie SL, Qiu X. High-level production of -linolenic acid in Brassica juncea using a 6 desaturase from Pythium irregulare. Plant Physiol 2002:35462. Huang X-M, Yang Q, Liu Z-H, Fan J-X, Chen X-L, Song J-Z, et al. Cloning and heterologous expression of a novel endoglucanase gene egVIII from Trichoderma viride in Saccharomyces cerevisiae. Appl Biochem Biotechnol 2010;162:10315. Huang Y-S, Chaudhary S, Thurmond JM, Bobik Jr EG, Yuan L, Chan GM, et al. Cloning of 12- and 6-desaturases from Mortierella alpina and recombinant production of linolenic acid in Saccharomyces cerevisiae. Lipids 1999;34:64959. Ibrahim AS, Edwards JEJ, Filler SG. Zygomycosis. In: Dismukes WE, Pappas PG, Sobel JD, editors. Clinical mycology. New York: Oxford University Press; 2003. p. 24151. Ichishima E, Taya N, Ikeguichi M, Chiba Y, Nakamura M, Kawabata C, et al. Molecular and enzymatic properties of recombinant 1,2--mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells. Biochem J 1999;339:58997. Imanaka H, Tanaka S, Feng B, Imamura K, Nakanishi K. Cultivation characteristics and gene expression proles of Aspergillus oryzae by membrane-surface liquid culture, shaking-ask culture and agar-plate culture. J Biosci Bioeng 2010;109:26773. Imazaka A, Tanaka A, Harimoto Y, Yamamoto M, Akimitsu K, Park P, et al. Contribution of peroxisomes to secondary metabolism and pathogenicity in the fungal plant pathogen Alternaria alternata. Eukaryot Cell 2010;9:68294. Ishida H, Matsumura K, Hata Y, Kawato A, Suginami K, Abe Y, et al. Establishment of a hyper-protein production system in submerged Aspergillus oryzae culture under tyrosinase-encoding gene (melO) promoter control. Appl Microbiol Biotechnol 2001;57:1317.
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx Ma LJ, Ibrahim AS, Skory C, Grabherr MG, Burger G, Butler M, et al. Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication author(s): source. PLoS Genet 2009;5(7). Article No: e1000549. Ma SM, et al. Enzymatic synthesis of aromatic polyketides using PKS4 from Gibberella fujikuroi. J Am Chem Soc 2007;129:106423. Mach RL, Zeilinger S. Regulation of gene expression in industrial fungi: Trichoderma. Appl Microbiol Biotechnol 2003;60:51522. Machida M. Progress of Aspergillus oryzae genomics. Adv Appl Microbiol 2002;51: 81-106. Machida M, Asai K, Sano M, Tanaka T, Kumagai T, Terai G, et al. Genome sequencing and analysis of Aspergillus oryzae. Nature 2005;438:115761. MacKenzie DA, Wongwathanarat P, Carter AT, Archer DB. Isolation and use of a homologous histone H4 promoter and a ribosomal DNA region in a transformation vector for the oil-producing fungus Mortierella alpina. Appl Environ Microbiol 2000;66: 465561. MacKenzie DA, Kraunsoe JAE, Chesshyre JA, Lowe G, Komiyama T, Fuller RS, et al. Aberrant processing of wild-type and mutant bovine pancreatic trypsin inhibitor secreted by Aspergillus niger. J Biotechnol 1998;63:13746. Maeda H, Sano M, Maruyama Y, Tanno T, Akao T, Totsuka Y, et al. Transcriptional analysis of genes for energy catabolism and hydrolytic enzymes in the lamentous fungus Aspergillus oryzae using cDNA microarrays and expressed sequence tags. Appl Microbiol Biotechnol 2004;65:7483. Mantyla A, Paloheimo M, Hakola S, Lindberg E, Leskinen S, Kallio J, et al. Production in Trichoderma reesei of three xylanases from Chaetomium thermophilum: a recombinant thermoxylanase for biobleaching of kraft pulp. Appl Microbiol Biotechnol 2007;76:37786. Maor R, Shirasu K. The arms race continues: battle strategies between plants and fungal pathogens. Curr