Department of Chemistry and Biochemistry, Molecular Biology Institute, UCLA-DOE Institute, University of California, Los Angeles, 90095, CA, USA.
Department of Biochemistry, Institute for Protein Design, University of Washington, Seattle, 98105, WA, USA.
Nat Commun. 2019 Feb 4;10(1):565. doi: 10.1038/s41467-019-08448-y.
Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.
天然化合物的类异戊二烯基化增加了结构多样性，改变了生物活性，并提高了治疗潜力。由于类异戊二烯基化合物的天然丰度通常较低，因此需要替代的生产方法。代谢工程使从廉价生物质中生产天然产物成为可能，但受到次级代谢途径的复杂性、中间产物和产物毒性以及底物可及性的限制。另一方面，酶催化的类异戊二烯基转移提供了极好的区域和立体特异性，但需要昂贵的异戊烯焦磷酸酯底物。在这里，我们开发了一种灵活的无细胞酶促类异戊二烯化系统，可从葡萄糖中生成异戊烯焦磷酸酯底物，从而对一系列天然产物进行类异戊二烯基化。该系统以>1 g/L 的浓度有效地生成大麻素前体大麻二酚酸 (CBGA) 和大麻二酚酸戊酸酯 (CBGVA)，并且通过单一酶促步骤可将前体转化为大麻二酚酸 (CBDA) 和大麻二酚酸戊酸酯 (CBDVA)。无细胞方法可能为难以在生物体中生产的化学品提供一种替代代谢工程的有力方法。
