Geranylgeranyl Diphosphate Synthase Research Articles

Exogenous Application of 5-Aminolevulinic Acid Promotes Coloration and Improves the Quality of Tomato Fruit by Regulating Carotenoid Metabolism.

5-Aminolevulinic acid (ALA) plays an important role in plant growth and development. It can also be used to enhance crop resistance to environmental stresses and improve the color and internal quality of fruits. However, there are limited reports regarding the effects of ALA on tomato fruit color and its regulatory mechanisms. Therefore, in this study, the effects of exogenous ALA on the quality and coloration of tomato fruits were examined. Tomato (Solanum lycopersicum “Yuanwei No. 1”) fruit surfaces were treated with different concentrations of ALA (0, 100, and 200 mg⋅L–1) on the 24th day after fruit setting (mature green fruit stage), and the content of soluble sugar, titratable acid, soluble protein, vitamin C, and total free amino acids, as well as amino acid components, intermediates of lycopene synthetic and metabolic pathways, and ALA metabolic pathway derivatives were determined during fruit ripening. The relative expression levels of genes involved in lycopene synthesis and metabolism and those involved in ALA metabolism were also analyzed. The results indicated that exogenous ALA (200 mg⋅L–1) increased the contents of soluble sugars, soluble proteins, total free amino acids, and vitamin C as well as 11 kinds of amino acid components in tomato fruits and reduced the content of titratable acids, thus improving the quality of tomato fruits harvested 4 days earlier than those of the control plants. In addition, exogenous ALA markedly improved carotenoid biosynthesis by upregulating the gene expression levels of geranylgeranyl diphosphate synthase, phytoene synthase 1, phytoene desaturase, and lycopene β-cyclase. Furthermore, exogenous ALA inhibited chlorophyll synthesis by downregulating the genes expression levels of Mg-chelatase and protochlorophyllide oxidoreductase. These findings suggest that supplementation with 200 mg⋅L–1 ALA not only enhances the nutritional quality and color of the fruit but also promotes early fruit maturation in tomato.

Plant geranylgeranyl diphosphate synthases: every (gene) family has a story.

Plant isoprenoids (also known as terpenes or terpenoids) are a wide family of primary and secondary metabolites with multiple functions. In particular, most photosynthesis-related isoprenoids (including carotenoids and chlorophylls) as well as diterpenes and polyterpenes derive from geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) enzymes in several cell compartments. Plant genomes typically harbor multiple copies of differentially expressed genes encoding GGPPS-like proteins. While sequence comparisons allow to identify potential GGPPS candidates, experimental evidence is required to ascertain their enzymatic activity and biological function. Actually, functional analyses of the full set of potential GGPPS paralogs are only available for a handful of plant species. Here we review our current knowledge on the GGPPS families of the model plant Arabidopsis thaliana and the crop species rice (Oryza sativa), pepper (Capsicum annuum) and tomato (Solanum lycopersicum). The results indicate that a major determinant of the biological role of particular GGPPS paralogs is the expression profile of the corresponding genes even though specific interactions with other proteins (including GGPP-consuming enzymes) might also contribute to subfunctionalization. In some species, however, a single GGPPS isoforms appears to be responsible for the production of most if not all GGPP required for cell functions. Deciphering the mechanisms regulating GGPPS activity in particular cell compartments, tissues, organs and plant species will be very useful for future metabolic engineering approaches aimed to manipulate the accumulation of particular GGPP-derived products of interest without negatively impacting the levels of other isoprenoids required to sustain essential cell functions.

