Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives

Process Biochemistry 50 (2015) 211–220

Contents lists available at ScienceDirect

Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Review

Heterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives Marta Zoglowek a , Peter S. Lübeck a , Birgitte K. Ahring a,b , Mette Lübeck a,∗ a b

Section for Sustainable Biotechnology, Aalborg University Copenhagen, A. C. Meyers Vænge 15, DK-2450 Copenhagen SV, Denmark Bioproducts, Sciences and Engineering Laboratory (BSEL), Washington State University Tri-Cities, 2710 Crimson Way, Richland 99354, WA, USA

a r t i c l e

i n f o

Article history: Received 10 September 2014 Received in revised form 5 December 2014 Accepted 11 December 2014 Available online 26 December 2014 Keywords: Cellobiohydrolase Heterologous expression Filamentous fungi On-site enzyme production Consolidated bioprocessing

a b s t r a c t Cellobiohydrolases are among the most important enzymes functioning in the hydrolysis of crystalline cellulose, significantly contributing to the efficient biorefining of recalcitrant lignocellulosic biomass into biofuels and bio-based products. Filamentous fungi are recognized as both well-known producers of commercial preparations of cellulolytic enzymes and efficient hosts for heterologous protein secretion. Thus, Aspergillus and Trichoderma species have been chosen as hosts for the heterologous expression of native or engineered enzymes aiming at the overproduction of single enzymes or as hosts for the secretion of multi-enzyme cocktails for on-site production in biorefineries, which is important for reducing the costs of biomass conversion. An even more interesting aspect is consolidated bioprocessing, in which a single fungus both hydrolyzes lignocellulose polymers and ferments the resulting sugars into valuable products. However, due to low cellobiohydrolase activities, certain fungi might be deficient with regard to enzymes of value for cellulose conversion, and improving cellobiohydrolase expression in filamentous fungi has proven to be challenging. In this review, we examine the effects of altering promoters, signal peptides, culture conditions and host post-translational modifications. For heterologous cellobiohydrolase production in filamentous fungi to become an industrially feasible process, the construction of site-integrating plasmids, development of protease-deficient strains and glycosylation engineering are obvious targets for constructing efficient enzyme producers. © 2015 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

5. 6. 7. 8.

Biomass recalcitrance and cellobiohydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Heterologous expression of cellobiohydrolases in filamentous fungi-challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Promoters for driving the heterologous expression of cellobiohydrolase genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Improving the secretion of heterologous cellobiohydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Fusion of cellobiohydrolases to signal peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Other fusion strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycosylation of heterologously expressed cellobiohydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proteolytic degradation of heterologously expressed cellobiohydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Overcoming proteolytic degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other post-translational modifications and quality control of heterologously expressed cellobiohydrolases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks and perspectives for industrial cellobiohydrolase production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +45 99402589; fax: +45 99402594. E-mail addresses: [email protected] (M. Zoglowek), [email protected] (P.S. Lübeck), [email protected] (B.K. Ahring), [email protected] (M. Lübeck). http://dx.doi.org/10.1016/j.procbio.2014.12.018 1359-5113/© 2015 Elsevier Ltd. All rights reserved.

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1. Biomass recalcitrance and cellobiohydrolases The core biorefinery concept, recapitulated in the recent European Biorefinery Vision 2030, includes the pretreatment, enzymatic conversion and refinement of lignocellulosic biomass into biobased materials, chemicals, energy, food ingredients and feed, leading to the development of sustainable production systems [1]. Indeed, lignocellulosic biomass such as agricultural and forestry byproducts, energy crops and biowaste will play a major role in renewable energy development, especially in countries with limited solar or wind resources [2]. Cellulose, together with other biomass components (hemicellulose and lignin), confers structure to the plants and defense against abiotic and biotic factors [3]. Cellulose is a homogenous linear crystalline biopolymer composed of ␤-d-glucose units covalently linked with (1→4)-␤-d-glycosidic bonds and also contains non-crystalline (amorphous) regions, though the exact proportions and distribution within plant tissues and cell walls have not been completely elucidated [4]. The cellulose crystal structure, kept together through a stable network of hydrogen bonds (intra-chain, inter-chain and inter-sheet) and van der Waals forces (inter-sheet), contributes significantly to biomass recalcitrance, which manifests as an inability to readily and economically release free soluble carbohydrates from the complex plant cell wall [5]. Recalcitrance is considered to be the bottleneck to harnessing biomass in the production of biofuels [6], which will help to eliminate our dependence on fossil energy. The molecular basis of recalcitrance has been investigated using molecular dynamics (MD) simulations by Gross et al. [6] and Beckham et al. [7]; however, the models should be further supported by experimental studies. An often-cited quintessential perspective on factors contributing to biomass recalcitrance and ways of overcoming them have been proffered by Himmel et al. [8]. Recalcitrance is initially overcome by mechanical and thermo-chemical pretreatment, which improves the accessibility of biomass for subsequent enzymatic conversion. However, such pretreatment often results in the release of inhibitory byproducts and lignin residues that hinder the subsequent enzymatic hydrolysis [9–11]. Therefore, it is necessary to use a combination of well-designed pretreatment methods and highly efficient enzymes to effectively address biomass recalcitrance [7]. Cellobiohydrolases (CBHs), which are classified into two glycosyl hydrolase (GH) families (6 and 7), are among the most important enzymes in the degradation of recalcitrant crystalline cellulose and are thus one of the main components in commercial enzyme cocktails used for biomass conversion. The commonly accepted hypothesis of CBH action involves binding to cellulose via the carbohydrate-binding module and cleaving cellobiose units from

either the reducing (GH7) or the non-reducing end (GH6) by decrystallizing and threading single cellulose chains into the tunnel of the catalytic module (Fig. 1), which is connected to the carbohydratebinding module via a flexible linker [7,12,13]. This processive mode of action, involving decrystallization, single cellulose chain threading, hydrolysis, and product expulsion, is considered to be one of the rate-limiting mechanisms in the enzymatic deconstruction of biomass [8]. In addition, there is a relationship between biomass recalcitrance and the efficiency of the enzyme in pulling single crystalline chains off the hydrophobic cellulose surface, and this could be influenced by the accurate design of pretreatment. However, the hydrolysis and cellobiose expulsion steps will not be affected by this process and depend exclusively on enzyme properties that might also be improved by engineering [7]. The precise structure–function relationship for CBHs and the process of enzymatic decrystallization is still not fully understood. Up-to-date MD simulations, revealing implications for CBH structure on cellulose hydrolysis, were performed by researchers at National Renewable Energy Laboratory (NREL) and co-scientists. This modeling confirmed that the aromatic residues of the CBH active site tunnel are responsible for cellodextrin ligand binding and processivity, i.e., either the cellulose substrate at the tunnel entry or the cellobiose product at the tunnel exit, with the cleavage of glycosidic bonds occurring between these steps. In the most recent research, differences in ligand binding between GH6 and GH7 CBHs were noted [14]. Linkers were also found to enhance the binding affinity of CBHs via non-specific binding to cellulose through their glycosylated regions near the carbohydrate-binding module [15]. In the same study, it was revealed that the linkers of CBHs belonging to GH6 and GH7 differ in length: the longer linkers of Cel6A contribute to its higher binding affinity compared to Cel7A. As a result, Cel6A exhibits lower processivity than Cel7A. Finally, the mechanisms of initial CBH binding to cellulose were investigated [16]. It was concluded that the binding of CBHs to cellulose is directional and is due to the preferential binding of carbohydrate-binding modules at specific cellulose sites, the recognition of reducing or non-reducing ends of cellulose and the structural arrangement of the catalytic residues that orient the cellulose chain inside the enzyme active site. Cellobiohydrolases Cel6A and Cel7A from the cellulolytic fungus Trichoderma reesei are the most widely studied CBHs, though such enzymes have also been evaluated in Penicillium spp. (P. verruculosum, P. pinophilum, P. funiculosum, P. echinulatum), Acremonium cellulolyticus, Chrysosporium lucknowense and Phanerochaete chrysosporium within the last few years [17,18]. Proteomic analyses of enzyme broths extracted from cultures of Aspergillus terreus, Phanerochaete carnosa and Neurospora crassa grown on

