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1.
Bosung Ku Jae-Cheol Jeong Benjamin N. Mijts Claudia Schmidt-Dannert Jonathan S. Dordick 《Applied microbiology》2005,71(11):6578-6583
The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4′-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoid-based pathways, could lead to the synthesis of novel carotenoids and their derivatives. 相似文献
2.
Farnesyl pyrophosphate (FPP) synthase catalyzes the biosynthesis of FPP, which is the precursors of sesquiterpenoids such as floral scent volatiles, from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). cDNA encoding wintersweet (Chimonanthus praecox L.) FPP synthase was isolated by the RT-PCR and RACE methods. The deduced amino acid sequence showed a high identity to plant FPP synthases. Expression of the gene in Escherichia coli yielded FPPS activity that catalyzed the synthesis of FPP as a main product. Tissue-specific and developmental analyses of the mRNA levels of CpFPPS and volatile sesquiterpenoids levels in C. praecox flowers revealed that the FPPS may play a regulatory role in floral volatile sesquiterpenoids of wintersweet. 相似文献
3.
Guo RT Ko TP Chen AP Kuo CJ Wang AH Liang PH 《The Journal of biological chemistry》2005,280(21):20762-20774
Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the consecutive condensation reactions of a farnesyl pyrophosphate (FPP) with eight isopentenyl pyrophosphates (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate that serves as a lipid carrier for peptidoglycan synthesis of bacterial cell wall. The structures of Escherichia coli UPPs were determined previously in an orthorhombic crystal form as an apoenzyme, in complex with Mg(2+)/sulfate/Triton, and with bound FPP. In a further search of its catalytic mechanism, the wild-type UPPs and the D26A mutant are crystallized in a new trigonal unit cell with Mg(2+)/IPP/farnesyl thiopyrophosphate (an FPP analogue) bound to the active site. In the wild-type enzyme, Mg(2+) is coordinated by the pyrophosphate of farnesyl thiopyrophosphate, the carboxylate of Asp(26), and three water molecules. In the mutant enzyme, it is bound to the pyrophosphate of IPP. The [Mg(2+)] dependence of the catalytic rate by UPPs shows that the activity is maximal at [Mg(2+)] = 1 mm but drops significantly when Mg(2+) ions are in excess (50 mm). Without Mg(2+), IPP binds to UPPs only at high concentration. Mutation of Asp(26) to other charged amino acids results in significant decrease of the UPPs activity. The role of Asp(26) is probably to assist the migration of Mg(2+) from IPP to FPP and thus initiate the condensation reaction by ionization of the pyrophosphate group from FPP. Other conserved residues, including His(43), Ser(71), Asn(74), and Arg(77), may serve as general acid/base and pyrophosphate carrier. Our results here improve the understanding of the UPPs enzyme reaction significantly. 相似文献
4.
New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species 总被引:6,自引:0,他引:6
The role of carotenoid genes crtB and crtE has been functionally assigned. These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens. Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation. In vitro reactions from extracts of E. coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP). These results strongly indicate that the crtE gene encodes GGPP synthase. Introduction of the crtB gene into A. tumefaciens led to the conversion of GGPP into phytoene. This activity was absent in similar transformants with the crtE gene. Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E. coli harboring both the crtB and crtE genes. 相似文献
5.
Yating Hu Yongjin J. Zhou Jichen Bao Luqi Huang Jens Nielsen Anastasia Krivoruchko 《Journal of industrial microbiology & biotechnology》2017,44(7):1065-1072
Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources. 相似文献
6.