Biosynthesis of pinene in purple non-sulfur photosynthetic bacteria

BackgroundPinene is a monoterpene, that is used in the manufacture of fragrances, insecticide, fine chemicals, and renewable fuels. Production of pinene by metabolic-engineered microorganisms is a sustainable method. Purple non-sulfur photosynthetic bacteria belong to photosynthetic chassis that are widely used to synthesize natural chemicals. To date, researches on the synthesis of pinene by purple non-sulfur photosynthetic bacteria has not been reported, leaving the potential of purple non-sulfur photosynthetic bacteria synthesizing pinene unexplored.ResultsRhodobacter sphaeroides strain was applied as a model and engineered to express the fusion protein of heterologous geranyl diphosphate synthase (GPPS) and pinene synthase (PS), hence achieving pinene production. The reaction condition of pinene production was optimized and 97.51 μg/L of pinene was yielded. Then, genes of 1-deoxy-d-xylulose 5-phosphate synthase, 1-deoxy-d-xylulose 5-phosphate reductoisomerase and isopentenyl diphosphate isomerase were overexpressed, and the ribosome binding site of GPPS-PS mRNA was optimized, improving pinene titer to 539.84 μg/L.Conclusions In this paper, through heterologous expression of GPPS-PS, pinene was successfully produced in R. sphaeroides, and pinene production was greatly improved by optimizing the expression of key enzymes. This is the first report on pinene produce by purple non-sulfur photosynthetic bacteria, which expands the availability of photosynthetic chassis for pinene production.

Synthetic Protein Scaffolds for Improving R-(-)-Linalool Production in Escherichia coli.

R-(-)-Linalool is widely used in the pharmaceutical, agrochemical, and fragrance industries; however, its applications are limited owing to low yield and high cost of production. To improve the production efficiency of R-(-)-linalool in Escherichia coli, three enzymes [E. coli-derived isopentenyl diphosphate isomerase, Abies grandis-derived geranyl diphosphate synthase, and Streptomyces clavuligerus-derived (3R)-linalool synthases] were physically colocalized to synthetic complexes using synthetic protein scaffolds of GTPase-binding domain, Src homology 3, and PSD95/DlgA/Zo-1. R-(-)-Linalool was produced at the highest concentration in the strain IGL114 containing a scaffold ratio of 1:1:4. By further optimizing the inducer, temperature, and glycerol concentration, the production titer of R-(-)-linalool in the shake flask was increased by approximately 10 times compared with that of the scaffold-free control and was 2.78 times the previously reported yield. The production in the fermenter was about 1.5 times the previous highest production. In general, the final strain accumulated 277.8 and 1523.2 mg/L R-(-)-linalool under the conditions of shake-flask and fed-batch fermentation, respectively. This study provides a foundation for the assembly of bacterial intracellular protein scaffolds.

Engineering Escherichia coli for production of geraniol by systematic synthetic biology approaches and laboratory-evolved fusion tags

Geraniol is a valuable monoterpene extensively used in the fragrance, food, and cosmetic industries. Increasing environmental concerns and supply gaps have motivated efforts to advance the microbial production of geraniol from renewable feedstocks. In this study, we first constructed a platform geraniol Escherichia coli strain by bioprospecting the key enzymes geranyl diphosphate synthase (GPPS) and geraniol synthase (GES) and selection of a host cell background. This strategy led to a 46.4-fold increase in geraniol titer to 964.3 mg/L. We propose that the expression level of eukaryotic GES can be further optimized through fusion tag evolution engineering. To this end, we manipulated GES to maximize flux towards the targeted product geraniol from precursor geranyl diphosphate (GPP) via the utilization of fusion tags. Additionally, we developed a high-throughput screening system to monitor fusion tag variants. This common plug-and-play toolbox proved to be a robust approach for systematic modulation of protein expression and can be used to tune biosynthetic metabolic pathways. Finally, by combining a modified E1* fusion tag, we achieved 2124.1 mg/L of geraniol in shake flask cultures, which reached 27.2% of the maximum theoretical yield and was the highest titer ever reported. We propose that this strategy has set a good reference for enhancing a broader range of terpenoid production in microbial cell factories, which might open new possibilities for the bio-production of other valuable chemicals.

Enhancing Geranylgeraniol Production by Metabolic Engineering and Utilization of Isoprenol as a Substrate in Saccharomyces cerevisiae.

The amount of geranylgeranyl diphosphate (GGPP) is vital for microbial production of geranylgeraniol (GGOH) in Saccharomyces cerevisiae. In this study, a GGPP synthase with stronger catalytic ability was used to increase the supply of GGPP, and an engineered strain producing 374.02 mg/L GGOH at the shake flask level was constructed. Then, by increasing the metabolic flux of the mevalonate (MVA) pathway and the supply of isopentenyl pyrophosphate (IPP), the titer was further increased to 772.98 mg/L at the shake flask level, and we achieved the highest GGOH titer to date of 5.07 g/L in a 5 L bioreactor. This is the first report on the utilization of isoprenol for increasing the amount of IPP and enhancing GGOH production in S. cerevisiae. In the future, these strategies and engineered strains can be used to enhance the production of other terpenoids in S. cerevisiae.