Fig. 1. Stereo representation showing the superimposition of the A. terreus cellobiohydrolase (At Cel7A) homology model and T. reesei cellobiohydrolase (Tr Cel7A) structure. At Cel7A and Tr Cel7A are shown in steel blue and red, respectively. The modeled cellulose chain entering into the catalytic cleft is shown in dark green. The putative catalytic residues of At Cel7A are shown in black. The figure was provided by Dr. Wimal Ubhayasekera. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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different carbon sources demonstrated CBH secretion in those organisms [19–21]. In addition to the above-mentioned species, other Trichoderma, Hypocrea, Volvariella, Talaromyces, and Thielavia species have been mentioned as alternative (to T. reesei) CBH producers [22]. An important aspect in overcoming biomass recalcitrance is synergy between enzymes [23]. In addition to exo–exo synergy in the degradation of cellulose from reducing and non-reducing ends, the exo–endo synergistic action of CBHs and endoglucanases (EGs), which generate a greater number of free crystal ends by degrading cellulose at random, was reported [24–26]. Additionally, the beta-glucosidases (BGLs) were found to be important in eliminating end-product inhibition in hydrolysis by degrading cellobiose into glucose [27]. Recently, the importance of synergy with enzymes belonging to auxiliary activities family 9, AA9 (formerly GH61), accounting for the oxidative disruption of the cellulose structure and facilitating subsequent degradation by glycosyl hydrolases, was stated [28]. In 2012, Novozymes A/S launched their stateof-the-art enzyme blend commercialized as Cellic CTec3, which consists of an efficient cellulase complex with improved BGLs, advanced AA9 enzymes and novel hemicellulases for biomass conversion into biofuel [29]. This innovation was followed by the establishment of commercial bioethanol production plants by Novozymes’ partners, such as Beta Renewables [30]. In addition to biofuel plants that depend on the delivery of commercial enzymes, biorefineries with on-site enzyme production could be developed to reduce the costs of enzyme supply. A further cost reduction is foreseen with consolidated bioprocessing (CBP), in which a single fungal biocatalyst hydrolyzes the polymers in lignocellulose while producing valuable products [31]. On-site enzyme production in single hosts or CBP requires that the fungal hosts or biocatalysts produce efficient cellobiohydrolases as well as other lignocellulosic degradation enzymes. This review evaluates heterologous CBH expression in filamentous fungi and discusses the main aspects that should be considered in heterologous protein expression, including promoters, signal peptides, culture conditions and host post-translational modifications. In addition, this review focuses on the challenges and improvements that need to be considered in research and development programs before the large-scale production of CBHs in fungal hosts for on-site enzyme production or CBP can become industrially feasible.

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in the heterologous expression of fungal CBHs. The heterologous expression of T. reesei Cel7A and Cel6A in Aspergillus nidulans and Aspergillus awamori was described in the late 1980s [41]. Since then, attempts have been made to produce various fungal CBHs in hosts such as Escherichia coli, yeast, filamentous fungi, insects and plants [31,42,43]. Insect cells and Aspergillus niger var. awamori expressed T. reesei Cel7A with properties that were closest to the native protein. However, insect cells are difficult to handle [44]. In the studies carried out by Lantz et al. [45] and Adney at al. [46], Aspergillus niger (and A. niger var. awamori) was successfully used to secrete recombinant T. reesei Cel6A and Cel7A, which were subjected to mutagenesis and screened for improved thermostability as well as enzymatic activity in these hosts. The recent progress in research on heterologous CBH expression in filamentous fungi is dictated by the need to discover novel highly efficient enzyme cocktails that would expedite biorefinery development and protein-engineered improvements, such as thermostability, of known CBHs. The overexpression of CBHs under strong inducible promoters would facilitate the production of high yields of those enzymes for biofuel industry. Among the goals of heterologous CBH expression in fungi is to produce well-adjusted enzyme cocktails in single hosts for lignocellulose degradation. Such strains with balanced homologous and heterologous enzyme activities, such as CBHs, EGs, and BGLs for on-site enzyme production, could eliminate the need for enzyme supplementation from other fungal fermentations [21,47]. Similar strategies would include either the heterologous expression of BGLs in efficient CBH producers [48–51] or the co-cultivation of compatible fungi producing high levels of each enzyme [21,52–54]. These approaches could consolidate enzyme production into a single bioreactor, thus reducing the cost of the necessary equipment. However, challenges with these two strategies arise if only a limited number of heterologous genes can be inserted into a single host [55], and that co-cultivation requires high compatibility among the applied strains [56]. In general, it could be hypothesized that the heterologous expression of CBHs is less efficient than the heterologous expression of other cellulases. For instance, the heterologous expression of a T. reesei EG, Cel7B, appeared to be slightly easier than the expression of a T. reesei CBH, Cel7A, in yeast or yeast-related hosts [57]. The heterologous production of fungal CBHs in yeast has already been evaluated by Haan et al. [31]. Therefore, this paper will mainly focus on reviewing the most significant studies on the heterologous expression of CBHs in filamentous fungi.

2. Heterologous expression of cellobiohydrolases in filamentous fungi-challenges The proteins used for different applications in food, feed, textile, pulp and paper industries as well as therapeutics are commonly produced in filamentous fungi. For a list (updated in 2009) of commercial enzymes, the majority of which are produced in both native and heterologous hosts [32], the reader is referred to The Association of Manufacturers and Formulators of Enzyme Products [33]. The application of filamentous fungi to the heterologous expression of fungal and non-fungal proteins has been extensively reviewed [34–37]. The advantages of using filamentous fungi over other hosts include a higher secretion capacity and efficient folding and post-translational modifications, such as glycosylation (without undesired hyper-glycosylation), which is well-suited for proteins of fungal origin. In addition, industrial fermentation systems are well-established for several fungal species. However, the heterologous production of certain proteins (especially those of non-fungal origin) at a comparable level to homologous production in g/l yield may be difficult to achieve [38,39]. In fact, the high production levels that are particularly desired in commercial enzyme production [40] were found to be one of the main challenges

3. Promoters for driving the heterologous expression of cellobiohydrolase genes In many cases, the heterologous expression of fungal enzymes previously reviewed [38] was controlled either by inducible promoters that were homologous to the expression host or by the constitutive A. nidulans glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter, which was reported to be functional in industrially used Penicillium and Aspergillus species [58] as well as other species, e.g., Trichoderma and Clonostachys [59,60]. The expression of the most prominent cellobiohydrolase, Cel7A (syn. CBH1), in T. reesei is regulated by the cbh1 promoter. As CBH1 can compose more than half of the total secreted protein from T. reesei, cbh1 is a very strong inducible promoter [61]. Such strong inducible promoters homologous to the expression host have been used for the heterologous expression of T. reesei, P. funiculosum, Aspergillus aculeatus or Melanocarpus albomyces cellobiohydrolase (cbh) genes [42,62–67]. In general, homologous strong promoters appear to result in higher expression yields than heterologous promoters [34,58].

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According to Zou et al. [68], the cbh1 promoter has also been used in other fungi, but attempts to use this promoter for the expression of T. reesei Cel7A in Aspergillus carbonarius did not appear to be successful [69], whereas the constitutive A. nidulans gpdA promoter was functional in A. carbonarius for expressing the heterologous A. terreus and T. reesei cbh genes. In these cases, expression was evidenced by high mRNA levels for all genes using RT-PCR, though the protein levels appeared to be low [21]. The gpdA promoter was also successfully used for expressing a laccase gene with a construct similar to that used for the cbh genes [70], as well as for expression of other genes in A. carbonarius [71,72]. Although it is assumed to be constitutively expressed, the activity of the gpdA promoter was reported to vary with the culture conditions, such as the carbon source and salt concentration [73–75]. In most of the reviewed studies, cbh genes were integrated at random sites into the chromosomal DNA of the heterologous host. However, random gene recombination will likely result in a few to multiple gene copies [62] integrated at loci differing in transcriptional efficiency and might subsequently cause transformant-to-transformant variations at the expressed protein level, as was reported for P. funiculosum Cel7A expressed in A. niger var. awamori [63]. Nonetheless, the correlation between mRNA transcription and the activity of heterologous CBHs was not investigated in detail in most of the reviewed studies. Additionally, the determined gene copy number and enzyme activity were not correlated [62,76]. Kubicek-Pranz et al. [76] assumed that the increase in activity observed for 3 out of 16 isolated transformants might have resulted from a transcriptionally efficient site of gene integration rather than from the number of gene copies; regardless, the hypothesis was not tested further. In general, successful heterologous expression depends on promoter efficiency, vector integration site, origin of the expressed protein, and the type of the host strain [77]. The final yield of heterologous enzymes might also be influenced by factors such as mRNA and intracellular protein stability, protein folding, post-translational modifications, secretion efficiency and proteolysis, which will be discussed below.

