毕赤酵母组成型表达脂肪酶及其高通量筛选方法

Constitutive expression of lipase in Pichia pastoris and the high throughput screening method

摘要:

【目的】获得组成型表达脂肪酶毕赤酵母, 建立利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因的有效方法。【方法】运用PCR技术从pGAPZαA表达载体上扩增得到GAP启动子片段, 插入到表达载体pPIC9K-proRCL中, 构建组成型表达载体pGAPK-proRCL。在保留含有同源双交换重组序列的诱导型启动子AOX1序列的基础上, 电转化后华根霉Rhizopus chinensis CCTCC M201021脂肪酶基因 proRCL表达盒在毕赤酵母基因组上发生双交换整合事件, 从而组成型表达单拷贝的华根霉脂肪酶基因。【结果】重组菌发酵144 h后, 脂肪酶最高酶活为130 U/ml。利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因。【结论】该方法将初筛时间从12 d缩短为3 d, 排除了多拷贝突变株的干扰, 为后续脂肪酶的定向进化及筛选奠定了基础。

Abstract:

[Objective] This study is aimed to constitutive express lipase in Pichia pastoris and establish an efficient method for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. [Methods] The GAP gene promoter was amplified from the plasmid of pGAPZαA and inserted into the inducible expression vector pPIC9K-proRCL resulting in constitutive expression of the lipase gene-proRCL. Based on the homologous recombinant region of AOX1 gene, proRCL from Rhizopus chinensis CCTCC M201021 was recombined into chromsome of P. pastoris GS115 by double-crossover integration event resulting in constitutive expression of the single-copy lipase gene from R. chinensis. [Results] The activity of lipase reached its peak (130 U/ml) after fermentation of 144 h. An efficient method was established for high-throughput screening constitutive expression lipase gene in P. pastoris using olive oil-rhodamine B plate. [Conclusion] The method was very convenient with advantages of shortened screening cycle from 12 d to 3 d without the interferences of multi-copy mutants. This method also established the foundation of the screening of library by directed evolution.