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Biotechnology Advances 26 (2008) 177 – 185 www.elsevier.com/locate/biotechadv

Research review paper

Genetic engineering of filamentous fungi — Progress, obstacles and future trends Vera Meyer ⁎ TU Berlin, Institut für Biotechnologie, Fachgebiet Mikrobiologie und Genetik, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany Received 28 August 2007; received in revised form 3 December 2007; accepted 4 December 2007 Available online 14 December 2007

Abstract Filamentous fungi are widely used in biotechnology as cell factories for the production of chemicals, pharmaceuticals and enzymes. In order to improve their productivities, genetic engineering strategies can be powerful approaches. Different transformation techniques as well as DNA- and RNA-based methods to rationally design metabolic fluxes have been developed for industrially important filamentous fungi. However, the lack of efficient genetic engineering approaches still forms an obstacle for a multitude of fungi producing new and commercially interesting metabolites. This review summarises the variety of options that have recently become available to introduce and control gene expression in filamentous fungi and discusses their advantages and disadvantages. Furthermore, important considerations that have to be taken into account to design the best engineering strategy will be discussed. © 2007 Elsevier Inc. All rights reserved. Keywords: Filamentous fungi; Genetic and metabolic engineering; Transformation; Gene targeting; ku70; Antisense; RNAi; Hammerhead ribozyme

Contents 1. Introduction . . . . . . . . . . . . . . . 2. Approaches to genetic transformation . . 3. Re-engineering gene targeting . . . . . . 4. RNA technologies for genetic engineering 5. Designing an engineering strategy . . . . 6. Conclusions and prospects . . . . . . . . References . . . . . . . . . . . . . . . . . . .

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1. Introduction The ability of filamentous fungi to grow on rather simple and inexpensive substrates as well as their capacity to produce a wide range of commercially interesting metabolites have attracted considerable interest to exploit them as production organisms in biotechnology. Nowadays, filamentous fungi are used in biotechnology as cell factories for a wide range of ⁎ Tel.: +49 30 314 72827; fax: +49 30 314 72922. E-mail address: [email protected]. 0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2007.12.001

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products. Diverse compounds ranging from simple organic acids to complex secondary metabolites are produced for the use in various market segments (Table 1). Due to their exceptional high capacity to express and secrete proteins, filamentous fungi have become indispensable for the production of enzymes of fungal and non-fungal origin. Currently, native or recombinant enzymes are mainly produced by Aspergillus niger, A. oryzae and Trichoderma reesei and also other strains are currently under development (Punt et al., 2002). In addition, filamentous fungi naturally produce an astonishing wealth of secondary metabolites and a few of them are

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V. Meyer / Biotechnology Advances 26 (2008) 177–185

Table 1 Selected examples of industrially important compounds produced by filamentous fungi Compound Acids Citric acid Itaconic acid Kojic acid Enzymes α-Amylase Chymosin Cellulase Cellobiohydrolase Glucoamylase

Organism

Main application areas

Aspergillus niger A. terreus A. oryzae

Food and beverage industry Polymer industry Food industry

A. niger, A. oryzae

Starch processing and food industry Food industry Textile, pulp and paper industry Textile, pulp and paper industry Starch processing industry Textile industry, Biosensor Textile, pulp and paper industry Food and detergent industry Food industry Food and detergent industry Food industry Food industry Textile, pulp, paper and bakery industry

A. niger Trichoderma viride, T. reesei T. viride, T. reesei

Glucose oxidase Laccase

A. phoenicis, Rhizopus delemar A. niger, A. oryzae Trametes versicolor

Lipases

A. niger, A. oryzae

Pectin lyase Proteases

T. reesei A. niger, A. oryzae, R. delemar A. niger, A. oryzae Mucor miehei T. reesei, T. konignii, A. niger

Phytase Rennin Xylanases Exopolysaccharides Scleroglucan Pullulan Schizophyllan PSK, PSP Secondary metabolites Cephalosporin Cyclosporin Ergot alkaloids Griseofulvin Lovastatin Penicillin Taxol Zeranol Others PUFA

Sclerotium rolfsii Aureobasidium pullulans Schizophyllum commune Tr. versicolor

Acremonium chrysogenum Tolypocladium nivenum Claviceps purpurea P. griseofulvum Monascus rubber, A. terreus P. chrysogenum Taxomyces andrenae Fusarium graminearum Mucor circinelloides

Panthothenic acid

F. oxysporum

Hydrophobin

T. reesei

Biomass

Agaricus bisporus F. venentatum (Quorn™)

Oil production, cosmetics industry Food and pharmaceutical industry Pharmaceutical industry Pharmaceutical industry

Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Pharmaceutical industry Livestock farming

Food, feed and pharmaceutical industry Food, feed and pharmaceutical industry Tissue engineering, nanotechnology Food industry Food industry

After (Bennett, 1998; Archer, 2000; Ooi and Liu, 2000; Willke and Vorlop, 2001; Adrio and Demain, 2003; Leathers, 2003; Linder et al., 2005; Polizeli et al., 2005; Olempska-Beer et al., 2006). PUFA: Polyunsaturated fatty acids.