4. Improving the secretion of heterologous cellobiohydrolases 4.1. Fusion of cellobiohydrolases to signal peptides Signal peptides (SPs), which are responsible for the entry of proteins into the secretory pathway, are also believed to initiate and assist protein folding [42,63] and are therefore very important for obtaining high yields of correctly folded heterologous enzymes. In yeast, the longer hydrophobic regions of SPs were found to be more efficiently recognized (than short regions) by the signal recognition particles (SRPs) [78] that are responsible for targeting the protein into the endoplasmic reticulum (ER) membrane [79]. As it is believed that SRPs recognize the particular conformation of the SP [80], it appears that even subtle differences between SPs can affect protein secretion. For example, the native SP of P. funiculosum, Cel7A, was reported to be significantly more efficient for the secretion of P. funiculosum Cel7A in A. niger var. awamori than nonnative A. niger glucoamylase (GA) SP, differing in length by only one amino acid [63,81]. In contrast, a non-native A. niger GA SP resulted in the secretion of relatively high levels of active heterologous T. reesei Cel7A when A. niger var. awamori was similarly used as the expression host [42]. These findings indicate that the efficiency of secretion likely depends on the accurate correlation between the SP, recombinant protein and the SRP system of the heterologous host. In the reviewed studies, the protein’s own native SP was found to be the most efficient in CBH secretion, whereas non-native SPs

caused changes to the secondary structure of the protein of different degrees [42] and secretion impairment [63]. Kanamasa et al. [64] speculated that the native SP of A. aculeatus CBH was the most suitable for the heterologous secretion of this enzyme in Aspergillus oryzae, reasoning by the fact that CBH is the most abundant protein efficiently produced by A. aculeatus. Another suggestion could be that the A. aculeatus CBH SP is extremely well recognized and processed by A. oryzae. In our laboratory, A. terreus and T. reesei CBHs were expressed using native signal peptides, despite the fact that their recognition by the A. carbonarius secretion system was difficult to predict, as only sparse knowledge is available for this in fungus. In the future, it could be interesting to study the fusion of heterologous cellobiohydrolases with the A. carbonarius betaglucosidase SP, which may facilitate successful secretion in this host. In general, when designing a fusion construct, proper cleavage of the SP after secretion should be considered, such that only the mature protein (without SP) accumulates in the culture broth [42]. Additionally, the introduction of a frameshift or additional amino acids between the SP and mature protein should be avoided due to their potential negative effect on folding and secretion [63]. 4.2. Other fusion strategies Generally, heterologous gene expression could be improved by fusion to a homologous gene encoding a highly secreted protein; this enhances mRNA stability, increases the efficiency of folding and secretion, and adds protection from proteolysis [34,35,39]. Among other fusion strategies, to enhance the hydrolytic potential of M. albomyces enzyme for the degradation of crystalline cellulose at elevated temperatures, the carbohydrate-binding module and linker region of T. reesei Cel7A was fused to the C-terminus of M. albomyces CBH lacking a native carbohydrate-binding module [67]. The recombinant fusion protein was expressed in T. reesei, resulting in an approximate 2-fold increase in the percentage of solubilized avicel compared to the wild-type M. albomyces enzyme expressed in T. reesei. A similar result was obtained by Szijártó et al. [65], though the improvement was less significant. Moreover, a fusion protein containing the catalytic modules of fungal CBHs and bacterial EGs, connected with a CBH linker, was developed in a Trichoderma host for increased cellulase enzyme yield and effectiveness in the synergistic degradation of crystalline cellulose [82]. In general, a CBH’s linker and carbohydrate-binding module fused to various biomass-degrading enzymes improves the hydrolysis of lignocellulose and increases protein stability as well as production yields [83,84]. Such fused enzymes having high activities of CBH and EG could be heterologously expressed in efficient BGL and AA9 producers to further improve single-host multi-enzyme production. The activities of heterologous CBHs have been evaluated in the hydrolysis of either various synthetic cellulosic substrates or complex biomasses, which likely differ in recalcitrance [7]. Table 1 presents the activities of heterologous CBHs expressed in various filamentous hosts under different promoters and with either the native or heterologous signal peptide. However, an accurate comparison could not be performed due to differences in the enzyme assays, substrates and units used; in addition, purified or crude enzyme preparations were used. Nevertheless, it could be observed that the activity varied from non-detectable to a near native level. 5. Glycosylation of heterologously expressed cellobiohydrolases Cellobiohydrolases are glycoproteins, undergoing both N-linked glycosylation of asparagine residues in their catalytic modules and O-linked glycosylation of serine/threonine residues in their

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Table 1 Heterologous expression of cellobiohydrolases. CBM = carbohydrate-binding module, MULac = 4-methylumbelliferyl ␤-d-lactoside, pNPC = p-nitrophenyl ␤-d-cellobioside, pNPL = p-nitrophenyl ␤-d-lactopyranoside, ND = non-detected. Origin/gene

T. reesei/ cel7a

T. reesei/ cel7a

Host strain

A. gossypii

A. niger var. awamori

Expression system

Promoter

Terminator

S. cerevisiae PGK1

S. cerevisiae PGK1

A. niger glucoamylase (GA)

A. niger trpC

Signal peptide

Assayed with substrate

Activity/% of cellulose hydrolysis

Culture conditions

Refs.

Native T. reesei Cel7A

MULac

ND

[57]

avicel

ND

Ashbya full medium (AFM) or synthetic complete defined (SCD) medium containing 2% glucose as the C-source and G418; cultivation at 30 ◦ C or 24 ◦ C, 200 rpm

Aspergillus glucoamylase

Pretreated yellow poplar

Approx. 92% of native Cel7A activity

[42]

native T. reesei Cel7A

Pretreated yellow poplar Pretreated corn stover

Approx. 92% of native Cel7A activity Approx. 62% of cellulose conversion at 120 h (60% for native Cel7A)

CM basal fermentation medium containing 5% soluble starch as the C-source; cultivation at 32 ◦ C, 225 rpm, for 4–5 days

CD-P medium [133] containing maltose as the C-source; cultivation at 30 ◦ C for 4 days

[62]

CM medium containing 1% glucose as the C-source and Zeo; cultivation at 29◦ C, 250 rpm, for 10 days

[63]

basal liquid minimal medium containing 1% glucose as the C-source; cultivation at 30 ◦ C for approx. 3 days

[64]

Complex nitrogen source medium containing lactose as the C-source [134]

[65]

T. reesei/ cel7a

A. oryzae

A. oryzae Taka-amylase (amyB)

Data not given

Data not given

avicel

0.0105 [␮mol/min/mg] 0.014 [␮mol/min/mg] for native Cel7A [132]

T. reesei/ cel6a

A. oryzae

A. oryzae Taka-amylase (amyB)

Data not given

Data not given

pNPC avicel

0.355 [␮mol/min/mg] 0.0205 [␮mol/min/mg] 0.027 [␮mol/min/mg] for native Cel6A [132] ND

P. funiculosum/ cel7a

A. niger var. awamori

A. niger glucoamylase (GA)

A. niger trpC

Native P. funiculosum Cel7A

pNPC

A. aculeatus/ cbhI

A. oryzae

P-no. 8142

A. oryzae agdA

pNPL

Approx. 0.013 [␮mol/min/mg] (max. activity during 10-day growth period)

Pretreated corn stover

Approx. 66% of cellulose conversion at 120 h (approx. 72% for native Cel7A) <0.002 [␮mol/min/mg] (during 10-day growth period) <0.002 [␮mol/min/mg] (during 10-day growth period)

A. niger glucoamylase

pNPL

T. reesei Cel7A

pNPL

native A. aculeatus CBHI

pNPL

approx. 40 [␮mol/min/l] (max. activity during approx. 3 days growth period)

Alkali-swollen cellulose

160 [U/l]

M. albomyces/cel7b

T. reesei

T. reesei cbh1

Data not given

Data not given

Avicel

Approx. 4% of cellulose conversion (at 24 h, 5 mg/g enzyme dosage)

M. albomyces/cel7b with T. reesei Cel7A CBM

T. reesei

T. reesei cbh1

Data not given

Data not given

Avicel

Approx. 5% of cellulose conversion (at 24 h, 5 mg/g enzyme dosage)

M. albomyces/ cel7b

T. reesei

T. reesei cbh1

T. reesei cbh1

Data not given

MULac

2.6 [MUL/ml]

Complex medium containing lactose as the C-source [135]; cultivation at 30 ◦ C, 250 rpm, for 7 days

[66]

