Geranylgeranyl Research Articles

Therapeutic targeting of the mevalonate-geranylgeranyl diphosphate pathway with statins overcomes chemotherapy resistance in small cell lung cancer.

Small cell lung cancer (SCLC) lacks effective treatments to overcome chemoresistance. Here we established multiple human chemoresistant xenograft models through long-term intermittent chemotherapy, mimicking clinically relevant therapeutic settings. We show that chemoresistant SCLC undergoes metabolic reprogramming relying on the mevalonate (MVA)-geranylgeranyl diphosphate (GGPP) pathway, which can be targeted using clinically approved statins. Mechanistically, statins induce oxidative stress accumulation and apoptosis through the GGPP synthase 1 (GGPS1)-RAB7A-autophagy axis. Statin treatment overcomes both intrinsic and acquired SCLC chemoresistance in vivo across different SCLC PDX models bearing high GGPS1 levels. Moreover, we show that GGPS1 expression is negatively associated with survival in patients with SCLC. Finally, we demonstrate that combined statin and chemotherapy treatment resulted in durable responses in three patients with SCLC who relapsed from first-line chemotherapy. Collectively, these data uncover the MVA-GGPP pathway as a metabolic vulnerability in SCLC and identify statins as a potentially effective treatment to overcome chemoresistance.

Perturbing electron carriers of green algae Chlamydomonas reinhardtii for efficient physiological and metabolic regulation

AbstractMicroalgae are widely used in the field of food bioengineering. There are relatively few studies on the synthesis of intracellular electron carriers in microalgae and their related metabolic effects. In this study, overexpression of Geranyl diphosphate: 4‐hydroxybenzoate geranyltransferase (lepgt), and the addition of 4‐hydroxybenzoic acid (4‐HB) as quinone ring precursor were adopted to regulate intracellular plastoquinone (PQ) and ubiquinone (UQ) levels in model algae Chlamydomonas reinhardtii. Small changes in PQ and UQ contents could regulate cell growth and metabolism. The expression of lepgt in C. reinhardtii significantly reduced the intracellular contents of PQ and UQ by 49.14% and 98.30%, respectively. The expression of lepgt observably reduced the photosystem activity and respiration rate and increased the contents of neutral lipid and starch by 63.68% and 19.42%, respectively. Exogenous low concentration of 4‐HB (1 mM) can effectively improve the contents of intracellular electron carriers, promote respiration, slightly reduce photosystem efficiency, and increase the maximum specific growth rate by 16.93%. It decreased the protein and oil contents by 15.48% and 15.10%, as well as changed the composition of long‐chain fatty acids. This paper provided a novel strategy for developing microalgal cell factories for future food production by perturbation on electron carriers of green algae.

Novel strategy to produce prenylated resveratrol by prenyltransferase iacE and evaluation of neuroprotective mechanisms

Prenylated resveratrols are drug candidates presented in many natural sources. They possess multiple pharmacological activities. Due to the limited abundance in nature and disadvantage of chemical synthesis, it is required to produce prenylated resveratrols by biological strategy. In this work, a prenyltransferase iacE from Pestalotiopsis fici was prepared by heterologous overexpression. It could catalyze 2-C-prenylation of resveratrol efficiently. Dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) could serve as prenyl donor. Glu97, Thr114 and Tyr413 were proved to be important residues in the active site by site-directed mutagenesis. The product was purified and identified to be 2-C-prenyl resveratrol by NMR. 2-C-prenyl resveratrol exerted neuroprotective activity through inhibiting ROS overproduction and improving the activities of antioxidant enzymes in HT22 cell. The genes involved in antioxidant defense were regulated, including upregulated CAT, SOD2, NFE2L2, GPx4, and downregulated PTGS2 and ACSL4. These results supplied a novel technique for prenylated resveratrols production.

Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule.

