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.

The re-emergence of natural products for drug discovery in the genomics era

“…The biosynthetic gene clusters of epothilones have been widely studied 166,167 . Recently, a 56 kb epothilone biosynthetic gene cluster was reassembled using unique restriction sites (which allowed for future module interchangeability) in the guanine-and cytosine-rich host Myxococcus xanthus 168 .…”

Principles of genetic circuit design

“…Synthetic regulation is also an important tool for the discovery of natural products, including pharmaceuticals, insecticides, and entirely new classes of chemicals. Accessing these products may require the coordinated expression of genes identified in sequence databases, intractable organisms, or “silent” gene clusters 17–22 . Moving outside of the fermenter, living cells could be programmed to serve as therapeutic agents that correct genetic disease (Fig.…”

Synthetic biology to access and expand nature's chemical diversity

“…Refactored BGCs can undergo wholesale swapping of genetic parts to optimize expression levels and increase titers. The concept of refactoring has started to be applied to BCGs 62,63 (Figure 2c) around the substitution of some synthetic regulatory elements and as the parts and tools improve, this will expand to include the complete elimination of native regulation.…”

“…The first is de novo synthesis where genes or entire clusters are chemically constructed, typically by synthesis companies 82 . The cost has dropped dramatically in the last decade and it is possible to order hundreds of individual genes or full clusters 43,63,83 . While the cost has declined significantly, it is still expensive to build large clusters and building comprehensive sets of clusters out of the sequence databases is prohibitive.…”