Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Genome-wide identification of the geranylgeranyl pyrophosphate synthase (GGPS) gene family involved in chlorophyll synthesis in cotton.

BACKGROUND: Geranylgeranyl pyrophosphate synthase (GGPS) is a structural enzyme of the terpene biosynthesis pathway that is involved in regulating plant photosynthesis, growth and development, but this gene family has not been systematically studied in cotton. RESULTS: In the current research, genome-wide identification was performed, and a total of 75 GGPS family members were found in four cotton species, Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium raimondii. The GGPS genes were divided into three subgroups by evolutionary analysis. Subcellular localization prediction showed that they were mainly located in chloroplasts and plastids. The closely related GGPS contains a similar gene structure and conserved motif, but some genes are quite different, resulting in functional differentiation. Chromosome location analysis, collinearity and selection pressure analysis showed that many fragment duplication events occurred in GGPS genes. Three-dimensional structure analysis and conservative sequence analysis showed that the members of the GGPS family contained a large number of α-helices and random crimps, and all contained two aspartic acid-rich domains, DDxxxxD and DDxxD (x is an arbitrary amino acid), suggesting its key role in function. Cis-regulatory element analysis showed that cotton GGPS may be involved in light response, abiotic stress and other processes. A GGPS gene was silenced successfully by virus-induced gene silencing (VIGS), and it was found that the chlorophyll content in cotton leaves decreased significantly, suggesting that the gene plays an important role in plant photosynthesis. CONCLUSIONS: In total, 75 genes were identified in four Gossypium species by a series of bioinformatics analysis. Gene silencing from GGPS members of G. hirsutum revealed that GGPS plays an important regulatory role in photosynthesis. This study provides a theoretical basis for the biological function of GGPS in cotton growth and development.

Identification and Functional Analysis of Two Novel Genes-Geranylgeranyl Pyrophosphate Synthase Gene (AlGGPPS) and Isopentenyl Pyrophosphate Isomerase Gene (AlIDI)-from Aurantiochytrium limacinum Significantly Enhance De Novo ß-Carotene Biosynthesis in Escherichia coli.

Precursor regulation has been an effective strategy to improve carotenoid production and the availability of novel precursor synthases facilitates engineering improvements. In this work, the putative geranylgeranyl pyrophosphate synthase encoding gene (AlGGPPS) and isopentenyl pyrophosphate isomerase encoding gene (AlIDI) from Aurantiochytrium limacinum MYA-1381 were isolated. We applied the excavated AlGGPPS and AlIDI to the de novo ß-carotene biosynthetic pathway in Escherichia coli for functional identification and engineering application. Results showed that the two novel genes both functioned in the synthesis of ß-carotene. Furthermore, AlGGPPS and AlIDI performed better than the original or endogenous one, with 39.7% and 80.9% increases in ß-carotene production, respectively. Due to the coordinated expression of the 2 functional genes, ß-carotene content of the modified carotenoid-producing E. coli accumulated a 2.99-fold yield of the initial EBIY strain in 12 h, reaching 10.99 mg/L in flask culture. This study helped to broaden current understanding of the carotenoid biosynthetic pathway in Aurantiochytrium and provided novel functional elements for carotenoid engineering improvements.

Heteromerization of short-chain trans-prenyltransferase controls precursor allocation within a plastidial terpenoid network.

Terpenes are the largest and most diverse class of plant specialized metabolites. Sesterterpenes (C25), which are derived from the plastid methylerythritol phosphate pathway, were recently characterized in plants. In Arabidopsis thaliana, four genes encoding geranylfarnesyl diphosphate synthase (GFPPS) (AtGFPPS1 to 4) are responsible for the production of GFPP, which is the common precursor for sesterterpene biosynthesis. However, the interplay between sesterterpenes and other known terpenes remain elusive. Here, we first provide genetic evidence to demonstrate that GFPPSs are responsible for sesterterpene production in Arabidopsis. Blockage of the sesterterpene pathway at the GFPPS step increased the production of geranylgeranyl diphosphate (GGPP)-derived terpenes. Interestingly, co-expression of sesterTPSs in GFPPS-OE (overexpression) plants rescued the phenotypic changes of GFPPS-OE plants by restoring the endogenous GGPP. We further demonstrated that, in addition to precursor (DMAPP/IPP) competition by GFPPS and GGPP synthase (GGPPS) in plastids, GFPPS directly decreased the activity of GGPPS through protein-protein interaction, ultimately leading to GGPP deficiency in planta. Our study provides a new regulatory mechanism of the plastidial terpenoid network in plant cells.