biotechnologically produced and became clinically significant drugs. The β-lactam group of antibiotics, including penicillin and cephalosporin, was the first group that benefited from the progress made in molecular techniques for filamentous fungi. The increasing knowledge of the biosynthesis and molecular genetics of β-lactam antibiotics led to new possibilities to rationally improve production strains and to engineer new biosynthesis pathways (Brakhage and Caruso, 2004). Further bioactive compounds produced by filamentous fungi and important for human welfare are, for example, cyclosporine A (immunosuppressive agent), lovastatin (cholesterol-lowering agent), taxol (anticancer agent) and griseofulvin (antifungal agent). Other fungal metabolites already produced commercially or potentially valuable in biotechnology involve exopolysaccharides such as pullulan suitable for coating foods or for improved delivery of therapeutic agents (Leathers, 2003) and hydrophobins useful as surface modifiers for (nano)technical and medical applications due to their ability to self-assemble at hydrophilic–hydrophobic interfaces into amphipathic films (Scholtmeijer et al., 2001; Hektor and Scholtmeijer, 2005). Although this is not a complete listing of industrially produced fungal-based compounds, it still reflects the metabolic versatility of filamentous fungi and their importance as cell factories in biotechnology. One important cornerstone for the future of fungal biotechnology will be the improvement of production strains at the molecular level. Genetic and metabolic engineering approaches to both natural and recombinant metabolite producing strains will be powerful tools for improving production levels, producing novel tailored compounds or directing the synthesis of desired products. However, this will only become feasible with the development of efficient methods to introduce and control gene expression in filamentous fungi. The objective of this review is to summarise and discuss the currently available options for genetic manipulation of filamentous fungi. Furthermore, the question of choosing the most appropriate strategy to genetically engineer a particular process will be addressed. For a specialised review that focuses on functional genomics with filamentous fungi, the readers are directed to (Weld et al., 2006). 2. Approaches to genetic transformation Genetic engineering can be a powerful approach for filamentous fungi in order to increase productivity and to minimise unwanted by-product formation. However, before engineering can become routine, introducing the desired genetic manipulation of the fungus of interest often represents a challenge. Foremost, the establishment of a suitable transformation method is not trivial for many fungi. Moreover, the mode and frequency of individual integration events resulting from homologous or illegitimate recombination is not only dependent on the transformation host itself but also on the applied transformation technique. Thus, designing an engineering strategy first requires consideration of the most suitable transformation method. Since the first report on successful protoplast-mediated transformation (PMT) of the yeast Saccharomyces cerevisiae (Hinnen et al., 1978), the use of protoplasts for transformation has been extended to several filamentous fungi (reviewed by

V. Meyer / Biotechnology Advances 26 (2008) 177–185

(Fincham, 1989; Ruiz-Diez, 2002)). However, the frequency of transformation is extremely low when compared to yields obtained with S. cerevisiae. In order to improve transformation of filamentous fungi, progress has been made over the last years that has resulted in the establishment of alternative methods for fungal transformation such as electroporation, biolistic transformation (reviewed by (Ruiz-Diez, 2002)) and Agrobacteriummediated transformation (AMT; reviewed by (Michielse et al., 2005b)). These methods have especially been shown to be valuable for fungal strains that do not form sufficient numbers of protoplasts or whose protoplasts do not regenerate sufficiently ((MacKenzie et al., 2004) and references therein). Table 2 summarises main features as well as advantages and disadvantages of these transformation techniques. Common to all four techniques is the necessity to optimise every method for the fungal strain of interest and often, only one or two of these methods can be applied to a particular species (Meyer et al., 2003). Importantly, AMT has been described to be an efficient transformation method for some fungi that were recalcitrant to the other methods described above (Michielse et al., 2005b). However, AMT has also been reported to be less successful or even fails to produce transformants, e.g. in A. niger (Michielse et al., 2005b). Hence, no general rule can be applied to predict the usefulness of a particular transformation technique for the fungus of interest. Instead, individual species have to be considered independently and the most appropriate method identified and optimised for each strain. Interestingly, mainly single-copy integration events were detected when AMT was used for the introduction of DNA into filamentous fungi such as A. awamori, A. giganteus, Calonectria morganii, F. oxysporum and Suillus bovines. In contrast, when these fungi were transformed by PMT, preferentially multicopy integration events were observed (de Groot et al., 1998; Malonek and Meinhardt, 2001; Mullins and Kang, 2001; Meyer et al., 2003). The impact of the transformation technique on the fate of the transforming DNA can thus have an important influence on the design of a metabolic engineering strategy for a

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given process. For example, when targeted integration or gene deletion is envisaged, AMT would be the method of choice. Contrary to this, PMT could be the optimal method when multiple copies of a gene of interest should integrate at random sites in the genome. It has been reported that such strategies (multiple integration of a gene encoding the desired protein) improved protein production in Aspergillus and Trichoderma (Verdoes et al., 1995; Lee et al., 1998; Askolin et al., 2001). Remarkably, AMT has also been shown to improve homologous recombination (HR) frequency in A. awamori (Michielse et al., 2005a). As discussed by Michielse et al. (Michielse et al., 2005b), one possible explanation for the increase in HR frequency obtained by AMT is the fact that A. tumefaciens delivers its DNA to the host as single-stranded DNA, whereas double-stranded DNA is transferred in the case of the other transformation methods. In S. cerevisiae, it has been shown that single-stranded DNA transforms cells at greater HR efficiency than that of double-stranded DNA, suggesting that the recombination machinery has a preferential affinity for single-stranded DNA (Simon and Moore, 1987). 3. Re-engineering gene targeting Targeted integration of genes to a genomic locus known to strengthen transcription is a strategy to improve expression of a protein of interest and thereby to maximise the capacity of a particular metabolic process. In addition, deletion of genes is often envisaged as a strategy for metabolic engineering in order to reduce metabolic fluxes of side pathways and to redirect fluxes to the product-forming pathway. However, gene targeting in filamentous fungi is often hampered by very low frequencies of HR and it is therefore not trivial to target a gene of interest to a particular genomic locus or to delete an endogenous gene. While in S. cerevisiae a minimal homologous fragment length of 30– 50 bp is sufficient to ensure a high yield of HR (Hua et al., 1997), several hundred basepairs up to several kilobases are necessary to achieve HR in filamentous fungi (Chevalet et al., 1992; Wu and

Table 2 Four common strategies for transformation of filamentous fungi Method Principle

