Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways

We report the successful cell-free reconstitution of two natural product biosynthetic pathways of divergent complexity and structural classes. We first constructed the teleocidin biosynthetic pathway using our BY-2 (tobacco) cell-free protein synthesis (CFPS) system. We discovered a direct interaction between TleA and MbtH, and showed that the BY-2 system is capable of producing more than 80 mg/L teleocidin B-3 with cofactor supplementation and ∼20 mg/L with no cofactors supplemented, demonstrating the high metabolic activity of the system. We then extended our methodology and report the first successful cell-free biosynthesis of UK-2 diol (precursor to the commercially valuable secondary metabolite UK-2A) from simple building blocks by refactoring a complex pathway of 10 proteins in the wheat germ CFPS system. We show that plant CFPS systems are suitable for reconstructing pathways and identifying the functions of uncharacterized genes linked to biosynthetic gene clusters and rate-limiting biosynthetic steps.