Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes

A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in <i>Streptomyces coelicolor</i>

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-β-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15-20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-β-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues

Structure–Function Mapping of Key Determinants for Hydrocarbon Biosynthesis by Squalene and Squalene Synthase-like Enzymes from the Green Alga <i>Botryococcus braunii</i> Race B

Squalene and botryococcene are branched-chain, triterpene compounds that arise from the head-to-head condensation of two molecules of farnesyl diphosphate to yield 1′–1 and 1′–3 linkages, respectively. The enzymes that catalyze their formation have attracted considerable interest from the medical field as potential drug targets and the renewable energy sector for metabolic engineering efforts. Recently, the enzymes responsible for botryococcene and squalene biosynthesis in the green alga <i>Botryococcus braunii</i> race B were characterized. To better understand how the specificity for the 1′–1 and 1′–3 linkages was controlled, we attempted to identify the functional residues and/or domains responsible for this step in the catalytic cascade. Existing crystal structures for the mammalian squalene synthase and <i>Staphylococcus</i> dehydrosqualene synthase enzymes were exploited to develop molecular models for the <i>B. braunii</i> botryococcene and squalene synthase enzymes. Residues within the active sites that could mediate catalytic specificity were identified, and reciprocal mutants were created in an attempt to interconvert the reaction product specificity of the enzymes. We report here the identification of several amino acid positions contributing to the rearrangement of the cyclopropyl intermediate to squalene, but these same positions do not appear to be sufficient to account for the cyclopropyl rearrangement to give botryococcene

How do early-life factors explain social inequalities in adolescent mental health? Findings from the UK Millennium Cohort Study.

BACKGROUND:Reducing inequalities in adolescent mental health is a public health priority, yet the pathways that link social conditions to mental health outcomes in the early years are unclear. We aimed to evaluate the extent to which early years risk factors explain social inequalities in adolescent mental health in the UK. METHODS:We analysed data from 6509 children captured in the UK Millennium Cohort Study. Mental health was assessed through the socioemotional behavioural problems at age 14 (Strengths and Difficulties Questionnaire). The main exposure was maternal education at birth, used as a measure of childhood socioeconomic conditions (SECs), and used to calculate the relative index of inequality. Using causal mediation analysis, we assessed how perinatal, individual child, family, peer relation and neighbourhood-level factors measured up to age 3-mediated the total effect (TE) of SECs on adolescent socioemotional behavioural problems, estimating the proportion mediated and natural indirect effect (NIE) via each block of mediators, and all mediators together. RESULTS:Children of mothers with no qualification were almost four times as likely to have socioemotional behavioural problems compared with degree plus level (relative risk (RR) 3.82, 95% CI 2.48 to 5.88). Overall, 63.9% (95% CI 50.2% to 77.6%) (NIE RR 1.97, 95% CI 1.63 to 2.37) of the TE (RR 4.40, 95% CI 3.18 to 6.07) of social inequalities on risk of adolescent socioemotional behavioural problems was mediated by early-life factors. CONCLUSIONS:About two-thirds of the social inequality in adolescent mental health was explained by early risk factors measured by age 3, highlighting the importance of public health interventions in this period

A BioBricks Metabolic Engineering Platform for the Biosynthesis of Anthracyclinones in Streptomyces coelicolor

Actinomycetes produce a variety of clinically indispensable molecules, such as antineoplastic anthracyclines. However, the actinomycetes are hindered in their further development as genetically engineered hosts for the synthesis of new anthracycline analogues due to their slow growth kinetics associated with their mycelial life cycle and the lack of a comprehensive genetic toolbox for combinatorial biosynthesis. In this report, we tackled both issues via the development of the BIOPOLYMER (BIOBricks POLYketide Metabolic EngineeRing) toolbox: a comprehensive synthetic biology toolbox consisting of engineered strains, promoters, vectors, and biosynthetic genes for the synthesis of anthracyclinones. An improved derivative of the production host Streptomyces coelicolor M1152 was created by deleting the matAB gene cluster that specifies extracellular poly-β-1,6-N-acetylglucosamine (PNAG). This resulted in a loss of mycelial aggregation, with improved biomass accumulation and anthracyclinone production. We then leveraged BIOPOLYMER to engineer four distinct anthracyclinone pathways, identifying optimal combinations of promoters, genes, and vectors to produce aklavinone, 9-epi-aklavinone, auramycinone, and nogalamycinone at titers between 15–20 mg/L. Optimization of nogalamycinone production strains resulted in titers of 103 mg/L. We structurally characterized six anthracyclinone products from fermentations, including new compounds 9,10-seco-7-deoxy-nogalamycinone and 4-O-β-d-glucosyl-nogalamycinone. Lastly, we tested the antiproliferative activity of the anthracyclinones in a mammalian cancer cell viability assay, in which nogalamycinone, auramycinone, and aklavinone exhibited moderate cytotoxicity against several cancer cell lines. We envision that BIOPOLYMER will serve as a foundational platform technology for the synthesis of designer anthracycline analogues.publishedVersio

Functional Identification of Valerena-1,10-diene Synthase, a Terpene Synthase Catalyzing a Unique Chemical Cascade in the Biosynthesis of Biologically Active Sesquiterpenes in Valeriana officinalis

Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes, found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate, the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root-specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, whereas VoTPS1 catalyzes the conversion of farnesyl diphosphate to valerena-1,10-diene. Using a yeast expression system, specific labeled [13C]acetate, and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl-containing sesquiterpenes.This research was originally published in Journal of Biological Chemistry. Yeo YS, Nybo SE, Chittiboyina AG, Weerasooriya AD, Wang YH, Gongora-Castillo E, Vaillancourt B, Buell CR, DellaPenna D, Celiz MD, Jones AD, Wurtele ES, Ransom N, Dudareva N, Shaaban KA, Tibrewal N, Chandra S, Smillie T, Khan IA, Coates RM, Watt DS, Chappell J. Functional identification of valerena-1.10-diene synthase. a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis. The Journal of Biological Chemistry. 2013; 288:31763-3173, doi: 10.1074/jbc.M112.415836. © the American Society for Biochemistry and Molecular Biology.</p