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1.
The filamentous fungus Penicillium paxilli contains two distinct geranylgeranyl diphosphate (GGPP) synthases, GgsA and GgsB (PaxG). PaxG and its homologues in Neotyphodium lolii and Fusarium fujikuroi are associated with diterpene secondary metabolite gene clusters. The genomes of other filamentous fungi including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Fusarium graminearum also contain two or more copies of GGPP synthase genes, although the diterpene metabolite capability of these fungi is not
known. The objective of this study was to understand the biological significance of the presence of two copies of GGPP synthases
in P. paxilli by investigating their subcellular localization. Using a carotenoid complementation assay and gene deletion analysis, we
show that P. paxilli GgsA and PaxG have GGPP synthase activities and that paxG is required for paxilline biosynthesis, respectively. In the ΔpaxG mutant background ggsA was unable to complement paxilline biosynthesis. A GgsA-EGFP fusion protein was localized to punctuate organelles and the
EGFP-GRV fusion protein, containing the C-terminus tripeptide GRV of PaxG, was localized to peroxisomes. A truncated PaxG
mutant lacking the C-terminus tripeptide GRV was unable to complement a ΔpaxG mutant demonstrating that the tripeptide is functionally important for paxilline biosynthesis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
2.
Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP),
which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence
was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves.
RT–PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid
accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase. 相似文献
3.
Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. It regulates the
formation of diterpenoid, such as tanshinones. We cloned a gene for GGPP synthase SmGGPPs involved in diterpenoid biosynthesis from Salvia miltiorrhiza. At 2,767 bp long, this gene comprises an intron and two exons that encode a polypeptide of 364 amino acid residues. Then
the 5′ flanking sequence of SmGGPPs was characterized by bioinformatics method. Deletion analysis of the promoter of SmGGPPs using tobacco plant displayed that the promoter was induced by heat and cold. To further search these cis-elements involved
in induction regulation in the 5′ flanking sequence of SmGGPPs, many putative cis-elements were predicted with the PlantCARE and PLACE databases. A group of putative cis-acting elements
are involved in induction regulation, including G-Box, WRKY, MYC and ATCT motifs. Real-time PCR analysis revealed that SmGGPPs is mainly expressed in the leaves and can also be induced by various factors, such as NaCl, wounding, high temperature, darkness,
pathogen, methyl jasmonate, abscisic acid, salicylic acid, and gibberellins. This study provides useful information for further
study of SmGGPPs and its regulator effect on the biosynthetic process of tanshinones so that researchers can improve the tanshinone contents
in S. miltiorrhiza. 相似文献
4.
Vallon T Ghanegaonkar S Vielhauer O Müller A Albermann C Sprenger G Reuss M Lemuth K 《Applied microbiology and biotechnology》2008,81(1):175-182
In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a
precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate
(FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ
extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl
diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression
vector.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
T. Vallon and S. Ghanegaonkar contributed equally to this work. 相似文献
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Sarma Rajeev Kumar Avanish Rai Dikki Pedenla Bomzan Krishna Kumar Andra Hemmerlin Varun Dwivedi Rucha C. Godbole Vitthal Barvkar Karuna Shanker H. B. Shilpashree Ankita Bhattacharya Attibele Ramamurthy Smitha Namratha Hegde Dinesh A. Nagegowda 《The Plant journal : for cell and molecular biology》2020,103(1):248-265
8.
Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis 总被引:5,自引:0,他引:5
Takaya A Zhang YW Asawatreratanakul K Wititsuwannakul D Wititsuwannakul R Takahashi S Koyama T 《Biochimica et biophysica acta》2003,1625(2):214-220
Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower. 相似文献
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10.
Yihua Ma Qingwen Chen Yaoyao Wang Fengxia Zhang Chengyuan Wang Guodong Wang 《植物学报(英文版)》2023,65(5):1170-1182
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 e... 相似文献
11.
Exploring diterpene metabolism in non‐model species: transcriptome‐enabled discovery and functional characterization of labda‐7,13E‐dienyl diphosphate synthase from Grindelia robusta
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Philipp Zerbe Selina M. Rodriguez Sibongile Mafu Angela Chiang Harpreet K. Sandhu Mark O'Neil‐Johnson Courtney M. Starks Jörg Bohlmann 《The Plant journal : for cell and molecular biology》2015,83(5):783-793
12.
Gao-Bin Pu Dong-Ming Ma Jian-Lin Chen Lan-Qing Ma Hong Wang Guo-Feng Li He-Chun Ye Ben-Ye Liu 《Plant cell reports》2009,28(7):1127-1135
This paper provides evidence that salicylic acid (SA) can activate artemisinin biosynthesis in Artemisia annua L. Exogenous application of SA to A. annua leaves was followed by a burst of reactive oxygen species (ROS) and the conversion of dihydroartemisinic acid into artemisinin.
In the 24 h after application, SA application led to a gradual increase in the expression of the 3-hydroxy-3-methylglutaryl
coenzyme A reductase (HMGR) gene and a temporary peak in the expression of the amorpha-4,11-diene synthase (ADS) gene. However, the expression of the farnesyl diphosphate synthase (FDS) gene and the cytochrome P450 monooxygenase (CYP71AV1) gene showed little change. At 96 h after SA (1.0 mM) treatment, the concentration of artemisinin, artemisinic acid and dihydroartemisinic
acid were 54, 127 and 72% higher than that of the control, respectively. Taken together, these results suggest that SA induces
artemisinin biosynthesis in at least two ways: by increasing the conversion of dihydroartemisinic acid into artemisinin caused
by the burst of ROS, and by up-regulating the expression of genes involved in artemisinin biosynthesis. 相似文献
13.
