产虫草素酿酒酵母工程菌株的构建与发酵优化

Construction and optimization of cordycepin-producing Saccharomyces cerevisiae

摘要:

虫草素作为药用真菌蛹虫草的主要活性成分，具有抗肿瘤、抗病毒等多种生理功能。现阶段虫草素主要通过蛹虫草液体发酵生产，但发酵周期长、生产强度低，制约了其大规模开发利用。文中在酿酒酵母Saccharomyces cerevisiae S288C中异源表达虫草素合成关键基因ScCNS1和ScCNS2，成功构建了产虫草素的酵母工程菌SHC16，发酵240 h虫草素产量可达67.32 mg/L；基因表达分析显示，发酵后期磷酸戊糖途径、嘌呤代谢及虫草素合成途径关键酶编码基因ZWF1、PRS4、ADE4、ScCNS1及ScCNS2表达水平显著上调。进一步地，通过优化发酵培养基组成，确定以50 g/L葡萄糖为初始底物结合一次补料、添加5 mmol/L Cu2+和1.0 g/L腺嘌呤为最适培养基组成。基于此在5 L搅拌发酵罐中开展补料分批发酵，144 h虫草素产量达到137.27 mg/L，生产强度达0.95 mg/(L·h)，较未优化发酵体系提高240%。

Abstract:

Cordycepin is the key active component of medicinal fungus Cordyceps militaris, and it shows multiple functional activities such as anti-tumor and anti-virus. Cordycepin was conventionally produced by liquid fermentation of C. militaris, but the long production cycle and the low productivity constrained its development and application. In this study, two key genes for cordycepin biosynthesis (ScCNS1 and ScCNS2) were introduced into Saccharomyces cerevisiae S288C, producing 67.32 mg/L cordycepin at 240 h. Analysis of gene expression profiles indicated that ZWF1, PRS4, ADE4, ScCNS1 and ScCNS2 which encode enzymes involved in pentose phosphate pathway, purine metabolism and cordycepin biosynthesis pathway, were significantly up-regulated in the late phage of fermentation. Optimization of fermentation medium determined that 50 g/L initial glucose followed by feeding, supplemented with 5 mmol/L Cu2+ and 1.0 g/L adenine were the best condition. Fed-batch fermentation using the engineered yeast in a 5 L stirred fermenter produced 137.27 mg/L cordycepin at 144 h, with a productivity up to 0.95 mg/(L·h) reached, which was 240% higher than that of the control.