04216nas a2200181 4500008004100000245013600041210006900177260000900246520351400255100002303769700002203792700002203814700002003836700002203856700000503878700002203883856012903905 2019 eng d00aEnhancing epi-cedrol production in escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi-cedrol synthase0 aEnhancing epicedrol production in escherichia coli by fusion exp cJULY3 a

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 mu M. 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.

1 aNavale, Govinda, R1 aSharma, Poojadevi1 aSaid, Madhukar, S1 aRamkumar, Sudha1 aDharne, Mahesh, S1 a1 aShinde, Sandip, S uhttps://library.ncl.res.in/content/enhancing-epi-cedrol-production-escherichia-coli-fusion-expression-farnesyl-pyrophosphate