Cloning and homologous characterization of geranylgeranyl pyrophosphate synthase (GGPPS) from Withania somnifera revealed alterations in metabolic flux towards gibberellic acid biosynthesis.

MAIN CONCLUSION: Overexpression of a novel geranylgeranyl pyrophosphate synthase gene (WsGGPPS) in planta resulted in increased levels of gibberellic acid and decrease in withanolide content. Withania somnifera (L.) Dunal, the herb from family Solanaceae is one of the most treasured medicinal plant used in traditional medicinal systems owing to its unique stockpile of pharmaceutically active secondary metabolites. Phytochemical and pharmacological studies in this plant were well established, but the genes affecting the regulation of biosynthesis of major metabolites were not well elucidated. In this study cloning and functional characterization of a key enzyme in terpenoid biosynthetic pathway viz. geranylgeranyl pyrophosphate synthase (EC 2.5.1.29) gene from Withania somnifera was performed. The full length WsGGPPS gene contained 1,104 base pairs that encode a polypeptide of 365 amino acids. The quantitative expression analysis suggested that WsGGPPS transcripts were expressed maximally in flower tissues followed by berry tissues. The expression levels of WsGGPPS were found to be regulated by methyl jasmonate (MeJA) and salicylic acid (SA). Amino acid sequence alignment and phylogenetic studies suggested that WsGGPPS had close similarities with GGPPS of Solanum tuberosum and Solanum pennellii. The structural analysis provided basic information about three dimensional features and physicochemical parameters of WsGGPPS protein. Overexpression of WsGGPPS in planta for its functional characterization suggested that the WsGGPPS was involved in gibberellic acid biosynthesis.

Production of ent-kaurene from lignocellulosic hydrolysate in Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides has emerged as a promising host for the production of bioproducts from lignocellulose, in part due to its ability to grow on lignocellulosic feedstocks, tolerate growth inhibitors, and co-utilize sugars and lignin-derived monomers. Ent-kaurene derivatives have a diverse range of potential applications from therapeutics to novel resin-based materials. RESULTS: The Design, Build, Test, and Learn (DBTL) approach was employed to engineer production of the non-native diterpene ent-kaurene in R. toruloides. Following expression of kaurene synthase (KS) in R. toruloides in the first DBTL cycle, a key limitation appeared to be the availability of the diterpene precursor, geranylgeranyl diphosphate (GGPP). Further DBTL cycles were carried out to select an optimal GGPP synthase and to balance its expression with KS, requiring two of the strongest promoters in R. toruloides, ANT (adenine nucleotide translocase) and TEF1 (translational elongation factor 1) to drive expression of the KS from Gibberella fujikuroi and a mutant version of an FPP synthase from Gallus gallus that produces GGPP. Scale-up of cultivation in a 2 L bioreactor using a corn stover hydrolysate resulted in an ent-kaurene titer of 1.4 g/L. CONCLUSION: This study builds upon previous work demonstrating the potential of R. toruloides as a robust and versatile host for the production of both mono- and sesquiterpenes, and is the first demonstration of the production of a non-native diterpene in this organism.

Inhibition of GGPPS1 attenuated LPS-induced acute lung injury and was associated with NLRP3 inflammasome suppression.

