Crop Health R&D, Corteva Agriscience, 9330 Zionsville Road, Indianapolis, Indiana 46268, United States.
ACS Synth Biol. 2024 Nov 15;13(11):3711-3723. doi: 10.1021/acssynbio.4c00560. Epub 2024 Oct 29.
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.
我们成功地重建了两条具有不同复杂性和结构类别的天然产物生物合成途径。我们首先使用我们的 BY-2（烟草）无细胞蛋白合成（CFPS）系统构建了 teleocidin 生物合成途径。我们发现了 TleA 和 MbtH 之间的直接相互作用，并表明 BY-2 系统能够在添加辅助因子的情况下生产超过 80 mg/L 的 teleocidin B-3，而在不添加辅助因子的情况下生产约 20 mg/L，这表明该系统具有很高的代谢活性。然后，我们扩展了我们的方法，并通过在小麦胚 CFPS 系统中重构 10 个蛋白质的复杂途径，首次成功地从简单的构建块中无细胞生物合成了 UK-2 二醇（商业上有价值的次生代谢物 UK-2A 的前体）。我们表明，植物 CFPS 系统适合于重建途径，并鉴定与生物合成基因簇和限速生物合成步骤相关的未鉴定基因的功能。
