Comprehensive Re-Engineering and Synthesis of a Complex Biosynthetic Gene Cluster

Studies of bacterial and fungal secondary metabolism have generally been limited to biosynthetic systems that are derived from cultivated organisms and are highly expressed. In addition, most natural product heterologous expression research still focuses on easily cultivated organisms, as source DNA is difficult to obtain or generate otherwise. Heterologous expression systems and basic transcriptional engineering have been used overcome some of these limitations. A robust, and industrially-scalable method for activating the expression of gene clusters from uncultivated or difficult-to-source organisms, however, has not been demonstrated.

Using low-cost gene synthesis and modern DNA assembly methods, it is now theoretically possible to design and fully-build complex gene clusters that have a high probability of being expressed. In practice, however, this procedure is severely 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.

Here, we describe the complete re-engineering, construction, and expression of a fully-synthetic complex biosynthetic gene cluster using DNA synthesis and artificial 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.