Inhibition of the mevalonate pathway using statins has been shown to be beneficial in the treatment of acute lung injury (ALI). Here, we investigated whether partial inhibition of this pathway by targeting geranylgeranyl pyrophosphate synthase large subunit 1 (GGPPS1), a catalase downstream of the mevalonate pathway, was effective at treating lung inflammation in ALI. Lipopolysaccharide (LPS) was intratracheally instilled to induce ALI in lung-specific GGPPS1-knockout and wild-type mice. Expression of GGPPS1 in lung tissues and alveolar epithelial cells was examined. The severity of lung injury and inflammation was determined in lung-specific GGPPS1 knockout and wild-type mice by measuring alveolar exudate, neutrophil infiltration, lung injury, and cell death. Change in global gene expression in response to GGPPS1 depletion was measured using mRNA microarray and verified in vivo and in vitro. We found that GGPPS1 levels increased significantly in lung tissues and alveolar epithelial cells in LPS-induced ALI mice. Compared with wild-type and simvastatin treated mice, the specific deletion of pulmonary GGPPS1 attenuated the severity of lung injury by inhibiting apoptosis of AECs. Furthermore, deletion of GGPPS1 inhibited LPS-induced inflammasome activation, in terms of IL-1ß release and pyroptosis, by downregulating NLRP3 expression. Finally, downregulation of GGPPS1 reduced the membrane expression of Ras-related protein Rab10 and Toll-like receptor 4 (TLR4) and inhibited the phosphonation of IκB. This effect might be attributed to the downregulation of GGPP levels. Our results suggested that inhibition of pulmonary GGPPS1 attenuated LPS-induced ALI predominantly by suppressing the NLRP3 inflammasome through Rab10-mediated TLR4 replenishment.

[Bioinformatics analysis of geranylgeranyl pyrophosphate synthase coding gene and amino acid sequence in Lamiaceae].

Geranylgeranyl pyrophosphate synthase enzyme is one of the key enzymes in the synthesis pathway of diterpenoid. Nine Lamiaceae genus GGPS synthase in Genebank was analyzed in this article. GGPS synthase the nucleic acid sequences and amino acid sequences, physicochemical properties, the signal peptide, leader peptides, transmembrane topological structure, hydrophobic, hydrophilic, subcellular localization, secondary structure, function domain, tertiary structure and evolutional relationship were predicted by using bioinformatics methods.Phylogenetic tree was constructed for the geranylgeranyl pyrophosphate synthase enzyme protein family. The results showed that GGPS amino acid sequence of the physical and chemical properties were basically identical, mainly hydrophilic protein, there existed chloroplast transit peptide, and no signal peptide and membrane structure domain, which mainly located in the chloroplast, the minor part located in mitochondria. The main secondary structures of the proteins are alpha helix and random coil. All these proteins have catalytic residues, aspartate-rich region, active site lid residues, substrate-Mg2+ binding site. The results provide theoretical reference for study on both the enzymatic characteristics of GGPS and the biosynthesis pathway of diterpenoid.

Heterologous expression of chloroplast-localized geranylgeranyl pyrophosphate synthase confers fast plant growth, early flowering and increased seed yield.

Geranylgeranyl pyrophosphate synthase (GGPS) is a key enzyme for a structurally diverse class of isoprenoid biosynthetic metabolites including gibberellins, carotenoids, chlorophylls and rubber. We expressed a chloroplast-targeted GGPS isolated from sunflower (Helianthus annuus) under control of the cauliflower mosaic virus 35S promoter in tobacco (Nicotiana tabacum). The resulting transgenic tobacco plants expressing heterologous GGPS showed remarkably enhanced growth (an increase in shoot and root biomass and height), early flowering, increased number of seed pods and greater seed yield compared with that of GUS-transgenic lines (control) or wild-type plants. The gibberellin levels in HaGGPS-transgenic plants were higher than those in control plants, indicating that the observed phenotype may result from increased gibberellin content. However, in HaGGPS-transformant tobacco plants, we did not observe the phenotypic defects such as reduced chlorophyll content and greater petiole and stalk length, which were previously reported for transgenic plants expressing gibberellin biosynthetic genes. Fast plant growth was also observed in HaGGPS-expressing Arabidopsis and dandelion plants. The results of this study suggest that GGPS expression in crop plants may yield desirable agronomic traits, including enhanced growth of shoots and roots, early flowering, greater numbers of seed pods and/or higher seed yield. This research has potential applications for fast production of plant biomass that provides commercially valuable biomaterials or bioenergy.

