title: Modular construction of a functional artificial epothilone polyketide pathway.
abstract: Natural products of microbial origin continue to be an important source of pharmaceuticals and agrochemicals exhibiting potent activities and often novel modes of action. Due to their inherent structural complexity chemical synthesis is often hardly possible, leaving fermentation as the only viable production route. In addition, the pharmaceutical properties of natural products often need to be optimized for application by sophisticated medicinal chemistry and/or biosynthetic engineering. The latter requires a detailed understanding of the biosynthetic process and genetic tools to modify the producing organism that are often unavailable. Consequently, heterologous expression of complex natural product pathways has been in the focus of development over recent years. However, piecing together existing DNA cloned from natural sources and achieving efficient expression in heterologous circuits represent several limitations that can be addressed by synthetic biology. In this work we have redesigned and reassembled the 56 kb epothilone biosynthetic gene cluster from Sorangium cellulosum for expression in the high GC host Myxococcus xanthus. The codon composition was adapted to a modified codon table for M. xanthus, and unique restriction sites were simultaneously introduced and others eliminated from the sequence in order to permit pathway assembly and future interchangeability of modular building blocks from the epothilone megasynthetase. The functionality of the artificial pathway was demonstrated by successful heterologous epothilone production in M. xanthus at significant yields that have to be improved in upcoming work. Our study sets the stage for future engineering of epothilone biosynthesis and production optimization using a highly flexible assembly strategy.
authors: Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Pharmaceutical Biotechnology, Saarland University , Saarbrücken, Germany.
authors: Corina
authors: C
authors: Osswald
authors: Gregor
authors: G
authors: Zipf
authors: Gisela
authors: G
authors: Schmidt
authors: Josef
authors: J
authors: Maier
authors: Hubert S
authors: HS
authors: Bernauer
authors: Rolf
authors: R
authors: Müller
authors: Silke C
authors: SC
authors: Wenzel
mesh_terms: D000465:Algorithms; D053898:Biosynthetic Pathways / Q000235:genetics; D001709:Biotechnology; D003062:Codon / Q000235:genetics; D034261:Epothilones / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005813:Genes, Synthetic; D005818:Genetic Engineering; D005810:Multigene Family; D009231:Myxococcales / Q000235:genetics / Q000378:metabolism; D016941:Myxococcus xanthus / Q000235:genetics / Q000378:metabolism; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism*; D061065:Polyketides / Q000378:metabolism; D058615:Synthetic Biology
chemical_list: D003062:Codon; D034261:Epothilones; D061065:Polyketides; D048630:Polyketide Synthases
keywords: artificial gene cluster; epothilone; heterologous expression; pathway engineering; polyketide biosynthesis; synthetic biology