Opin Microbiol 2005;8:399404. Marahiel MA. Multidomain enzymes involved in peptide synthesis. FEBS Lett 1992;307:403. Maras M, De Bruyn A, Vervecken W, Uusitalo J, Penttila M, Busson R, et al. In vivo synthesis of complex N-glycans by expression of human N-acetylglucosamyltransferase 1 in the lamentous fungus Trichoderma reesei. FEBS Lett 1999a;452:36570. Maras M, Saelens X, Laroy W, Piens K, Claeyssens M, Fiers W, et al. In vitro conversion of the carbohydrate moiety of fungal glycoproteins to mammalian-type oligosaccharidesevidence for N-acetylglucosaminetransferase-1-accepting glycans from Trichoderma reesei. Eur J Biochem 1997;249:7017. Maras M, van Die I, Contreras R, van den Hondel CAMJJ. Filamentous fungi as production organisms for glycoproteins of bio-medical interest. Glycoconj J 1999b;16:99-107. Martin JA, Murphy RA, Power RFG. Cloning and expression of fungal phytases in genetically modied strains of Aspergillus awamori. J Ind Microbiol Biotechnol 2003;30: 56876. Martin JF. Clusters of genes for the biosynthesis of antibioticsregulatory genes and overproduction of pharmaceuticals. J Ind Microbiol Biotechnol 1992;9:7390. Martn JF, Liras P. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu Rev Microbiol 1989;43: 173206. Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, et al. Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nature Biotechnol 2008;26:55360. Martinez D, Larrondo LF, Putnam N, Gelpke MD, Huang K, Chapman J, et al. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature Biotechnol 2004;22:695700. Maruyama J, Ohnuma H, Yoshikawa A, Kadokura H, Nakajima H, Kitamoto K. Production and product quality assessment of human hepatitis B virus-preS2 antigen in submerged and solid state cultures of A. oryzae. J Biosci Bioeng 2000;90:11820. Masai K, Maruyama J, Nakajima H, Sakamoto K, Akita O, Kitamoto K. Analysis of differential gene expression across regions of the Aspergillus oryzae mycelium. Fungal Genet Newslett 2005;52:161. Matsuzaki F, Shimizu M, Wariishi H. Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysogenum exposed to exogenous benzoic acid. J Proteome Res 2008;7:234250. Meesapyodsuk D, Reed DW, Covello PS, Qiu X. Primary structure, regioselectivity, and evolution of the membrane-bound fatty acid desaturases of Claviceps purpurea. J Biol Chem 2007;282:201919. Meyer HW. Molds in oor dust and building-related symptoms in adolescent school children. Indoor Air 2004;14:6572. Meyer V. Genetic engineering of lamentous fungi-progress, obstacles and future trends. Biotechnol Adv 2008;26:17785. Michaelson LV, Lazarus CM, Grifths G, Napier JA. Isolation of a 5-fatty acid desaturase gene from Mortierella alpina. J Biol Chem 1998;273:190559. Minetoki T, Kumagai C, Gomi K, Kitamoto K, Takahashi K. Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efcient expression of Aspergillus oryzae amylase-encoding genes. Appl Microbiol Biotechnol 1998;50:45967. Monk BC, Goffeau A. Outwitting multidrug resistance to antifungals. Science 2008;321: 3679. Monsanto. Microbial sequence database. http://microbial.cereon.com2001. Moralejo FJ, Cardoza RE, Gutierrez S, Lombrana M, Fierro F, Martin JF. Silencing of the Aspergillopepsin B (pepB) gene of Aspergillus awamori by antisense RNA expression or protease removal by gene disruption results in a large increase in thaumatin production. Appl Environ Microbiol 2002;68:35509. Moriguchi T, Kezuka Y, Nonaka T, Ebizuka Y, Fujii I. Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release. J Biol Chem 2010;285: 1563743.
