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

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.
Competing Interests: The authors have declared no conflict of interest.
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