Dataset Information

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.

SUBMITTER: Navale GR

PROVIDER: S-EPMC6999565 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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

Navale Govinda R GR Sharma Poojadevi P Said Madhukar S MS Ramkumar Sudha S Dharne Mahesh S MS Thulasiram H V HV Shinde Sandip S SS

Engineering in life sciences 20190725 9

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 clon ...[more]

PMID: 32625036

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Project description:Human farnesyl pyrophosphate synthase (hFPPS) catalyzes the production of the 15-carbon isoprenoid farnesyl pyrophosphate. The enzyme is a key regulator of the mevalonate pathway and a well-established drug target. Notably, it was elucidated as the molecular target of nitrogen-containing bisphosphonates, a class of drugs that have been widely successful against bone resorption disorders. More recently, research has focused on the anticancer effects of these inhibitors. In order to achieve increased non-skeletal tissue exposure, we created phenylaminopyridine bisphosphonates (PNP-BPs) that have bulky hydrophobic side chains through a structure-based approach. Some of these compounds have proven to be more potent than the current clinical drugs in a number of antiproliferation assays using multiple myeloma cell lines. In the present work, we characterized the binding of our most potent PNP-BPs to the target enzyme, hFPPS. Co-crystal structures demonstrate that the molecular interactions designed to elicit tighter binding are indeed established. We carried out thermodynamic studies as well; the newly introduced protein-ligand interactions are clearly reflected in the enthalpy of binding measured, which is more favorable for the new PNP-BPs than for the lead compound. These studies also indicate that the affinity of the PNP-BPs to hFPPS is comparable to that of the current drug risedronate. Risedronate forms additional polar interactions via its hydroxyl functional group and thus exhibits more favorable binding enthalpy; however, the entropy of binding is more favorable for the PNP-BPs, owing to the greater desolvation effects resulting from their large hydrophobic side chains. These results therefore confirm the overall validity of our drug design strategy. With a distinctly different molecular scaffold, the PNP-BPs described in this report represent an interesting new group of future drug candidates. Further investigation should follow to characterize the tissue distribution profile and assess the potential clinical benefits of these compounds.

Project description:The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor carotenoid biosynthesis in X. dendrorhous.

Dataset Information

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

Publications

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

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