Background
MenH (2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase) is a key enzyme in the biosynthesis of menaquinone, catalyzing an unusual 2,5-elimination of pyruvate from 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate. 相似文献14.
Bacterial phytoene synthase: molecular cloning,expression, and characterization of Erwinia herbicola phytoene synthase 总被引:1,自引:0,他引:1
Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene. 相似文献
15.
Metabolic engineering of microorganisms is an alternative and attractive route for production of valuable terpenoids that are usually extracted from plant sources. Tanshinones are the bioactive components of Salvia miltiorrhizha Bunge, which is a well‐known traditional Chinese medicine widely used for treatment of many cardiovascular diseases. As a step toward microbial production of tanshinones, copalyl diphosphate (CPP) synthase, and normal CPP kaurene synthase‐like genes, which convert the universal diterpenoid precursor geranylgeranyl diphosphate (GGPP) to miltiradiene (an important intermediate of the tanshinones synthetic pathway), was introduced into Saccharomyces cerevisiae, resulting in production of 4.2 mg/L miltiradiene. Improving supplies of isoprenoid precursors was then investigated for increasing miltiradiene production. Although over‐expression of a truncated 3‐hydroxyl‐3‐methylglutaryl‐CoA reductase (tHMGR) and a mutated global regulatory factor (upc2.1) gene did improve supply of farnesyl diphosphate (FPP), production of miltiradiene was not increased while large amounts of squalene (78 mg/L) were accumulated. In contrast, miltiradiene production increased to 8.8 mg/L by improving supply of GGPP through over‐expression of a fusion gene of FPP synthase (ERG20) and endogenous GGPP synthase (BTS1) together with a heterologous GGPP synthase from Sulfolobus acidocaldarius (SaGGPS). Auxotrophic markers in the episomal plasmids were then replaced by antibiotic markers, so that engineered yeast strains could use rich medium to obtain better cell growth while keeping plasmid stabilities. Over‐expressing ERG20‐BTS1 and SaGGPS genes increased miltiradiene production from 5.4 to 28.2 mg/L. Combinatorial over‐expression of tHMGR‐upc2.1 and ERG20‐BTS1‐SaGGPS genes had a synergetic effects on miltiradiene production, increasing titer to 61.8 mg/L. Finally, fed‐batch fermentation was performed, and 488 mg/L miltiradiene was produced. The yeast strains engineered in this work provide a basis for creating an alternative way for production of tanshinones in place of extraction from plant sources. Biotechnol. Bioeng. 2012; 109: 2845–2853. © 2012 Wiley Periodicals, Inc. 相似文献
16.
Guoyin Kai Pan Liao Tong Zhang Wei Zhou Jing Wang Hui Xu Yuanyun Liu Lin Zhang 《Biotechnology and Bioprocess Engineering》2010,15(2):236-245
Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP),
which is a key precursor for diterpenes including tanshinone. In this study, a full-length cDNA encoding GGPPS was isolated
from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmGGPPS (GenBank Accession No. FJ643617). The full-length cDNA of SmGGPPS was 1,234 bp containing a 1,092 bp open reading frame (ORF) encoding a polypeptide of 364 amino acids. Analysis of SmGGPPS genomic DNA revealed that it contained 2 exons and 1 intron. Bioinformatics analyses revealed that the deduced SmGGPPS had extensive homology with other plant GGPPSs contained all 5 conserved domains and functional aspartate-rich motifs of
the prenyltransferases. Molecular modeling showed that SmGGPPS is a new GGPPS with a spatial structure similar to other plant GGPPSs. Phylogenetic tree analysis indicated that SmGGPPS belongs to the plant GGPPS super-family and has the closest relationship with GGPPS from Nicotiana attenuate. The functional identification in Escherichia coli showed that SmGGPPS could accelerate the biosynthesis of carotenoid, demonstrating that SmGGPPS encoded a functional protein. Expression pattern analysis implied that SmGGPPS expressed higher in leaves and roots, weaker in stems. The expression of SmGGPPS could be up-regulated by Salicylic acid (SA) in leaves and inhibited by methyl jasmonate (MeJA) in 3 tested tissues, suggesting
that SmGGPPS was elicitor-responsive. This work will be helpful to understand more about the role of SmGGPPS involved in the tanshinones biosynthesis pathway and metabolic engineering to improve tanshiones production in S. miltiorrhiza. 相似文献
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Govinda R. Navale Poojadevi Sharma Madhukar S. Said Sudha Ramkumar Mahesh S. Dharne H. V. Thulasiram Sandip S. Shinde 《Engineering in Life Science》2019,19(9):606-616
Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi‐cedrol synthase (AaECS). The stop codon at the N‐terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐epi‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The Km values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes. 相似文献
19.
Chien-Tsun Kuan Jinli Chang Jan-Eric Mansson Jianjun Li Charles Pegram Pam Fredman Roger E McLendon Darell D Bigner 《BMC developmental biology》2010,10(1):114