| Modular polyketide synthases (PKSs) are large, multifunctional enzymes responsible for synthesizing many useful natural products such as erythromycin, rapamycin, and avermectin. Due to the structural complexity of polyketide natural products, industrial scale production normally relies on fermentation processes rather than chemical synthesis. Consequently, methods used to produce novel polyketide analogues would be useful because they could be integrated into the fermentation process. In this work, we have attempted to "redesign" the biosynthetic machinery in new organisms, capable of making polyketides not accessible through traditional, synthetic chemistry.; S. avermitilis normally uses isobutyryl CoA and 2-methylbutyryl CoA as starter units to produce the avermectin family of compounds along with branched-chain fatty acids. In order to study the role of precursors in fatty acid and polyketide biosynthesis, the fatty acid profiles of the S. avermitilis branched-chain alpha-keto acid dehydrogenase (bkd) mutant were examined in detail. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant was shown not to have an obligate growth requirement for these precursors. In addition, previous experiments have shown that when exogenous branched-chain fatty acids were fed to the bkd mutant, a wide variety of avermectin analogs could be generated. These precursor-directed biosynthetic studies eventually led to the development of doramectin, produced by addition of cyclohexanecarboxylic acid (CHC) to the S. avermitilis bkd mutant. Interestingly, whereas S. avermitilis requires exogenous CHC in order to produce doramectin, Streptomyces collinus is capable of producing; CHC during ansatrienin A biosynthesis. In this work, we have identified a gene cluster associated with CHC biosynthesis in S. collinus. Expression of this gene cassette in a bkd mutant of S. avermitilis constitutes a functional CHC biosynthetic pathway, and allows for production of doramectin in the absence of exogenous CHC. In order to expand the use of this technology past avermectin biosynthesis, we went on to use components from the pikromycin PKS from Streptomyces venezuelae to construct several hybrid PKSs designed to utilize CHC. |
