Substrate tolerance of module 6 of the epothilone synthetase.
The epothilone synthetase is a decamodular megasynthase responsible for the biosynthesis of a class of polyketide natural products with clinically promising antitumor activity. Recently, we developed a system comprised of modules 6-9 of the epothilone synthetase for the precursor-directed biosynthesis of epothilones in Escherichia coli [Boddy, C. N., Hotta, K., Tse, M. L., Watts, R. E., and Khosla, C. (2004) J. Am. Chem. Soc. 126, 7436-7437]. To systematically explore the biosynthetic potential of this system, we have now investigated the ability of the crucial first module in this engineered pathway, EpoD-M6, to accept, elongate, and process unnatural substrates. EpoD-M6 was expressed, purified, and demonstrated to accept both acyl-CoA and acylSNAC substrates. Of the substrates that were tested, octanoylSNAC and 3-octenoylSNAC proved to be excellent substrates in addition to the more complex natural substrate. Thus, this polyketide synthase module showed considerable tolerance, a feature that bodes well for the precursor-directed biosynthesis of epothilone analogues and related complex polyketides.
Tse
Martha Lovato
ML
Department of Chemistry, Stanford University, Stanford, California 94305, USA.
Watts
R Edward
RE
Khosla
Chaitan
C
D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D009231:Myxococcales / Q000201:enzymology; D048630:Polyketide Synthases / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D021122:Protein Subunits; D048630:Polyketide Synthases