Adaptive changes in geranylgeranyl pyrophosphate synthase gene expression level under ethanol stress conditions in Oenococcus oeni.

AIMS: The aim of this study was to investigate the effect of ethanol exposure on the expression level of geranylgeranyl pyrophosphate synthase gene involved in the metabolism of Oenococcus oeni to probe the mechanisms of ethanol tolerance correlated with adaptive changes. METHODS AND RESULTS: The evaluation of ten potential internal control genes and the comparative study of their stability were performed to select the most stable internal controls for the normalization of expression data. The expression level analysis by qPCR and changes after exposure to ethanol stresses highlighted a significant increase in the presence of higher ethanol concentrations. CONCLUSIONS: The analysis of results suggest that O. oeni adjusts the expression of genes to adapt to stress conditions and the high expression level of ggpps would allow a flow of isoprenoid precursors towards the carotenoids and related pathways to stabilize bacterial cell membranes, improving the cell membrane disturbances and preventing cell death induced by ethanol. SIGNIFICANCE AND IMPACT OF THE STUDY: The involvement of ggpps gene in physiological changes of bacterial behaviour confirmed the exposure to stress requires the activation of defence mechanism to be more tolerant to adverse conditions. Improving the knowledge of stress tolerance and adaptation mechanisms of O. oeni is essential to enhance the efficiency of the malolactic starter in wine and to obtain the development of starters able to survive to direct inoculation with a large benefit for wine technology.

Engineering Yarrowia lipolytica for Efficient Synthesis of Geranylgeraniol.

Geranylgeraniol (GGOH) is a crucial component in fragrances and essential oils, and a valuable precursor of vitamin E. It is primarily extracted from the oleoresin of Bixa orellana, but is challenged by long plant growth cycles, severe environmental pollution, and low extraction efficiency. Chemically synthesized GGOH typically comprises a mix of isomers, making the separation process both challenging and costly. Advancements in synthetic biology have enabled the construction of microbial cell factories for GGOH production. In this study, Yarrowia lipolytica was engineered to efficiently synthesize GGOH by expressing heterologous phosphatase genes, enhancing precursor supplies of farnesyl diphosphate, geranylgeranyl pyrophosphate, and acetyl-CoA, and downregulating the squalene synthesis pathway by promoter engineering. Additionally, optimizing fermentation conditions and reducing reactive oxygen species significantly increased the GGOH titer to 3346.47 mg/L in a shake flask. To the best of our knowledge, this is the highest reported GGOH titer in shaking flasks to date, setting a new benchmark for terpenoid production.

Gibberellic acid 3 enhanced the anticancer activity of Abeliophyllum distichum adventitious roots by activating the diterpenoid biosynthesis pathway.

The industrial value of various plants has been improved through the of plant cell culture systems with elicitation. In this study, the adventitious root of Abeliophyllum distichum (AdAR) was treated with gibberellic acid 3 (GA3) to improve its anticancer property. The hexane fraction of the GA3-treated A. distichum adventitious root exhibited a stronger cytotoxic activity against A549 cells than the hexane fraction of AdAR. Through GC/MS and principal component analysis, we identified ferruginol and sugiol as anticancer compounds, which were induced by GA3 treatment in AdAR. Gene expression analysis combined with functional characterisation suggests that the GA3 treatment increased the transcription of geranylgeranyl pyrophosphate synthases and copalyl diphosphate synthase, which led to the accumulation of diterpenoids, including ferruginol and sugiol. Overall, these findings can contribute to the advancement of metabolic engineering for enhancing the biosynthesis of active diterpenoids, and facilitate the large-scale production of bioactive compounds sourced from A. distichum.

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 â€‹mg â€‹L-1 of limonene at a rate of 8.2 â€‹mg â€‹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.

Relationship between ß-Carotene Accumulation and Geranylgeranyl Pyrophosphate Synthase in Different Species of Dunaliella.

