A Synechococcus leopoliensis SAUG 1402-1 operon harboring the 1-deoxyxylulose 5-phosphate synthase gene and two additional open reading frames is functionally involved in the dimethylallyl diphosphate synthesis

Experiments have been performed to prove the existence and the functionality of the novel mevalonate independent 1-deoxyxylulose 5-phosphate isoprenoid biosynthesis pathway in cyanobacteria. For this purpose, a segment of the 1-deoxyxylulose 5-phosphate synthase gene (dxs) was amplified from Synechococcus leopoliensis SAUG 1402-1 DNA via PCR using oligonucleotides for conserved regions of dxs. Subsequent hybridization screening of a genomic cosmid library of S. leopoliensis with this segment has led to the identification of an 18.7 kbp segment of the S. leopoliensis genome on which a dxs homologous gene and two adjacent open reading frames organized in one operon could be localized by DNA sequencing. The three genes of the operon were separately expressed in Escherichia coli, proving that the identified cyanobacterial dxs is functionally involved in the formation of dimethylallyl diphosphate, one basic intermediate of isoprenoid biosynthesis.     

Cloning and characterization of 1-deoxy-D-xylulose 5-phosphate synthase from Streptomyces sp. Strain CL190, which uses both the mevalonate and nonmevalonate pathways for isopentenyl diphosphate biosynthesis

In addition to the ubiquitous mevalonate pathway, Streptomyces sp. strain CL190 utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. The initial step of this nonmevalonate pathway is the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) by condensation of pyruvate and glyceraldehyde 3-phosphate catalyzed by DXP synthase. The corresponding gene, dxs, was cloned from CL190 by using PCR with two oligonucleotide primers synthesized on the basis of two highly conserved regions among dxs homologs from six genera. The dxs gene of CL190 encodes 631 amino acid residues with a predicted molecular mass of 68 kDa. The recombinant enzyme overexpressed in Escherichia coli was purified as a soluble protein and characterized. The molecular mass of the enzyme was estimated to be 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 130 kDa by gel filtration chromatography, suggesting that the enzyme is most likely to be a dimer. The enzyme showed a pH optimum of 9.0, with a V(max) of 370 U per mg of protein and K(m)s of 65 microM for pyruvate and 120 microM for D-glyceraldehyde 3-phosphate. The purified enzyme catalyzed the formation of 1-deoxyxylulose by condensation of pyruvate and glyceraldehyde as well, with a K(m) value of 35 mM for D-glyceraldehyde. To compare the enzymatic properties of CL190 and E. coli DXP synthases, the latter enzyme was also overexpressed and purified. Although these two enzymes had different origins, they showed the same enzymatic properties.     

Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle

Potato tubers were engineered to express a bacterial gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in order to investigate the effects of perturbation of isoprenoid biosynthesis. Twenty-four independent transgenic lines out of 38 generated produced tubers with significantly elongated shape that also exhibited an early tuber sprouting phenotype. Expression analysis of nine transgenic lines (four exhibiting the phenotype and five showing a wild-type phenotype) demonstrated that the phenotype was strongly associated with dxs expression. At harvest, apical bud growth had already commenced in dxs-expressing tubers whereas in control lines no bud growth was evident until dormancy was released after 56-70 d of storage. The initial phase of bud growth in dxs tubers was followed by a lag period of approximately 56 d, before further elongation of the developing sprouts could be detected. Thus dxs expression results in the separation of distinct phases in the dormancy and sprouting processes. In order to account for the sprouting phenotype, the levels of plastid-derived isoprenoid growth regulators were measured in transgenic and control tubers. The major difference measured was an increase in the level of trans-zeatin riboside in tubers at harvest expressing dxs. Additionally, compared with controls, in some dxs-expressing lines, tuber carotenoid content increased approximately 2-fold, with most of the increase accounted for by a 6-7-fold increase in phytoene.     

