里氏木霉Cel5A基因优化及其在毕赤酵母中的高效表达

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

摘要:

内切纤维素酶Cel5A缺乏是限制纤维素酶制剂高效酶解天然纤维素的关键因素。本文尝试构建高效表达里氏木霉Cel5A的毕赤酵母重组菌株以弥补目前Cel5A的天然分泌不足，通过基因密码子偏好性优化里氏木霉Cel5A基因和构建表达载体pPIC9K-eg2，并将其电转入毕赤酵母GS115以构建重组子，利用浓度梯度平板和摇瓶发酵筛选获得一株高产毕赤酵母Pichia pastoris菌株GS115-EG Ⅱ。重组酶的酶学性质分析显示，该酶分子量50 kDa、最适pH (pH 4.5) 略有降低及最适反应温度为60 ℃，专一性地作用于非结晶纤维素，与天然里氏木霉Cel5A并无明显区别。通过摇瓶发酵的初步优化，该菌摇瓶培养条件：培养温度28 ℃、起始pH 5.0、接种量2%、每24 h添加甲醇1.5% (V/V)、每24 h添加山梨醇4 g/L及吐温80添加4 g/L，发酵192 h重组酶酶活达到24.0 U/mL。进一步上罐 (5 L) 发酵180 h，该重组酶Cel5A酶活高达270.9 U/mL，蛋白含量达到4.16 g/L。重组毕赤酵母P. pastoris GS115-EG Ⅱ是一株适合于外源表达Cel5A的工程菌，该重组酶可替代天然分泌Cel5A适用于当前酶基生物炼制模式下木质纤维素基质高效水解中。

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.