Abstract
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.
Highlights
Microbial production is a useful alternative to natural extraction for prenylated natural products, but comes with many challenges such as the need to divert carbon flux from central metabolism and product toxicity to name a few
The acetyl-CoA is assembled into the prenyl-donor compound, GPP, via the mevalonate pathway using the ATP and NADPH produced from glycolysis
To expand the capabilities of our synthetic biochemistry platform we developed a prenylating system that employs a nonspecific prenylating enzyme such as NphB, AtaPT, or NovQ to produce an array of prenyl-compounds derived from glucose[29,30,31]
Summary
That effectively generate and recycle high energy cofactors (ATP, NAD(P)H) so that they can be used many times. We have previously reported a flexible enzymatic purge valve and rheostat for regulating the supply of reducing equivalents and ATP26–28, allowing us to build systems that run for many days and produce high titers of isobutanol and terpenes. We employ these concepts to develop cell-free production of a variety of prenylated compounds. We use glucose as a feedstock to produce GPP and optimize the system for the high-titer production of the cannabinoid compounds CBGA and cannabigerovarinic acid (CBGVA)
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