Improved Bioconversion of 15-Fluoro-6-deoxyerythronolide B to 15-Fluoro-erythromycin A by Overexpression of the eryK Gene in Saccharopolyspora erythraea

摘要

The bioconversion of a 6-deoxyerythronolide B analogue to the corresponding erythromycin A analogue(R-EryA)by a Saccharopolyspora erythraea mutant lacking the ketosynthase in the first polyketide synthase module was significantly improved by changing fluxes at a key branch point affecting the erythromycin congener distribution.This was achieved by integrating an additional copy of the eryK gene into the chromosome under control of the eryAIp promoter.Real-time PCR analysis of RNA confirmed higher expression of eryK in.the resulting strain,S.erythraea K301-105B,compared to its parent.In shake flasks,K301-105B produced less of the shunt product 15-fluoro-erythromycin B(15F-EryB),suggesting a shift in congener distribution toward the desired product,15-fluoro-erythromycin A(15F-EryA).In bioreactor studies,K301-105B produced 1.3 g/L of 15F-EryA with 75-80% molar yield on fed precursor,compared with 0.9 g/L 15F-EryA with 50-55% molar yield on fed precursor by the parent strain.At higher precursor feed rates,K301-105B produced 3.5 g/L of 15F-EryA while maintaining 75-80% molar yield on fed precursor.