Production of natural rubber by Parthenium argentaum (guayule) requires increased yield for economic sustainability. An RNAi gene silencing strategy was used to engineer isoprenoid biosynthesis by downregulation of squalene synthase (SQS), such that the pool of farnesyl diphosphate (FPP) substrate might instead be available to initiate natural rubber synthesis. Downregulation of SQS resulted in significantly reduced squalene and slightly increased rubber, but not in the same tissues nor to the same extent, partially due to an apparent negative feedback regulatory mechanism that downregulated mevalonate pathway isoprenoid production, presumably associated with excess geranyl pyrophosphate levels. A detailed metabolomics analysis of isoprenoid production in guayule revealed significant differences in metabolism in different tissues, including in active mevalonate and methylerythritol phosphate pathways in stem tissue, where rubber and squalene accumulate. New insights and strategies for engineering isoprenoid production in guayule were identified.

Recent Advancements in Gibberellic Acid Formulation Techniques and Economics of Use in Agriculture

Gibberellins comprises of a broader family of plant advancement hormones found during the 1930s, which are synthesized by implies of the terpenes course from the geranylgeranyl diphosphate and incorporate a crucial structure formed by an ent-gibberellane tetracyclic skeleton. Among them Gibberellic acid (GA3 ), which acts as a characteristic plant development controller, especially for stem extending, seed germination, and extended common item degree. It can be gotten from plants, organisms, and microbes. There are as well a couple of reports roughly microalgae GA3 makers. Life forms, especially Gibberella fujikuroi, are favored for GA3 generation through submerged maturing or solid-state development. Various factors may impact its generation, a few of which are related to the control and scale-up of maturing parameters. Particular GA3 items are open on the advertise. They can be found in liquid, powder, tablet, beads and granule dosage forms containing because it were GA3 or a mix of other normal dynamic gibberellins, which can be employed in agriculture, horticulture & floriculture crops.

Correction to: A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms.

Correction to: A Single Arabidopsis Gene Encodes Two Differentially Targeted Geranylgeranyl Diphosphate Synthase Isoforms.

Knockout of GGPPS1 restrains rab37-mediated autophagy in response to ventilator-induced lung injury.

Mechanical ventilation may cause ventilator-induced lung injury (VILI) in patients requiring ventilator support. Inhibition of autophagy is an important approach to ameliorate VILI as it always enhances lung injury after exposure to various stress agents. This study aimed to further reveal the potential mechanisms underlying the effects of geranylgeranyl diphosphate synthase large subunit 1 (GGPPS1) knockout and autophagy in VILI using C57BL/6 mice with lung-specific GGPPS1 knockout that were subjected to mechanical ventilation. The results demonstrate that GGPPS1 knockout mice exhibit significantly attenuated VILI based on the histologic score, the lung wet-to-dry ratio, total protein levels, neutrophils in bronchoalveolar lavage fluid, and reduced levels of inflammatory cytokines. Importantly, the expression levels of autophagy markers were obviously decreased in GGPPS1 knockout mice compared with wild-type mice. The inhibitory effects of GGPPS1 knockout on autophagy were further confirmed by measuring the ultrastructural change of lung tissues under transmission electron microscopy. In addition, knockdown of GGPPS1 in RAW264.7 cells reduced cyclic stretch-induced inflammation and autophagy. The benefits of GGPPS1 knockout for VILI can be partially eliminated through treatment with rapamycin. Further analysis revealed that Rab37 was significantly downregulated in GGPPS1 knockout mice after mechanical ventilation, while it was highly expressed in the control group. Simultaneously, Rab37 overexpression significantly enhances autophagy in cells that are treated with cyclin stretch, including GGPPS1 knockout cells. Collectively, our results indicate that GGPPS1 knockout results in reduced expression of Rab37 proteins, further restraining autophagy and VILI.

