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

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p PIC9K-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-EGⅡ。重组酶的酶学性质分析显示,该酶分子量50 k Da、最适p H(p H 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p H 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/m L。进一步上罐(5 L)发酵180 h,该重组酶Cel5A酶活高达270.9 U/m L,蛋白含量达到4.16 g/L。重组毕赤酵母P.pastoris GS115-EGⅡ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。

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 Cel5 A in Pichia pastoris by constructing a recombinant strain.With the gene optimization based on codon bias,and the construction of expression vector p PIC9K-eg2,the optimized gene was electro-transformed into P.pastoris GS115 to form transformants.Then,a high Cel5 A 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 Cel5 A reacted optimally at p H 4.5 and 60 ℃,with 50 k Da of molecular weight,preferentially degrading amorphous cellulose.Recombinant Cel5 A was not significantly different from the native T.reesei Cel5 A.Moreover,a shake-flask fermentation of the recombinant strain was optimized as below：incubation temperature 28 ℃,initial p H 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/m L of the Cel5 A after 192 h fermentation.When incubated in a 5 L fermentation,Cel5 A enzyme activity reached 270.9 U/m L 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 Cel5 A.And the recombinant Cel5 A can be used as an alternative to the native T.reesei Cel5 A in development of a commercially relevant enzyme based biorefinery process.