Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw

Fang, Hao; Xia, Liming

doi:10.1016/j.biombioe.2015.04.014

Cited by 36 publications

(35 citation statements)

References 31 publications

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“…The species of EG family is very numerous. In previous, some EG and its gene from various microbe were reported (Fang and Xia 2015b; Lhotak et al 1988; Murray et al 2003). Some EG gene was further overexpressed in Saccharomyces cerevisiae since S. cerevisiae preliminary post modification ability (Akcapinar et al 2011; Huang et al 2010; Qin et al 2008; Wang and Zhang 2003).…”

Section: Introductionmentioning

confidence: 99%

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

et al. 2016

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Endo-1,4-β-d-glucanase (EG), as a key constituent of cellulase taking the responsibility of cutting β-1,4 glycosidic bonds, plays the essential role in the process of degrading cellulose by cellulase. Cloning and expressing the EG gene is important to the cellulase research and application. In this work, a novel EG gene was cloned from Trichoderma virens ZY-01, which was a cellulase secreting microbe isolated by our laboratory. The DNA sequence showed that the length of the cloned EG is 1069 bp, which had 95.2% similarity to the EG IV from T. viride AS 3.3711. Further, the expression vector pET-32a-EG was constructed and was successfully heterologously expressed in Escherichia coli. The expression product was purified with Ni2+ affinity chromatography and its enzymatic properties were investigated. The SDS-PAGE showed the target protein is 39 kDa, which is consistent with the translated result from the DNA sequence. The kinetic parameter for the expression product was Km = 13.71 mg/mL and Vmax=0.51 μmol/min·mL. The optimal reaction pH and temperature was pH = 7.0 and T = 40 °C, which is similar to the native EG produced by Trichoderma virens ZY-01. It provides the foundation for the endo-1,4-β-d-glucanase further evolution and application.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0282-0) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

et al. 2016

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“…The vector pPIC9K, harboring a‐factor signal peptide sequence and the methanol‐inducible alcohol oxidase gene ( AOX1 ) that is a strong promoter controlling recombinant gene expression, was employed as expression vector of cbh1 gene in P. pastoris . This vector is a gift from Professor Menghao Cai at East China University of Science and Technology …”

Section: Methodsmentioning

confidence: 99%

“…The two rounds of screening were conducted in chemostat at 30°C for 2–3 days. The transformants appearing on the YPD agar plates with the higher G418 concentration were preferred because the higher resistance indicated the larger number of insertions or integrations of recombinant DNA fragments into yeast genome in theory …”

Section: Methodsmentioning

confidence: 99%

“…Pichia pastoris is an excellent host for expression and production of heterologous proteins because it has the strong capability of producing large amounts of proteins in low cost, attributable to it is a strong promoter of P AOX1 that can be induced by cheap substrate methanol . Thus, it has been widely used as a host to produce a range of proteins including enzymes, antibodies, cytokines, plasma proteins, and hormones …”

Section: Introductionmentioning

confidence: 99%

“…In this work, a cellobiohydrolase gene ( cbh1 ) cloned from the genomic DNA of the N. crassa strain (FGSC 2489) was expressed in P. pastoris under the control of P AOX1 . Firstly, numerous P. pastoris transformants were screened to choose those with high resistance to selective marker, which is proportional to Avicelase activity . Secondly, the enzymatic properties of the recombinant cellobiohydrolase I (CBHI) produced by the engineered P. pastoris was investigated.…”

Section: Introductionmentioning

confidence: 99%

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Heterologous expression of Neurospora crassa cbh1 gene in Pichia pastoris resulted in production of a neutral cellobiohydrolase I

Yang

Deng

Zhao

et al. 2019

Biotechnology Progress

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The high production cost of cellulase is one of the limitations that hinder the commercialization of lignocellulose-based biorefineries. As one of the important cellulases, Neurospora crassa cellulase is not so intensively investigated as T. reesei cellulase.In this study, the cbh1gene (NCU07340) cloned from N. crassa was expressed in Pichia pastoris under the control of alcohol oxidase 1 (AOX1) promotor. Six transformants with the highest resistance to G418 were selected by two rounds of transformant screening, among which the most robust producer of the recombinant cellobiohydrolase I (CBHI) has an Avicelase activity of 0.22 U/mL. After fermentation optimization, it was improved to 0.30 U/mL. Interestingly, the optimal temperature and pH of the recombinant CBHI were 60 C and 7.2, respectively, and it was quite stable within the wide ranges of temperature and pH. This work is a good example for the future improvement and optimization of N. crassa cellulase.

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Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

Sun

Yang

Bai

et al. 2017

J of Chemical Tech & Biotech

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BACKGROUND: The deficiency of family 5 endoglucanase (Cel5A) and family 6 cellobiohydrolase (Cel6A) has become a key limiting factor on cellulase enzymatic hydrolysis in bioprocessing of cellulosic biomass. To improve the production of Trichoderma reesei Cel5A / Cel6A, a Vitreoscilla hemoglobin (VHb) gene was tried to co-express extracellularly for the first time with Cel5A / Cel6A in Pichia pastoris GS115.

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Heterologous expression and production of Trichoderma reesei cellobiohydrolase II in Pichia pastoris and the application in the enzymatic hydrolysis of corn stover and rice straw

Cited by 36 publications

References 31 publications

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

Cloning a novel endo-1,4-β-d-glucanase gene from Trichoderma virens and heterologous expression in E. coli

Heterologous expression of Neurospora crassa cbh1 gene in Pichia pastoris resulted in production of a neutral cellobiohydrolase I

Enhanced heterologous expression of Trichoderma reesei Cel5A/Cel6A in Pichia pastoris with extracellular co‐expression of Vitreoscilla hemoglobin

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