Studies of bacterial and fungal secondary metabolism have generally been limited to biosynthetic systems that are derived from cultivated organisms and are highly expressed. This is primarily due to the challenges associated with sourcing and maninpulating DNA from rare or uncultivated organisms. If source material is available, in the form of clone libraries or genomic DNA, DNA assembly methods can be easily applied to engineer the expression of complex gene clusters. In many cases, however, source material is difficult to obtain. Using gene synthesis and modern DNA assembly methods, it is now possible to source and engineer complex gene clusters, from challenging environments, such that they are consistently expressed. In practice, however, this procedure is limited by the current capabilities of commercial DNA synthesis providers. Namely, high GC-content, homopolymers, and regions with secondary structure cannot be synthesized. This limits the range of natural product hosts, transcriptional machinery (promoters, terminators) and biosynthetic gene clusters that can benefit from this approach.<br/> <br/>Here, we describe the complete re-engineering, construction, and expression of a fully-synthetic complex biosynthetic gene cluster using DNA synthesis and synthetic promoters. In constructing and characterizing this gene cluster, we have uncovered rules that can be applied toward the de novo DNA synthesis and robust expression of complex gene clusters. This approach enables the more systematic study of natural product biosynthesis by enabling fully in silico analysis, design, and construction of gene clusters from the uncultivated majority and rare source organisms. We anticipate that, as the cost of DNA synthesis continues to drop, more natural product gene clusters will be fully syntheiszed and engineered based on these principles moving forward, circumventing the need for original source material.

Comprehensive re-engineering and synthesis of a complex biosynthetic gene cluster

251st National Meeting (2016)

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