Advantage

Disadvantage

PMT

Different cell types (spores, germlings, hyphal tissue) can be used

Particular batch of lytic enzyme alters TR Requires regeneration procedure Copy number of DNA insertions is often high Various parameters during co-cultivation affect TR More time-consuming than the other methods

Preparation of protoplasts using cell wall-degrading enzymes Uptake of DNA is achieved by the addition of PEG and CaCl2

AMT

EP

BT

A. tumefaciens carries two vectors (the binary vector containing the DNA of interest between the 24 bp border repeat and the T-vector containing the virulence region important for DNA transfer) DNA transfer is achieved during co-cultivation of A. tumefaciens with the fungus Reversible membrane permeabilisation induced by local application of electric pulses mediates DNA uptake Particles (tungsten, gold) are coated with DNA and become accelerated at high velocity into cells

Different cell types (spores, germlings, hyphal tissue) can be used Copy number of DNA insertions is low Improves targeted integration

Different cell types (spores, germlings) can be Often requires protoplast formation used to render cells competent Simple and cheap method Recipient cells can retain their cell walls (no Requires special equipment pre-treatment necessary)

After (Fincham, 1989; Ruiz-Diez, 2002; Michielse et al., 2005b). PMT: Protoplast-mediated transformation; AMT: Agrobacterium-mediated transformation; EP: Electroporation; BT: Biolistic transformation; TR: transformation rate.

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Fig. 1. HR-dependent frequency in A. niger wild type (closed diamonds) and ΔkusA (closed squares) recipient strains (Meyer et al., 2007).

Linz, 1993; Shiotani and Tsuge, 1995; Weidner et al., 1998; Young et al., 1998; Fernandez-Martin et al., 2000; Yu et al., 2004; Meyer et al., 2007). Still, the frequency of HR in filamentous fungi is very low (usually between 0 and 30% (Asch and Kinsey, 1990; Bird and Bradshaw, 1997; Meyer et al., 2002; Meyer et al., 2007)), is significantly species-dependent (Kwon-Chung et al., 1998) and its occurrence is strongly dependent on the transcriptional status of the targeted genomic locus (Bird and Bradshaw, 1997). Hence, a multitude of transformants have usually to be analysed in order to identify the correct one, a process which makes fungal transformation tedious and laborious. One approach to overcome this limitation is the use of strains which are defective for the nonhomologous end joining pathway (NHEJ). In eukaryotes, integration of a DNA fragment into the genome requires the action of a double-strand break repair mechanism. The two major pathways, HR and NHEJ, are conserved through evolution and have been described to mediate double-strand break repair (Dudasova et al., 2004; Krogh and Symington, 2004). HR involves interaction between homologous sequences and thus leads to targeted integration. In contrast, NHEJ mediates ligation of DNA strands sharing no homology and hence results in random integration. The HR pathway depends on the Rad52 epistasis group, whereas the NHEJ pathway depends on the Ku heterodimer (Ku70/Ku80protein complex) and the DNA ligase IV-Xrcc4 complex (Dudasova et al., 2004; Krogh and Symington, 2004). According to the ‘gatekeeper’ model (Haber, 1999), both pathways compete with each other. When Rad52p binds to the ends of the introduced DNA, the DNA ends will be processed by HR, however, when Ku binds, the DNA becomes integrated via the NHEJ pathway. In filamentous fungi, like in other multicellular organisms and in contrast to S. cerevisiae, the NHEJ-pathway seems to be dominant over the HR-pathway. Most recent studies in non-Saccharomyces yeasts and filamentous fungi revealed that by deleting components of the NHEJ-pathway, the random integration of DNA fragments can be strongly reduced. For example, deletion of the Ku70 homologue in A. niger (KusA) dramatically improved homologous integration efficiency and reached more than 80% compared to 7% in the wild-type background, when only 500 bp homologous flanks were used (Fig. 1). Similarly, mutants deficient for Ku70, Ku80 or Lig4 homologues gave rise to high HR frequencies in other fungal

systems (Table 3) such as A. nidulans (Nayak et al., 2006), A. oryzae (Takahashi et al., 2006), A. fumigatus (da Silva Ferreira et al., 2006; Krappmann et al., 2006), A. sojae (Takahashi et al., 2006), Claviceps purpurea (Haarmann et al., 2008), Cryptococcus neoformans (Goins et al., 2006), Kluyveromyces lactis (Kooistra et al., 2004), Magnaporthe grisea (Villalba et al., 2008), Neurospora crassa (Ninomiya et al., 2004; Ishibashi et al., 2006) and Sordaria macrospora (Pöggeler and Kück, 2006). Hence, mutants defective for NHEJ offer the opportunity to make targeted genetic alterations in filamentous fungi more straightforward and less time-consuming and will serve as a powerful tool for genetic engineering. In this context, however, it is important to stress that the Ku proteins are important to maintain telomere length in yeast and plants and are necessary to ensure chromosome stability in mammals (Boulton and Jackson, 1996; Bailey et al., 1999; Bundock et al., 2002; Riha et al., 2002). Congruently, phenotypic analysis of fungal strains defective in NHEJ demonstrated that these strains showed higher susceptibility to various toxins and irradiation (Müller et al., 2001; Ninomiya et al., 2004; da Silva Ferreira et al., 2006; Meyer et al., 2007). To bypass this problem, fungal strains in which proteins of the NHEJ machinery are transiently silenced could be an attractive option (Nielsen et al., in press; Ueno et al., 2007). 4. RNA technologies for genetic engineering The possibility to encourage or to silence a given metabolic pathway is not only restricted to DNA-based approaches. An alternative strategy circumventing the need for gene targeting and in particular gene deletion would be the use of RNA-based methods that silence gene expression post-transcriptionally. These tools are especially valuable when (i) gene targeting approaches fail, (ii) multiple copies of a gene of interest are present in the genome and/or (iii) isogenes might compensate for the knockout of the deleted gene (Akashi et al., 2005). Three RNA-based methods – antisense RNA, hammerhead ribozymes and RNA interference – have been shown to be valuable tools for gene silencing in eukaryotes (Müller and Stahl, 2004). Table 4 Table 3 Examples of gene targeting frequencies obtained in wild-type and Δku70 strains Strain

Length of homologous flanks (bp) Reference 100

A. fumigatus

wt: 0% mut: 75% A. nidulans wt: n.d. mut: n.d. A. niger wt: 4% mut: 18% A. sojae wt: n.d. mut: 0% N. crassa wt: 2% mut: 10% S. macrospora wt: n.d. mut: n.d.

