Chapter 14
Polyketides Downloaded from pubs.acs.org by YORK UNIV on 12/06/18. For personal use only.
Novel Polyketides from Genetic Engineering (••• and Lessons We Have Learned from Making Them) Leonard Katz, Jonathan Kennedy, Sarah C. Mutka, John R. Carney, Karen S. MacMillan, and Sumati Murli Kosan Biosciences, Inc., 3832 Bay Center Place, Haywood, C A 94545
Knowledge of the domain organization of type I modular polyketide synthases (PKS) usually allows accurate prediction of the structure of the corresponding polyketide. Genetic engineering of such PKSs can be employed to produce novel compounds of predicted structure. Genetic engineering of the epothilone P K S has given unexpected results that have revealed subtleties of the biosynthesis not previously understood. In one instance, an unprecedented mechanism was uncovered.
Epothilone is a mixed peptide-polyketide produced from the myxobacterium Sorangium cellulosum (1) that inhibits proliferation of eukaryotic cells through the stabilization of microtubules (2). Epothilone D , as well as a number of derivatives of various epothilone congeners (Fig. 1), are currently in clinical trials for the treatment of various cancers. The compounds are made from a peptide synthetase (PS)-polyketide synthase (PKS) whose genes have been cloned, sequenced (3, 4) and expressed in various heterologous hosts (4-7). Organization of the genes, their corresponding proteins and constituent modules and domains, are shown in Fig. 1. As in the case of many type I modular polyketide synthases, the epothilone (epo) P K S has been engineered to produce novel analogs. Some of the analogs thus produced were what would be predicted from current understanding of polyketide biosynthetic pathways. Other changes to the P K S resulted in the production of novel compounds not predicted by standard polyketide biosynthesis models. Analysis of these compounds, discussed in this chapter, revealed aspects of the biosynthesis of epothilone not previously understood. 200
© 2007 American Chemical Society
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epoA epoB EpoA
epoC EpoB
epoD
epoE
epoF
EpoC
KSy AT ACFv C A(cys) T Ox> KS mmAT DH KR ACP> Load Module 1 Module 2 EpoD KS mAT KR ACP KS AT KR ACP KS mAT DH ER KR ACP KS mmAT DH ER KR ACP/ Module 3 Module 4 Module 5 Module 6 EpoE EpoF KS mmAT KR ACP KS mmAT MT AC^> KS mAT KR ACP TE)> Module 7 Module 8 Module 9
R Epothilone C H Epothilone D CH
3
R Epothilone A H Epothilone B CH
3
Figure 1. Domain organization of the epothilone PKS and structures of epothilone congeners. The top line shows the gene organization and gene names. The boxed segment below shows the organization of the domains and modules corresponding to each gene. Domain abbreviations: ACP, acyl carrier protein; AT, acyltransferase; mAT and mmAT, AT domain specifying malonate and methylmalonate, respectively; DH, dehydratase; ER, enoylreductase; MT, C-methyltransferase; KR, /3-ketoreductase; KS, (i-ketoacyl ACP synthase; KSy, KS domain containing tyrosine residue in the active site; TE, thioesterase.
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Biosynthesis of Epothilone Epothilone is produced from the progressive condensation of acyl thioesters employing the P K S shown schematically in Fig. 1. Synthesis starts with the loading of malonyl CoA on the A T domain of EpoA, followed by its transfer to the A C P domain and decarboxylation through the action of the K S y domain, leaving acetyl-ACP. EpoB is a peptide synthetase specific for cysteine. Condensation between the acetyl moiety on EpoA and cysteine on EpoB produces the acetyl-cysteinyl depsipeptide, and subsequent internal cyclization and oxidation produce the methylthiazole group found in the fmished compounds. Condensation between the methylthiazole and the malonyl-ACP on module 2 (EpoC) produces a triketide. The P-carbonyl of the triketide is subsequently reduced to an O H group through the function of the K R domain in EpoC. This decarboxylase (Claisen) condensation resembles the condensations that take place during fatty acid biosynthesis. Subsequent progressive condensations catalyzed by modules 3 through 9 result in production of the decaketide which undergoes internal lactonization to generate epothilone C or epothilone D . Module 8 contains a methyltransferase domain that adds a second methyl group to C-2 of the acyl chain formed after the 8 condensation step to produce the geminal dimethyl found in the finished compounds. Epothilones A and B are produced from epothilones C and D through the action of the epoxidase EpoK, produced from a gene that is adjacent to the P K S encoding genes. th
The stepwise biosynthesis of epothilone follows a predictable route through the epo PS-PKS, but two aspects of the synthesis are not explained by the domain organization. It is clear that both epothilones C and D, which differ in the composition of the side chain at C - l 2 , are produced from the same P K S , although only a single compound is produced from each round of biosynthesis. The A T domain in module 4 (epo AT4), therefore, must be able to utilize as a substrate either malonyl CoA (to produce epothilone C) or methylmalonyl C o A (to produce epothilone D). In general, A T domains in bacterial type I modular PKSs are highly specific for a single substrate, and epo AT4 represents the only known example of an A T domain with a lack of malonate-methylmalonate selectivity. B y sequence comparisons, epo A T 4 resembles the malonatespecifying A T domains (
