# Rational Design and Modification of NphB for Cannabinoids Biosynthesis
## 3 Discussion  
Using computational simulations, we refined NphB to accommodate various substrates, substantially increasing yields of different cannabinoids through one-step catalysis by NphB. Previous studies have focused on enhancing the product specificity and kinetic performance of NphB to increase the yield of CBGA [21; 41]. Mutants such as Y288A_G286S or Y288V_A232S dramatically improved NphB kinetics; such point mutations also rendered almost exclusive production of CBGA, leading to 21-fold increase in CBGA titer (744 mg/L) [26; 28]. Using a continuous reaction setup in a cell-free system also increased the CBGA titer to 1.25 g/L [27]. Furthermore, the product specificity for CBGA was enhanced by the Q295F mutation, which increased the in vivo activity in E. coli by 20-fold [42]. Building on this work, we conducted detailed mutagenesis and screening of critical NphB enzyme sites. We identified several key amino acid residues that are pivotal for the enzyme's activity and specificity. These residues include Q161, S214, H290, G286, S212, and F213. By introducing mutations at these sites, either individually or in various combinations, we have achieved differentiated enhancements in the production of several cannabinoids, including CBG, CBGA, CBGVA, and CBGV. Each mutation or combination of mutations was strategically chosen based on their potential to alter the enzyme's spatial configuration or its dynamic interaction with specific substrates. The analysis of the catalytic mechanisms of different NphB mutants lays a solid foundation for future enhancements of the NphB enzyme. Our results provide novel insights into enzyme catalytic mechanisms and lay the groundwork for identifying even higher-activity variants in the future. Based on the reaction mechanism, mutations that stabilize substrate orientation, stabilize the intermediate, and promote substrate entry and exit can influence the enzyme's activity from different perspectives.  
Cannabinoids continue to be investigated for their prospective therapeutic utilities, with over 113 distinct scaffolds identified thus far [43]. While CBG has attracted substantial attention, other cannabinoids also demonstrate promising medicinal attributes. As additional cannabinoids emerge, innovative bioproduction platforms become imperative.  
Figure 5: Structural and substrate-binding pocket size comparison between the wild-type (**A**) and the W4 mutant (**B**) variant.  
Traditionally, the biosynthetic pathway for cannabinoids involves the catalysis of Olivetol and GPP by the enzyme NphB to produce CBGA. Subsequently, using CBGA as a substrate, other cannabinoids such as THCA, CBDA, and CBCA can be synthesized under the catalysis of other enzymes. We developed an innovative approach leveraging NphB's capabilities in response to the growing diversity of cannabinoids and their potential applications. We have engineered NphB to perform a one-step catalysis that directly produces various cannabinoids, bypassing the need for subsequent enzymatic transformations typically required in traditional pathways. This streamlined approach not only significantly enhances the efficiency of cannabinoid synthesis but also improves carbon utilization within the biosynthetic pathways.  
By reducing the enzymatic steps involved, we minimize the metabolic burden on the host systems and enhance the overall yield and purity of the desired cannabinoids. Our approach has fundamentally enhanced the efficiency of cannabinoid synthesis and improved carbon utilization within the biosynthetic pathways. There is potential to transplant these modified NphB enzymes into living systems or incorporate them into multi-enzyme, cell-free configurations for the industrial production of cannabinoids from readily available precursors.