Screening and characterization of methylation tailoring gene-mcbD in marinacarboline biosynthesis

Abstract:

[Objective] The aim of this study was to screen the methylation tailoring gene-mcbD in marinacarboline biosynthesis pathway from the genomic sequence of Marinactinospora thermotolerans SCSIO 00652, and to characterize McbD both in vitro and in vivo.[Methods] Orf03255 (McbD) was selected by phylogenetic analysis of the methyltransferases from M. thermotolerans SCSIO 00652 with MfnG (D/L-Tyr methyltransferase) using Mega6. The mcbD gene was amplified from the M. thermotolerans SCSIO 00652 genomic DNA, cloned into pET28a(+) vector, and expressed in E. coli BL21 (DE3); the McbD protein was purified with Ni-NTA affinity chromatography. The McbD-mediated enzymatic reaction was performed with marinacarboline B as substrate and detected with HPLC. The ΔmcbD mutant was constructed with PCR-targeting genetic manipulation system. Difference in fermentation extracts between M. thermotolerans SCSIO 00652 wild-type and ΔmcbD was analyzed with HPLC.[Results] The N-His tagged McbD was successfully expressed in E. coli BL21 (DE3) in soluble form, and purified by Ni-NTA affinity chromatography; further biochemical reaction showed that McbD can catalyze the transformation of marinacarboline B to marinacarboline C with SAM. The ΔmcbD inactivated mutant completely abolished the production of marinacarboline C, and accumulated marinacarboline B.[Conclusion] McbD, responsible for the methylation tailoring of marinacarboline, is indispensable in marinacarboline biosynthetic pathway.