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

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.

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.

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.

We confirmed that PieB2 is function as a SAM dependent methyltransferase in the biosynthetic gene cluster of piericidin A1.