1-Deoxy-D-xylulose 5-phosphate synthase, the gene product of open reading frame (ORF) 2816 and ORF 2895 in Rhodobacter capsulatus

In eubacteria, green algae, and plant chloroplasts, isopentenyl diphosphate, a key intermediate in the biosynthesis of isoprenoids, is synthesized by the methylerythritol phosphate pathway. The five carbons of the basic isoprenoid unit are assembled by joining pyruvate and D-glyceraldehyde 3-phosphate. The reaction is catalyzed by the thiamine diphosphate-dependent enzyme 1-deoxy-D-xylulose 5-phosphate synthase. In Rhodobacter capsulatus, two open reading frames (ORFs) carry the genes that encode 1-deoxy-D-xylulose 5-phosphate synthase. ORF 2816 is located in the photosynthesis-related gene cluster, along with most of the genes required for synthesis of the photosynthetic machinery of the bacterium, whereas ORF 2895 is located elsewhere in the genome. The proteins encoded by ORF 2816 and ORF 2895, 1-deoxy-D-xylulose 5-phosphate synthase A and B, containing a His(6) tag, were synthesized in Escherichia coli and purified to greater than 95% homogeneity in two steps. 1-Deoxy-D-xylulose 5-phosphate synthase A appears to be a homodimer with 68 kDa subunits. A new assay was developed, and the following steady-state kinetic constants were determined for 1-deoxy-D-xylulose 5-phosphate synthase A and B: K(m)(pyruvate) = 0.61 and 3.0 mM, K(m)(D-glyceraldehyde 3-phosphate) = 150 and 120 microM, and V(max) = 1.9 and 1.4 micromol/min/mg in 200 mM sodium citrate (pH 7.4). The ORF encoding 1-deoxy-D-xylulose 5-phosphate synthase B complemented the disrupted essential dxs gene in E. coli strain FH11.     

Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato

The genetic manipulation of both the mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3-hydroxymethylglutaryl CoA (hmgr-1) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) genes, respectively. Transgenic plants containing an additional hmgr-1 from Arabidopsis thaliana, under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4-fold), but IPP-derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6-fold), which was inherited in the next generation. Phytoene and beta-carotene exhibited the greatest increases (2.4- and 2.2-fold, respectively). Extra-plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.     

The sorbitol phosphotransferase system is responsible for transport of 2-C-methyl-D-erythritol into Salmonella enterica serovar typhimurium

2-C-methyl-D-erythritol 4-phosphate is the first committed intermediate in the biosynthesis of the isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. Supplementation of the growth medium with 2-C-methyl-D-erythritol has been shown to complement disruptions in the Escherichia coli gene for 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme that synthesizes the immediate precursor of 2-C-methyl-D-erythritol 4-phosphate. In order to be utilized in isoprenoid biosynthesis, 2-C-methyl-D-erythritol must be phosphorylated. We describe the construction of Salmonella enterica serovar Typhimurium strain RMC26, in which the essential gene encoding 1-deoxy-D-xylulose 5-phosphate synthase has been disrupted by insertion of a synthetic mevalonate operon consisting of the yeast ERG8, ERG12, and ERG19 genes, responsible for converting mevalonate to isopentenyl diphosphate under the control of an arabinose-inducible promoter. Random mutagenesis of RMC26 produced defects in the sorbitol phosphotransferase system that prevented the transport of 2-C-methyl-D-erythritol into the cell. RMC26 and mutant strains of RMC26 unable to grow on 2-C-methyl-D-erythritol were incubated in buffer containing mevalonate and deuterium-labeled 2-C-methyl-D-erythritol. Ubiquinone-8 was isolated from these cells and analyzed for deuterium content. Efficient incorporation of deuterium was observed for RMC26. However, there was no evidence of deuterium incorporation into the isoprenoid side chain of ubiquinone Q8 in the RMC26 mutants.     

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).     

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.     

Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in Escherichia coli enhances lycopene production

Isopentenyl diphosphate (IPP) is the common, five-carbon building block in the biosynthesis of all carotenoids. IPP in Escherichia coli is synthesized through the nonmevalonate pathway, which has not been completely elucidated. The first reaction of IPP biosynthesis in E. coli is the formation of 1-deoxy-D-xylulose-5-phosphate (DXP), catalyzed by DXP synthase and encoded by dxs. The second reaction in the pathway is the reduction of DXP to 2-C-methyl-D-erythritol-4-phos- phate, catalyzed by DXP reductoisomerase and encoded by dxr. To determine if one or more of the reactions in the nonmevalonate pathway controlled flux to IPP, dxs and dxr were placed on several expression vectors under the control of three different promoters and transformed into three E. coli strains (DH5alpha, XL1-Blue, and JM101) that had been engineered to produce lycopene. Lycopene production was improved significantly in strains transformed with the dxs expression vectors. When the dxs gene was expressed from the arabinose-inducible araBAD promoter (P(BAD)) on a medium-copy plasmid, lycopene production was twofold higher than when dxs was expressed from the IPTG-inducible trc and lac promoters (P(trc) and P(lac), respectively) on medium-copy and high-copy plasmids. Given the low final densities of cells expressing dxs from IPTG-inducible promoters, the low lycopene production was probably due to the metabolic burden of plasmid maintenance and an excessive drain of central metabolic intermediates. At arabinose concentrations between 0 and 1.33 mM, cells expressing both dxs and dxr from P(BAD) on a medium-copy plasmid produced 1.4-2.0 times more lycopene than cells expressing dxs only. However, at higher arabinose concentrations lycopene production in cells expressing both dxs and dxr was lower than in cells expressing dxs only. A comparison of the three E. coli strains transformed with the arabinose-inducible dxs on a medium-copy plasmid revealed that lycopene production was highest in XL1-Blue.     