19
Muller D, Stahl U, Meyer V. Application of hammerhead ribozymes in lamentous fungi. J Microbiol Meth 2006;65:58595. Muller WH, Bovenberg RA, Groothuis MH, Kattevilder F, Smaal EB, van der Voort LH, et al. Involvement of microbodies in penicillin biosynthesis. Biochim Biophys Acta 1992;1116:2103. Muller WH, van der Krift TP, Krouwer AJJ, Wosten HAB, van der Voort L, Smaal EB, et al. localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum. EMBO 1991;10:48995. Nakajima K, Asakura T, Maruyama J, Morita Y, Oike H, Shimizu-Ibuka A, et al. Extracellular production of neoculin, a sweet-tasting heterodimeric protein with tastemodifying activity by Aspergillus oryzae. Appl Environ Microbiol 2006;72:371623. Nevalainen H, Souminen P, Taimisto K. On the safety of Trichoderma reesei. J Biotechnol 1994;37:193200. Nevalainen KMH, Te'o VS, Bergquist PL. Heterologous protein expression in lamentous fungi. Trends Biotechnol 2005;23:46874. Ngiam C, Jeenes DA, Punt PJ, van den Hondel CAMJJ, Archer DA. Characterization of a foldase, protein disulde isomerase A in the protein secretory pathway of Aspergillus niger. Appl Environ Microbiol 2000;66:77582. Nuesch J, Heim J, Treichler H-J. The biosynthesis of sulfur-containing -lactam antibiotics. Annu Rev Microbiol 1987;41:5175. Nyyssonen E, Penttila M, Harkki M, Saloheimo A, Knowles JK, Keranen S. Efcient production of antibody fragments by the lamentous fungus Trichoderma reesei. Biotechnology 1993;11:5915. O'Donnell D, Wang L, Xu J, Ridgway D, Gu T, Moo-Young M. Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity. Biochem Eng 2001;8:18793. Oda K, Kakizono D, Yamada O, Lefuji H, Akita O, Iwashita K. Proteomic analysis of extracellular proteins of Aspergillus oryzae grown under submerged and solid-state culture conditions. Appl Environ Microbiol 2006;72:344857. Ouyang J, Yan M, Kong D, Xu L. A complete protein pattern of cellulose and hemicellulase genes in the lamentous fungus Trichoderma reesei. Biotechnol J 2006;1: 126674. Papagianni M, Young MM. Protease secretion in glucoamylase producer Aspergillus niger cultures: fungal morphology and inoculum effects. Proc Biochem 2002;l37: 12718. Papagiannopoulos P, Andrianopoulos A, Sharp JA, Davis MA, Hynes MJ. The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters. Mol Gen Genet 1996;251:41221. Paper JM, Scott-Craig JS, Adhikari ND, Cuomo CA, Walton JD. Comparative proteomics of extracellular proteins in vitro and in planta from the pathogenic fungus Fusarium graminearum. Proteomics 2007;7:317183. Paul D, Pandey G, Jain RK. Suicidal genetically engineered microorganisms for bioremediation: need and perspectives. Bioessays 2005:56373. Pel HJ, de Winde JH, Archer DB, Dyer PS, Hofmann G, Schaap PJ, et al. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nature Biotechnol 2007;25:22131. Penttila M. Heterologous protein production in Trichoderma. In: Harman GE, Kubicek CP, editors. Trichoderma and Gliocladium. London: Taylor and Francis; 1998. p. 36582. Pisanelli I, Kujawa M, Gschnitzer D, Spaduit O, Seiboth B, Peterbauer C. Heterologous expression of an Agaricus meleagris pyranose dehydrogenase-encoding gene in Aspergillus spp. and characterization of the recombinant enzyme. Appl Microbiol Biotechnol 2010;86:599606. Poidevin L, Levasseur A, Paes G, Navarro D, Heiss-Blanquet S, Asther M, et al. Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications. Letts Appl Microbiol 2009;49:6738. Punt PJ, van Biezen N, Conesa A, Albers A, Mangnus J, van den Hondel C. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol 2002;20:2006. Punt PJ, van Gemeren IA, Drint-Kuijvenhoven A, Hessing JGM, van Muijlwijk-Harteveld GM, Beijersbergen A, et al. Analysis of the role of the gene bipA, encoding the major endoplasmic reticulum chaperone protein in the secretion of homologous and heterologous proteins in black Aspergilli. Appl Microbiol Biotechnol 1998;50:44754. Punt PJ, Schuren FHJ, Lehmbeck J, Christens J, Hjort C, van den Hondel C. Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes. Fungal Genet Biol 2008;45:15919. Qin Y, Ying SH, Chen Y, Shen ZC, Feng MG. Integration of insecticidal Vip3Aa1 into Beauveria bassiana enhances fungal virulence to Spodoptera litura larvae by cuticle and per os infection. Appl Environ Microbiol 2010;76:46118. Radzio R, Kueck U. Synthesis of biotechnologically relevant heterologous proteins in lamentous fungi. Proc Biochem 1997;32:52937. Ratledge C. Lipids in fatty acids. In: Rose AH, editor. Primary products of metabolism. London: Academic Press; 1978. p. 26396. Roberts IN, Jeenes DJ, Mackenzie DA, Wilkinson AP, Summer IG, Archer DB. Heterologous gene expression in A. niger: A glucoamylaseporcine pancreatic phospholipase A2 fusion protein is secreted and processed to yield mature enzyme. Gene 1992;122:15561. Robin J, Jakobsen M, Beyer M, Noorman H, Nielsen J. Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7aminodeacetoxycephalosporanic acid production. Appl Microbiol Biotechnol 2001;57:35762. Robin J, Lettier G, McIntyre M, Noorman H, Nielsen J. Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: growth yields and morphological characterization. Biotechnol Bioeng 2003a;83:3618.
