Approaches to stabilization of interdomain recomb

Authors: Z Hu R P Desai Y Volchegursky T Leaf E Woo P Licari D V Santi C R Hutchinson R McDaniel

Publish Date: 2003/03/01

Volume: 30, Issue: 3, Pages: 161-167

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Abstract

Regions of extremely high sequence identity are recurrent in modular polyketide synthase PKS genes Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites We present two different solutions for circumventing intraplasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor In one example a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions Both methods resulted in PKS complexes capable of 6deoxyerythronolide B analogue biosynthesis in S coelicolor CH999 permitting reproducible scaleup to at least 5l stirredtank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs

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