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Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C 10 ) and farnesyl pyrophosphate (C 15 ) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (K m ) and catalytic efficiency (k cat /K m ) values of 16.72 ± 1.32 µM and 9.57 × 10 -3  µM -1  s -1 , respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx 8 W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase., (© 2021. The Author(s).)

Vanda 'Tan Chay Yan', a beautiful golden-orange orchid with mild fragrance, is valuable in the floral industry. In this study, the volatile compounds from the flowers were profiled and a transcriptome library was created to search for fragrance-related transcripts. Volatiles from the tepals were captured using a SPME device with PDMS-coated fibre for GC-MS analysis. A transcriptome library was constructed and sequenced using the Illumina MiSeq Desktop Sequencer, de novo assembled using CLC Genomic Workbench version 6.0. BLASTx analysis, functional annotations and gene mapping of the annotated genes to the KEGG database were carried out using the Blast2go Pro software. Twelve compounds were identified in the volatiles including monoterpenes (93.19%), sesquiterpenes (4.32%) and traces of other compounds (2.49%) whereby cis-β-ocimene topped the list as the major constituent (88.41%). De novo assembly of the transcriptome generated a total of 34,027 sequences from which 20 fragrance-related transcripts in the terpenoid and benzenoid/phenylpropanoid biosynthetic pathways were identified. Combined information garnered from the volatiles and fragrance-related transcripts highlights the basic information on the fragrance constituents and biosynthetic pathways of this orchid which are useful for orchid breeders in selection of parents for new hybrids with fragrance as their value-added characteristic for the floriculture industry. [ABSTRACT FROM AUTHOR]

Isoprenoids are a large, diverse group of secondary metabolites which has recently raised a renewed research interest due to genetic engineering advances, allowing specific isoprenoids to be produced and characterized in heterologous hosts. Many researches on metabolic engineering of heterologous hosts for increased isoprenoid production are focussed on Escherichia coli and yeasts. E. coli, as most prokaryotes, use the 2-C-methyl- d-erythritol-4-phosphate (MEP) pathway for isoprenoid production. Yeasts on the other hand, use the mevalonate pathway which is commonly found in eukaryotes. However, Lactococcus lactis is an attractive alternative host for heterologous isoprenoid production. Apart from being food-grade, this Gram-positive prokaryote uses the mevalonate pathway for isoprenoid production instead of the MEP pathway. Previous studies have shown that L. lactis is able to produce sesquiterpenes through heterologous expression of plant sesquiterpene synthases. In this work, we analysed the gene expression of the lactococcal mevalonate pathway through RT- qPCR to successfully engineer L. lactis as an efficient host for isoprenoid production. We then overexpressed the mvk gene singly or co-expressed with the mvaA gene as an attempt to increase β-sesquiphellandrene production in L. lactis. It was observed that co-expression of mvk with mvaA doubled the amount of β-sesquiphellandrene produced. [ABSTRACT FROM AUTHOR]