500

1000

wt: 2% mut: 84% wt: n.d. mut: 89% wt: 7% mut: 88% wt: n.d. mut: 14% wt: 9% mut: 91% wt: n.d. mut: n.d.

wt: 10% mut: 96% wt: n.d. mut: 92% wt: 19% mut: 95% wt: n.d. mut: 71% wt: 21% mut: 100% wt: 4% mut: 100%

(Krappmann et al., 2006) (Nayak et al., 2006) (Meyer et al., 2007) (Takahashi et al., 2006) (Ninomiya et al., 2004) (Pöggeler and Kück, 2006)

wt: wild-type; mut: mutant strain deleted for the Ku70 homologue; n.d.: not determined.

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Table 4 RNA-based tools for gene silencing in eukaryotes Method

Principle

Advantage

Disadvantage

AS

Expressed antisense RNA hybridises by Watson-Crick pairing to the target RNA Translation or processing of the target mRNA becomes inhibited Small catalytic active RNA molecule hybridises with the target RNA (two recognition arms are complementary to the target RNA) Sequence-specific cleavage of the target RNA Double stranded RNA is diced into siRNAs (21-25 bps long) siRNAs hybridise with the target RNA and guide it to a silencing complex (RISC) that degrades the target RNA

Simple and inexpensive

Sometimes low affinity to target RNA due to secondary structures of the antisense molecule

High specificity

Laborious Difficult to predict the best target site Sometimes low activity Requires the activity of endogenous protein complexes Instability possible

HHR

RNAi

High activity

After (Müller and Stahl, 2004). AS: Antisense RNA; HHR: Hammerhead ribozymes; RNAi: RNA interference; siRNA: small interfering RNA. For detailed information see text.

outlines the main principles of the tools and their advantages and disadvantages. Successful gene silencing using artificial antisense constructs have been reported for filamentous fungi (Zheng et al., 1998; Kitamoto et al., 1999; Bautista et al., 2000; Ngiam et al., 2000; Moralejo et al., 2002; Lombrana et al., 2004; Blanco and Judelson, 2005). For example, an antisense construct displaying homology to two different genes coding for extracellular carboxypeptidases was transferred by PMT into the genome of A. oryzae. The construct, found to be randomly integrated in multiple copies into the genome, resulted in about 70% reduced protease activities. This antisense strategy improved the capacities of A. oryzae as host for heterologous protein expression as more stable and higher level of human lysozyme were detected (Zheng et al., 1998). Similarly, antisense silencing of the protease aspergillopepsin B achieved a reduction of 10–70% of protease levels and resulted in a 30% increase in heterologous thaumatin production in A. awamori (Moralejo et al., 2002). Remarkably, there has been no antisense-mediated reduction of gene expression to zero levels reported to date, indicating that the efficiency of this strategy is limited. However, in view of the fact that a complete knockdown of a particular gene can be lethal to the fungus of interest, attenuation of its expression by the antisense tool can still be a powerful approach. For instance, the wide-domain transcription factor CreA, the key component of carbon catabolite repression in Aspergillus (Dowzer and Kelly, 1991; Ruijter and Visser, 1997; Shroff et al., 1997), negatively regulates a number of industrially important enzymes. As shown by Bautista et al. (Bautista et al., 2000), partial suppression of creA expression in A. nidulans by its antisense molecule (about 50% reduced expression was estimated) yielded in partial alleviation of glucose repression and thereby in a substantial increase of the productivities of intra- and extracellular glucoserepressible enzymes. Most importantly, growth characteristics of A. nidulans were not affected. Catalytic RNA molecules, also referred to as ribozymes, offer an alternative strategy for gene silencing approaches. The hammerhead ribozyme is the smallest, best-known and most widely used class of RNA-based enzymes (Müller and Stahl, 2004; Akashi et al., 2005). The substrate-recognition arms can be engineered so that the arms are complementary to any chosen

RNA target, enabling the ribozyme to bind to its target. The cleavage of the substrate RNA occurs immediately adjacent to a “NUX“ triplet, where N represents any base and X can be A, U, or C (Shimayama et al., 1995). The applicability of a ribozymebased technology as tool for effective post-transcriptional suppression of gene expression has been shown for bacterial, yeast, plant and mammalian systems (Bussiere et al., 2003; Akashi et al., 2005; Isaacs et al., 2006). Most recently, a proof of principle for the use of hammerhead ribozymes to control gene expression in filamentous fungi was given (Müller et al., 2006). As model organism, A. giganteus was used as for this fungus gene deletions were not feasible due to the lack of HR (Meyer et al., 2002). A reporter-based model system using the β-glucuronidase transcript (uidA) as the target mRNA was employed. This system was used to validate the activity of seven in silico selected hammerhead ribozymes targeting different sites within the uidA mRNA. All ribozymes tested were able to reduce the reporter activity in A. giganteus (up to a maximum of 100%), demonstrating that ribozyme technology is indeed a useful tool for fungal metabolic engineering and can also be used to completely silence a gene of interest. However, several limiting conditions have to be considered. First, target sites within the 5'-region of the substrate mRNA seem to be preferentially recognized as uidA expression was completely abolished in these cases. When ribozymes targeting the 3'-region of uidA were tested, expression of the target gene was reduced to only about 20–50%. Secondly, it cannot be guaranteed that the in silico predicted target site is indeed recognised and cleaved by the tailored ribozyme in vivo as other uidA target sites than the predicted ones were recognised under in vivo conditions for two out of seven ribozymes tested. It thus seems that, as reported for other organisms, it is difficult to predict the actual in vivo ribozyme binding sites by in silico methods (Akashi et al., 2005). Finally, RNA interference (RNAi) has been shown to be remarkably potent to silence fungal genes post-transcriptionally (Nakayashiki, 2005; Nakayashiki et al., 2005). In this silencing mechanism, double-stranded RNA with homology to the gene of interest becomes expressed in the fungal host strain and triggers degradation of its cognate RNA via 21–25 bps long small interfering RNA (siRNA) molecules. A prerequisite for this method is that the fungal strain comprises components of

