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

Abstract:

[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.