基于组合优化策略在毕赤酵母中高效表达 杜邦嗜热菌脂肪酶High-efficient expression of Thermomyces dupontii lipase in Pichia pastoris based on combinatorial optimization strategy

杜邦嗜热菌脂肪酶(LIP1)是一种在洗涤行业、生物柴油制备、油脂改性等领域具有良好应用潜力的碱性脂肪酶，然而其天然产量极低，难以满足产业化需求。采用组合优化策略筛选出高效表达LIP1的毕赤酵母重组菌株。首先，构建毕赤酵母密码子偏好性优化的LIP1基因，通过高浓度G418抗性平板和BMMY-罗丹明B定性平板筛选出脂肪酶活性较高的重组菌株；其次，利用信号肽优化和分子伴侣共表达优化筛选得到一株高效表达的重组毕赤酵母菌株GS115/pPIC9K-Mss-SSA4-LIP1，采用碱滴定法测定其酶活,采用比色法测定其底物特异性。结果表明，经组合优化策略筛选出的菌株在摇瓶和5 L发酵罐中发酵最高分泌酶活分别达到1 136 U/mL和12 150 U/mL。底物特异性结果显示重组脂肪酶LIP1最适底物为C8链长的对硝基苯酚酯。综上所述，基于组合优化策略实现了LIP1在毕赤酵母中的高效表达，为未来LIP1的产业化奠定基础。 Thermomyces dupontii lipase (LIP1) is an alkaline lipase, which posses good application potential in washing industry, biodiesel preparation, oil modification, etc. However, the natural productivity of LIP1 is extremely too low to meet the requirements of industrial application. A combinatorial optimization strategy was applied for the screening of Pichia pastoris strain highly expressing LIP1. Firstly, a Pichia pastoris prefered codon optimized LIP1 gene was constructed, and the recombinant strain with higher LIP1 gene copy numbers was screened through high-concentration G418 plates and BMMY-rhodamine B qualitative plates. Moreover, through signal peptides optimization and chaperone co-expression optimization, a high-productivity recombinant Pichia postoris strain GS115/pPIC9K-Mss-SSA4-LIP1 was obtained.The enzyme activity was determined by alkali titration,and the substrate specificity was determined by colorimetric assay. The results showed that the highest secretive activity of the strain after combinatorial optimization strategy could reach1 136 U/mL in flask fermentation and 12 150 U/mL in 5 L fermenter, The substrate specificity result showed that the most suitable substrate for Pichia pastoris recombinanted LIP1 was p-nitrophenol ester with eight carbons.In conclusion, the high-efficient expression of LIP1 in Pichia pastoris can be realized based on the combinatorial optimization strategy, which will lay the foundation for the future industrialization of LIP1.