Terpenoid biosynthesis and the stereochemistry of enzyme-catalysed allylic addition-elimination reactions. 总被引:1,自引:0,他引:1
D E Cane C Abell P H Harrison B R Hubbard C T Kane R Lattman J S Oliver S W Weiner 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1991,332(1263):123-129
Allylic addition-elimination reactions are widely used in the enzyme-catalysed formation of terpenoid metabolites. It has earlier been shown that the isoprenoid chain elongation reaction catalysed by farnesyl pyrophosphate synthase involving successive condensations of dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophosphate (GPP) with isopentenyl pyrophosphate (IPP) corresponds to such an SE' reaction with net syn stereochemistry for the sequential electrophilic addition and proton elimination steps. Studies of the enzymic cyclization of farnesyl pyrophosphate (FPP) to pentalenene have now established the stereochemical course of two additional biological SE' reactions. Incubation of both (9R)- and (9S)-[9-3H,4,8-14]FPP with pentalenene synthase and analysis of the resulting labelled pentalenene has revealed that H-9re of FPP becomes H-8 of pentalenene, while H-9si undergoes net intramolecular transfer to the adjacent carbon, becoming H-1re (H-1 alpha) of pentalenene, as confirmed by subsequent experiments with [10-2H, 11-13C]FPP. These results correspond to net anti-stereochemistry in the intramolecular allylic addition-elimination reaction. The stereochemical course of a second SE' reaction has now been examined by analogous incubations of (4S,8S)-[4,8-3H,4,8-14C]FPP and (4R,8R)-[4,8-3H, 4.8-14C]FPP with pentalenene synthase. Determination of the distribution of label in the derived pentalenenes showed stereospecific loss of the original H-8si proton. Analysis of the plausible conformation of the presumed reaction intermediates revealed that the stereochemical course of the latter reaction cannot properly be described as either syn or anti, since cyclization and subsequent double bond formation require significant internal motions to allow proper overlap of the scissile C-H bond with the developing carbocation. 相似文献
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The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids. 相似文献
9.
Purification and characterization of farnesyl pyrophosphate synthase from Capsicum annuum 总被引:5,自引:0,他引:5
Farnesyl pyrophosphate synthase (FPP) displaying dimethylallyl transferase activity (EC 2.5.1.1) and geranyl transferase activity (EC 2.5.1.10) was purified from Capsicum fruits. This prenyltransferase has a molecular mass of 89,000 +/- 5000 Da resulting from the association of two apparently identical subunits having a molecular mass of 43,000 +/- 2000 Da. Antibodies raised against Capsicum FPP synthase selectively blocked the transferase activity. Analysis of the immunological relationships between FPP synthase and geranylgeranyl pyrophosphate synthase (EC 2.5.1.1, EC 2.5.1.10 and EC 2.5.1.30) revealed that these two enzymes though performing the same mechanism of catalysis and accepting identical substrates have different antigenic determinants. Thus, in connection to previous work, this immunological study suggests that Capsicum FPP is strictly located in the extraplastidial compartment. 相似文献
10.
Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of eight molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphate (FPP) to generate C(55) undecaprenyl pyrophosphate. We investigated the kinetics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were found. In the presence of excess FPP, the intermediates (C(25)-C(50)) accumulated. Under a condition with enzyme and FPP in excess of IPP, instead of C(20)-geranylgeranyl pyrophosphate, C(20), C(25), and C(30) were the major products. The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Triton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k(cat) value matched the rate constant of each IPP condensation obtained from the enzyme single-turnover experiments. This suggested that the IPP condensation rather than product release was the rate-limiting step in the presence of Triton. In the absence of Triton, the intermediates formed and disappeared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting. This was further supported by a burst product formation. Judging from the accumulation level of C(55), C(60), and C(65), their dissociation from the enzyme cannot be too slow and an even slower enzyme conformational change with a rate of 0.001 s(-1) might govern the UPPs reaction rate under the steady-state condition in the absence of Triton. 相似文献
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Santalum album L. commonly known as East-Indian sandal or chandan is a hemiparasitic tree of family santalaceae. Santalol is a bioprospecting molecule present in sandalwood and any effort towards metabolic engineering of this important moiety would require knowledge on gene regulation. Santalol is a sesquiterpene synthesized through mevalonate or non-mevalonate pathways. First step of santalol biosynthesis involves head to tail condensation of isopentenyl pyrophosphate (IPP) with its allylic co-substrate dimethyl allyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP; C10 — a monoterpene). GPP upon one additional condensation with IPP produces farnesyl pyrophosphate (FPP; C15 — an open chain sesquiterpene). Both the reactions are catalyzed by farnesyl diphosphate synthase (FDS). Santalene synthase (SS), a terpene cyclase catalyzes cyclization of open ring FPP into a mixture of cyclic sesquiterpenes such as α-santalene, epi-β-santalene, β-santalene and exo bergamotene, the main constituents of sandal oil. The objective of the present work was to generate a comprehensive knowledge on the genes involved in santalol production and study their molecular regulation. To achieve this, sequences encoding farnesyl diphosphate synthase and santalene synthase were isolated from sandalwood using suppression subtraction hybridization and 2D gel electrophoresis technology. Functional characterization of both the genes was done through enzyme assays and tissue-specific expression of both the genes was studied. To our knowledge, this is the first report on studies on molecular regulation, and tissue-specific expression of the genes involved in santalol biosynthesis. 相似文献
14.
