Production of aklanoic acid, an early intermediate of daunorubicin (dnr) biosynthesis in Streptomyces peucetius, by hybrid type II polyletide synthase (PKS) genes was studied as a function of the order and type of genes cloned in a plasmid vector and expressed from the ermE* promoter. Compared with expression of the daunorubicin polyketide synthase (dps) genes in the normal order from their native promoter, the dnrG monooxygenase, dpsA and dpsB ketoreductase and dpsF cyclase genes, when cloned in this order in Streptomyces lividans, produced aklanoic acid and SEK-43. The latter compound is made from acetyl-coenzyme A instead of propionyl-coenzyme A as the starter unit and does not have the characteristic fused ring aromatic system of aklanoic acid. Replacement of either dpsG with its tcmM homolog or dnrG with the Streptomyces glaucescens tcmJ PKS gene resulted in little or no aklanoic acid production. In contrast, addition of the dpsH gene to the tcmJ, dpsA, dpsB, tcmM, dpsE and dpsF gene cassette restored cyclization pattern of aklanoic acid. The reason for the ability of dpsH to override the negative effect of tcmM and restore both the proper choice of starter unit and cyclization pattern is unknown, but overall results indicate that iterative type II PKSs can function aberrantly, perhaps as a function of abnormal protein stoichiometries or interactions. Currently, we are investigating structures of compounds when these gene cassettes are expressed in other mutants.
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