Improved bioconversion of 15-fluoro-6-deoxyerythronolide B to 15-fluoro-erythromycin A by overexpression of the eryK Gene in Saccharopolyspora erythraea.
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.
Desai
Ruchir P
RP
Kosan Biosciences, Inc., 3825 Bay Center Place, Hayward, California 94545, USA. desai@kosan.com
Rodriguez
Eduardo
E
Galazzo
Jorge L
JL
Licari
Peter
P
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics*; D001711:Biotransformation; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics*; D004917:Erythromycin / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D016930:Saccharopolyspora / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D013045:Species Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C038315:erythronolide B; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C078882:EryK protein, Saccharopolyspora erythraea