Chonglong Wang Sang‐Hwal Yoon Asad Ali Shah Young‐Ryun Chung Jae‐Yean Kim Eui‐Sung Choi Jay D. Keasling Seon‐Won Kim 《Biotechnology and bioengineering》2010,107(3):421-429
Farnesol (FOH) production has been carried out in metabolically engineered Escherichia coli. FOH is formed through the depyrophosphorylation of farnesyl pyrophosphate (FPP), which is synthesized from isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) by FPP synthase. In order to increase FPP synthesis, E. coli was metabolically engineered to overexpress ispA and to utilize the foreign mevalonate (MVA) pathway for the efficient synthesis of IPP and DMAPP. Two‐phase culture using a decane overlay of the culture broth was applied to reduce volatile loss of FOH produced during culture and to extract FOH from the culture broth. A FOH production of 135.5 mg/L was obtained from the recombinant E. coli harboring the pTispA and pSNA plasmids for ispA overexpression and MVA pathway utilization, respectively. It is interesting to observe that a large amount of FOH could be produced from E. coli without FOH synthase by the augmentation of FPP synthesis. Introduction of the exogenous MVA pathway enabled the dramatic production of FOH by E. coli while no detectable FOH production was observed in the endogenous MEP pathway‐only control. Biotechnol. Bioeng. 2010;107: 421–429. © 2010 Wiley Periodicals, Inc. 相似文献
15.
Yu-Jun Zhao Xin Chen Meng Zhang Ping Su Yu-Jia Liu Yu-Ru Tong Xiu-Juan Wang Lu-Qi Huang Wei Gao 《PloS one》2015,10(5)
Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products. 相似文献
16.
The prenylation of proteins utilizes the polyisoprenyl pyrophosphates (FPP) and geranylgeranyl pyrophosphate (GGPP) as prenyl donors. These polyisoprenoids are also precursors to ubiquinone and dolichol synthesis. We have previously described the geranylgeranylation of rab 1b from labeled mevalonate in rabbit reticulocyte lysates (Khosravi-Far, R., Lutz, R. J., Cox, A. D., Conroy, L., Bourne, J. R., Sinensky, M., Balch, W. E., Buss, J. C., and Der, C. J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6264-6268). We now directly demonstrate the incorporation of mevalonate into FPP and GGPP in rabbit reticulocyte cytosol. High pressure liquid chromatography analysis reveals that only all-trans-E,E,E-GGPP, the prenyl donor for in vivo protein geranylgeranylation, is synthesized. Incubations with recombinant H-ras and rab1b result in an increased synthesis of farnesyl and geranylgeranyl derivatives, respectively. The increase is wholly accounted for by protein-incorporated polyisoprenoids with no change in the polyisoprenyl pyrophosphate pools. Further, GGPP inhibits its own synthesis, without affecting FPP synthesis, with half-maximal inhibition at approximately 3 microM GGPP. Inhibition of FPP synthesis by the inhibition of isopentenyl isomerase causes a dramatic increase in isopentenyl pyrophosphate synthesis. FPP also inhibits conversion of mevalonate into FPP. These findings indicate that these polyisoprenyl pyrophosphates can down-regulate their own synthesis in vitro, and this regulation may control the levels of these polyisoprenoids in vivo. 相似文献
17.