M. albomyces/ cel7b

T. reesei

T. reesei cbh1

Data not given

Native M. albomyces Cel7B

Avicel

Approx. 1% solubilized avicel at 45 ◦ C Approx. 1.2% solubilized avicel at 70 ◦ C

Complex cellulase inducing medium containing lactose as the C-source; cultivation for 5 days [136]

[67]

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Table 1 (Continued) Origin/gene

Host strain

Expression system

Signal peptide

Assayed with substrate

Activity/% of cellulose hydrolysis

Promoter

Terminator

M. albomyces/cel7b with T. reesei Cel7A CBM

T. reesei

T. reesei cbh1

Data not given

Native M. albomyces Cel7B

Avicel

Approx. 2% solubilized avicel at 45 ◦ C Approx. 1.8% solubilized avicel at 70 ◦ C

T. reesei/ cel6a

T. reesei

Data not given

Data not given

Data not given

Avicel

Approx. 2.3 reducing sugar [g/l] (approx. 1 [g/l] for native enzyme mixture)

Filter paper strips

0.74 [U/mg] (0.42 [U/mg] for native enzyme mixture)

linkers [85]. Although glycosylation sites and pathways are relatively conserved in filamentous fungi [46,86–88], the extent of attached glycans varies among strains [86,89–91] and species [92], typically with different numbers of mannose residues attached. Glycosylation also varies with culture conditions [85,90]. Moreover, T. reesei Cel7A and Cel6A (together with Cel7B and Cel45A) were found to be glycosylated to a higher extent than T. reesei Cel12A or BGLI when heterologously expressed in A. oryzae [62]. In general, glycosylation affects a protein’s structure and subsequently its function and stability [93]. Therefore, it is of particular relevance to consider glycosylation in relation to protein expression in heterologous hosts. The over-glycosylation of heterologous CBHs is less extensive in filamentous fungi than in yeast, though the attachment of additional glycans, typically identified as either higher molecular weight (MW ) on SDS-PAGE and/or the dispersion of western blotting protein bands, was also observed in most of the tested fungal hosts. For instance, a maximum of three mannose residues are typically located at the N-glycosylation sites of native T. reesei Cel7A, whereas up to twenty residues were detected in T. reesei Cel7A when expressed in A. niger var. awamori [92]. Among the reviewed studies, native-like glycosylation, which was evidenced by an identical MW to the experimental or calculated MW of the native proteins was reported only for either A. aculeatus CBH expressed in A. oryzae [64] or M. albomyces CBH produced in T. reesei [65]. In the remaining examples, differences in glycosylation patterns caused either a decrease in enzyme activity toward crystalline cellulose, decrease in protein stability and/or increase in non-productive cellulose binding. For example, the over-glycosylation of T. reesei CBHs heterologously expressed in A. oryzae resulted in a 22–36% increase in MW and an approximately 25% loss in activity toward avicel compared to the native proteins [62]. Similarly, the sixfold higher amount of N-glycans (10% difference in MW ) on T. reesei Cel7A expressed in A. niger var. awamori caused a 29% reduction in activity toward bacterial cellulose [92]. The greatest changes were caused by alterations in N-glycosylation, as reported for either T. reesei or P. funiculosum CBHs expressed in A. niger var. awamori [46] or T. reesei CBH expressed in Ashbya gossypii [57]. A detailed investigation of the role of particular Nlinked glycans led to a conclusion that of three CBHs glycosylation sites, N384 of T. reesei and either N45, N388 or N430 of P. funiculosum CBH largely affected the enzymatic hydrolysis of crystalline cellulose [46]. However, the nature of this impact was still not completely revealed, and it was suggested that the over-glycosylation of those sites negatively influenced either the access of the catalytic module to the crystalline cellulose surface, enzyme processivity (by hindering the accommodation of new crystals), enzyme binding (by interacting with a linker), or protein stability. The removal

Culture conditions

Refs.

Mandels-Andreotti medium [137] containing 1% lactose as the C-source; cultivation at 28 ◦ C, 250 rpm, for an appropriate time

[76]

of those N-glycosylation sites was demonstrated to significantly increase recombinant enzyme activity compared to the wild-type glycosylation applied in the expression host A. niger var. awamori [46]. Moreover, the addition of a new N194 glycosylation site in P. funiculosum CBH facilitated high rates of cellulose conversion for longer hydrolysis times. In other studies, it was shown that the Olinked glycosylation of T. reesei CBH (both Cel6A and Cel7A) linkers and/or the cellulose-binding surface of carbohydrate-binding modules type I (CBM I) enhances the binding affinity of the enzyme to cellulose crystals [15,94]. Thus, there is a dual aspect to consider: on the one hand, glycosylation can impair heterologous CBH expression; on the other hand, glycosylation could be used as a tool for CBH engineering in a heterologous system to improve an enzyme’s mode of action in cellulose hydrolysis. The latter has been reviewed by Beckham et al. [95]. We have recently constructed a version of Cel7A from T. reesei in which the glycosylation site recommended by Adney et al. [46] was removed and expressed the gene in A. carbonarius, resulting in a slight improvement in expression and activity (unpublished results). In general, the negative influence of glycosylation on heterologous CBHs can be overcome either by enzymatic deglycosylation, restoring most of the native activity, or by synergy with additional enzymes, such as EGs, which will complement the debilitated activity of over-glycosylated proteins [92]. Deglycosylation is however not feasible at an industrial scale due to the reduced protein solubility and potential of aggregate formation [95,96], which might be especially problematic in enzymatic cellulose hydrolysis at a high solid substrate content to minimize enzyme costs [95,97]. Additionally, the application of extra EGs to complement the debilitated activity of glycosylated CBHs should be avoided to limit external enzyme supplementation and develop cost-effective biorefineries [98]. Another strategy includes the heterologous expression of CBHs in glycosylation-deficient strains, as was reported for a Saccharomyces cerevisiae mutant, in which the knockout of glycosylation-related genes resulted in significantly higher activity of heterologously expressed P. chrysosporium exocellulase [99]. In contrast, the inhibition of O-glycosylation in T. reesei [100] and mutation of O-glycosylation in A. nidulans [101] impaired native glycoprotein secretion, indicating that the exact correlation between glycosylation and protein secretion should be investigated before applying glycosylation-deficient strains in heterologous protein expression. Even small changes in the number of glycans attached (a single glycan) were reported to significantly influence enzyme performance [94]. In addition, differences in glycosylation patterns might also result from a variety of glycantrimming enzyme sets, which are specific for each expression host and can be modulated by culture conditions such as pH [85,91].

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Thus, it is important to use a reliable control system of glycosylation alterations during heterologous enzyme production. Recently, Gupta et al. [102] proposed a simple, rapid and automated method to determine the batch-to-batch consistency of the glycosylation patterns of heterologous CBHs.

6. Proteolytic degradation of heterologously expressed cellobiohydrolases Another reason for impaired heterologous protein production in filamentous fungi is proteolytic degradation, which causes both low yields of in vivo production and a further reduction in protein levels during downstream processing [103]. Proteolysis mainly accounts for the poor expression of non-fungal proteins [37] but has not been investigated in detail in reviewed studies on the heterologous expression of CBHs in filamentous fungi. In our laboratory, although either A. terreus or T. reesei cbhs were readily expressed in A. carbonarius, as shown by relatively high mRNA levels, they failed to be secreted in high amounts [21]. We speculated that proteolytic degradation could be one of the reasons for this result, possibly together with other factors such as incorrect folding, post-translational processing and impairment of intracellular transport. Similarly, the discrepancy between high mRNA levels and undetectable extracellular protein reported for the heterologous expression of Phlebia radiata lignin peroxidase in T. reesei was explained by the probable proteolytic degradation of misfolded protein (in addition to likely problems in mRNA translation) [104]. Essentially, it appears that a protein’s susceptibility to proteolysis depends on its sensitivity and varies from protein-to-protein, regardless of its fungal or non-fungal origin. Among cellulases, CBHs (Cel7A and Cel6A) of T. reesei are more sensitive to proteolytic degradation than either T. reesei EG (Cel7B) or BGL when secreted by the T. reesei QM 9414 mutant, which possesses high protease activity [105]. The in vitro studies carried out by Hagspiel et al. [105] and Dunne [106] showed that the acid proteases of T. reesei only degraded native CBHs to a limited extent. The degree of proteolysis might be influenced by glycosylation (especially of CBH linkers), which protects protein from degradation [43,107]. Thus, heterologous proteins with attached non-native glycans that are possibly also incorrectly folded might be more prone to protease attack. T. reesei Cel7A expressed in A. oryzae was proteolytically degraded during later stages of cultivation, as shown by the disappearance of an SDS-PAGE protein band using 6- and 7-day-old cultures [62]. Similarly, the enzyme activity of T. reesei Cel7A expressed in A. niger var. awamori using a non-native A. niger glucoamylase signal peptide was lost after 72 h of cultivation due to protease degradation [42]. In addition to their undesired influence on cellulase yields, intracellular proteases are hypothesized to positively control enzyme release, activity, stability and posttranslational modifications, resulting in the production of enzyme isoforms [105,106,108], and the type and extent of these proteolytic modifications might vary in different heterologous hosts [109]. Protease degradation was found to be decreased, inhibited or even eliminated by (1) the manipulation of bioprocessing parameters such as fungal morphology, cell immobilization, culture conditions, (2) the addition of protease inhibitors, or (3) the construction of protease-deficient strains [103,110–113]. However, none of these methods was shown to completely eliminate proteolysis. The fine-tuning of bioprocessing conditions was the focus of the review by Wang et al. [111], in which it was evaluated that a high concentration of ammonium and glucose restricts the secretion of extracellular proteases. Regardless, this carbon source cannot be applied if the aim is to produce heterologous CBHs together with other native cellulases because the expression (most likely regulated by native inducible promoters) of the latter

