Enhancing epi‐cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi‐cedrol synthase

Abstract Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase ( SaFPPS ) individually with terpene synthase and Artemisia annua Epi ‐cedrol synthase ( AaECS ). The stop codon at the N‐terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐ epi ‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K m values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi ‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi ‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi ‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.