杜松烯合成酶是棉酚合成途径中的关键酶,催化(E,E)-法呢基焦磷酸(FPP)环化形成(+)-δ-杜松烯。从陆地棉Y18R中克隆分离了杜松烯合成酶基因(GhCdn),该基因的基因组序列为2 700 bp,具有6个内含子,剪切后其ORF为1 665 bp,编码554个氨基酸,该基因属于杜松烯合成酶C亚家族。应用Overlap PCR方法将其上的2个Hind Ⅲ 酶切位点钝化后,将GhCdn基因连接到表达载体pBI121上,构建出分别由组成型启动子CaMV 35S和绿色组织高效启动子Psbp驱动的2个植物表达载体pGBI-CaMV 35S-GhCdn和pGBI-Psbp-GhCdn。通过农杆菌介导法转化棉花下胚轴并进行组织培养,获得了9个35S转基因阳性愈伤系和2个P转基因阳性愈伤系。经检测,阳性愈伤组织中GhCdn基因的mRNA表达量和棉酚含量均有所增加,但35S系普遍高于P系。本研究为通过基因工程手段提高棉花组织器官中的棉酚含量提供了依据。 相似文献
18.
Shin-Ichiro Suye Keiichi Kamiya Takuo Kawamoto Atsuo Tanaka 《Biocatalysis and Biotransformation》2013,31(1):23-28
Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 u h. 相似文献
19.
Shin-Ichiro Suye Keiichi Kamiya Takuo Kawamoto Atsuo Tanaka 《Biocatalysis and Biotransformation》2002,20(1):23-28
Bioconversion of cinnamyl alcohol to cinnamaldehyde was carried out in an aqueous-organic two-phase reaction system by the repeated use of horse liver alcohol dehydrogenase (HLADH) and NAD + with coenzyme regeneration. Both HLADH and the coenzyme were efficiently entrapped in the aqueous phase, while the substrate was supplied successively from the organic phase and the product was accumulated in the organic phase. Optimum conditions for cinnamaldehyde production in the aqueous-organic two-phase system were also examined, including substrate concentration, pH, and organic solvent type. Under suitable conditions, both HLADH and NAD + in the aqueous-organic two-phase system could be reused, and NAD + cycling numbers of 3040 were obtained after repeated operation for 40 λh. 相似文献
20.
Xixian Chen Congqiang Zhang Ruiyang Zou Kang Zhou Gregory Stephanopoulos Heng Phon Too 《PloS one》2013,8(11)
In vitro synthesis of chemicals and pharmaceuticals using enzymes is of considerable interest as these biocatalysts facilitate a wide variety of reactions under mild conditions with excellent regio-, chemo- and stereoselectivities. A significant challenge in a multi-enzymatic reaction is the need to optimize the various steps involved simultaneously so as to obtain high-yield of a product. In this study, statistical experimental design was used to guide the optimization of a total synthesis of amorpha-4,11-diene (AD) using multienzymes in the mevalonate pathway. A combinatorial approach guided by Taguchi orthogonal array design identified the local optimum enzymatic activity ratio for Erg12:Erg8:Erg19:Idi:IspA to be 100∶100∶1∶25∶5, with a constant concentration of amorpha-4,11-diene synthase (Ads, 100 mg/L). The model also identified an unexpected inhibitory effect of farnesyl pyrophosphate synthase (IspA), where the activity was negatively correlated with AD yield. This was due to the precipitation of farnesyl pyrophosphate (FPP), the product of IspA. Response surface methodology was then used to optimize IspA and Ads activities simultaneously so as to minimize the accumulation of FPP and the result showed that Ads to be a critical factor. By increasing the concentration of Ads, a complete conversion (∼100%) of mevalonic acid (MVA) to AD was achieved. Monovalent ions and pH were effective means of enhancing the specific Ads activity and specific AD yield significantly. The results from this study represent the first in vitro reconstitution of the mevalonate pathway for the production of an isoprenoid and the approaches developed herein may be used to produce other isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) based products. 相似文献