217

enzymes will be repressed at a high glucose level, which is known to inhibit cellulolytic enzyme production. Both alkaline and acid proteases could be responsible for the degradation of heterologous proteins in filamentous fungi, and the predominant production of these proteases depends on the species [103]. The activity of acidic proteases was shown to decrease steadily at a pH greater than 3.0 and was significantly lower between pH 5.0 and 6.0 for A. niger culture broth [114]. The activity of alkaline proteases could be reduced by gene disruption [115].

6.1. Overcoming proteolytic degradation Proteolytic degradation occurs mainly during fungal growth and is caused by the combined action of intracellular and extracellular proteases. Thus, protease inhibitors should be added during the process of cultivation. However, the low stability of protease inhibitors limits their application to small-scale protein expression and would not be feasible in industrial processes [37]. Additionally, it was demonstrated that the use of accurate inhibitors for different types of proteases is required [116]. The construction of protease-deficient strains of Aspergillus, Trichoderma and Myceliophthora sp. for application in the heterologous expression of polypeptides, including cellulolytic enzymes, has been described [117–120]. Protease-deficient strains were also reported in the successful production of other heterologous fungal proteins such as lignin-degrading enzymes and lipases [121,122]. Nonetheless, the disruption of protease genes might lead to an undesirable reduction of strain capacity in industrial protein production [111].

7. Other post-translational modifications and quality control of heterologously expressed cellobiohydrolases The formation of disulfide bridges is another post-translational modification that is impacted by heterologous expression in eukaryotic and prokaryotic hosts [44,123]. In general, the disulfide bonds formed between two cysteine residues during protein folding are responsible for protein stability. For example, the engineered disulfide bridges of Talaromyces emersonii CBH that was expressed in S. cerevisiae improved enzyme thermostability compared to the wildtype T. emersonii enzyme; this allowed for efficient microcrystalline cellulose hydrolysis at elevated temperatures [124]. The engineering of disulfide bridges should be considered for the production of highly thermostable CBHs, but the engineering needs to be targeted, such that the overall structure of the enzyme is not substantially altered, which might consequently result in misfolding of the expressed protein and its degradation within the endoplasmic reticulum [34,35]. The heterologous expression of multidomain proteins containing multiple disulfide bridges is generally more complex than the expression of single-domain proteins due to their complicated folding (W. Ubhayasekera, pers. comm.). Cellobiohydrolases contain numerous disulfide bridges and possess a two-domain structure, comprising the catalytic module and the carbohydrate-binding module connected by a flexible linker; thus, their folding in heterologous hosts might be problematic. In addition to glycosylation and proteolytic degradation, improper folding likely accounts for difficulties in heterologous CBH production. The expression of heterologous proteins in filamentous fungi could be improved by modulating protein quality control mechanisms: the unfolded protein response (UPR) and endoplasmic reticulum-associated protein response (ERAD). It was shown that the constitutive induction of UPR as well as the overexpression of UPR components increased the expression of heterologous laccase and manganese peroxidase in A. niger [125,126].

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8. Concluding remarks and perspectives for industrial cellobiohydrolase production The heterologous expression of well-known T. reesei CBHs and less characterized enzymes from P. funiculosum, A. aculeatus and M. albomyces has been reported in industrially exploited filamentous fungal strains such as A. niger var. awamori, A. oryzae, T. reesei or A. gossypii. The construction of efficient expression hosts is a complex process [35] and typically involves random mutagenesis followed by further optimization of desired features using genetic engineering, including the targeted deletion of proteases [34]. In comparison to other enzymes, the heterologous expression of CBHs appears to be challenging. A detailed understanding of heterologous CBH expression in filamentous fungi is important for the development of optimal key enzymes for the conversion of recalcitrant biomass in biorefineries. Moreover, the general findings from such studies could be applied to the heterologous production of other lignocellulolytic enzymes [46]. By exploiting Trichoderma and Aspergillus species in industrial protein production [127,128], strong promoter variants have been successfully isolated and applied, for example, the T. reesei cbh1 or A. niger glucoamylase promoters [34] that were the most commonly applied in the reviewed studies. The limited number of vectors available for heterologous protein expression in filamentous fungi compared to a wide variety of vectors for E. coli and S. cerevisiae is a potential limiting factor for achieving high yields of heterologously expressed enzymes [35,112]. For example, features such as autonomous replication, which are present in bacterial and yeast vectors are not available in a broad range of fungal species [129]. The construction of plasmids that integrate at specific highly transcribed chromosomal loci by homologous recombination should be further advanced to eliminate the negative effect of the integration site [130]. Strategies for site-specific integration, including Agrobacterium tumefaciensmediated DNA transformation and strain engineering, have been previously described [35,130]. Here, we describe that promoters that are homologous to the host and the native signal peptides of expressed proteins are the most efficient for heterologous CBH expression and secretion. For the industrial-scale production of heterologous CBHs with high activity toward recalcitrant crystalline cellulose and with broad thermostability, fungal expression strains should be designed with optimal glycosylation and limited proteolysis. Due to the complexity of glycosylation pathways and its important role in the secretion of homologous proteins in filamentous fungi, the engineering (addition or deletion) of CBH glycosylation sites [46] appears to be a more efficient strategy than modification of the glycosylation system of the heterologous host for achieving improved cellulose conversion. Although the construction of protease-deficient strains is already well established, to our knowledge, the effect of applying protease-deficient strains to heterologous CBH expression has not been reported. Finally, the efficient heterologous CBH expression system should encourage the development of the protein engineering that is crucial for designing improved enzymes for the conversion of pretreated biomass since it has been found that the pretreatment of lignocellulosic biomass results in cellulose with properties that are different from those of natural plant cellulose, for which CBHs originally evolved [7]. Further improvement in heterologous CBH production could be facilitated by various genomic and proteomic strategies, enabling the discovery of novel CBHs, identification of protease genes for the construction of protease-deficient strains, detection of highly secreted proteins for the construction of fusion partners [131], determination of efficient promoters [34], investigations of accessory proteins, such as AA9 family enzymes, for their influence on the efficiency of CBHs and other cellulases, and finally analysis of protein glycosylation for the engineering of heterologous hosts [87]