Escherichia coli tRNA 2-selenouridine synthase SelU selects its prenyl substrate to accomplish its enzymatic function

Bacterial tRNA 2-selenouridine synthase (SelU) in vitro converts S2U-RNA to its selenium analog (Se2U-RNA) in a two-step process: (i) geranylation of S2U-RNA (with geranyl pyrophosphate, gePP), and (ii) selenation of the resulting geS2U-RNA (with the selenophosphate anion, SePO33−). Using an S2U-containing anticodon stem-loop fragment derived from tRNALys (S2U-RNA) and recombinant SelU with an MBP tag, we found that only geranyl (C10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C5), dimethylallyl (C5), farnesyl (C15) and geranylgeranyl (C20) are not. Interestingly, methyl (C1)− and C5−, C10−, and C15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by SelU, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl ≥ farnesyl > dimethylallyl ≫ methyl. Microscale thermophoresis showed an affinity between gePP and SelU in the micromolar range, while the other pyrophosphates tested, such as isopentenyl, dimethylallyl, farnesyl and geranylgeranyl, either did not bind to the protein or their binding affinity was above 1 mM. These results agree well with the in silico analysis, with gePP being the best binding substrate (the lowest relative free energy of binding (ΔG) and a small solvent-accessible surface area (SASA)). These results suggest that SelU has high substrate specificity for the prenylation reaction (only gePP is accepted), whereas there is little discrimination for the selenation reaction. We therefore suggest that only gePP and the geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs, as it is found in the bacterial system.

Phycocyanin Fusion Constructs for Heterologous Protein Expression Accumulate as Functional Heterohexameric Complexes in Cyanobacteria.

Overexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein "P" of interest is expressed as a fusion with the abundant CpcB β-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous "P" protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (α,β*P)3CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, β*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (α,β*P)3CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per β-subunit. (iii) The (α,β*P)3CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,β*P)3CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon α-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (α,β*P)3CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,β)3 compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest.

First report of triterpenes pathway in Calotropis procera revealed to accumulate beta-amyrin

The major reports on Calotropis procera (C. procera) indicated the importance of this plant as a resource of pharmaceutically active ingredients as well as its medical advantages. β-amyrin (BA) is a significant substance in this plant and has a pharmacological effects in some frameworks, like focal and fringe sensory system, digestive and immune systems. In this study, the impact of sunlight before and after irrigation on the BA production in C. procera is studied its pathway with involved eight key enzymes. The eight enzymes' genes were characterized and successfully submitted to NCBI; AAS (acc.no. KU997645) for α-amyrin synthase, BAS (acc.no. MW976955) for β-amyrin synthase, SE (acc.no. MW976956) for squalene epoxidase, SS (acc.no. MW976957) for squalene synthase, GPPS, (acc.no. MW976958) for geranyl pyrophosphate synthase, FPPS (acc.no. MW976959) for farnasyl pyrophosphate synthase, CAS1, (acc.no. MZ00598) for cycloartenol synthase1 and LS (acc.no. MZ005982) for lupeol synthase. qRT-PCR analysis revealed high expression levels of GPPS, FPPS, SS, SE, and BAS genes at all times specially midday. Otherwise, CAS1, LS and BAS expression levels were very low at all daylight periods. The UPLC β-amyrin data are in accordance with qRT-PCR results. This indicates that triterpenes biosynthetic pathway in C. procera is going to β-amyrin accumulation with the highest level at midday.

Quality Parameters, Empirical and Kinetic Models of Lycopene and Beta-carotene Bioformation in Tomatoes (Solanum lycopersicum)

This study modelled a kinetic for estimating geranyl geranyl pyrophosphate (GGPP) concentration in the carotenoid pathway in tomatoes (Solanum lycopersicon). Kinetics of bioformation of carotenoids in tomatoes of different cultivars (Cherry-Nasmata, VAR-10 and 4-lobes) at different ripening stages and conditions served as a model system in estimating the GGPP concentration in the kinetic model. Physicochemical parameters, lycopene and beta-carotene contents were assessed and compared under the two ripening conditions using standard laboratory procedures. The solid contents in the three cultivars of tomato range from 5.61% to 6.85% and 5.83% to 7.16% at field and ambient temperature ripening, respectively, the pH values were all in the acidic region. Highest lycopene and beta-carotene concentrations were observed in 4-lobes cultivar at field ripening. The obtained data subjected to empirical and kinetic modeling favoured the exponential models as a first order kinetics for both lycopene and beta-carotene bioformation in Cherry-Nasmata and 4-lobes cultivars. This suggests that beta-carotene bioformation is dependent on the pathway of lycopene in the tomato cultivars under study. The quality parameters of the tomato cultivars showed that ripening conditions have influence on their quality contents and the GGPP concentrations of some of the tomatoes were successfully estimated using the kinetic model.