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the RNAi silencing machinery, such as RNA-dependent RNA polymerase (RdRP), dicer-like proteins and the RNA-induced silencing complex (RISC) (Nakayashiki, 2005). Remarkably, some fungal strains, e.g. Ustilago maydis, Candida albicans and S. cerevisiae, lack components of the RNAi silencing machinery, indicating that this tool is not applicable for these organisms (Nakayashiki, 2005). However, specific inhibition of gene expression by RNAi has been shown to be suitable for a multitude of filamentous fungi such as A. nidulans (Hammond and Keller, 2005), A. fumigatus (Mouyna et al., 2004; Bromley et al., 2006; Henry et al., 2007; Khalaj et al., 2007), A. oryzae (Yamada et al., 2007), Bipolaris oryzae (Moriwaki et al., 2007), Colletotrichum lagenarium (Nakayashiki et al., 2005), Coprinus cinereus (Namekawa et al., 2005; Wälti et al., 2006), F. solani (Ha et al., 2006), Magnaporthe oryzae (Kadotani et al., 2003; Kadotani et al., 2004; Nakayashiki et al., 2005), Mucor circinelloides (Nicolas et al., 2007), N. crassa (Goldoni et al., 2004) and Schizophyllum commune (de Jong et al., 2006). Usually, plasmid constructs have been integrated into fungal genomes that express self-complementary hairpin RNA molecules (encoded by an inverted repeat which is interrupted by a spacer sequence). It has also been shown that simply addition of synthetic siRNA molecules to the culture medium of A. nidulans can result in specific suppression of the corresponding target gene (Khatri and Rajam, 2007). Similar to the antisense strategy, RNAi-induced silencing of fungal gene expression was most often found to be incomplete (maximal reduction up to 10% of wild type level) and full knockout phenotypes were seldom observed. However, as most recently reported, overexpression of the RNAi construct either by increased copy numbers or by the use of a strong, inducible promoter can lead to complete repression of gene expression (Khalaj et al., 2007). A striking disadvantage of RNAi-based gene silencing, however, could be an instability of the silencing construct (Goldoni et al., 2004). As shown for A. fumigatus, about 50% of the transformants lost the RNAi construct or parts of it after prolonged cultivation of the transformants (Goldoni et al., 2004; Henry et al., 2007). As proposed by the authors, one possible explanation for this phenomenon might be the loss of one of the inverted repeats after the first mitotic events. This could suggest that problems might be associated with this tool, however, the molecular mechanisms responsible for RNA-mediated gene silencing in filamentous fungi are not yet fully understood. Hence, much more basic knowledge is required to fully exploit the RNAi tool to customise fungal metabolic versatility. Filamentous fungi such as N. crassa and A. nidulans provide excellent experimental model systems to unravel the molecular machinery involved in fungal gene silencing (Nakayashiki, 2005; Nakayashiki et al., 2005; Fulci and Macino, 2007).

fully silence a gene or is it rather advisable to partially downregulate a gene of interest in order to circumvent detrimental effects for the fungal host? Is it possible to silence a group of genes simultaneously by using only a single genetic modification? Alternatively, is overexpression of a particular gene envisaged and is it thus advisable to integrate multiple copies of the gene into the genome? Each of these considerations will call for a different strategy in order to introduce the best genetic modification for metabolic engineering. Fig. 2A exemplarily highlights some genetic engineering approaches that could be considered in case of overexpressing a gene of interest. In this case, at least three strategies can be followed: i) PMT is suggested as transformation method when heterologous integrations of multiple gene copies are envisaged. ii) AMT is the method of choice when a single copy of the gene should integrate via homologous recombination to a specific genomic locus. If, however, AMT is not applicable for a certain fungus, iii) PMT of a NHEJ-deficient recipient strain could be used instead. For this strategy, the use of a strain in which the NHEJ machinery is transiently silenced is recommended. Fig. 2B depicts two main strategies that could be mapped out when the intention is to either partially or completely downregulate a gene of interest. i) Antisense or RNAi constructs can by introduced by PMT into the fungus of interest, whereby multiple copies of these constructs ectopically integrate into the genome. As the efficiency of both RNA-based methods is limited, partial gene silencing can be achieved and even controlled. This strategy is recommended when the target gene is essential for fungal growth. ii) For a

5. Designing an engineering strategy The molecular toolbox for genetic engineering of filamentous fungi has been considerably enlarged over the last years. In order to design the most appropriate engineering strategy for a particular process, all available options for genetic modification must be considered carefully. For example, is the intention to

Fig. 2. Guidelines for the design of genetic engineering approaches for filamentous fungi aiming at overexpression (A) or downregulation (B) of a gene of interest. PMT: Protoplast-mediated transformation; AMT: Agrobacteriummediated transformation; NHEJ: nonhomologous end joining pathway.