and enzyme activity [95]. Overcoming these barriers will pave the way for the development of efficient on-site enzyme producers or fungal strains for consolidated bioprocessing to directly convert biomass into useful products. Acknowledgements This work was financially supported by the Danish Council for Strategic Research (MycoFuelChem, project no. 0603-00499B). The authors would like to acknowledge Dr. Wimal Ubhayasekera for the preparation of Fig. 1. References [1] Star-COLIBRI. Joint European biorefinery vision for 2030; 2011. Available from: http://www.star-colibri.eu/publications/star-colibri-publications/ [2] Macqueen D. Bundles of energy: the case for renewable biomass energy: natural resource issues No. 24. International Institution for Environment and Development: London; 2011. [3] Sticklen MB. Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 2008;9:433–43. [4] Ruel K, Nishiyama Y, Joseleau J. Crystalline and amorphous cellulose in the secondary walls of Arabidopsis. Plant Sci 2012;193–194:48–61. [5] Ritter S. News of the week: a fast track to green gasoline. Chem Eng News 2008;86:10. [6] Gross AS, Chu J. On the molecular origins of biomass recalcitrance: the interaction network and solvation structures of cellulose microfibrils. J Phys Chem B 2010;114:13333–41. [7] Beckham GT, Matthews JF, Peters B, Bomble YJ, Himmel ME, Crowley MF. Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs. J Phys Chem B 2011;115:4118–27. [8] Himmel ME, Ding S, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 2007;315:804–7. [9] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol 2002;83:1–11. [10] Palonen H, Tjerneld F, Zacchi G, Tenkanen M. Adsorption of Trichoderma reesei CBH I and EG II and their catalytic domains on steam pretreated softwood and isolated lignin. J Biotechnol 2004;107:65–72. [11] Öhgren K, Bura R, Saddler J, Zacchi G. Effect of hemicellulose and lignin removal on enzymatic hydrolysis of steam pretreated corn stover. Bioresour Technol 2007;98:2503–10. [12] Divne C, Ståhlberg J, Teeri TT, Jones TA. High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A˚ long tunnel of cellobiohydrolase I from Trichoderma reesei. J Mol Biol 1998;275:309–25. [13] Xi J, Du W, Zhong L. Probing the interaction between cellulose and cellulase with a nanomechanical sensor. In: Van De Ven TGM, editor. Cellulose – medical, pharmaceutical and electronic applications. Rijeka: InTech; 2013., ISBN 978-953-51-1191-7, http://dx.doi.org/10.5772/50285. [14] Taylor CB, Payne CM, Himmel ME, Crowley MF, McCabe C, Beckham GT. Binding site dynamics and aromatic–carbohydrate interactions in processive and non-processive family 7 glycoside hydrolases. J Phys Chem B 2013;117:4924–33. [15] Payne CM, Resch MG, Chen L, Crowley MF, Himmel ME, Taylor LE, Sandgren M, Ståhlberg J, Stals I, Tan Z. Glycosylated linkers in multimodular lignocellulosedegrading enzymes dynamically bind to cellulose. Proc Natl Acad Sci U S A 2013;110:14646–51. [16] GhattyVenkataKrishna P, Alekozai E, Beckham G, Schulz R, Crowley M, Uberbacher E, Cheng X. Initial recognition of a cellodextrin chain in the cellulose-binding tunnel may affect cellobiohydrolase directional specificity. Biophys J 2013;104:904–12. [17] Gusakov AV. Alternatives to Trichoderma reesei in biofuel production. Trends Biotechnol 2011;29:419–25. ˜ IG, Vasella A, Ståhlberg J, Mowbray SL. Structures of [18] Ubhayasekera W, Munoz Phanerochaete chrysosporium Cel7D in complex with product and inhibitors. FEBS J 2005;272:1952–64. [19] Phillips CM, Iavarone AT, Marletta MA. Quantitative proteomic approach for cellulose degradation by Neurospora crassa. J Proteome Res 2011;10:4177–85. [20] Mahajan S, Master ER. Proteomic characterization of lignocellulose-degrading enzymes secreted by Phanerochaete carnosa grown on spruce and microcrystalline cellulose. Appl Microbiol Biotechnol 2010;86:1903–14. [21] Kolasa M. Enzymes for efficient and cost–effective hydrolysis of pre-treated biomass. Copenhagen: Aalborg University; 2014 [Ph. D. thesis]. [22] Adney WS, Himmel ME, Decker SR, Knoshaug EP, Nimlos MR, Crowley MF, Jeoh T. Cellobiohydrolase I. enzymes. U.S Patent Application 12/123,352. Washington, DC: U.S. Patent and Trademark Office; 2008. [23] Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes – factors affecting enzymes, conversion and synergy. Biotechnol Adv 2012;30:1458–80.

M. Zoglowek et al. / Process Biochemistry 50 (2015) 211–220 [24] Wood TM, McCrae SI. The cellulase of Trichoderma koningii. Purification and properties of some endoglucanase components with special reference to their action on cellulose when acting alone and in synergism with the cellobiohydrolase. Biochem J 1978;171:61–72. [25] Wood TM, McCrae SI. Synergism between enzymes involved in the solubilization of native cellulose. Adv Chem Ser 1979;181:181–209. [26] Ryu DDY, Kim C, Mandels M. Competitive adsorption of cellulase components and its significance in a synergistic mechanism. Biotechnol Bioeng 1984;26:488–96. [27] Sternberg D, Vuayakumar P, Reese E. ␤-glucosidase: microbial production and effect on enzymatic hydrolysis of cellulose. Can J Microbiol 1977;23:139–47. [28] Quinlan RJ, Sweeney MD, Leggio LL, Otten H, Poulsen JN, Johansen KS, Krogh KB, Jørgensen CI, Tovborg M, Anthonsen A. Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A 2011;108:15079–84. [29] Novozymes A/S. Cellulosic ethanol. Novozymes Cellic CTec3. (Application sheet No.2012-01394-03); 2012. Avialable from: http://www.bioenergy. novozymes.com/en/cellulosic-ethanol/Cellic-HTec3/Documents/CE APP Cellic Ctec3.pdf [30] Novozymes World’s first advanced biofuels facility opens; 2013. Retrieved from: http://www.novozymes.com/en/news/news-archive/Pages/World%E2 %80%99s-first-advanced-biofuels-facility-opens.aspx [31] Haan R, Kroukamp H, van Zyl JH, van Zyl WH. Cellobiohydrolase secretion by yeast: current state and prospects for improvement. Process Biochem 2012;48:1–12. [32] Wells AS, Finch GL, Michels PC, Wong JW. Use of enzymes in the manufacture of active pharmaceutical ingredients – a science and safety-based approach to ensure patient safety and drug quality. Org Process Res Dev 2012;16:1986–93. [33] Amfep List of commercial enzymes; 2009. Retrieved from: http://www. amfep.org/content/list-enzymes [34] Nevalainen KMH, Te’o VSJ, Bergquist PL. Heterologous protein expression in filamentous fungi. Trends Biotechnol 2005;23:468–74. [35] Fleißner A, Dersch P. Expression and export: recombinant protein production systems for Aspergillus. Appl Microbiol Biotechnol 2010;87:1255–70. [36] Ward OP. Production of recombinant proteins by filamentous fungi. Biotechnol Adv 2012;30:1119–39. [37] Wiebe MG. Stable production of recombinant proteins in filamentous fungiproblems and improvements. Mycologist 2003;17:140–4. [38] Jeenes DJ, Mackenzie DA, Roberts IN, Archer DB. Heterologous protein production by filamentous fungi. Biotechnol Genet Eng Rev 1991;9:327–68. [39] Gouka R, Punt P, Van Den Hondel C. Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol 1997;47:1–11. [40] Archer DB. Enzyme production by recombinant Aspergillus. Bioprocess Technol 1994;19:373–93. [41] Berka RM, Barnett CC. The development of gene expression systems for filamentous fungi. Biotechnol Adv 1989;7:127–54. [42] Adney WS, Chou Y, Decker SR, Ding S, Baker JO, Kunkel G, Vinzant TB, Himmel ME. Heterologous expression of Trichoderma reesei 1, 4-ß-d-glucan cellobiohydrolase (Cel 7A). Am Chem Soc 2003;855:403–37. [43] Taylor IILE, Dai Z, Decker SR, Brunecky R, Adney WS, Ding S, Himmel ME. Heterologous expression of glycosyl hydrolases in planta: a new departure for biofuels. Trends Biotechnol 2008;26:413–24. [44] Boer H, Teeri TT, Koivula A. Characterization of Trichoderma reesei cellobiohydrolase Cel7A secreted from Pichia pastoris using two different promoters. Biotechnol Bioeng 2000;69:486–94. [45] Lantz SE, Goedegebuur F, Hommes R, Kaper T, Kelemen BR, Mitchinson C, Wallace L, Ståhlberg J, Larenas EA. Hypocrea jecorina CEL6A protein engineering. Biotechnol Biofuels 2010;3:20. [46] Adney WS, Jeoh T, Beckham GT, Chou Y, Baker JO, Michener W, Brunecky R, Himmel ME. Probing the role of N-linked glycans in the stability and activity of fungal cellobiohydrolases by mutational analysis. Cellulose 2009;16:699–709. [47] Bower BS, Larenas EA. Heterologous and homologous cellulase expression system. US Patent 20,100,323,426. Washington, DC: U.S. Patent and Trademark Office; 2010. [48] Ma L, Zhang J, Zou G, Wang C, Zhou Z. Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens. Enzyme Microb Technol 2011;49:366–71. [49] Dashtban M, Qin W. Overexpression of an exotic thermotolerant ␤glucosidase in Trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw. Microb Cell Fact 2012;11:1–15. [50] Nakazawa H, Kawai T, Ida N, Shida Y, Kobayashi Y, Okada H, Tani S, Sumitani J, Kawaguchi T, Morikawa Y. Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus ␤-glucosidase 1 for efficient biomass conversion. Biotechnol Bioeng 2012;109:92–9. [51] Sørensen A, Lübeck M, Lübeck PS, Ahring BK. Fungal beta-glucosidases. A bottleneck in industrial use of lignocellulosic materials. Biomolecules 2013;3:612–31. [52] Wen Z, Liao W, Chen S. Production of cellulase/␤-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure. Process Biochem 2005;40:3087–94. [53] Ghose T, Panda T, Bisaria V. Effect of culture phasing and mannanase on production of cellulase and hemicellulase by mixed culture of Trichoderma reesei D 1–6 and Aspergillus wentii pt 2804. Biotechnol Bioeng 1985;27: 1353–61.