Several geranylgeranyl diphosphate synthase isoforms supply metabolic substrates for carotenoid biosynthesis in tomato.

Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway. Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms. The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de-etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR-Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued. Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform.

Cholesterol metabolic enzyme Ggpps regulates epicardium development and ventricular wall architecture integrity in mice

During embryonic heart development, the progenitor cells in the epicardium would migrate and differentiate into noncardiomyocytes in myocardium and affect the integrity of ventricular wall, but the underlying mechanism has not been well studied. We have found that myocardium geranylgeranyl diphosphate synthase (Ggpps), a metabolic enzyme for cholesterol biosynthesis, is critical for cardiac cytoarchitecture remodelling during heart development. Here, we further reveal that epicardial Ggpps could also regulate ventricular wall architecture integrity. Epicardium-specific deletion of Ggpps before embryonic day 10.5 (E10.5) is embryonic lethal, whereas after E13.5 is survival but with defects in the epicardium and ventricular wall structure. Ggpps deficiency in the epicardium enhances the proliferation of epicardial cells and disrupts cell‒cell contact, which makes epicardial cells easier to invade into ventricular wall. Thus, the fibroblast proliferation and coronary formation in myocardium were found enhanced that might disturb the coronary vasculature remodelling and ventricular wall integrity. These processes might be associated with the activation of YAP signalling, whose nuclear distribution is blocked by Ggpps deletion. In conclusion, our findings reveal a potential link between the cholesterol metabolism and heart epicardium and myocardium development in mammals, which might provide a new view of the cause for congenital heart diseases and potential therapeutic target in pathological cardiac conditions.

Computer-Aid Directed Evolution of GPPS and PS Enzymes.

Pinene, a natural active monoterpene, is widely used as a flavoring agent, perfume, medicine, and biofuel. Although genetically engineered microorganisms have successfully produced pinene, to date, the biological yield of pinene is much lower than that of semiterpenes (isoprene) and sesquiterpenes (farnesene). In addition to the low heterologous expression of geranyl pyrophosphate synthase (GPPS) and pinene synthase (PS), cytotoxicity due to accumulation of the monoterpene also limits the production of pinene in microorganisms. In this study, we attempted to use two strategies to increase the biological yield of pinene. By deleting the random coils of GPPS and PS alone or in combination, a strain with a 335% yield increase was obtained. Additionally, upon computer-guided molecular modeling and docking of GPPS with isopentenyl pyrophosphate (IPP), its substrate, the key sites located within the catalytic pocket for substrate binding, was predicted. After screening, a strain harboring the T273R mutation of GPPS was selected among a batch of mutations of the key sites with a 154% increase in pinene yield.

Geranylgeranyl diphosphate synthase deficiency hyperactivates macrophages and aggravates lipopolysaccharide-induced acute lung injury

Macrophage activation is a key contributing factor for excessive inflammatory responses of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Geranylgeranyl diphosphate synthase (GGPPS) plays a key role in the development of inflammatory diseases. Our group previously showed that GGPPS in alveolar epithelium have deleterious effects on acute lung injury induced by LPS or mechanical ventilation. Herein, we examined the role of GGPPS in modulating macrophage activation in ALI/ARDS. We found significant increased GGPPS expression in alveolar macrophages in patients with ARDS compared with healthy volunteers and in ALI mice induced by LPS. GGPPS-floxed control (GGPPSfl/fl) and myeloid-selective knockout (GGPPSfl/flLysMcre) mice were then generated. Interestingly, using an LPS-induced ALI mouse model, we showed that myeloid-specific GGPPS knockout significantly increased mortality, aggravated lung injury, and increased the accumulation of inflammatory cells, total protein, and inflammatory cytokines in BALF. In vitro, GGPPS deficiency upregulated the production of LPS-induced IL-6, IL-1β, and TNF-α in alveolar macrophages, bone marrow-derived macrophages (BMDMs), and THP-1 cells. Mechanistically, GGPPS knockout increased phosphorylation and nuclear translocation of NF-κB p65 induced by LPS. In addition, GGPPS deficiency increased the level of GTP-Rac1, which was responsible for NF-κB activation. In conclusion, decreased expression of GGPPS in macrophages aggravates lung injury and inflammation in ARDS, at least partly by regulating Rac1-dependent NF-κB signaling. GGPPS in macrophages may represent a novel therapeutic target in ARDS.