Incomplete Hydrogenation by Geranylgeranyl Reductase from a Proteobacterial Phototroph Halorhodospira halochloris, Resulting in the Production of Bacteriochlorophyll with a Tetrahydrogeranylgeranyl Tail.

ABSTRACTLight harvesting and charge separation are functions of chlorophyll and bacteriochlorophyll pigments. While most photosynthetic organisms use (bacterio)chlorophylls with a phytyl (2-phytenyl) group as the hydrophobic isoprenoid tail, Halorhodospira halochloris, an anoxygenic photosynthetic bacterium belonging to Gammaproteobacteria, produces bacteriochlorophylls with a unique 6,7,14,15-tetrahydrogeranylgeranyl (2,10-phytadienyl) tail. Geranylgeranyl reductase (GGR), encoded by the bchP gene, catalyzes hydrogenation at three unsaturated C=C bonds of a geranylgeranyl group, giving rise to the phytyl tail. In this study, we discovered that H. halochloris GGR exhibits only partial hydrogenation activities, resulting in the tetrahydrogeranylgeranyl tail formation. We hypothesized that the hydrogenation activity of H. halochloris GGR differed from that of Chlorobaculum tepidum GGR, which also produces a pigment with partially reduced hydrophobic tails (2,6-phytadienylated chlorophyll a). An engineered GGR was also constructed and demonstrated to perform only single hydrogenation, resulting in the dihydrogeranylgeranyl tail formation. H. halochloris original and variant GGRs shed light on GGR catalytic mechanisms and offer prospective bioengineering tools in the microbial production of isoprenoid compounds.IMPORTANCE Geranylgeranyl reductase (GGR) catalyzes the hydrogenation of carbon–carbon double bonds of unsaturated hydrocarbons of isoprenoid compounds, including α-tocopherols, phylloquinone, archaeal cell membranes, and (bacterio)chlorophyll pigments in various organisms. GGRs in photosynthetic organisms, including anoxygenic phototrophic bacteria, cyanobacteria, and plants perform successive triple hydrogenation to produce chlorophylls and bacteriochlorophylls with a phytyl chain. Here, we demonstrated that the GGR of a gammaproteobacterium Halorhodospira halochloris catalyzed unique double hydrogenation to produce bacteriochlorophylls with a tetrahydrogeranylgeranyl tail. We also constructed a variant enzyme derived from H. halochloris GGR that performs only single hydrogenation. The results of this study provide new insights into catalytic mechanisms of multiposition reductions by a single enzyme.

A cytochrome P450 superfamily gene, IbCYP82D47, increases carotenoid contents in transgenic sweet potato

The cytochrome P450 superfamily (CYP450) is one of the largest protein families in plants, and its members play diverse roles in primary and secondary metabolic biosynthesis. In this study, the CYP450 family gene IbCYP82D47 was cloned from the high carotenoid line HVB-3 of sweet potato (Ipomoea batatas). The IbCYP82D47 protein harbored two transmembrane domains and dynamically localized between plastid stroma and membrane. Overexpression of IbCYP82D47 not only increased total carotenoid, lutein, zeaxanthin and violaxanthin contents by 32.2–48.0%, 10.5–13.3%, 40.2–136% and 82.4–106%, respectively, but also increased the number of carotenoid globules in sweet potato storage roots. Furthermore, genes associated with the carotenoid biosynthesis (IbDXS, IbPSY, IbLCYE, IbBCH, IbZEP) were upregulated in transgenic sweet potato. In addition, IbCYP82D47 physically interacts with geranylgeranyl diphosphate synthase 12 (IbGGPPS12). Our findings suggest that IbCYP82D47 increases carotenoid contents by interacting with the carotenoid biosynthesis related protein IbGGPPS12, and influencing the expressions of carotenoid biosynthesis related genes in transgenic sweet potato.