219

[54] Fang H, Zhao C, Song X. Optimization of enzymatic hydrolysis of steamexploded corn stover by two approaches: response surface methodology or using cellulase from mixed cultures of Trichoderma reesei RUT-C30 and Aspergillus niger NL02. Bioresour Technol 2010;101:4111–9. [55] Bryant C. The economics of enzyme production; 2010. Retrieved from: http://www.bioenergy.novozymes.com/en/learn-more/webinars/Documents/ Theeconomicsofonsiteenzymeproduction.pdf [56] Gutierrez-Correa M, Portal L, Moreno P, Tengerdy RP. Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane bagasse. Bioresour Technol 1999;68:173–8. [57] Ribeiro O, Wiebe M, Ilmen M, Domingues L, Penttilä M. Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii. Appl Microbiol Biotechnol 2010;87:1437–46. [58] Radzio R, Kück U. Synthesis of biotechnologically relevant heterologous proteins in filamentous fungi. Process Biochem 1997;32:529–39. [59] Thrane C, Lübeck M, Green H, Degefu Y, Allerup S, Thrane U, Jensen DF. A tool for monitoring Trichoderma harzianum: I. Transformation with the GUS gene by protoplast technology. Phytopathol 1995;85:1428–35. [60] Lübeck M, Knudsen I, Jensen B, Thrane U, Janvier C, Funck Jensen D. GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies. Mycol Res 2002;106: 815–26. [61] Keränen S, Penttilä M. Production of recombinant proteins in the filamentous fungus Trichoderma reesei. Curr Opin Biotechnol 1995;6:534–7. [62] Takashima S, Iikura H, Nakamura A, Hidaka M, Masaki H, Uozumi T. Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties. J Biotechnol 1998;65:163–71. [63] Chou YC, Adney WS, Decker SR, Baker JO, Kunkel G, Templeton DW, Himmel ME. Cloning and heterologous expression of the gene encoding a family 7 glycosyl hydrolase from Penicillium funiculosum. ACS Symp Ser 2004;889:170–93. [64] Kanamasa S, Mochizuki M, Takada G, Kawaguchi T, Sumitani J, Araii M. Overexpression of Aspergillus aculeatus cellobiohydrolase I in Aspergillus oryzae. J Biosci Bioeng 2003;95:627–9. [65] Szijártó N, Siika-aho M, Tenkanen M, Alapuranen M, Vehmaanperä J, Réczey K, Viikari L. Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces. J Biotechnol 2008;136: 140–7. [66] Haakana H, Miettinen-Oinonen A, Joutsjoki V, Mäntylä A, Suominen P, Vehmaanperä J. Cloning of cellulase genes from Melanocarpus albomyces and their efficient expression in Trichoderma reesei. Enzyme Microb Technol 2004;34:159–67. [67] Voutilainen SP, Boer H, Alapuranen M, Jänis J, Vehmaanperä J, Koivula A. Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B. Appl Microbiol Biotechnol 2009;83:261–72. [68] Zou G, Shi S, Jiang Y, van den Brink J, de Vries RP, Chen L, Zhang J, Ma L, Wang C, Zhou Z. Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering. Microb Cell Fact 2012;11:21. [69] Grønbech M [M. Sc. thesis] Consolidated bioprocessing: expression of cellobiohydrolase 1 from Trichoderma reesei in Aspergillus carbonarius with a wildtype and mutated promoter. Copenhagen: Aalborg University; 2013. [70] Szocs-Puskas T [M. Sc. thesis] Laccase for improving the lignocellulolytic capacity of Aspergillus carbonarius. Copenhagen: Aalborg University; 2013. [71] Crespo-Sempere A, López-Pérez M, Martínez-Culebras P, González-Candelas L. Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes. Int J Food Microbiol 2011;148:135–40. [72] Hansen NB, Lübeck M, Lübeck PS. Advancing USER cloning into simple USER and nicking cloning. J Microbiol Methods 2014;96:42–9. [73] El-Enshasy H, Hellmuth K, Rinas U. GpdA-promoter-controlled production of glucose oxidase by recombinant Aspergillus niger using nonglucose carbon sources. Appl Biochem Biotechnol 2001;90:57–66. [74] Gellissen G. Production of recombinant proteins - novel microbial and eukaryotic expression systems. Weinheim: Wiley-VCH; 2006. [75] Redkar RJ, Herzog RW, Singh NK. Transcriptional activation of the Aspergillus nidulans gpdA promoter by osmotic signals. Appl Environ Microbiol 1998;64:2229–31. [76] Kubicek-Pranz EM, Gruber F, Kubicek CP. Transformation of Trichoderma reesei with the cellobiohydrolase II gene as a means for obtaining strains with increased cellulase production and specific activity. J Biotechnol 1991;20:83–94. [77] Van den Hondel CA, Punt PJ, van Gorcom RF. Heterologous gene expression in filamentous fungi. More gene manipulations in fungi. San Diego: Academic Press; 1991. p. 396–428. [78] Rothe C, Lehle L. Sorting of invertase signal peptide mutants in yeast dependent and independent on the signal-recognition particle. Eur J Biochem 1998;252:16–24. [79] Ng D, Brown JD, Walter P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 1996;134:269–78. [80] Matoba S, Ogrydziak DM. Another factor besides hydrophobicity can affect signal peptide interaction with signal recognition particle. J Biol Chem 1998;273:18841–7. [81] Chou YC, Adney WS, Decker SR, Baker JO, Himmel ME. Heterologous expression, purification, and characterization of a cellobiohydrolase from Penicillium funiculosum. NREL Report No. PO-510-34326; 2003. p. 1. Retrieved from: http://www1.eere.energy.gov/bioenergy/pdfs/34326.pdf

