Modular Construction of a Functional Artificial Epothilone Polyketide Pathway

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.