20
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx Swift RJ, Wiebe MG, Robson GD, Trinci APJ. Recombinant glucoamylase production by Aspergillus niger B1 in chemostat and pH-autostat cultures. Fungal Genet Biol 1998;25:1009. Szewczyk E, Chiang YM, Oakley CE, Davidson AD, Wang CC, Oakley BR. Identication and characterization of the asperthecin gene cluster of Aspergillus nidulans. Appl Environ Microbiol 2008;74:760712. Tailor MJ, Richardson T. Application of microbial enzymes in food systems and in biotechnology. Adv Appl Microbiol 1979;25:7-35. Takuchi M, Ichishima E. A 155 K acid carboxypeptidase O from Aspergillus oryzae. Agric Biol Chem 1986;50:6338. te Biesebeke R, Ruijter G, Rahardjo YSP, Hoogschagen MJ, Heerikhuisen M, Levin A, et al. Aspergillus oryzae in solid-state and submerged fermentations. FEMS Yeast Res 2002;2:2458. Tian C, Beeson WT, Iaverone AT, Sun J, Marletta MA, Cate JH, Glass NL. Systems anaslysis of plant cell wall degradation by the model lamentous fungus Neurospora crassa. Proc Natl Acad Sci USA 2009;106:2215762. Toida J, Fukuzawa M, Kobayashi G, Ito K, Sekiguchi J. Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli. FEMS Microbiol Lett 2000;189:15964. Torres NV, Voit EO, Gonzalez-Alcon C. Optimization of nonlinear biotechnological processes with linear programming: application to citric acid production by Aspergillus niger. Biotechnol Bioeng 1996;49:24758. Tronsmo A, Klemsdal SS, Hayes CK, Lorito M, Harman GE. The role of hydrolytic enzymes produced by Trichoderma harzianum in biological control of plant diseases. In: Suominen P, Reinikainen T, editors. Proc 2nd Tricel Symposium on Trichoderma Cellulases and other Hydrolases, 8. Found Biotechnol Indust Fermenation Res; 1993. p. 15968. Tsang A, Butler G, Powlowski, Panisko EA, Baker SE. Analytical and computational approaches to dene the Aspergillus niger secretome. Fungal Genet Biol 2009;46 (Suppl 1):S15360. Tsuchiya K, Nagasjhiam T, Yamamoto Y, Gomi K, Kitamoto K, Umagai C. High level secretion of calf chymosin using a glucoamylase prochymosin fusion gene in Aspergillus oryzae. Biosci Biotechnol Biochem 1994;58:8959. Uusitalo JM, Nevalainen KM, Harkki AM, Knowles JK, Penttila ME. Enzyme production by recombinant Trichoderma reesei strains. J Biotechnol 1991;17:3549. Vaheri MP, Vaheri MEO, Kauppinen VS. Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol. Eur J Appl Microbiol Biotechnol 1979;8:7380. van de Sandt EJAX, de Vroom E. Innovations in cephalosporin and penicillin production: painting the antibiotics industry green. Chim Oggi-Chem Today 2000;18: 725. van den Berg M, Albang R, Albermann K, Badger JH, Daran J-M, Driessen AJM, et al. Genome sequencing and analysis of the lamentous fungus Penicillium chrysogenum. Nature Biotechnol 2008;26:11618. van den Brink HJ, Petersen SG, Rahbek-Nielsen H, Hellmuth K, Harboe M. Increased production of chymosin by glycosylation. J Biotechnol 2006;125:30410. van den Hombergh JPTW, van de Vondervoort PJI, Fraissinet L, Visser J. Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol 1997;15:25663. Van Dijck PWM, Selten GCM, Hempenius RA. On the safety of a new generation of DSM Aspergillus niger enzyme production strains. Regulat Toxicol Pharmacol 2003;38: 2735. van Gemeren IA, Punt PJ, Drint-Kuyvenhoven A, Broekhuijsen MP, Hoog AV, Beijersbergen A, et al. The ER chaperone encoding bipA gene of black Aspergilli is induced by heat shock and unfolded proteins. Gene 1997;198:4352. Venegas-Caleron M, Sayanova O, Napier JA. An alternative to sh oils: metabolic engineering