Recombinant Protein Stability in Cyanobacteria.

The living cell possesses extraordinary molecular and biochemical mechanisms by which to recognize and efficiently remove foreign, damaged, or denatured proteins. This essential function has been a barrier to the overexpression of recombinant proteins in most expression systems. A notable exception is the overexpression in E. coli of recombinant proteins, most of which, however, end-up as "inclusion bodies", i.e., cytoplasmic aggregates of proteins that are inaccessible to the cell's proteasome. "Fusion constructs as protein overexpression vectors" proved to be unparalleled in their ability to cause substantial accumulation of recombinant proteins from plants, animals, and bacteria, as soluble proteins in unicellular cyanobacteria. Recombinant protein levels in the range of 10-20% of the total cellular protein can be achieved. The present work investigated this unique property in the context of recombinant protein stability in Synechocystis sp. PCC 6803 by developing and applying an in vivo cellular tobacco etch virus cleavage system with the objective of separating the target heterologous proteins from their fusion leader sequences. The work provides new insights about the overexpression, cellular stability, and exploitation of transgenes with commercial interest, highly expressed in a cyanobacterial biofactory. The results support the notion that eukaryotic plant- and animal-origin recombinant proteins are unstable, when free in the cyanobacterial cytosol but stable when in a fusion configuration with a highly expressed cyanobacterial native or heterologous protein. Included in this analysis are recombinant proteins of the plant isoprenoid biosynthetic pathway (isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase), the human interferon protein, as well as prokaryotic proteins (tetanus toxin fragment C and the antibiotic resistance genes kanamycin and chloramphenicol). The future success of synthetic biology approaches with cyanobacteria and other systems would require overexpression of pathway enzymes to attain product volume, and the work reported in this paper sets the foundation for such recombinant pathway enzyme overexpression.

In vivo evaluation of combination therapy targeting the isoprenoid biosynthetic pathway

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.

Specific decorations of 17-hydroxygeranyllinalool diterpene glycosides solve the autotoxicity problem of chemical defense inNicotiana attenuata

The native diploid tobacco Nicotiana attenuata produces abundant, potent anti-herbivore defense metabolites known as 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) whose glycosylation and malonylation biosynthetic steps are regulated by jasmonate signaling. To characterize the biosynthetic pathway of HGL-DTGs, we conducted a genome-wide analysis of uridine diphosphate glycosyltransferases (UGTs) and identified 107 family-1 UGT members. The transcript levels of three UGTs were highly correlated with the transcript levels two key HGL-DTG biosynthetic genes: geranylgeranyl diphosphate synthase (NaGGPPS) and geranyllinalool synthase (NaGLS). NaGLS’s role in HGL-DTG biosynthesis was confirmed by virus-induced gene silencing. Silencing the Uridine diphosphate (UDP)-rhamnosyltransferase gene UGT91T1 demonstrated its role in the rhamnosylation of HGL-DTGs. In vitro enzyme assays revealed that UGT74P3 and UGT74P4 use UDP-glucose for the glucosylation of 17-hydroxygeranyllinalool (17-HGL) to lyciumoside I. Plants with stable silencing of UGT74P3 and UGT74P5 were severely developmentally deformed, pointing to a phytotoxic effect of the aglycone. The application of synthetic 17-HGL and silencing of the UGTs in HGL-DTG-free plants confirmed this phytotoxic effect. Feeding assays with tobacco hornworm (Manduca sexta) larvae revealed the defensive functions of the glucosylation and rhamnosylation steps in HGL-DTG biosynthesis. Glucosylation of 17-HGL is therefore a critical step that contributes to the resulting metabolites’ defensive function and solves the autotoxicity problem of this potent chemical defense.