220

M. Zoglowek et al. / Process Biochemistry 50 (2015) 211–220

[82] Bower BS, Larenas EA, Mitchinson C. Exo-endo cellulase fusion protein. U.S. Patent No. 8,097,445. Washington, DC: U.S. Patent and Trademark Office; 2012. [83] Levasseur A, Navarro D, Punt PJ, Belaïch J, Asther M, Record E. Construction of engineered bifunctional enzymes and their overproduction in Aspergillus niger for improved enzymatic tools to degrade agricultural by-products. Appl Environ Microbiol 2005;71:8132–40. [84] Punt PJ, Levasseur A, Visser H, Wery J, Record E. Fungal protein production: design and production of chimeric proteins. Annu Rev Microbiol 2011;65:57–69. [85] Stals I, Sandra K, Geysens S, Contreras R, Van Beeumen J, Claeyssens M. Factors influencing glycosylation of Trichoderma reesei cellulases. I: postsecretorial changes of the O-and N-glycosylation pattern of Cel7A. Glycobiology 2004;14:713–24. [86] Eriksson T, Stals I, Collén A, Tjerneld F, Claeyssens M, Stålbrand H, Brumer H. Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma reesei) Cel7B catalytic module. Eur J Biochem 2004;271:1266–76. [87] Deshpande N, Wilkins MR, Packer N, Nevalainen H. Protein glycosylation pathways in filamentous fungi. Glycobiology 2008;18:626–37. [88] Lommel M, Strahl S. Protein O-mannosylation. Conserved from bacteria to humans*. Glycobiology 2009;19:816–28. [89] Harrison MJ, Nouwens AS, Jardine DR, Zachara NE, Gooley AA, Nevalainen H, Packer NH. Modified glycosylation of cellobiohydrolase I from a high cellulase-producing mutant strain of Trichoderma reesei. Eur J Biochem 1998;256:119–27. [90] Hui JP, Lanthier P, White TC, McHugh SG, Yaguchi M, Roy R, Thibault P. Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry. J Chromatogr B 2001;752:349–68. [91] Stals I, Sandra K, Devreese B, Van Beeumen J, Claeyssens M. Factors influencing glycosylation of Trichoderma reesei cellulases. II: N-glycosylation of Cel7A core protein isolated from different strains. Glycobiology 2004;14:725–37. [92] Jeoh T, Michener W, Himmel ME, Decker SR, Adney WS. Implications of cellobiohydrolase glycosylation for use in biomass conversion. Biotechnol Biofuels 2008;1:10. [93] Imperiali B, O’Connor SE. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr Opin Chem Biol 1999;3:643–9. [94] Taylor CB, Talib MF, McCabe C, Bu L, Adney WS, Himmel ME, Crowley MF, Beckham GT. Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module. J Biol Chem 2012;287:3147–55. [95] Beckham GT, Dai Z, Matthews JF, Momany M, Payne CM, Adney WS, Baker SE, Himmel ME. Harnessing glycosylation to improve cellulase activity. Curr Opin Biotechnol 2012;23:338–45. [96] Wang C, Eufemi M, Turano C, Giartosio A. Influence of the carbohydrate moiety on the stability of glycoproteins. Biochem 1996;35:7299–307. [97] Humbird D, Mohagheghi A, Dowe N, Schell DJ. Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover. Biotechnol Prog 2010;26:1245–51. [98] Banerjee G, Scott-Craig JS, Walton JD. Improving enzymes for biomass conversion: a basic research perspective. Bioenergy Res 2010;3: 82–92. [99] Wang T, Huang C, Chen H, Ho P, Ke H, Cho H, Ruan S, Hung K, Wang I, Cai Y. Systematic screening of glycosylation-and trafficking-associated gene knockouts in Saccharomyces cerevisiae identifies mutants with improved heterologous exocellulase activity and host secretion. BMC Biotechnol 2013;13: 71. [100] Kubicek C, Panda T, Schreferl-Kunar G, Gruber F, Messner R. O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei. Can J Microbiol 1987;33:698–703. ´ ˛ ´ [101] Perlinska-Lenart U, Kurzatkowski W, Janas P, Kopinska A, Palamarczyk G, Kruszewska JS. Protein production and secretion in an Aspergillus nidulans mutant impaired in glycosylation. Acta Biochim Pol 2005;52:195–205. [102] Gupta R, Baldock SJ, Fielden PR, Grieve BD. Capillary zone electrophoresis for the analysis of glycoforms of cellobiohydrolase. J Chromatogr A 2011;1218:5362–8. [103] van den Hombergh JP, van de Vondervoort Peter J, Fraissinet-Tachet L, Visser J. Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol 1997;15:256–63. [104] Saloheimo M, Barajas V, Niku-Paavola M, Knowles JK. A lignin peroxidaseencoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei. Gene 1989;85:343–51. [105] Hagspiel K, Haab D, Kubicek CP. Protease activity and proteolytic modification of cellulases from a Trichoderma reesei QM 9414 selectant. Appl Microbiol Biotechnol 1989;32:61–7. [106] Dunne C. Relationship between extracellular proteases and the cellulase complex of Trichoderma reesei. In: Enzyme engineering. New York: Springer; 1982. p. 355–6. [107] Gustavsson M, Lehtiö J, Denman S, Teeri TT, Hult K, Martinelle M. Stable linker peptides for a cellulose-binding domain-lipase fusion protein expressed in Pichia pastoris. Protein Eng 2001;14:711–5. [108] Knowles J, Lehtovaara P, Teeri T. Cellulase families and their genes. Trends Biotechnol 1987;5:255–61. [109] Eneyskaya EV, Kulminskaya AA, Savel’ev AN, Savel’eva NV, Shabalin KA, Neustroev KN. Acid protease from Trichoderma reesei: limited proteolysis of fungal carbohydrases. Appl Microbiol Biotechnol 1999;52:226–31.

[110] 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. [111] Wang L, Ridgway D, Gu T, Moo-Young M. Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations. Biotechnol Adv 2005;23:115–29. [112] Sharma R, Katoch M, Srivastava P, Qazi G. Approaches for refining heterologous protein production in filamentous fungi. World J Microbiol Biotechnol 2009;25:2083–94. [113] Culleton H, McKie V, de Vries RP. Physiological and molecular aspects of degradation of plant polysaccharides by fungi: what have we learned from Aspergillus? Biotechnol J 2013;8:884–94. [114] 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 J 2001;8:187–93. [115] Zhang G, Zhu Y, Wei D, Wang W. Enhanced production of heterologous proteins by the filamentous fungus Trichoderma reesei via disruption of the alkaline serine protease SPW combined with a pH control strategy. Plasmid 2014;71:16–22. [116] Haab D, Hagspiel K, Szakmary K, Kubicek CP. Formation of the extracellular proteases from Trichoderma reesei QM 9414 involved in cellulase degradation. J Biotechnol 1990;16:187–98. [117] Lehmbeck J. Alkaline protease deficient filamentous fungi. EP Patent 0,894,126. Munich, Germany: European Patent Office; 2006. [118] Connelly M, Brody H. Methods for producing biological substances in enzymedeficient mutants of Aspergillus niger. US Patent 8,445,283. Washington, DC: U.S. Patent and Trademark Office; 2013. [119] Maiyuran S, Jang A, Brown K, Merino S, Shasky J, Abbate E. Methods for producing polypeptidies in protease-deficient mutants of Trichoderma. US Patent 20,120,288,892. Washington, DC: U.S. Patent and Trademark Office; 2012. [120] Visser H, Joosten V, Punt PJ, Gusakov AV, Olson PT, Joosten R, Bartels J, Visser J, Sinitsyn AP, Emalfarb MA. Research development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1. Ind Biotechnol 2011;7:214–23. [121] Conesa A, van den Hondel CA, Punt PJ. Studies on the production of fungal peroxidases in Aspergillus niger. Appl Environ Microbiol 2000;66:3016–23. [122] Shu Z, Jiang H, Lin R, Jiang Y, Lin L, Huang J. Technical methods to improve yield, activity and stability in the development of microbial lipases. J Mol Catal B 2010;62:1–8. [123] Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C. Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Fact 2008;7:11. [124] Voutilainen SP, Murray PG, Tuohy MG, Koivula A. Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity. Protein Eng Des Sel 2010;23:69–79. [125] Conesa A, Jeenes D, Archer DB, van den Hondel Cees AMJJ, Punt PJ. Calnexin overexpression increases manganese peroxidase production in Aspergillus niger. Appl Environ Microbiol 2002;68:846–51. [126] Valkonen M, Ward M, Wang H, Penttilä M, Saloheimo M. Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl Environ Microbiol 2003;69:6979–86. [127] Peterson R, Nevalainen H. Trichoderma reesei RUT-C30-thirty years of strain improvement. Microbiol 2012;158:58–68. [128] Lubertozzi D, Keasling JD. Developing Aspergillus as a host for heterologous expression. Biotechnol Adv 2009;27:53–75. [129] Nevalainen H, Peterson R. Making recombinant proteins in filamentous fungiare we expecting too much? Front Microbiol 2014;5:75. [130] 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. [131] Han MJ, Kim NJ, Lee SY, Chang HN. Extracellular proteome of Aspergillus terreus grown on different carbon sources. Curr Genet 2010;56:369–82. [132] Tomme P, Van Tilbeurgh H, Pettersson G, Van Damme J, Vandekerckhove J, Knowles J, Teeri T, Claeyssens M. Studies of the cellulolytic system of Trichoderma reesei QM 9414. Eur J Biochem 1988;170:575–81. [133] Tsuchiya K, Nagashima T, Yamamoto Y, Gomi K, Kitamoto K, Kumagai C, Tamura G. High level secretion of calf chymosin using a glucoamylaseprochymosin fusion gene in Aspergillus oryzae. Biosci Biotechnol Biochem 1994;58:895–9. [134] Joutsjoki VV, Torkkeli TK, Nevalainen KH. Transformation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: production of a heterologous glucoamylase by Trichoderma reesei. Curr Genet 1993;24:223–8. [135] Suominen PL, Mantyla AL, Karhunen T, Hakola S, Nevalainen H. High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes. Mol Gen Genet 1993;241:523–30. [136] Paloheimo M, Mäntylä A, Kallio J, Suominen P. High-yield production of a bacterial xylanase in the filamentous fungus Trichoderma reesei requires a carrier polypeptide with an intact domain structure. Appl Environ Microbiol 2003;69:7073–82. [137] Mandels M, Andreotti R. Problems and challenges in the cellulose to cellulase fermentation. Proc Biochem 1978;13:6–13.