Gene optimization and efficient expression of Trichoderma reesei Cel5A in Pichia pastoris.

Renhui Bai; Yunbo Zhang; Chundi Wang; Feiyang Zhang; Zhe Zhang; Fubao Sun; Zhenyu Zhang

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Gene optimization and efficient expression of Trichoderma reesei Cel5A in Pichia pastoris.

Author: Renhui BAI ¹ ; Yunbo ZHANG ¹ ; Chundi WANG ¹ ; Feiyang ZHANG ² ; Zhe ZHANG ² ; Fubao SUN ¹ ; Zhenyu ZHANG ¹
Author Information

1. Laboratory of Industrial Biotechnology of Department of Education, Jiangnan University, Wuxi 214122, Jiangsu, China.
2. State Key Laboratory of Motor Vehicle Biofuel Technology, Henan Tianguan Group Co., Ltd, Nanyang 473000, Henan, China.
Publication Type:Journal Article
Keywords: AOX1 promoters; CMC enzyme activity; Pichia pastoris GS115-EG Ⅱ; Trichoderma reesei Cel5A gene; codon bias; fermentation optimization
From: Chinese Journal of Biotechnology 2016;32(10):1381-1394
CountryChina
Language:Chinese
Abstract: Deficient activity of endo-1,4-beta-glucanase II (Cel5A) secreted by Trichoderma reesei is one of the challenges involved in effective cellulase saccharification of cellulosic substrates. Therefore, we expressed Cel5A in Pichia pastoris by constructing a recombinant strain. With the gene optimization based on codon bias, and the construction of expression vector pPIC9K-eg2, the optimized gene was electro-transformed into P. pastoris GS115 to form transformants. Then, a high Cel5A activity producing recombinant, namely P. pastoris GS115-EG Ⅱ, was selected on G-418 resistant plates, followed by shake-flask cultivation. Enzyme characterization showed that the recombinant Cel5A reacted optimally at pH 4.5 and 60 ℃, with 50 kDa of molecular weight, preferentially degrading amorphous cellulose. Recombinant Cel5A was not significantly different from the native T. reesei Cel5A. Moreover, a shake-flask fermentation of the recombinant strain was optimized as below: incubation temperature 28 ℃, initial pH 5.0, inoculum volume 2%, methanol addition (per 24 h) 1.5% (V/V), sorbitol addition (per 24 h) 4 g/L and Tween 80 4 g/L. Under above optimized condition, the recombinant produced 24.0 U/mL of the Cel5A after 192 h fermentation. When incubated in a 5 L fermentation, Cel5A enzyme activity reached 270.9 U/mL at 180 h, with 4.16 g/L of the total protein. The study indicates that the recombinant strain P. pastoris GS115-EG Ⅱ is extremely suitable for heterologous expression of T. reesei cellulase Cel5A. And the recombinant Cel5A can be used as an alternative to the native T. reesei Cel5A in development of a commercially relevant enzyme based biorefinery process.