[Function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1].

Objective: In order to obtain piericidin intermediates of low toxicity by metabolic engineering, we studied the function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1. Methods: The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var. Hangzhouwanensis was constructedby double crossover recombination. The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid pET28a for overexpressing N-(His)6-tag PieB2 in E. coli BL21(DE3)/pLysE. The recombinant PieB2 was purified by affinity chromatography via AKTA FPLC system. The PieB2 catalyzed reactions were performed using SAM and demethyl-piericidinas substrates. Results: The disruption mutant LQ-9 produced demethyl-piericidininstead of piereicidin A1, which was restored by in trans complementation of the pieB2 gene. The N-(His)6-tag PieB2 was expressed in E. coli in soluble form and was successfully purified via Ni2+ mediated affinity chromatography. In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidininto piereicidin A1 in the presence of SAM. The demethyl-piericidin intermediat showed an attractive biological activities as well as piericidin A1. Conclusion: We confirmed that PieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.