Cloning and Heterologous Expression Optimization of Homoserine Acetyltransferase Gene from Pleurotus ostreatus

Objective:In order to further improve the yield of enzymatic synthesis of methionine, a candidate gene was cloned from Pleurotus ostreatus. Methods:Homoserine acetyltransferase gene hta was cloned from Pleurotus ostreatus by reverse transcription PCR. The expression vector pET32a-hta containing gene hta constructed by restriction enzymes was transformed into E. coli BL21 and the induction conditions were optimized. The expression vectors pET32a-hta and pET22b-hta were subsequently transformed into E. coli engineered strains Rosetta, Origami B and Rosetta gami plysS, respectively, to investigate the expression of the enzyme in different combinations of vectors and hosts. Results:Results of analysis of ExPAS-ProtParam tool, PSIPRED, SWISS-MODEL and other softwares showed that the sequence coded a polypeptide with 445 amino acids, PI was 5.93 and mainly contained helical, random curling and extending secondary structures. The expression of soluble protein reached the highest level in the engineered strain BL21/pET32a-hta after 16 h induction with 0.2 mmol/L IPTG in 25℃. Results in HPLC showed that the highest specific activity was 2.2 mU/mg in the engineering bacteria Rosetta/pET32a-hta. Conclusion:After optimization of induction conditions and the expression system, the expression level and specific activity of HTA increased, which laid a foundation for further large-scale production of methionine.