ReviewHeterologous expression of cellobiohydrolases in filamentous fungi – An update on the current challenges, achievements and perspectives
Section snippets
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 bio-based 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
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
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,
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
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 linkers [85]. Although glycosylation sites and pathways are relatively conserved in filamentous fungi [46], [86], [87], [88], the extent of attached glycans varies among strains [86], [89], [90], [91] and species [92], typically with different numbers of mannose residues attached. Glycosylation also varies
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
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
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
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.
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