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

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.