Heterologous Production of Epothilone C and D in Escherichia coli

Abstract

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.

In Vivo Manipulation of the Bleomycin Biosynthetic Gene Cluster in Streptomyces verticillus ATCC15003 Revealing New Insights into Its Biosynthetic Pathway

“…To date, many other microbial producers of important natural products have been reported to be genetically inaccessible or extremely hard to manipulate (32)(33)(34). However, the availability of an efficient genetic system is one of the key prerequisites for successful engineering of natural product biosynthetic pathways to generate new biologically active derivatives using combinatorial biosynthesis.…”

The Chemistry and Biology of Epothilones—The Wheel Keeps Turning

“…[7,57]) and the development of heterologous expression systems for epothilones and individual derivatives. [7,58,59] A detailed discussion of these achievements, however, is beyond the scope of this review article (with a few specific exceptions) as is the enormous amount of clinical data that have emerged from the various clinical trials. For these areas the reader is referred to a number of excellent recent review articles dealing with the corresponding subject (see references cited above).…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…After preliminary researches, [208] the first heterologous total biosynthesis of epothilone was achieved in E. coli by Kosan Biosciences. [209] Because the enzymes were large enough to be insoluble in the cytoplasm, chaperones were coexpressed to enhance the protein solubility. Chaperones, proteins that aid in polypeptide folding and solubility, have been actively used for solubilizing large PKSs.…”