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complete gene silencing (if the target gene does not have an essential function for the fungus), three alternative possibilities could be considered: Either AMT or PMT using a transiently deficient NHEJ recipient strain can be used to knock out a gene of interest. Another option is to make use of the ribozyme technology with which a complete gene knock down can be adjusted. 6. Conclusions and prospects Thanks to considerable insights into fungal genetics and biology, the molecular and technical toolboxes available nowadays to optimize industrial fungal strains are impressive. What more is to come? Most of the techniques and designing options described above have been successfully established and validated for only a few dozen of fungal strains and the lack of efficient genetic engineering strategies forms still an obstacle for a multitude of fungi producing commercially interesting metabolites. To fully explore their biotechnological capacities, these constraints have to be solved. The knowledge of genetic engineering strategies accumulated so far will provide a valuable framework and opens new avenues for fungi where no genetic manipulation has been achieved yet. Furthermore, the availability of fungal genomes and their better understanding will open new doors to better engineer industrial host strains and will certainly contribute to the removal of bottlenecks. Moreover, new product classes will definitely be developed and manufactured. However, the postgenomic era also calls for new approaches to substantially understand the metabolism, growth and phenotype of filamentous fungi. Genome-wide transcription profiling, proteomics and the reconstruction of the complete metabolic networks will provide valuable in-depth insights into cellular processes of filamentous fungi. In consequence, the classical approach followed in fungal biotechnology, i.e. the focus on individual genes or proteins, will be expanded by global approaches, all aiming at the understanding of the interaction of the complete set of cellular components. This concept of ‘systems biology’ and the knowledge deduced will significantly improve industrial fermentation processes and will further increase the value of filamentous fungi in the future of biotechnology. References Adrio JL, Demain AL. Fungal biotechnology. Int Microbiol 2003;6:191–9. Akashi H, Matsumoto S, Taira K. Gene discovery by ribozyme and siRNA libraries. Nat Rev Mol Cell Biol 2005;6:413–22. Archer DB. Filamentous fungi as microbial cell factories for food use. Curr Opin Biotechnol 2000;11:478–83. Asch DK, Kinsey JA. Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene. Mol Gen Genet 1990;221:37–43. Askolin S, Nakari-Setala T, Tenkanen M. Overproduction, purification, and characterization of the Trichoderma reesei hydrophobin HFBI. Appl Microbiol Biotechnol 2001;57:124–30. Bailey SM, Meyne J, Chen DJ, Kurimasa A, Li GC, Lehnert BE, et al. DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc Natl Acad Sci U S A 1999;96:14899–904. Bautista LF, Aleksenko A, Hentzer M, Santerre-Henriksen A, Nielsen J. Antisense silencing of the creA gene in Aspergillus nidulans. Appl Environ Microbiol 2000;66:4579–81.

183

Bennett JW. Mycotechnology: the role of fungi in biotechnology. J Biotechnol 1998;66:101–7. Bird D, Bradshaw R. Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 1997;255:219–25. Blanco FA, Judelson HS. A bZIP transcription factor from Phytophthora interacts with a protein kinase and is required for zoospore motility and plant infection. Mol Microbiol 2005;56:638–48. Boulton SJ, Jackson SP. Identification of a Saccharomyces cerevisiae Ku80 homologue: roles in DNA double strand break rejoining and in telomeric maintenance. Nucleic Acids Res 1996;24:4639–48. Brakhage AA, Caruso ML. Biotechnical genetics of antibiotic biosynthesis. In: Esser K, editor. The Mycota II. Genetics and Biotechnology. SpringerVerlag; 2004. p. 317–53. Bromley M, Gordon C, Rovira-Graells N, Oliver J. The Aspergillus fumigatus cellobiohydrolase B (cbhB) promoter is tightly regulated and can be exploited for controlled protein expression and RNAi. FEMS Microbiol Lett 2006;264:246–54. Bundock P, van Attikum H, Hooykaas P. Increased telomere length and hypersensitivity to DNA damaging agents in an Arabidopsis KU70 mutant. Nucleic Acids Res 2002;30:3395–400. Bussiere F, Ledu S, Girard M, Heroux M, Perreault JP, Matton DP. Development of an efficient cis–trans–cis ribozyme cassette to inactivate plant genes. J Plant Biotechnol 2003;1:423–35. Chevalet L, Tiraby G, Cabane B, Loison G. Transformation of Aspergillus flavus: construction of urate oxidase-deficient mutants by gene disruption. Curr Genet 1992;21:447–53. da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T, et al. The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell 2006;5:207–11. de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 1998;16:839–42. de Jong JF, Deelstra HJ, Wösten HA, Lugones LG. RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune. Appl Environ Microbiol 2006;72:1267–9. Dowzer CE, Kelly JM. Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans. Mol Cell Biol 1991;11:5701–9. Dudasova Z, Dudas A, Chovanec M. Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol Rev 2004;28:581–601. Fernandez-Martin R, Cerda-Olmedo E, Avalos J. Homologous recombination and allele replacement in transformants of Fusarium fujikuroi. Mol Gen Genet 2000;263:838–45. Fincham JR. Transformation in fungi. Microbiol Rev 1989;53:148–70. Fulci V, Macino G. Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa. Curr Opin Microbiol 2007;10:199–203. Goins CL, Gerik KJ, Lodge JK. Improvements to gene deletion in the fungal pathogen Cryptococcus neoformans: absence of Ku proteins increases homologous recombination, and co-transformation of independent DNA molecules allows rapid complementation of deletion phenotypes. Fungal Genet Biol 2006;43:531–44. Goldoni M, Azzalin G, Macino G, Cogoni C. Efficient gene silencing by expression of double stranded RNA in Neurospora crassa. Fungal Genet Biol 2004;41:1016–24. Ha YS, Covert SF, Momany M. FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani. Eukaryot Cell 2006;5:1036–42. Haarmann T, Lorenz N, Tudzynski P. Use of a nonhomologous end joining deficient strain (Δku70) of the ergot fungus Claviceps purpurea for identification of a nonribosomal peptide synthetase gene involved in ergotamine biosynthesis. Fungal Genet Biol 2008;45:35–44. Haber JE. DNA repair. Gatekeepers of recombination. Nature 1999;398:665–7. Hammond TM, Keller NP. RNA silencing in Aspergillus nidulans is independent of RNA-dependent RNA polymerases. Genetics 2005;169:607–17. Hektor HJ, Scholtmeijer K. Hydrophobins: proteins with potential. Curr Opin Biotechnol 2005;16:434–9. Henry C, Mouyna I, Latge JP. Testing the efficacy of RNA interference constructs in Aspergillus fumigatus. Curr Genet 2007;51:277–84.

