Mutational analysis of the myxovirescin biosynthetic gene cluster reveals novel insights into the functional elaboration of polyketide backbones

It has been proposed that two acyl carrier proteins (ACPs)-TaB and TaE-and two 3-hydroxy-3-methylglutoryl synthases (HMGSs)-TaC and TaF-could constitute two functional ACPHMGS pairs (7681W and TaE/TaF) responsible for the incorporotion of acetate and propionote units into the myxovirescin A scaffold, leading to the formation of beta-methyl and beta-ethyl groups, respectively. It has been suggested that three more proteins-TaX and TaY which are members of the superfamily of enoyl-CoA hydratoses (ECHs), and a variant ketosynthose (KS) TaK-are shared between two ACP-HMGS pairs, to give the complete set of enzymes required to perform the beta-alkylations. The beta-methyl branch is presumably further hydroxylated (by TaH) and methylated to produce the methoxymethyl group observed in myxovirescin A. To substantiate this hypothesis, a series of gene-deletion mutants were created, and the effects of these mutations on myxovirescin production were examined. As predicted, taB and AtaE ACP mutants revealed similar phenotypes to their associated HMGS mutants JtaC and JtaF, respectively, thus providing direct evidence for the role of TaE/ToF in the formation of the beta-e ethyl branch and implying a role for TaB/TaC in the formation of the beta-methyl group. Production of myxovirescin A was dramatically reduced in a JtaK mutant and abolished in both the JtaX and the JtaY mutant backgrounds. Analysis of a JtaH mutant confirmed the role of the cytochrome P450 TaH in hydroxylation of the beta-methyl group. Taken together, these experiments support a model in which the discrete ACPs TaB and TaE are compatible only with their associated HMGSs TaC and TaF, respectively, and function in a substrate-specific manner. Both TaB and TaC are essential for myxovirescin production, and the TaB/TaC pair can rescue antibiotic production in the absence of either TaE or TaF Finally, the reduced level of myxovirescin production in the JtaE mutant, relative to the AtaF strain, suggests an additional function of the ToE ACP.