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-pyrophosphate02505nas a2200265 4500008004100000022001400041245016800055210006900223260000800292300001200300490000700312520152900319653003101848653001101879653002501890653002201915653002001937100003701957700003801994700002202032700001802054700002002072700002202092856012502114 2023 eng d a1479-262100aTargeted metabolite profiling and de novo transcriptome sequencing reveal the key terpene synthase genes in medicinally important plant, Couroupita guianensis Aubl0 aTargeted metabolite profiling and de novo transcriptome sequenci cDEC a558-5700 v213 a

The Lecythidaceae family tree, Couroupita guianensis Aubl, popularly known as Nagpushpa, is a widely cultivated ornamental tree with several uses in traditional medicine. The tree is revered as highly sacred in Indian traditional culture due to its uniquely shaped, fragrant flowers. Considering the significance, we were prompted to carry out the metabolite and transcriptome analysis of Nagapushpa. The flower, petals, stamen, stem and leaf of C. guianensis were metabolically profiled, and it was discovered that the flower tissue contained the highest terpenoid reservoir. A number of terpenoid pathway transcripts were also found in the flower tissue after transcriptome profiling. KEGG pathway mapping was carried out to correlate transcript sequences with the biosynthesis of different types of terpenes. We were able to clone three full-length terpene synthase gene candidates, i.e. monoterpene ocimene synthase, diterpene ent-kaurene synthase and sesquiterpene farnesene synthase. The transcript expression of selected terpene synthase genes was also verified in flower tissue. These cloned sequences were used for in silico structural investigations and protein function prediction at the level of 3D structure. The data presented in this study provide a comprehensive resource for the metabolic and transcriptomic profiles of C. guianensis. The study paves the way towards the elucidation of terpene biosynthetic pathway in C. guianensis and heterologous production of useful terpenoids in the future.

10aCouroupita guianensis Aubl10aflower10aMetabolite profiling10aterpene synthases10aTranscriptomics1 aThulasiram, Hirekodathakallu, V.1 aKaregaonkar, Shrikant, Jagannathr1 aSharma, Poojadevi1 aKumar, Ashish1 aRamkumar, Sudha1 aPandreka, Avinash uhttps://library.ncl.res.in/content/targeted-metabolite-profiling-and-de-novo-transcriptome-sequencing-reveal-key-terpene