Limonene production in microalgal photoautotrophic cultivation / Produção de limoneno em cultivo fotoautotrófico microalgal

There is a worldwide consensus in the food industry that aims to replace synthetic ingredients with natural ingredients for health promotion. The industrial relevance of microalgae as sources of extensive spectrum of bioproducts and as promising raw materials for the production of natural additives is constantly growing. Limonene, due to its proven safety, makes it widely exploitable in the food, cosmetics and pharmaceutical industries, for exhibiting vast biological activity, with beneficial effects on health. Some microalgae produce this biocompound from the isoprenoid pathway through the action of the enzyme limonene synthase (LMS) from geranyl-pyrophosphate (GPP). Therefore, the aim of the study was to investigate the formation of the volatile compound limonene, derived from six microalgae, from photoautotrophic cultivation, as well as to evaluate its odor properties. The experiment was carried out in a bioreactor with BG11 medium. The incubation conditions used were 25°C, photon flux density of 25 μmol.m-2.s-1, aeration of 1VVM (air volume per volume of medium per minute) and light cycle of 24:0h (bright:dark). Volatiles were isolated by headspace solid phase microextraction, separated by gas chromatography, identified by mass spectrometry (SPME-GC-MS) and their odor significance was evaluated based on the NIST spectrum library. Limonene was identified in the volatile fraction of the six microalgae, contributing positively to the aroma (citrus and mint), demonstrating high biotechnological capacity to obtain an alternative of this natural biocompound with commercial viability, with the microalgae Scenedesmus bijuga being the one with the greatest quantitative potential.

Geranylgeranyl diphosphate synthase inhibitor and proteasome inhibitor combination therapy in multiple myeloma

BackgroundMultiple myeloma (MM) remains an incurable malignancy, despite the advent of therapies such as proteosome inhibitors (PIs) that disrupt protein homeostasis and induce ER stress. We have pursued inhibition of geranylgeranyl diphosphate synthase (GGDPS) as a novel mechanism by which to target protein homeostasis in MM cells. GGDPS inhibitors (GGSI) disrupt Rab geranylgeranylation, which in turn results in perturbation of Rab-mediated protein trafficking, leading to accumulation of intracellular monoclonal protein, induction of ER stress and apoptosis. Our lead GGSI, RAM2061, has demonstrated favorable pharmacokinetic properties and in vivo efficacy. Here we sought to evaluate if combination therapy with GGSI and PI would result in enhanced disruption of the unfolded protein response (UPR) and increase anti-MM efficacy.MethodsMTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with bortezomib in human MM cells. The effects of RAM2061 and/or PI (bortezomib or carfilzomib) on markers of UPR and apoptosis were evaluated by a combination of immunoblot (ATF4, IRE1, p-eIF2a, cleaved caspases and PARP), RT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and flow cytometry (Annexin-V). Induction of immunogenic cell death (ICD) was assessed by immunoblot (HMGB1 release) and flow cytometry (calreticulin translocation). Cell assays were performed using both concurrent and sequential incubation with PIs. To evaluate the in vivo activity of GGSI/PI, a flank xenograft using MM.1S cells was performed.ResultsIsobologram analysis of cytotoxicity data revealed that sequential treatment of bortezomib with RAM2061 has a synergistic effect in MM cells, while concurrent treatment was primarily additive or mildly antagonistic. The effect of PIs on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was dependent on timing of the PI exposure. Combination treatment with RAM2061 and bortezomib enhanced activation of ICD pathway markers. Lastly, combination treatment slowed MM tumor growth and lengthened survival in a MM xenograft model without evidence of off-target toxicity.ConclusionWe demonstrate that GGSI/PI treatment can potentiate activation of the UPR and apoptotic pathway, as well as induce upregulation of markers associated with the ICD pathway. Collectively, these findings lay the groundwork for future clinical studies evaluating combination GGSI and PI therapy in patients with MM.

