Enhanced cellulase production from <i>Trichoderma reesei</i> Rut‐C30 by engineering with an artificial zinc finger protein library

Zhang, Fei; Bai, Feng‐Wu; Zhao, Xin‐Qing

doi:10.1002/biot.201600227

Cited by 38 publications

(20 citation statements)

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“…In summary, chimeric transcription activators CX C ‐S and CX C ‐L were constructed to combine the specificity of ClrB DNA‐binding domain for cellulase gene promoters and the constitutive transcriptional activation ability of XlnR A871V C‐terminal fragment . Overexpression of these chimeric regulators conferred deregulated expression of cellulase in P. oxalicum .…”

Section: Resultsmentioning

confidence: 99%

Constitutive Expression of Chimeric Transcription Factors Enables Cellulase Synthesis under Non‐Inducing Conditions in Penicillium oxalicum

Gao

Xia

et al. 2017

Biotechnology Journal

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Industrial production of cellulase by filamentous fungi is largely dependent on cellulose, which serves as a natural inducer of cellulase expression. However, insoluble cellulose is unfavorable to submerged fermentation and thus limits the production level of cellulase. The possibility of cellulase production under non-inducing conditions is explored in Penicillium oxalicum by overexpressing two chimeric transcription factors. The chimeric transcription factors contain the DNA binding domain of cellulase transcriptional activator ClrB linked to the C-terminal sequences of XlnR , a constitutively active mutant of hemicellulase transcriptional activator. The obtained recombinant mutants exhibited dramatically improved basal production of cellulase, which was not observed with the overexpression of intact ClrB. When cultivated in a complex cellulosic medium, one of these mutants, OE-CX -S-1, displayed a 7.3-fold increase in cellulase production (2.8 U mL ) relative to the parent strain. The results demonstrate that the dependence of cellulase synthesis on cellulose could be reduced by the overexpression of artificially designed chimeric transcription factors, and offers a potential strategy to engineer fungal strains for improving cellulase production.

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Section: Resultsmentioning

confidence: 99%

Constitutive Expression of Chimeric Transcription Factors Enables Cellulase Synthesis under Non‐Inducing Conditions in Penicillium oxalicum

Gao

Xia

et al. 2017

Biotechnology Journal

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“…The AZFPs were designed to be composed of four zinc fingers as a DNA-binding domain (DBD) followed by an AD of Gal4p (Gal4 AD ). We have modified the library and successfully obtained mutants with enhanced cellulase production in T. reesei Rut-C30 (Zhang et al, 2016). However, Gal4 AD in the AZFPs originates from budding yeast Saccharomyces cerevisiae, and it remains unknown whether exogenous transcriptional regulation domains can efficiently recruit protein complex to initiate transcription.…”

Section: Introductionmentioning

confidence: 99%

Engineering the Effector Domain of the Artificial Transcription Factor to Improve Cellulase Production by Trichoderma reesei

Meng

Zhang

Wang

et al. 2020

Front. Bioeng. Biotechnol.

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Filamentous fungal strains of Trichoderma reesei have been widely used for cellulase production, and great effort has been devoted to enhancing their cellulase titers for the economic biorefinery of lignocellulosic biomass. In our previous studies, artificial zinc finger proteins (AZFPs) with the Gal4 effector domain were used to enhance cellulase biosynthesis in T. reesei, and it is of great interest to modify the AZFPs to further improve cellulase production. In this study, the endogenous activation domain from the transcription activator Xyr1 was used to replace the activation domain of Gal4 of the AZFP to explore impact on cellulase production. The cellulase producer T. reesei TU-6 was used as a host strain, and the engineered strains containing the Xyr1 and the Gal4 activation domains were named as T. reesei QS2 and T. reesei QS1, respectively. Compared to T. reesei QS1, activities of filter paper and endoglucanases in crude cellulase produced by T. reesei QS2 increased 24.6 and 50.4%, respectively. Real-time qPCR analysis also revealed significant up-regulation of major genes encoding cellulase in T. reesei QS2. Furthermore, the biomass hydrolytic performance of the cellulase was evaluated, and 83.8 and 97.9% more glucose was released during the hydrolysis of pretreated corn stover using crude enzyme produced by T. reesei QS2, when compared to the hydrolysis with cellulase produced by T. reesei QS1 and the parent strain T. reesei TU-6. As a result, we proved that the effector domain in the AZFPs can be optimized to construct more effective artificial transcription factors for engineering T. reesei to improve its cellulase production.

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“…An artificial transcription activator containing the two DNA-binding domains from CREI and ACEI and an effector domain from ACEII can regulate gene expression in T. reesei [ 20 ]. Zhang et al [ 21 ] screened a mutant T. reesei strain U3 with enhanced cellulase production by transforming an artificial zinc finger protein library. Similarly, an artificial transcription activator linking the CREI-binding domain with the C-terminus of XYRI was shown to improve cellulase production in the recombinant strain T. reesei zxy-2 with glucose as the sole carbon source [ 22 ].…”

Section: Introductionmentioning

confidence: 99%

Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators

Zhang

Wang

et al. 2018

Microb Cell Fact

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BackgroundCellulase can convert lignocellulosic feedstocks into fermentable sugars, which can be used for the industrial production of biofuels and chemicals. The high cost of cellulase production remains a challenge for lignocellulose breakdown. Trichoderma reesei RUT C30 serves as a well-known industrial workhorse for cellulase production. Therefore, the enhancement of cellulase production by T. reesei RUT C30 is of great importance.ResultsTwo sets of novel minimal transcriptional activators (DBDace2-VP16 and DBDcre1-VP16) were designed and expressed in T. reesei RUT C30. Expression of DBDace2-VP16 and DBDcre1-VP16 improved cellulase production under induction (avicel or lactose) and repression (glucose) conditions, respectively. The strain TMTA66 under avicel and TMTA139 under glucose with the highest cellulase activities outperformed other transformants and the parental strain under the corresponding conditions. For TMTA66 strains, the highest FPase activity was approximately 1.3-fold greater than that of the parental strain RUT C30 at 120 h of cultivation in a shake flask using avicel as the sole carbon source. The FPase activity (U/mg biomass) in TMTA139 strains was approximately 26.5-fold higher than that of the parental strain RUT C30 at 72 h of cultivation in a shake flask using glucose as the sole carbon source. Furthermore, the crude enzymes produced in the 7-L fermenter from TMTA66 and TMTA139 supplemented with commercial β-glucosidase hydrolyzed pretreated corn stover effectively.ConclusionsThese results show that replacing natural transcription factors with minimal transcriptional activators is a powerful strategy to enhance cellulase production in T. reesei. Our current study also offers an alternative genetic engineering strategy for the enhanced production of industrial products by other fungi.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0926-7) contains supplementary material, which is available to authorized users.

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Enhanced cellulase production from Trichoderma reesei Rut‐C30 by engineering with an artificial zinc finger protein library

Cited by 38 publications

References 46 publications

Constitutive Expression of Chimeric Transcription Factors Enables Cellulase Synthesis under Non‐Inducing Conditions in Penicillium oxalicum

Constitutive Expression of Chimeric Transcription Factors Enables Cellulase Synthesis under Non‐Inducing Conditions in Penicillium oxalicum

Engineering the Effector Domain of the Artificial Transcription Factor to Improve Cellulase Production by Trichoderma reesei

Enhanced cellulase production in Trichoderma reesei RUT C30 via constitution of minimal transcriptional activators

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