# Rational Design and Modification of NphB for Cannabinoids Biosynthesis
## 2 Results  
### Screening and Characterization of NphB for CBG Biosynthesis  
To enhance the diversity of products, a comparative study was conducted on the structural differences of various cannabinoids. Rational design led to the selection and use of non-olivetolic acid (non-oa) substrates as alternatives to olivetolic acid (oa) for conjugation with geranyl pyrophosphate (GPP) mediated by NphB. This strategy facilitated the creation of novel cannabinoid scaffolds not derived directly from cannabigerolic acid (CBGA) but synthesized by NphB. These include cannabigerol (CBG) from olivetol (oli), cannabigerovarin (CBGV) from 5-propyl-1,3-benzenediol (ol3), and cannabigerovarin acid (CBGVA) from 2,4-dihydroxy-6-propylbenzoic acid (oa3) (Figure 1A). The primary objective of this study was to synthesize a spectrum of cannabinoids, with a particular focus on the biosynthesis of CBG, demonstrating the utilization of oli and GPP as precursors in NphB-catalyzed reactions.  
To identify potential sequences for our synthetic biology research, the previously reported aromatic prenyltransferase Orf2 from Streptomyces sp. strain CL190 was used as the query sequence to perform a homology search in the NCBI database [25; 30]. We selected a total of 100 prenyltransferase sequences with more than 30% identity (Figure S1). Based on the gene tree, we delineated four discrete clades and selected 3 to 5 representative sequences from each clade for further analysis. Finally, a total of 16 prenyltransferase sequences were selected (Figure 1B), followed by a streamlined renaming process for these selected sequences (Table S1).  
The efficiency of CBG synthesis from the various sequences was analyzed (Figure 1C), where the sequences N3, N2, and N5 showed relatively high activities, yielding 5.8 mg/L, 5.6 mg/L, and 4.2 mg/L of CBG, respectively. The wild-type enzyme demonstrated peak activity at 30 \({}^{\circ}\)C with a 5 mM concentration of magnesium ions in a pH 8.0 buffered solution (Figure S2A-D). To boost the wild-type enzyme's catalytic activity for CBG, we downloaded the protein structure model of the NphB sequence (1 ZB6_A) from the Protein data bank (PDB) database and utilized Rosetta to dock the necessary substrates and cofactors to the active site [36], followed by the optimization of the docked structure (Figure S3) [37; 38; 39; 40]. Our focus was on the residues lining the substrate-binding pocket (Figure 1D), which are critical for the enzyme's interaction with its substrate. Conformations were deemed plausible based on two criteria: the distance between the substrate reactive site and the \(\alpha\)-C of GPP within 5 A and a negative docking score (binding energy). The most reasonable structure for further analysis was selected based on empirical experience. Superimposition of docking results with the NphB-GSPP-1,6-DHN complex confirmed the reliability of our findings, illustrating that the oli occupied a similar area within the catalytic pocket, as seen with 1,6-DHN [24; 25]. The measured distance from the C5 of oli to the C1 of GPP was measured at 5.1 A, which is comparable to the 4.2 A observed in the complex structure of NphB with 1,6-DHN and GSPP. By combining the docking results of the N3 model with substrate interaction and kinetic analysis, we further engineered N3 to improve its catalytic efficiency in producing CBG.