Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

Sun, Fubao; Bai, Renhui; Yang, Huimin; Wang, Fei; He, Jing; Wang, Chundi; Tu, Maobing

doi:10.1016/j.enzmictec.2016.07.004

Cited by 16 publications

(21 citation statements)

References 33 publications

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“…M30794.1), was synthesized in Shanghai Xuguan Biotechnological Development Co, Ltd (Shanghai, China), and preserved in our laboratory. Two recombinants, P. pastoris GS115‐ Cel5A and P. pastoris GS115‐ Cel6A , were constructed in our previous work . Plasmid pPICZαA was purchased from Invitrogen (CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…The expression vector pPICZαA‐ vgb was linearized by restriction digestion with Sac I and kept at –20 °C for use. The operation procedure on constructing the VHb co‐expression transformants was made with the competent cell recombinants, P. pastoris GS115‐ Cel5A and P. pastoris GS115‐ Cel6A as described previously …”

Section: Methodsmentioning

confidence: 99%

“…The HCDFB fermentation of two co‐expression recombinants was carried out with 1.5 L working volume in a 5‐L fermenter (WMC‐9005A/T, Wanmuchun, Shanghai). The experimental procedure was implemented according to our earlier publications . The inoculum of 100 mL was transferred into the fermentor after the seed was grown to OD 600 = 15 at 30 °C in YPD medium.…”

Section: Methodsmentioning

confidence: 99%

“…successfully expressed the codon optimized Trichoderma reesei Cel5A in Pichia pastoris and constructed the recombinant host capable of producing a maximum CMCase activity of 2 358 U mL ‐1 with 1% methanol induction for 72 h. Researchers have argued that the Pichia pastoris host appears a desirable candidate for the expression . Just recently, we also attempted the expression of codon optimized Trichoderma reesei Cel5A / Cel6A in Pichia pastoris GS115 under the AOX1 promoter . With a high‐cell‐density fed‐batch (HCDFB) fermentation, two recombinants P. pastoris GS115‐ Cel5A / Cel6A presented high biomass and secretion level, producing 270 U mL ‐1 of Cel5A with 650 of OD 600 and 1100 U L ‐1 of Cel6A CMCase with 550 of OD 600 , respectively, suggesting that HCDFB cultivation is a favorable way to express the cellulase at high levels in the methanol inducible yeast.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

Self Cite

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“…Potential bottlenecks are the codon usage of the heterologous gene (Sun et al, 2016), the gene copy number, efficient transcription and translation, processing and folding and final secretion out of the cell and protein turnover by proteolysis (Hohenblum et al, 2004; Aw and Polizzi, 2013). Among them, a commonly used approach to improve the expression level in P. pastoris is to increase the copy number of the expression cassette, which was shown to be effective in many cases (Clare et al, 1991; Marx et al, 2009; Nordén et al, 2011; Aw and Polizzi, 2013).…”

Section: Introductionmentioning

confidence: 99%

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

Parashar

Satyanarayana

2017

Front. Microbiol.

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Recombinant chimeric α-amylase (Ba-Gt-amy) has been produced extracellularly in Pichia pastoris under AOX promoter. Clones of P. pastoris with multiple gene copies have been generated by multiple transformations and post-transformational vector amplification, which led to 10.7-fold enhancement in α-amylase titre as compared to a clone with a copy of the gene. The recombinant P. pastoris integrated eight copies of Ba-Gt-amy in the genome of P. pastoris, as revealed by real time PCR data analysis. Heterologous protein expression as well as mRNA level of Ba-Gt-amy was higher in multi-copy clone than that with single copy. The pure Ba-Gt-amy expressed in P. pastoris is a glycoprotein of 75 kDa, which is optimally active at pH 4.0 and 60°C with T1/2 of 40 min at 70°C. The Kinetic parameters and end product analysis suggested that glycosylation has no effect on catalytic properties of Ba-Gt-amy. The enzyme saccharifies soluble as well as raw starches efficiently and generates maltose and maltooligosaccharides, thus, useful in baking and sugar syrup industries. The strategy for generating multi-copy clones is being reported for the first time, which could be useful in enhancing the production of other recombinant proteins.

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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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Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

Cited by 16 publications

References 33 publications

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

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

Production of Chimeric Acidic α-Amylase by the Recombinant Pichia pastoris and Its Applications

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

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