184

V. Meyer / Biotechnology Advances 26 (2008) 177–185

Hinnen A, Hicks JB, Fink GR. Transformation of yeast. Proc Natl Acad Sci U S A 1978;75:1929–33. Hua SB, Qiu M, Chan E, Zhu L, Luo Y. Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. Plasmid 1997;38:91–6. Isaacs FJ, Dwyer DJ, Collins JJ. RNA synthetic biology. Nat Biotechnol 2006;24:545–54. Ishibashi K, Suzuki K, Ando Y, Takakura C, Inoue H. Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci U S A 2006;103:14871–6. Kadotani N, Nakayashiki H, Tosa Y, Mayama S. RNA silencing in the phytopathogenic fungus Magnaporthe oryzae. Mol Plant Microbe Interact 2003;16:769–76. Kadotani N, Nakayashiki H, Tosa Y, Mayama S. One of the two Dicer-like proteins in the filamentous fungi Magnaporthe oryzae genome is responsible for hairpin RNA-triggered RNA silencing and related small interfering RNA accumulation. J Biol Chem 2004;279:44467–74. Khalaj V, Eslami H, Azizi M, Rovira-Graells N, Bromley M. Efficient downregulation of alb1 gene using an AMA1-based episomal expression of RNAi construct in Aspergillus fumigatus. FEMS Microbiol Lett 2007;270:250–4. Khatri M, Rajam MV. Targeting polyamines of Aspergillus nidulans by siRNA specific to fungal ornithine decarboxylase gene. Med Mycol 2007;45: 211–20. Kitamoto N, Yoshino S, Ohmiya K, Tsukagoshi N. Sequence analysis, overexpression, and antisense inhibition of a beta-xylosidase gene, xylA, from Aspergillus oryzae KBN616. Appl Environ Microbiol 1999;65:20–4. Kooistra R, Hooykaas PJ, Steensma HY. Efficient gene targeting in Kluyveromyces lactis. Yeast 2004;21:781–92. Krappmann S, Sasse C, Braus GH. Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joiningdeficient genetic background. Eukaryot Cell 2006;5:212–5. Krogh BO, Symington LS. Recombination proteins in yeast. Annu Rev Genet 2004;38:233–71. Kwon-Chung KJ, Goldman WE, Klein B, Szaniszlo PJ. Fate of transforming DNA in pathogenic fungi. Med Mycol 1998;36(Suppl 1):38–44. Leathers TD. Biotechnological production and applications of pullulan. Appl Microbiol Biotechnol 2003;62:468–73. Lee DG, Nishimura-Masuda I, Nakamura A, Hidaka M, Masaki H, Uozumi T. Overproduction of alpha-glucosidase in Aspergillus niger transformed with the cloned gene aglA. J Gen Appl Microbiol 1998;44:177–81. Linder MB, Szilvay GR, Nakari-Setala T, Penttila ME. Hydrophobins: the proteinamphiphiles of filamentous fungi. FEMS Microbiol Rev 2005;29:877–96. Lombrana M, Moralejo FJ, Pinto R, Martin JF. Modulation of Aspergillus awamori thaumatin secretion by modification of bipA gene expression. Appl Environ Microbiol 2004;70:5145–52. MacKenzie DA, Jeenes DJ, Archer DB. Filamentous fungi as expression systems for heterologous proteins. In: Esser K, editor. The Mycota II. Genetics and Biotechnology. Springer-Verlag; 2004. p. 289–315. Malonek S, Meinhardt F. Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic ascomycete Calonectria morganii. Curr Genet 2001;40:152–5. Meyer V, Arentshorst M, El-Ghezal A, Drews AC, Kooistra R, van den Hondel CA, et al. Highly efficient gene targeting in the Aspergillus niger kusA mutant. J Biotechnol 2007;128:770–5. Meyer V, Müller D, Strowig T, Stahl U. Comparison of different transformation methods for Aspergillus giganteus. Curr Genet 2003;43:371–7. Meyer V, Wedde M, Stahl U. Transcriptional regulation of the antifungal protein in Aspergillus giganteus. Mol Genet Genomics 2002;266:747–57. Michielse CB, Arentshorst M, Ram AF, van den Hondel CA. Agrobacteriummediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genet Biol 2005a;42:9–19. Michielse CB, Hooykaas PJ, van den Hondel CA, Ram AF. Agrobacteriummediated transformation as a tool for functional genomics in fungi. Curr Genet 2005b;48:1–17. 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;68:3550–9. Moriwaki A, Ueno M, Arase S, Kihara J. RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae. FEMS Microbiol Lett 2007;269:85–9. Mouyna I, Henry C, Doering TL, Latge JP. Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett 2004;237:317–24. Müller D, Stahl U. RNA: a powerful tool for recombination and regulated expression of genes. In: Esser K, editor. Progress in Botany, vol. 66. Springer-Verlag; 2004. p. 31–45. Müller D, Stahl U, Meyer V. Application of hammerhead ribozymes in filamentous fungi. J Microbiol Methods 2006;65:585–95. Müller EM, Locke EG, Cunningham KW. Differential regulation of two Ca(2+) influx systems by pheromone signaling in Saccharomyces cerevisiae. Genetics 2001;159:1527–38. Mullins ED, Kang S. Transformation: a tool for studying fungal pathogens of plants. Cell Mol Life Sci 2001;58:2043–52. Nakayashiki H. RNA silencing in fungi: mechanisms and applications. FEBS Lett 2005;579:5950–7. Nakayashiki H, Hanada S, Nguyen BQ, Kadotani N, Tosa Y, Mayama S. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol 2005;42:275–83. Namekawa SH, Iwabata K, Sugawara H, Hamada FN, Koshiyama A, Chiku H, et al. Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by doublestranded RNA-mediated gene silencing. Microbiology 2005;151:3669–78. Nayak T, Szewczyk E, Oakley CE, Osmani A, Ukil L, Murray SL, et al. A versatile and efficient gene-targeting system for Aspergillus nidulans. Genetics 2006;172:1557–66. Ngiam C, Jeenes DJ, Punt PJ, van den Hondel CA, Archer DB. Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger. Appl Environ Microbiol 2000;66:775–82. Nicolas FE, de Haro JP, Torres-Martinez S, Ruiz-Vazquez RM. Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects. Fungal Genet Biol 2007;44:504–16. Nielsen J.B., Nielsen M.L., Mortensen U.H. Transient disruption of nonhomologous end-joining facilitates targeted genome manipulations in the filamentous fungus Aspergillus nidulans. Fungal Genet Biol in press [Epub ahead of print]. doi:10.1016/j.fgb.2007.07.003. Ninomiya Y, Suzuki K, Ishii C, Inoue H. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining. Proc Natl Acad Sci U S A 2004;101:12248–53. Olempska-Beer ZS, Merker RI, Ditto MD, DiNovi MJ. Food-processing enzymes from recombinant microorganisms — a review. Regul Toxicol Pharmacol 2006;45:144–58. Ooi VE, Liu F. Immunomodulation and anti-cancer activity of polysaccharideprotein complexes. Curr Med Chem 2000;7:715–29. Pöggeler S, Kück U. Highly efficient generation of signal transduction knockout mutants using a fungal strain deficient in the mammalian ku70 ortholog. Gene 2006;378:1–10. Polizeli ML, Rizzatti AC, Monti R, Terenzi HF, Jorge JA, Amorim DS. Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 2005;67:577–91. Punt PJ, van Biezen N, Conesa A, Albers A, Mangnus J, van den Hondel CA. Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol 2002;20:200–6. Riha K, Watson JM, Parkey J, Shippen DE. Telomere length deregulation and enhanced sensitivity to genotoxic stress in Arabidopsis mutants deficient in Ku70. EMBO J 2002;21:2819–26. Ruijter GJ, Visser J. Carbon repression in Aspergilli. FEMS Microbiol Lett 1997;151:103–14. Ruiz-Diez B. Strategies for the transformation of filamentous fungi. J Appl Microbiol 2002;92:189–95. Scholtmeijer K, Wessels JG, Wösten HA. Fungal hydrophobins in medical and technical applications. Appl Microbiol Biotechnol 2001;56:1–8. Shimayama T, Nishikawa S, Taira K. Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. Biochemistry 1995;34:3649–54.

