# Rational Design and Modification of NphB for Cannabinoids Biosynthesis
## 2 Results
### Engineering of NphB for Cannabinoids Biosynthesis  
Based on the docking results of the enzyme with the substrates, 54 residues within a 6 A range of oli and GPP were selected for preliminary analysis as part of the substrate-binding pocket. Conservation analysis led to the exclusion of 10 conserved residues from mutation considerations (Figure 2A). In the reaction between oli and GPP, the pyrophosphate moiety of GPP engages in hydrogen bonding with the side chains of residues K119, N173, Y175, Y216, R228, and K284, thereby securing its position (Figure 2C). The preynl chain of GPP is embedded in a hydrophobic environment, surrounded by hydrophobic amino acid residues. Olivetol forms hydrogen bonds with Y288 and is stabilized by \(\pi\)-\(\pi\) stacking with the phenyl ring of F213 (Figure 2D). While residues critical for stabilizing GPP's pyrophosphate group were excluded from the mutagenesis targets to avoid any destabilization of GPP, Y288, and F213, impacting both GPP and Olivetol, are still considered viable targets for mutational analysis. To further narrow down the target sites, virtual saturation mutagenesis was performed on the residues within the substrate-binding pocket using Rosetta 2022.08. The change in binding energy for each mutant was used to assess their potential impact on enzyme activity (Figure 2B). A decrease in binding energy indicates a more stable substrate-enzyme complex, which is favorable for catalysis. Consequently, the sites with increased binding energy among all 20 residues were eliminated. Considering the aforementioned criteria, we identified V47, V49, F107, A108, S212, F213, S214, Q161, F176, S177, T269, V271, C286, Y288, and H290 for our mutational experiments. Figure 1: Screening of preynyltransferase orthologous to NphB. (**A**) Yield routes of various cannabinoids by NphB. (**B**) Gene tree for the selected homologous sequences to the previously reported NphB from Streptomyces sp. strain CL190. The GenBank accession: AB187169. (**C**) The CBG yield spectrum of different sequences. The error bars represent standard deviation. Measurements were conducted in triplicate. (**D**) A protein model for the N3 homolog, the pink region delineates the substrate-binding pocket. We designed a dual-site combinatorial saturation screening for efficient screening of candidate residues. Considering that the selected mutation residues are either adjacent or very close in spatial position, this basis was used for the dual-site combinatorial saturation screening. We first used a pyrophosphate assay kit for high-throughput preliminary screenings, then cultured and purified clones showing elevated fluorescence values. We then used the purified enzymes to confirm their activity. The first round of screening identified the mutant WT-Q161R/S214H (named W1) as showing the most significant increase in CBG catalytic activity, boosting CBG production from 2.42 mg/L to 16.23 mg/L. Interestingly, the WT-S212A/F213S mutant exhibited good catalytic activity for CBGVA, possibly due to the smaller side chains of the mutated residues allowing the reaction chamber to accommodate larger substrates. Subsequent screenings continued based on W1 and WT-S212A/F213S, focusing on sites with beneficial mutations from the first round. Further mutations based on W1 led to strains W2 (W1-V49C/H290D) and W5 (W1-F176D/S177T/Y288V). Both strains achieved nearly identical CBG yields of approximately 18.62 mg/L. Further mutations based on the W2 strain led to the creation of strain W3 (W2-V47D/G286C), which marked  
Figure 2: Rational engineering of NphB enzyme. (**A**) Conservation analysis of residues within the NphB active sites. (**B**) Gibbs free energy heatmap of residues within the NphB active sites. The black dots represent each mutation site, and the average change in binding energy after virtual saturation mutagenesis. (**C**) Interaction analysis between geranyl diphosphate (GPP) and surrounding residues. (**D**) Interaction analysis between Olivetol and surrounding residues.