Transcriptome and volatile compounds profiling analyses provide insights into the molecular mechanism underlying the floral fragrance of tree peony

Tree peony (Paeonia suffruticosa Andr.) is a popular ornamental plant around the world, which is known for its distinctively large and fragrant flowers. However, the underlying molecular mechanism of floral fragrance synthesis in tree peony remains poorly understood. In this study, integrative volatile profiling and RNA sequencing were conducted in four wild tree peony species: Paeonia ostii, Paeonia rockii, Paeonia delavayi and Paeonia lutea. A total of 67 floral volatiles were identified through gas chromatography-mass spectrometry (GC–MS) analysis. Terpenes were most abundant volatiles in P. ostii and P. rockii, while benzenoids content were relatively higher in P. delavayi and P. lutea. Transcriptome data revealed 17,967 differential expression genes (DEGs), 116 and 147 of which were found be related to the accumulation of terpenes and benzenoids, respectively. Specifically, 1-deoxy-d-xylulose 5-phosphate synthase, geranyl pyrophosphate synthase, farnesyl pyrophosphate synthase and terpene synthase may be the major regulators of floral terpenes biosynthesis in P. ostii and P. rockii, whilst 3-deoxy-7-phosphoheptulonate synthase, phenylalanine ammonialyase, cinnamate: CoA ligase and short-chain dehydrogenases/reductases may control the production of benzenoids production in P. delavayi and P. lutea. Taken together, results from this study sheds light on the molecular mechanism of fragrance variation among different tree peony species and also provides a valuable resource to investigate floral fragrance formation in tree peony.

Enhanced limonene production in a fast-growing cyanobacterium through combinatorial metabolic engineering

Terpenoids are a large and diverse group of natural products with commercial applications. Microbial production of terpenes is considered as a feasible approach for the stable supply of these complex hydrocarbons. Cyanobacteria, photosynthetic prokaryotes, are attractive hosts for sustainable bioproduction, because these autotrophs require only light and CO2 for growth. Despite cyanobacteria having been engineered to produce a variety of compounds, their productivities of terpenes are generally low. Further research is needed to determine the bottleneck reactions for enhancing terpene production in cyanobacteria. In this study, we engineered the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce a commercially-used terpenoid, limonene. We identified a beneficial mutation in the gene encoding geranylgeranyl pyrophosphate synthase crtE, leading to a 2.5-fold increase in limonene production. The engineered strain produced 16.4 ​mg ​L−1 of limonene at a rate of 8.2 ​mg ​L−1 day−1, which is 8-fold higher than limonene productivities previously reported in other cyanobacterial species. Furthermore, we employed a combinatorial metabolic engineering approach to optimize genes involved in the upstream pathway of limonene biosynthesis. By modulating the expression of genes encoding the enzymes in the MEP pathway and the geranyl pyrophosphate synthase, we showed that optimization of the expression level is critical to enhance limonene production in cyanobacteria.

Integrated Transcriptomic and Metabolomic analysis reveals a transcriptional regulation network for the biosynthesis of carotenoids and flavonoids in \u2018Cara cara\u2019 navel Orange

BackgroundCarotenoids and flavonoids are important secondary metabolites in plants, which exert multiple bioactivities and benefits to human health. Although the genes that encode carotenogenesis and flavonoid biosynthetic enzymes are well characterized, the transcriptional regulatory mechanisms that are related to the pathway genes remain to be investigated. In this study, ‘Cara cara’ navel orange (CNO) fruit at four development stages were used to identify the key genes and TFs for carotenoids and flavonoids accumulation.ResultsIn this study, CNO was used to investigate the profiles of carotenoids and flavonoids by a combination of metabolomic and transcriptomic analyses. The important stage for the accumulation of the major carotenoid, lycopene was found to be at 120 days after florescence (DAF). The transcripts of five carotenogenesis genes were highly correlated with lycopene contents, and 16, 40, 48, 24 and 18 transcription factors (TFs) were predicted to potentially bind 1-deoxy-D-xylulose-5-phosphate synthase (DXS1), deoxyxylulose 5-phosphate reductoisomerase (DXR), geranylgeranyl diphosphate synthase (GGPPS2), phytoene synthase (PSY1) and lycopene β-cyclase (LCYB) promoters, respectively. Narirutin was the most abundant flavonoid in the flesh at the early stages, 60 DAF was the most important stage for the accumulation of flavonoids, and 17, 22, 14, 25, 24 and 16 TFs could potentially bind phenylalanine ammonia-lyase (PAL-1 and PAL-4), 4-Coumarate-CoA ligase (4CL-2 and 4CL-5), chalcone synthase (CHS-1) and chalcone isomerase (CHI) promoters, respectively. Furthermore, both sets of 15 candidate TFs might regulate at least three key genes and contribute to carotenoids/flavonoids accumulation in CNO fruit. Finally, a hierarchical model for the regulatory network among the pathway genes and TFs was proposed.ConclusionsCollectively, our results suggest that DXS1, DXR, GGPPS2, PSY1 and LCYB genes were the most important genes for carotenoids accumulation, while PAL-1, PAL-4, 4CL-2, 4CL-5, CHS-1 and CHI for flavonoids biosynthesis. A total of 24 TFs were postulated as co-regulators in both pathways directly, which might play important roles in carotenoids and flavonoids accumulation in CNO fruit.

