Abstract
It has been proposed that two acyl carrier proteins (ACPs)—TaB and TaE—and two 3‐hydroxy‐3‐methylglutaryl synthases (HMGSs)—TaC and TaF—could constitute two functional ACP‐HMGS pairs (TaB/TaC and TaE/TaF) responsible for the incorporation of acetate and propionate units into the myxovirescin A scaffold, leading to the formation of β‐methyl and β‐ethyl groups, respectively. It has been suggested that three more proteins—TaX and TaY, which are members of the superfamily of enoyl‐CoA hydratases (ECHs), and a variant ketosynthase (KS) TaK—are shared between two ACP‐HMGS pairs, to give the complete set of enzymes required to perform the β‐alkylations. The β‐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, Δ__taB__ and Δ__taE__ ACP mutants revealed similar phenotypes to their associated HMGS mutants Δ__taC__ and Δ__taF__, respectively, thus providing direct evidence for the role of TaE/TaF in the formation of the β‐ethyl branch and implying a role for TaB/TaC in the formation of the β‐methyl group. Production of myxovirescin A was dramatically reduced in a Δ__taK__ mutant and abolished in both the Δ__taX__ and the Δ__taY__ mutant backgrounds. Analysis of a Δ__taH__ mutant confirmed the role of the cytochrome P450 TaH in hydroxylation of the β‐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 Δ__taE__ mutant, relative to the Δ__taF__ strain, suggests an additional function of the TaE ACP.