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 translation initiation region (from +1 to +78), and constructed the corresponding variant. The formation of hairpin structure was significantly 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 extract 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 protein, and favor protein folding correctly to efficiently improve the enzyme specific activity and biotransformation function.
Alcohol Oxidoreductases ; biosynthesis ; chemistry ; genetics ; Base Sequence ; Biocatalysis ; Candida ; enzymology ; Catalysis ; Escherichia coli ; genetics ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Nucleic Acid Conformation ; Peptide Chain Initiation, Translational ; RNA, Messenger ; chemistry ; Recombinant Proteins ; biosynthesis ; genetics ; Stereoisomerism