Elsevier

Process Biochemistry

Volume 50, Issue 2, February 2015, Pages 211-220
Process Biochemistry

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

https://doi.org/10.1016/j.procbio.2014.12.018Get rights and content

Highlights

  • Heterologous expression of CBHs in single hosts may produce multi-enzyme cocktails.

  • Activity of heterologously expressed CBHs is (often) lower than of the native enzyme.

  • Native inducible promoters are most successful for heterologous expression of CBHs.

  • Over-glycosylation of heterologously produced CBHs decreases enzyme activity.

  • Modulating protein quality control mechanisms in the host could improve CBH secretion.

Abstract

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.

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.

References (137)

  • M.G. Wiebe

    Stable production of recombinant proteins in filamentous fungi-problems and improvements

    Mycologist

    (2003)
  • R.M. Berka et al.

    The development of gene expression systems for filamentous fungi

    Biotechnol Adv

    (1989)
  • L. Ma et al.

    Improvement of cellulase activity in Trichoderma reesei by heterologous expression of a beta-glucosidase gene from Penicillium decumbens

    Enzyme Microb Technol

    (2011)
  • Z. Wen et al.

    Production of cellulase/β-glucosidase by the mixed fungi culture Trichoderma reesei and Aspergillus phoenicis on dairy manure

    Process Biochem

    (2005)
  • H. Fang et al.

    Optimization of enzymatic hydrolysis of steam-exploded 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)
  • M. Gutierrez-Correa et al.

    Mixed culture solid substrate fermentation of Trichoderma reesei with Aspergillus niger on sugar cane bagasse

    Bioresour Technol

    (1999)
  • R. Radzio et al.

    Synthesis of biotechnologically relevant heterologous proteins in filamentous fungi

    Process Biochem

    (1997)
  • M. Lübeck et al.

    GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies

    Mycol Res

    (2002)
  • S. Keränen et al.

    Production of recombinant proteins in the filamentous fungus Trichoderma reesei

    Curr Opin Biotechnol

    (1995)
  • S. Takashima et al.

    Overproduction of recombinant Trichoderma reesei cellulases by Aspergillus oryzae and their enzymatic properties

    J Biotechnol

    (1998)
  • S. Kanamasa et al.

    Overexpression of Aspergillus aculeatus cellobiohydrolase I in Aspergillus oryzae

    J Biosci Bioeng

    (2003)
  • N. Szijártó et al.

    Hydrolysis of amorphous and crystalline cellulose by heterologously produced cellulases of Melanocarpus albomyces

    J Biotechnol

    (2008)
  • H. Haakana et al.

    Cloning of cellulase genes from Melanocarpus albomyces and their efficient expression in Trichoderma reesei

    Enzyme Microb Technol

    (2004)
  • A. Crespo-Sempere et al.

    Development of a green fluorescent tagged strain of Aspergillus carbonarius to monitor fungal colonization in grapes

    Int J Food Microbiol

    (2011)
  • N.B. Hansen et al.

    Advancing USER cloning into simple USER and nicking cloning

    J Microbiol Methods

    (2014)
  • E.M. Kubicek-Pranz et al.

    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)
  • C.A. Van den Hondel et al.

    Heterologous gene expression in filamentous fungi. More gene manipulations in fungi

    (1991)
  • S. Matoba et al.

    Another factor besides hydrophobicity can affect signal peptide interaction with signal recognition particle

    J Biol Chem

    (1998)
  • Y.C. Chou et al.

    Heterologous expression, purification, and characterization of a cellobiohydrolase from Penicillium funiculosum. NREL Report No. PO-510-34326

    (2003)
  • J.P. Hui et al.

    Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry

    J Chromatogr B

    (2001)
  • B. Imperiali et al.

    Effect of N-linked glycosylation on glycopeptide and glycoprotein structure

    Curr Opin Chem Biol

    (1999)
  • C.B. Taylor et al.

    Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module

    J Biol Chem

    (2012)
  • G.T. Beckham et al.

    Harnessing glycosylation to improve cellulase activity

    Curr Opin Biotechnol

    (2012)
  • Star-COLIBRI

    Joint European biorefinery vision for 2030

    (2011)
  • D. Macqueen

    Bundles of energy: the case for renewable biomass energy: natural resource issues No. 24

    (2011)
  • M.B. Sticklen

    Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol

    Nat Rev Genet

    (2008)
  • S. Ritter

    News of the week: a fast track to green gasoline

    Chem Eng News

    (2008)
  • A.S. Gross et al.

    On the molecular origins of biomass recalcitrance: the interaction network and solvation structures of cellulose microfibrils

    J Phys Chem B

    (2010)
  • G.T. Beckham et al.

    Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs

    J Phys Chem B

    (2011)
  • M.E. Himmel et al.

    Biomass recalcitrance: engineering plants and enzymes for biofuels production

    Science

    (2007)
  • J. Xi et al.

    Probing the interaction between cellulose and cellulase with a nanomechanical sensor

  • C.B. Taylor et al.

    Binding site dynamics and aromatic–carbohydrate interactions in processive and non-processive family 7 glycoside hydrolases

    J Phys Chem B

    (2013)
  • C.M. Payne et al.

    Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose

    Proc Natl Acad Sci U S A

    (2013)
  • W. Ubhayasekera et al.

    Structures of Phanerochaete chrysosporium Cel7D in complex with product and inhibitors

    FEBS J

    (2005)
  • C.M. Phillips et al.

    Quantitative proteomic approach for cellulose degradation by Neurospora crassa

    J Proteome Res

    (2011)
  • S. Mahajan et al.

    Proteomic characterization of lignocellulose-degrading enzymes secreted by Phanerochaete carnosa grown on spruce and microcrystalline cellulose

    Appl Microbiol Biotechnol

    (2010)
  • M. Kolasa

    Enzymes for efficient and cost–effective hydrolysis of pre-treated biomass

    (2014)
  • Adney WS, Himmel ME, Decker SR, Knoshaug EP, Nimlos MR, Crowley MF, Jeoh T. Cellobiohydrolase I. enzymes. U.S Patent...
  • T.M. Wood et al.

    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)
  • T.M. Wood et al.

    Synergism between enzymes involved in the solubilization of native cellulose

    Adv Chem Ser

    (1979)
  • Cited by (0)

    View full text