Combination treatment with statins and bezafibrate induces myotoxicity via inhibition of geranylgeranyl pyrophosphate biosynthesis and Rho activation in L6 myoblasts and myotube cells.

Statins and fibrates are frequently used to treat hyperlipidemia; however, these drugs may have adverse effects such as rhabdomyolysis. The incidence of rhabdomyolysis due to fibrates and statins is low (0.0028-0.0096%) when administered as monotherapy, however it increases to 0.015-0.021% when the drugs are used in combination. The mechanism underlying myotoxicity induced by the combination of statins and fibrates is yet unclear. Here, we investigated the mechanisms underlying induced myotoxicity in rat myoblasts L6 and differentiated L6 cells (myotubes) using a combination of statins and fibrates. We found that cell death induced by a combination of fluvastatin or simvastatin with bezafibrate or fenofibrate in L6 myoblasts and myotubes was mediated by inhibition of geranylgeranyl pyrophosphate (GGPP) production. Additionally, the drug combination inhibited Rho activation in L6 myoblasts and myotube cells. In L6 myoblasts, the combination of statins and bezafibrate enhanced p27 expression and induced G1 arrest and apoptosis. Furthermore, combined treatment suppressed Akt activation and enhanced Bim expression in L6 myotubes but did not affect extracellular regulated protein kinase 1/2 activation. These results suggested that combined administration of statins and fibrates induced death of L6 myoblasts and myotube cells by inhibiting GGPP biosynthesis and Rho pathway activation. Supplementation with GGPP may be therapeutically beneficial for preventing myotoxicity associated with combined statin and fibrates treatment.

Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2–7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Removal of lycopene substrate inhibition enables high carotenoid productivity in Yarrowia lipolytica

Substrate inhibition of enzymes can be a major obstacle to the production of valuable chemicals in engineered microorganisms. Here, we show substrate inhibition of lycopene cyclase as the main limitation in carotenoid biosynthesis in Yarrowia lipolytica. To overcome this bottleneck, we exploit two independent approaches. Structure-guided protein engineering yields a variant, Y27R, characterized by complete loss of substrate inhibition without reduction of enzymatic activity. Alternatively, establishing a geranylgeranyl pyrophosphate synthase-mediated flux flow restrictor also prevents the onset of substrate inhibition by diverting metabolic flux away from the inhibitory metabolite while maintaining sufficient flux towards product formation. Both approaches result in high levels of near-exclusive β-carotene production. Ultimately, we construct strains capable of producing 39.5 g/L β-carotene at a productivity of 0.165 g/L/h in bioreactor fermentations (a 1441-fold improvement over the initial strain). Our findings provide effective approaches for removing substrate inhibition in engineering pathways for efficient synthesis of natural products.

Synthesis and Evaluation of Structurally Diverse C-2-Substituted Thienopyrimidine-Based Inhibitors of the Human Geranylgeranyl Pyrophosphate Synthase.

Novel analogues of C-2-substituted thienopyrimidine-based bisphosphonates (C2-ThP-BPs) are described that are potent inhibitors of the human geranylgeranyl pyrophosphate synthase (hGGPPS). Members of this class of compounds induce target-selective apoptosis of multiple myeloma (MM) cells and exhibit antimyeloma activity in vivo. A key structural element of these inhibitors is a linker moiety that connects their (((2-phenylthieno[2,3-d]pyrimidin-4-yl)amino)methylene)bisphosphonic acid core to various side chains. The structural diversity of this linker moiety, as well as the side chains attached to it, was investigated and found to significantly impact the toxicity of these compounds in MM cells. The most potent inhibitor identified was evaluated in mouse and rat for liver toxicity and systemic exposure, respectively, providing further optimism for the potential value of such compounds as human therapeutics.