产顺式-4-L-羟脯氨酸工程菌的构建及转化条件的优化

【目的】构建产顺式-4-L-羟脯氨酸(cis-4-Hyp)的工程菌并优化其转化条件。【方法】通过调整大肠杆菌的密码子偏好性以及mRNA二级结构对顺式-4-L-脯氨酸羟化酶(cis-P4H)基因进行优化,构建该基因的表达菌株。采用Ni-NTA亲和层析柱分离纯化cis-P4H,测定cis-P4H的酶活和稳定性。然后采用全细胞催化法制备cis-4-Hyp,通过单因素试验和正交试验对相关的转化条件进行优化。【结果】构建了一株产cis-4-Hyp的工程菌,cis-P4H的比活为2.65 U/mg,半衰期为2.32 h。经过条件优化后,采用OD600为0.9时加入IPTG获得的工程菌菌体构建转化体系,在转化体系pH 6.5,转化温度为31°C,转化时间为60 h时,L-脯氨酸转化率最高达到83.33%。【结论】研究获得的工程菌及转化条件具有良好的工业应用前景。

Construction of an engineered strain producing cis-4-L-hydroxyproline and optimization of its biotransformation

[Objective] To obtain an engineered strain producing cis-4-L-hydroxyproline and optimize the related transformation conditions. [Methods] The DNA sequence encoding cis-4-proline hydroxylase (cis-P4H) was adjusted based on the codon bias of Escherichia coli and mRNA secondary structure, an engineered strain expressing the optimized cis-P4H gene was constructed. The cis-P4H was purified by Ni-NTA column and its activity and stability were analysed. Whole-cell catalysis was used for the production of cis-4-Hyp, then the related transformation conditions were optimized by single factor experiment and orthogonal experiment. [Results] An engineered strain was obtained to produce cis-4-Hyp. The specific activity of cis-P4H was 2.65 U/mg and the half-life period of cis-P4H was 2.32 h. When the optical density (OD600) of the cells reached up to 0.9, IPTG was added for inducing protein expression, then the cells were used for transformation system. The transformation rate from L-proline to cis-4-Hyp was more than 83.33% at the optimized condition (pH 6.5, 31 °C, 60 h). [Conclusion] The engineered strain and related transformation conditions have a good prospect for industrial application.