of oil-seed crops to produce omega-3 long chain polyunsaturated fatty acids. Prog Lipid Res 2010;49:10819. Verdoes JC, Punt PJ, Burlingame R, Bartels J, Van Dijk R, Slump E, et al. A dedicated vector for efcient library construction and high throughput screening in the hyphal fungus Chrysosporium lucknowense. Ind Biotechnol 2007;3:4857. von Dohren H. A survey of nonribosomal peptide synthetase (NRPS) genes in Aspergillus nidulans. Fungal Genet Biol 2009;46(Suppl 1):S4552. Wan X, Zhang YB, Wang P, Jiang M. Molecular cloning and expression analysis of a delta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can accumulate high levels of gamma-linolenic acid. J Microbiol 2011;49:1514. Wang JW, Zheng LP, Tan RX. Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in Taxus yunnanensis suspension cells. Appl Microbiol Biotechnol 2007a;75:118390. Wang L, Darin R, Tingyue G, Moo-Young M. Effects of process parameters on heterologous protein production in Aspergillus niger fermentation. J Chem Technol Biotechnol 2003;78:125966. Wang L, Ridgway D, Gu T, Moo-Young M. Bioprocessing strategies to improve heterologous protein production in lamentous fungal fermentations. Biotechnol Advances 2005;23:11529. Wang SW, Ma X, Ping WX, Zhou DP. Research advances on taxol production by microbe fermentation. Microbiology 2007b;34:5615. Wang YC, Guo BH, Miao ZQ, Tang KX. Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI). FEMS Microbiol Lett 2007c;273:2539. Wang Z-Y, Soanes DM, Kershaw MJ, Talbot NJ. Functional analysis of lipid metabolism in Magnaporthe grisea reveals a requirement for peroxisomal fatty acid -oxidation during appressorium-mediated plant infection. Mol Plant Microbe Interact 2007d;20:47591. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 1971;93:23257.
Robin J, Bonneau S, Schipper D, Noorman H, Nielsen J. Inuence of the adipate and dissolved oxygen concentrations on the beta-lactam production during continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus. Metab Eng 2003b;5:428. Rodgers CJ, Blanford CF, Giddens SR, Skamnioto P, Armstrong FA, Gurr SJ. Designer laccases: a vogue for high-potential fungal enzymes. Trends Biotechnol 2010;28: 6372. Romaine PC, Chen X. Methods and compositions for highly efcient transformation of lamentous fungi. United States Patent Application 20020070007. 2002. Ruijter GJG, Kubicek CP, Visser J. Production of organic acids by fungi. Mycota 2002;10: 21320. Ruijter GJG, Panneman H, Visser J. Overexpression of phosphofructokinase and pyruvate kinase in citric acid producing Aspergillus niger. Biochim Biophys Acta 1997;1334:31726. Sakaki T, Munetsuna E. Enzyme systems for biodegradation of polychlorinated dibenzo-p-dioxins. Appl Microbiol Biotechnol 2010;88:2330. Sakuradani E, Kobayashi M, Shimizu S. 6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungusgene cloning and its heterologous expression in a fungus, Aspergillus. Gene 1999;238:44553. Sanchez JF, Chiang YM, Szewczyk E, Davidson AD, Ahuja M, Oakley CE, et al. Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans. Mol Biosyst 2009;6:58793. Saruwatari T, Praseuth AP, Sato M, Torikai K, Noguchi H, Watanabe K. A comprehensive overview on genomically directed assembly of aromatic polyketides and macrolide lactones using fungal megasynthases. J Antibiotics 2011;64:9-17. Schirmbck M, Lorito M, Wang YL, Hayes CK, Arisan-Atac I, Scala F, et al. Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi. Appl Environ Microbiol 1994;60:436470. Schmoll M, Kubicek CP. Regulation of Trichoderma cellulose formation: lessons in molecular biology from an industrial fungus. A review. Acta Microbiol Immunol Hung 2003;50:12545. Schmoll M, Seibel C, Kotlowski C, Vendt FWG, Liebmann B, Kubicek C. Recombinant production of an Aspergillus bidulans class 1 hydrophobin (DewA) in Hypocrea jecorina (Trichoderma reesei) is promoter dependent. Appl Microbiol Biotechnol 2010;88:95-103. Schoen C, Reichard U, Monod M, Kratzin HD, Ruchel R. Molecular cloning of an extracellular aspartic proteinase from Rhizopus microsporus and evidence for its expression during infection. Med Mycol 2002;40:6171. Schuster A, Schmoll M. Biology and biotechnology of Trichoderma. Appl Microbiol Biotechnol 2010;87:78799. Schuster E, Dunn-Coleman N, Frisvad JC, van Dijck PWM. On the safety of Aspergillus nigera review. Appl Microbiol Biotechnol 2002;59:42635. Segal BH, Walsh TJ. Current approaches to diagnosis and treatment of invasive aspergillosis. Am J Respir Crit Care Med 2006;173:70717. Seiboth B, Gamauf C, Pail M, Hartl L, Kubicek CP. The D-xylose reductase of Hypocrea jecorina is the major aldose reductase in pentose and D-galactose catabolism and necessary for beta-galactosidase and cellulase induction by lactose. Mol Microbiol 2007;66:890900. Sharma R, Katoch M, Srivastava P, Qazi G. Approaches for rening heterologous protein production in lamentous fungi. World J Microbiol Biotechnol 2009;25:208394. Shen B. Polyketide biosynthesis beyond the type I, II and III polyketide synthase paradigms. Curr Opin Chem Biol 2003;7:28595. Shimizu S, Ogawa J, Kataoka M, Kobayashi M. Screening of novel microbial enzymes for the production of biologically and chemically useful compounds. Adv Biochem Eng Biotech 1997;58:4587. Siewers V, Chen X, Huang L, Zhang J, Nielsen J. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae. Metab Eng 2009;11:3917. Sims AH, Robson GD, Hoyle DC, Oliver SG, Turner G, Prade RA, et al. Use of expressed sequence tag analysis and cDNA microarrays of the lamentous fungus Aspergillus nidulans. Fungal Genet Biol 2004;41:199212. Smith DJ, Earl AJ, Turner G. The multifunctional peptide synthetase performing the rst step of penicillin biosynthesis in Penicillium chrysogenum is a 421 073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases. EMBO J 1990;9:274350. Spencer JA, Jeenes DJ, MacKenzie DA, Hyanie DT, Archer DB. Determinants of the delity of processing glucoamylase-lysozyme fusions by Aspergillus niger. Eur J Biochem 1998;258:10712. Spreer A, Ruchel R, Reichard U. Characterization of an extracellular subtilisin protease of Rhizopus microsporus and evidence for its expression during invasive rhinoorbital mycosis. Med Mycol 2006;44:72331. Sriram S, Ray RC. Trichoderma: systematics, molecular taxonomy and agricultural and industrial applications. In: Ray RC, editor. Microbial Biotechnology in Agriculture and Aquaculture, Vol 1. Eneld: Science Publishers; 2005. p. 33576. St Leger RJ, Joshi L, Bidochka MJ, Roberts DW. Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc Natl Acad Sci USA 1996;93:634954. Stachelhaus T, Marahiel MA. Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis. FEMS Microbiol Lett 1995;125:3-14. Stachelhaus T, Schneider A, Marahiel MA. Engineered biosynthesis of peptide antibiotics. Biochem Pharmacol 1996;52:17786. Sternberg D, Mandels GR. Induction of cellulolytic enzymes in Trichoderma reesei by sophorose. J Bacteriol 1979;139:7619. Stewart P, Whitwam RE, Kersten PJ, Cullen D, Tien M. Efcient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae. Appl Microbiol Biotechnol 1996;62:8604.