To study the relationship between ß-carotene synthesis and geranylgeranyl pyrophosphate synthase (GGPS) activity, 15 species of Dunaliella were used to determine the changes in photosynthetic pigment contents, chlorophyll fluorescence parameters, ß-carotene content, and GGPS activity. By observing the morphology and size of 15 species of Dunaliella, D8 has the largest individual algal cell and D9 has the smallest individual. Growth was relatively slow during days one through seven. After about eight days, the cells entered the logarithmic growth period and grew rapidly to a high density. After about 45 days, they entered a mature period, and growth slowed down. The contents of chlorophyll, carotenoids, and ß-carotene increased during growth. D1 has the highest accumulation of ß-carotene, and GGPS enzyme activity has a positive linear relationship with the ß-carotene synthesis content. Phylogenetic analysis showed that the GGPS proteins of the 15 species were highly homologous, and the GGPS protein was not part of the membrane.

Isolation, expression, and functional analysis of the geranylgeranyl pyrophosphate synthase (GGPPS) gene from Liriodendron tulipifera.

Terpenoids are important secondary metabolites in plants and are involved in stress responses and pollinator attraction. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key synthase in the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway of terpenoid synthesis, catalyzing the synthesis of diterpenoids. Liriodendron tulipifera is a nectar plant in North America. Little is known about the key genes involved in the biosynthetic pathways of terpenoids, the precursors of most compounds related to nectar, fragrance and coloring in flowers in L. tulipifera. In this study, the LtuGGPPS2 gene and its promoter (LtuGGPPS2-pro) were cloned from L. tulipifera. The results of sequence alignment showed that the LtuGGPPS2 gene is highly homologous to GGPPS genes of other plants. Subcellular localization analysis showed that the LtuGGPPS2 protein localizes to chloroplasts, suggesting that the LtuGGPPS2 gene is probably related to carotenoid and chlorophyll synthesis. Based on tissue expression profiles revealed by RT-qPCR, the expression level of the LtuGGPPS2 gene was highest in petals. These results were consistent with the changes in volatile and nonvolatile terpenoids in the flowers of L. tulipifera. GUS staining to examine the LtuGGPPS2 promoter indicated that it is responsive to hormones. Overexpression of the LtuGGPPS2 gene increased the carotenoid content and GGPPS enzyme activity in Arabidopsis thaliana, indicating that LtuGGPPS2 is the key terpenoid synthase in the flowers of L. tulipifera. Our findings lay a foundation for further functional analysis of the LtuGGPPS2 gene and deeper investigation of the terpenoid biosynthetic pathway in L. tulipifera.

Evidence for a role of geranylgeranylation in renal angiomyolipoma and renal epithelioid angiomyolipoma.

Research on mevalonate kinase deficiency has revealed that it may lead to the development of renal angiomyolipomas (RAMLs). Thus, it was suspected that geranylgeranyl pyrophosphate synthase (GGPPS), a key enzyme in the mevalonate pathway, may be involved in the development of RAMLs. In the present study, the expression of GGPPS in RAMLs and renal epithelioid angiomyolipomas (REAs) was assessed, and paraffin embedded specimens from 60 patients, including 9 cases with REA and 51 cases with RAML, were examined. Immunoreactivity was evaluated semi-quantitatively according to the intensity of staining and the percentage of positively stained cells. The results indicated that GGPPS was predominantly present in the cytoplasm, and REA tissues exhibited higher expression of GGPPS in the cytoplasm compared with RAML tissues. It was also identified that GGPPS was upregulated in TSC2-null cells, and inhibition of GGPPS could induce apoptosis of TSC2-null cells by autophagy. In conclusion, the increased expression of GGPPS in RAMLs and REAs indicated that mevalonate pathways may be involved in disease progression. GGPPS may serve as a potential therapeutic target and the current results may provide a novel therapeutic strategy for RAML and lymphangioleiomyomatosis.

Isoprenoids and tau pathology in sporadic Alzheimer's disease.

