Construction of a cellulase hyper-expression system in Trichoderma reesei by promoter and enzyme engineering

Abstract

BackgroundTrichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes.In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus.ResultsThe modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover (PCS) was dramatically increased by 40% at 55°C and 169% at 60°C when its enzyme mixture was used in the hydrolysis.ConclusionsThis study shows that optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes in T. reesei.

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

“…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].…”

How to achieve high-level expression of microbial enzymes

“…For example, the strong cbh1 (cellobiohydrolase I) promoter of T. reesei was used in the heterologous expression of cellulase. 170 Lv et al constructed two T. reesei expression vectors, pWEF31 and pWEF3, which are useful for the large-scale expression of the gene encoding the enzyme. 171 Three endoxylanase genes (Ct xyn11A, Ct xyn11B and Ct xyn11C) from the thermophilic fungus Chaetomium thermophilum CBS 730.95 were expressed in T. reesei under the control of the strong T. reesei cel7A (chh1) promoter.…”

Recombinant hyperthermophilic enzyme expression in plants: a novel approach for lignocellulose digestion

“…The most obvious is the relatively low level of in situ protein expression/accumulation observed in most studies (Table S1 in the supplementary material online), in which an order-of-magnitude increase in expression level is required for complete conversion of biomass [8,33]. In addition to the obvious requirement for careful codon optimization [8,46,59] and/or codon harmonization [69], the further development of combinations of transit peptides and promoters (strong promoters and chimeric promoters), with or without promoter engineering, may be effective strategies [52,70]. Carbohydrate-binding modules can also be used to target better the recombinant enzymes and ultimately improve biomass digestion efficiency [45].…”