mRNA翻译起始区二级结构优化提高(R)-羰基还原酶的表达及催化效率

Improved expression and catalytic efficiency of (R)-carbonyl reductase in Escherichia coli by secondary structure optimization of mRNA translation initiation region

摘要:

为了提高近平滑假丝酵母(Candida parapsilosis CCTCC M203011)的(R)-羰基还原酶在大肠杆菌中的表达水平及催化效率, 对酶编码基因mRNA翻译起始区中+1~+78区进行二级结构的优化, 并构建了相应的突变体。优化后mRNA翻译起始区的发夹结构明显减少, 自由能显著下降(由原始的-9.5 kcal/mol降至-5.0 kcal/mol), 使酶蛋白的表达水平及粗酶比活力分别比优化前提高了4~5倍和61.9%。在高底物2-羟基苯乙酮浓度(5.0 g/L)下, 优化突变株不对称转化效率较高, 产物(R)-苯基乙二醇的光学纯度和产率分别为93.1% e.e.和81.8%, 比优化前提高了27.5%和40.5%。研究结果表明: 优化mRNA翻译起始区的二级结构, 克服蛋白翻译启动的空间位阻, 不仅能促进翻译的顺利进行, 使目标蛋白得到高效表达, 而且有利于蛋白空间结构的正确折叠, 有效提高酶蛋白活力及生物催化功能。

Abstract:

To improve the expression level and catalytic efficiency of (R)-carbonyl reductase from Candida parapsilosis in Escherichia coli, we optimized the mRNA secondary structure of (R)-carbonyl reductase gene in translational initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was signi?cantly reduced and the Gibbs free energy was dramatically decreased from -9.5 kcal/mol to -5.0 kcal/mol after optimization. As a result, the expression level of (R)-carbonyl reductase in the variant was increased by 4-5 times and its specific activity in cell-free extracts was enhanced by 61.9% compared to the wild-type strain. When using the whole cells as catalyst and 2-hydroxyacetophenone as substrate with a high concentration of 5.0 g/L, the variant showed excellent performance to give (R)-1-phenyl-1, 2-ethanediol with optical purity of 93.1% enantiomeric excess and a yield of 81.8%, which were increased by 27.5% and 40.5% respectively than those of the wild-type. In conclusion, the optimization of mRNA secondary structure in translation initiation region can overcome the steric hindrance of translation startup, promote translation smoothly to acquire high expression of target protei1n, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.