A cell-free platform for the prenylation of natural products and application to cannabinoid production

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.

Acceptor substrate determines donor specificity of an aromatic prenyltransferase: expanding the biocatalytic potential of NphB

“…Understanding how these structural variations in the donor affect activity with a desired acceptor will be crucial to further develop NphB as a biocatalyst. Thus far, the enzyme has been utilized in biocatalytic systems almost exclusively for its ability to geranylate diverse aromatic acceptors (Qian et al 2019;Valliere et al 2019;Zirpel et al 2017). However, the donor promiscuity established in this work opens up the possibility of utilizing NphB for the late-stage modification of aromatic compounds.…”

“…Most of the flavonoids were geranylated at C6 of the A ring (ortho to both hydroxy groups) and on the C7-hydroxy oxygen (Kumano et al 2008;Kuzuyama et al 2005), though a chrysin analog generated by Shindo et al (2011) with 2,3-dihydroxylation of the C ring was geranylated para to the 2′-hydroxy moiety on this ring. Finally, the resorcinol derivatives underwent C-geranylation at C2 or C4, ortho to both or one hydroxy group respectively (Kumano et al 2008;Qian et al 2019;Valliere et al 2019). As for non-GPP donors, NphB has been shown to utilize a terminal azido-substituted GPP derivative with 1,6-DHN, installing it ortho to the C6 hydroxy group, as well as farnesyl pyrophosphate (FPP) with 1,6-DHN and unreported regiospecificity (Kuzuyama et al 2005;Xiao et al 2009).…”

“…Each known site of geranylation is highlighted with a blue "G" valuable cannabinoids. This initial utility was further expanded in 2019 when NphB was implemented in a multienzyme, "synthetic biochemistry" platform for the generation of various cannabinoids (Valliere et al 2019). The optimized system, which included an NphB variant (G286SY288A) engineered to produce cannabigerolic acid (CBGA) almost exclusively from olivetolic acid, produced 1.25 g/L of this cannabinoid over 24 h. Furthermore, an in vivo system was published in the same year that produced geranylated phenolic acids, including CBGA, in Escherichia coli using an engineered NphB variant (G286S) (Qian et al 2019).…”

Interface Engineering in Multiphase Systems toward Synthetic Cells and Organelles: From Soft Matter Fundamentals to Biomedical Applications

“…[440] Based on this work, another cell-free enzymatic prenylating system is developed to generate an array of natural products with high yield, such as cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA), from glucose. [441] Moreover, the cascade reactions have been conceptualized to design multicompartment therapeutic carriers, where the physiological conditions of tumor microenvironments, such pH and high permeability, are exploited to activate cascade reactions to kill cancer cells cooperatively. [129,442,443] Enzymatic reactions in confinements are highly efficient and selective, but how the superactivity of enzymes depends on confinement is not well understood.…”

Structural organization of biocatalytic systems: the next dimension of synthetic metabolism

“…These synthetic in vitro metabolic networks (SIVMNs) are drafted based on physical and chemical principles, assembled from individual enzymes and further optimized in several rounds [17,18]. Examples are the formation of polyhydroxybutyrate from glucose through a 18-enzyme network [19], a synthetic pathway that converts glucose in more than 20 steps into geranyl pyrophosphate, which in turn is used to prenylate different compounds [20], as well as a synthetic pathway ('CETCH cycle') for the continuous capture and conversion of CO 2 that was constructed from 17 different enzymes, including three re-engineered enzymes [18].…”