Structure-Guided Engineering of Prenyltransferase NphB for High-Yield and Regioselective Cannabinoid Production

Abstract

Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years. This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa, dubbed the green rush. In transgenic synthetic biology applications, NphB is a promiscuous prenyltransferase from Streptomyces sp. often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid (CBGA), the key precursor to many other cannabinoids. However, its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA. Herein, we demonstrated a detailed and extensive computational structure-guided approach in identifying target residues of mutation for engineering NphB for optimal CBGA production. Our comprehensive computational workflow has led to the discovery of several highly regiospecific variants that produce CBGA exclusively, with the best-performing V49W/Y288P variant having a 13.6-fold yield improvement, outperforming all previous work on NphB enzyme engineering. We subsequently investigated the effects of these mutations by X-ray crystallographic studies of the mutant variants and performed molecular dynamics simulations to uncover an interplay of a H-bonding network and an optimal ligand orientation that favors the CBGA production over the side product. Collectively, this study not only recapitulates the utility of computational tools in informing and accelerating experimental design but also contributes to a better understanding of molecular mechanisms that govern enzyme regioselectivity and readily aids in cannabinoid synthetic biology production for future research into maximizing their therapeutic potential.

Generation of Cannabigerolic Acid Derivatives and Their Precursors by Using the Promiscuity of the Aromatic Prenyltransferase NphB

“…These energetically favorable interactions further favor the rotation of the resorcylate core by the G286S/Y288A variant. This is also supported by a comparison with Lim et al, who concluded that a hydrogen bond between OH4 and Tyr288 leads to an unfavorable orientation for CBGA‐C5 production [22] …”

“…The most important feature of the proposed binding mode is that the resorcylate core and therefore the prenylation position C3 is located close to C1 of the GSPP and the side chain is oriented towards the entrance of the binding pocket. In X‐ray structures, dockings, and MD simulations, a distance of 3 to 4.5 Å between the two atoms was identified [9,13,22] . To evaluate whether a substrate is likely to be converted by NphB, the distance between the C3 of the resorcylate core and C1 of GSPP was calculated for each pose.…”

“…Molecular docking . Docking experiments were performed using GOLD [22] . The protein structure (PDB ID: 1ZDW) were prepared using UCSF Chimera [23] .…”

Biocatalytic Friedel‐Crafts Reactions

“…The potential of aPTases to produce biologically active compounds has recently been furthered in their application toward improving heterologous cannabinoid biosynthesis [66] and in the chemoenzymatic synthesis of daptomycin analogues with activity modulation [67] . The discussion on this diverse enzyme family provided here is far from exhaustive, and for further information we direct the reader to recent reviews on the topic [46,68] .…”

Mechanism-Based Redesign of GAP to Activate Oncogenic Ras

“…28 This validates an approach that targets oncogenic Ras by restoring its activity, instead of modulating the signaling by the inhibition of downstream effectors. Nature tailored enzymes to be highly efficient and selective; 29 computational design principles are established to develop catalysts and enzymes, 30,31 exploiting structural 32 and dynamical 33 information to optimize reactivity.…”