Precursor-directed production of erythromycin analogs by Saccharopolysporaerythraea

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 deg rees) Saccharopolyspora erythraea strains to produce 13-substituted erythro mycin analogs. This direct feeding process potentially represents a simplif ied production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concent ration up to a precursor-specific saturation level. However, the rate of pr oduct formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Stre ptomyces coelicolor. Several strategies were pursued to address the issue o f these high diketide consumption rates: (1) elucidation of the locale of d iketide degradation, (2) addition of beta -oxidation inhibitors to the cult ures, and (3) addition of a sacrificial diketide enantiomer to occupy putat ive degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythrom ycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs t o a large-scale production system for these compounds. (C) 2001 John Wiley & Sons, Inc.