Functional Characterization of a New Bifunctional Terpene Synthase LpNES1 from a Medicinal Plant Laggera pter odonta.

Laggera pterodonta, known in China as 'Choulingdan' for its stimulous odor, has long been used as traditional herbal medicine. The essential oil of L. pterodonta, which exhibits various pharmacological activities, is a rich resource of monoterpenes and sesquiterpenes. To date, however, the terpene synthases responsible for their production remain unknown. In present study, a new terpene synthase gene (LpNES1) was identified from L. pterodonta, transcript level of which was significantly upregulated in response to methyl jasmonate treatment. Recombinant LpNES1 could synthesize (E)-nerolidol and minor β-farnesene from farnesyl diphosphate and linalool from geranyl diphosphate in vitro. Whereas, only sesquiterpenes including (E)-nerolidol and minor β-farnesene were released when LpNES1 was reconstituted in yeast, even coexpressed with a geranyl diphosphate synthase (ERG20WW). Combined with subcellular localization experiment, the result indicated that the cytosol-targeted LpNES1 was responsible for (E)-nerolidol biosynthesis exclusively in L. pterodonta. Additionally, the expression level of LpNES1 gene was more prominent in floral buds than that in other tissues. LpNES1 characterized in present study not only lays the molecular foundation for sesquiterpene biosynthesis of L. pterodonta, but provides a key element for further biosynthesis of bioactive compound in microbes.

Накопление нейтральных липидов и β-каротина дрожжами Yarrowia lipolytica на среде с сахарозой в качестве источника углерода

Recombinant Yarrowia lipolytica yeast has been used as a host strain for the expression of the Saccharomyces cerevisiae ScSUC2 gene and Y. lipolytica YlHXK1 gene encoding invertase and hexakinase, respectively. The expression was carried out on the background of the enhanced pathway of the synthesis of neutral lipids. This allowed the yeast to efficiently utilize glucose as a single carbon source. The engineered strain accumulated neutral lipids in amount of 50.7% of biomass dry weight, when cultured in tubes in minimal medium with nitrogen limitation and high sucrose content. The next metabolic engineering step was the use of the CRISPR-Cas9 editing system to introduce a heterologous β-carotene synthesis pathway by the expression of the Mucor circinelloides genes CarRP and CarB, encoding the bifunctional enzyme phytoene synthase/licopene-β-cyclase (carRP) and phytoene dehydrogenase (carB), as well as an increase in the expression level of the Y. lipolytica YlGGS1 gene, encoding geranyl diphosphate synthase enzyme. The β-carotene production on a sucrose-containing medium was shown for the first time; it amounted for 24.0 mg/g dry biomass, or 406.9 mg/L, on the 5th day of cultivation in tubes in a rich medium with a sucrose portioned supply (50 g/L). The corresponding values for a rich medium with a higher sucrose concentration (90 g/L) were 21.9 mg/g dry biomass and 625.8 mg/L. Yarrowia lipolytica, β-carotene, sucrose, CRISPR-Cas9 The plasmids pdKu70Yl-URA3, pLTet-SP-CAT, pARS-Cre-reverse, pMW-att-Cm and pGPD1Yl were kindly provided by Ph.D. I.A. Laptev ("Kurchatov Institute"-GOSNIIGENETIKA NRC). The work was carried out using the equipment of the Multipurpose Scientific Facility of the "Russian State Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»-GOSNIIGENETIKA. The work was supported by state assignment no. 14 of 25.07.2012 AAAA-A20-120093090016-9. The quantitative analysis of lipids and β-carotene was carried out by V.A. Zolottsev within the framework of the "Long-Term Program of Fundamental Scientific Research in the Russian Federation (2021-2030)".

