Identification Of Genes Involved In Post-pks Tailoring Steps In Antibiotic Piericidin A1 Biosynthesis

Piericidin A1 is a kind of ?-pyridone antibiotics that can be produced by various Streptomyces strains. Piericidin A1 shows strong biological activities especially to fungal and some insects with a minimum inhibitory concentration, as a potent inhibitory of coenzyme Q in mitochondrial respiration.In order to obtain piericidin intermediates of low toxicity by metabolic engineering, function of methyltransferase gene pieB2 in the biosynthetic cluster of piericidin A1 was investigated. The methyltransferase pieB2 gene disrupted Streptomyces piomogeues var.Hangzhouwanensis was constructed by double crossover recombination.The disruption mutant LQ-9 was able to produce demethyl-piericidin instead of piereicidin A1, which was restored by in trans complementation of the pieB2 gene. This new compound's structure was confirmed by NMR. The methyltransferase gene pieB2 was also PCR amplified and cloned into the plasmid pET28 a for overexpressing N-(His)6-tag PieB2 in E. coli BL21(DE3). The N-(His)6-tag PieB2 was expressed in E. coli in soluble form and was successfully purified via Ni+2mediated affinity chromatography. The PieB2 catalyzed reactions were performed using SAM and demethyl-piericidin as substrates. In vitro biochemical experiments showed that PieB2 could convert demethyl-piericidin into 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.Another methyltransferase gene pie B1 in the biosynthetic cluster of piericidin A1 was investigated too. The methyltransferase pieB1 gene disrupted Streptomyces piomogeues var. Hangzhouwanensis was constructed by double crossover recombination. The disruption mutant LQ-8 was able to produce a new compound instead of piereicidin A1, this new compound's structure was confirmed by NMR. The methyltransferase gene pieB1 was also PCR amplified and cloned into the plasmids for overexpressing, but the recombinant Pie B1 was failed to be purified in E. coli BL21(DE3). Then we feed the product separated from LQ-9 into LQ-5, The disruption mutant LQ-5 was able to produce piericidin A1. We confirmed that PieB1 is function as methyltransferase in the biosynthetic gene cluster of piericidin A1.The regulatory gene pieR gene disrupted Streptomyces piomogeues var. Hangzhouwanensis was constructed by double crossover recombination. The disruption mutant ZZY-3 decreased the ability to produce piericidin A1, proving pieR plays a positive regulation role in the biosynthetic of piericidin A1.