The mevalonate pathway has been described to play a key role in Alzheimer's disease (AD) physiopathology. Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are nonsterol isoprenoids derived from mevalonate, which serve as precursors to numerous human metabolites. They facilitate protein prenylation; hFPP and hGGPP synthases act as gateway enzymes to the prenylation of the small guanosine triphosphate (GTP)ase proteins such as RhoA and cdc42 that have been shown to facilitate phospho-tau (p-Tau, i.e., protein tau phosphorylated) production in the brain. In this study, a significant positive correlation was observed between the synthases mRNA prevalence and disease status (FPPS, p < 0.001, n = 123; GGPPS, p < 0.001, n = 122). The levels of mRNA for hFPPS and hGGPPS were found to significantly correlate with the amount of p-Tau protein levels (p < 0.05, n = 34) and neurofibrillary tangle density (p < 0.05, n = 39) in the frontal cortex. Interestingly, high levels of hFPPS and hGGPPS mRNA prevalence are associated with earlier age of onset in AD (p < 0.05, n = 58). Together, these results suggest that accumulation of p-Tau in the AD brain is related, at least in part, to increased levels of neuronal isoprenoids.

Characterization and Functional Identification of a Gene Encoding Geranylgeranyl Diphosphate Synthase from Dunaliella bardawil.

Geranylgeranyl diphosphate synthase (GGPS) catalyzes the biosynthesis of geranylgeranyl diphosphate, a key precursor for carotenoid biosynthesis. In this study, a full-length cDNA encoding GGPS from Dunaliella bardawil (DbGGPS) was isolated by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of DbGGPS was 1814 bp, containing a 1074 bp ORF encoding 357 amino acids with a calculated mass of 38.88 kDa. Analysis of DbGGPS genomic DNA revealed that it contained 10 exons and 9 introns. It was predicted that DbGGPS possessed a 48 amino acid transit peptide at its N terminus. Bioinformatic analysis revealed that DbGGPS was a member of a group of polyprenyltransferases with five conserved domains and two highly conserved aspartate-rich motifs. Using heterologous expression, carotenoid complementation assay, and gene deletion analysis, it was shown that the coding region of DbGGPS encodes a functional GGPS. This provides new gene sources for carotenoid genetic engineering.

De novo transcriptome analysis of Liriodendron chinense petals and leaves by Illumina sequencing.

Liriodendron chinense (Hemsl.) Sarg is an endangered species and occupies a pivotal position in phylogenetic studies of flowering plants, while its genomic resources are limited. In this study, we performed transcriptome sequencing for L. chinense petals and leaves using the Illumina paired-end sequencing technique. Approximately 17.02-Gb clean reads were obtained, and de novo assembly generated 87,841 unigenes, with an average length of 778 bp. Of these, there were 65,535 (74.61%) unigenes with significant similarity to publically available plant protein sequences. There were 3386 genes identified as significant differentially expressed between petals and leaves, among them 2969 (87.68%) were up-regulated and 417 (12.31%) down-regulated in petals. Metabolic pathway analysis revealed that 25 unigenes were predicted to be responsible for the biosynthesis of carotenoids, with 7 genes differentially expressed between these two tissues. This report is the first to identify genes associated with carotenoid biosynthesis in Liriodendron and represents a valuable resource for future genomic studies on the endangered species L. chinense.

Cloning and prokaryotic expression of CDS region from GGPPS gene of Panax notoginseng / 中草药

Objective: In order to study the function of geranylgeranyl pyrophosphate synthase (GGPPS) gene, the CDS nucleotide sequence of GGPS was cloned from Panax notoginseng, and its prokaryotic expression was performed. Methods: The primers were designed according to the reported GGPPS gene sequence in Genbank, and the coding sequence was obtained by RT-PCR. The prokaryotic expression vector was constructed and transformed into Escherichia coli BL21 for the expression under the induction of isopropyl β-D-1-thiogalactopyranoside (IPTG). Results: The CDS of GGPS gene had a full length of 1 032 bp coding for 343 amino acids. Results of SDS-PAGE showed that a 29 000—44 000 protein was achieved and the recombinant protein was mainly in the form of insoluble inclusion body. Conclusion: The CDS nucleotide sequence of GGPPS gene was successfully cloned, and the stable prokaryotic expression was established. This study will provide a foundation for the further functional researches of GGPPS gene in P. notoginseng.