Isolation and expression of five genes in the mevalonate pathway of the Chinese white pine beetle, Dendroctonus armandi (Curculionidae: Scolytinae).

The Chinese white pine beetle Dendroctonus armandi (Tsai and Li) is a significant pest of the Qinling and Bashan Mountains pine forests of China. The Chinese white pine beetle can overcome the defences of Chinese white pine Pinus armandi (Franch) through pheromone-assisted aggregation that results in a mass attack of host trees. We isolated five full-length complementary DNAs encoding mevalonate pathway-related enzyme genes from the Chinese white pine beetle (D. armandi), which are acetoacetyl-CoA thiolase (AACT), geranylgeranyl diphosphate synthase (GGPPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MPDC), and phosphomevalonate kinase (PMK). Bioinformatic analyses were performed on the full-length deduced amino acid sequences. Differential expression of these five genes was observed between sexes, and within these significant differences among topically applied juvenile hormone III (JH III), fed on phloem of P. armandi, tissue distribution, and development stage. Mevalonate pathway genes expression were induced by JH III and feeding.

Transcriptome analysis for molecular landscaping of genes controlling diterpene andrographolide biosynthesis in Andrographis paniculata (Burm . f.) Nees.

Kalmegh [Andrographis paniculata (Burm. f.) Nees.] is one of the essential medicinal plants due to an important terpenoid, i.e. andrographolide possesses immense therapeutic and pharmacological uses. The experiment was performed to elucidate the expression of candidate genes associated with andrographolide biosynthesis. Based on results obtained in chromatography for andrographolide content analysis of six genotypes, two contrast genotypes, i.e. IC-520361 (maximum andrographolide content—2.33%) and Anand Local (lowest andrographolide content—1.01%) were selected for the transcriptome analysis. A total of 1.04 Gb of raw data were produced using MiSeq Illumina platform, in which IC 520361 generated 645 million base pairs sequence along with 4,524,251 raw reads and Anand Local produced 419 million base pairs sequence along with 3,021,316 raw reads. The combined assembly of high quality reads generated for both the samples had 33,247,454 bp of total assembled bases and 38,292 of transcripts. The GC percent of assembled transcripts was 44.79%, an average read length was 800 bp and N50 value was 1186 bp. Species-specific distribution using BLAST X (Nr), showed the highest Blast hits with Sesamum indicum. Out of 23,346 transcripts, 87% of transcripts annotated in UniProt KB (Universal Protein Resource KnowledgeBase) database and only 0.21% of transcripts were annotated in TAIR (The Arabidopsis Information Resources). Biological processes gene ontology classified based on Blast2GO showed, out of 6853 transcripts, 1370 of transcripts were represented by terpenoid biosynthetic pathway, which involved in secondary metabolite andrographolide biosynthesis. The heat map showed 1016 transcripts were differentially expressed between two kalmegh genotypes, in which nine important differentially expressed transcripts related to MEP (2C methyl-d-erythritol 4-phosphate) and MVA (Mevalonic acid) andrographolide biosynthesis pathways such as, geranyl diphosphate synthase small subunit, Isopentenyl-diphosphate delta-isomerase i-like, 4, 13-hydroxy-3-methylglutaryl-coenzyme a reductase etc. were upregulated in IC 520361 as compared to Anand Local, which were validated through RT-qPCR. The highest expression of gene 13-hydroxy-3-methylglutaryl-coenzyme a reductase (HMGR) was reported, which is responsible for accumulation of andrographolide in leaf. This comparative transcriptome analysis confirmed the expression level of genes were higher in accession IC 520361 as compare to Anand Local related to andrographolide biosynthesis pathways i.e. MEP and MVA. These up-regulated genes could be over-expressed to enhance the andrographolide content using genetic engineering of these metabolic pathways. It will also give an idea to the breeder for development of molecular markers for direct screening of the genotypes.Electronic supplementary materialThe online version of this article (10.1007/s13205-020-02511-y) contains supplementary material, which is available to authorized users.