O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx Ward M, Wilson LJ, Kodama KH, Rey MW, Berka RM. Improved production of chymosin in Aspergillus by expression as a glucoamylase-chymosin fusion. Bio-Technology 1990;8:43540. Ward OP, Qin WM, Dhanjoon J, Ye J, Singh A. Physiology and biotechnology of Aspergillus. Adv Appl Microbiol 2006:1-75. Ward OP, Rao MB, Kulkarni A. Proteases. In: Schaechter M, editor. Encyclopedia of microbiology Vol 1. Oxford: Elsevier; 2009. p. 495511. Ward OP, Singh A. Omega-3/6 fatty acids: alternative sources of production. Process Biochem 2005;40:362752. Ward OP. Bioprocessing. Milton Keynes: Open University Press; 1991. Ward OP. Fermentation biotechnology: principles, processes and products. Milton Keynes: Open University Press; 1989. Ward OP. Proteases. Comprehensive Biotechnology 2nd ed. Moo-Young M, editor. Vol. 3. 2011. pp 571582. Ward PP, May GS, Headon DR, Conneely OM. An inducible expression system for the production of human lactoferrin in A. nidulans. Gene 1992;122:21923. Ward PP, Peddington CS, Cunnighham GA, Zhou X, Wyatt RD, Conneely OM. A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Biotechnology 1995;13:498503. Watanabe CM, Wilson D, Linz JE, Townsend CA. Demonstration of the catalytic roles and evidence for the physical association of type I fatty acid synthases and a polyketide synthase in the biosynthesis of aatoxin B1. Chem Biol 1996;3:4639. Weber G, Schrgendorfer K, Schneider-Scherzer E, Leitner E. The peptide synthetase catalyzing cyclosporin production in Tolypocladium niveum is encoded by a giant 45.8-kilobase open reading frame. Curr Genet 1994;26:1205. Wong P, Walter M, Lee W, Mannhaupt G, Munsterkotter M, Mewes H-W, et al. FGDB: revisiting the genome annotation of the plant pathogen Fusarium graminearum. Nucleic Acid Res 2011;39:D6379. Xu J, Wang L, Ridgway D, Gu T, Moo-Young M. Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth. Biotechnol Prog 2000;16:2227.
21
Yamada O, Ikeda R, Ohkita Y, Hayashi R, Sakamoto K, Akita O. Gene silencing by RNA interference in the koji mold Aspergillus oryzae. Biosci Biotechnol Biochem 2007;71:13844. Yoon J, Aishan T, Maruyama J, Kitamoto K. Enhanced production and secretion of heterologous proteins by the lamentous fungus Aspergillus oryzae via disruption of the vacuolar protein sorting receptor gene Aovps10. Appl Environ Microbiol 2010;76:571827. Yoon J, Maruyama J, Kitamoto K. Disruption of ten protease genes in the lamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 2011;89:74759. Yoshida T, Kato Y, Asada Y, Nakajima T. Filamentous fungus Aspergillus oryzae has two types of alpha-1,2-mannosidase, one of which is a microsomal enzyme that removes a single mannose residue from Man9GlcNAc2. Glycoconj J 2000;17:7458. Zarrin M, Leeder AC, Turner G. A rapid method for promoter exchange in Aspergillus nidulans using recombinant PCR. Fungal Genet Biol 2005;42:18. Zhang S, Sakuradani E, Ito K, Shimizu S. Identication of a novel bifunctional 12/15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#8222. FEBS Lett 2007;581:3159. Zhang W, Li Y, Tang Y. Engineered biosynthesis of bacterial aromatic polyketides in Escherichia coli. Proc Natl Acad Sci USA 2008;105:206838. Zheng XF, Kobayashi Y, Takeuchi A. Construction of a low-serinetypecarboxypeptidase-producing mutant of Aspergillus oryzae by the expression of antisense RNA and its use as a host for heterologous protein secretion. Appl Microbiol Biotechnol 1998;49:3944. Zhou X, Zhu H, Liu L, Lin J, Tang K. A review: recent advances and future prospects of taxol-producing endophytic fungi. Appl Microbiol Biotechnol 2010;86:170717. Zhou XW, Wei YM, Zhu HF, Wang ZN, Lin J, Liu L, Tang KX. Protoplast formation, regeneration and transformation from the taxol-producing fungus Ozonium sp. Afr J Biotechnol 2008;7:201724.

(2011) Production of Ant Proteins by Filamentous Fungi

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Contents lists available at SciVerse ScienceDirect

Production of recombinant proteins by lamentous fungi

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

Non-ribosomally synthesized antibiotics and producing hosts

Taxol-producing strains Common transformation methods for lamentous fungi

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

O.P. Ward / Biotechnology Advances xxx (2011) xxxxxx

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