V. Meyer / Biotechnology Advances 26 (2008) 177–185 Shiotani H, Tsuge T. Efficient gene targeting in the filamentous fungus Alternaria alternata. Mol Gen Genet 1995;248:142–50. Shroff RA, O'Connor SM, Hynes MJ, Lockington RA, Kelly JM. Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans. Fungal Genet Biol 1997;22:28–38. Simon JR, Moore PD. Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae. Mol Cell Biol 1987;7:2329–34. Takahashi T, Masuda T, Koyama Y. Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae. Mol Genet Genomics 2006;275:460–70. Ueno K, Uno J, Nakayama H, Sasamoto K, Mikami Y, Chibana H. Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata. Eukaryot Cell 2007;6:1239–47. Verdoes JC, Punt PJ, van den Hondel CA. Molecular genetic strain improvement for the overproduction of fungal proteins by filamentous fungi. Appl Microbiol Biotechnol 1995;43:195–205. Villalba F, Collemare J, Landraud P, Lambou K, Brozek V, Cirer B, et al. Improved gene targeting in Magnaporthe grisea by inactivation of MgKU80 required for non-homologous end joining. Fungal Genet Biol 2008;45:68–75. Wälti MA, Villalba C, Buser RM, Grunler A, Aebi M, Kunzler M. Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression of homologous hairpin RNAs. Eukaryot Cell 2006;5:732–44.

185

Weidner G, d'Enfert C, Koch A, Mol PC, Brakhage AA. Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'monophosphate decarboxylase. Curr Genet 1998;33:378–85. Weld RJ, Plummer KM, Carpenter MA, Ridgway HJ. Approaches to functional genomics in filamentous fungi. Cell Res 2006;16:31–44. Willke T, Vorlop KD. Biotechnological production of itaconic acid. Appl Microbiol Biotechnol 2001;56:289–95. Wu TS, Linz JE. Recombinational inactivation of the gene encoding nitrate reductase in Aspergillus parasiticus. Appl Environ Microbiol 1993;59: 2998–3002. 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;71:138–44. Young C, Itoh Y, Johnson R, Garthwaite I, Miles CO, Munday-Finch SC, Scott B. Paxilline-negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration. Curr Genet 1998;33:368–77. Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, Scazzocchio C. Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 2004;41:973–81. Zheng XF, Kobayashi Y, Takeuchi M. Construction of a low-serine-typecarboxypeptidase-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;49:39–44.