三角褐指藻dxs基因克隆与诱导表达

为研究6种外源因素处理对三角褐指藻(Phaeodactylum tricornutum)1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)基因表达的影响,通过高通量转录组测序技术获得三角褐指藻dxs基因cDNA全长序列,并对其进行生物信息学分析。研究结果表明,三角褐指藻dxs基因cDNA全长2476 bp, ORF全长2193 bp,编码730个氨基酸,具有高度保守的ThDP结合位点和转酮醇酶结构域。三角褐指藻DXS蛋白为亲水性稳定蛋白,相对分子质量(M_w)为79.31 kD,理论等电点为6.65,具有信号肽、跨膜区域、卷曲螺旋和TM-螺旋等。系统进化树分析结果表明, DXS蛋白进化树分为高等植物和藻类2个分支,高等植物DXS蛋白进一步分为DXS1和DXS2两支,藻类DXS蛋白聚类为3个分支,分别为硅藻门、红藻门和绿藻门分支,三角褐指藻聚类在硅藻门分支上。诱导表达调控结果表明,三角褐指藻dxs基因受到茉莉酸甲酯(MeJA)、花生四烯酸(AA)、硫酸铈铵(ACS)、光合诱导素(PIF)、光合抑制剂(DCMU)和光质等6种外源因素的诱导调控。在100μmol/L MeJA...     

Evidence of a role for LytB in the nonmevalonate pathway of isoprenoid biosynthesis

It is proposed that the lytB gene encodes an enzyme of the deoxyxylulose-5-phosphate (DOXP) pathway that catalyzes a step at or subsequent to the point at which the pathway branches to form isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A mutant of the cyanobacterium Synechocystis strain PCC 6803 with an insertion in the promoter region of lytB grew slowly and produced greenish-yellow, easily bleached colonies. Insertions in the coding region of lytB were lethal. Supplementation of the culture medium with the alcohol analogues of IPP and DMAPP (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) completely alleviated the growth impairment of the mutant. The Synechocystis lytB gene and a lytB cDNA from the flowering plant Adonis aestivalis were each found to significantly enhance accumulation of carotenoids in Escherichia coli engineered to produce these colored isoprenoid compounds. When combined with a cDNA encoding deoxyxylulose-5-phosphate synthase (dxs), the initial enzyme of the DOXP pathway, the individual salutary effects of lytB and dxs were multiplied. In contrast, the combination of lytB and a cDNA encoding IPP isomerase (ipi) was no more effective in enhancing carotenoid accumulation than ipi alone, indicating that the ratio of IPP and DMAPP produced via the DOXP pathway is influenced by LytB.     

Identification of gcpE as a novel gene of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid biosynthesis in Escherichia coli

The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis is essential in most eubacteria and plants and has remarkable biotechnological interest. However, only the first steps of this pathway have been determined. Using bioinformatic and genetic approaches, we have identified gcpE as a novel gene of the MEP pathway. The distribution of this gene in bacteria and plants strictly parallels that of the gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, which catalyses the first committed step of the MEP pathway. Our data demonstrate that the gcpE gene is essential for the MEP pathway in Escherichia coli and indicate that this gene is required for the trunk line of the isoprenoid biosynthetic route.     

pMB9 plasmids bearing the Salmonella typhimurium his operon and gnd gene

A plasmid containing the entire Salmonella typhimurium his operon was constructed from plasmid pM89 and an EcoRI fragment of phi 80 his imm lambda DNA. The recombinant pST41 also includes the glucose 6-phosphate dehydrogenase (gnd) gene and has one EcoRI endonuclease cleavage site in the integrated fragment. This plasmid served as a source for the construction of two additional plasmids, one carrying the OGDC-region of the his operon and the other a CBHAFIE segment of the his gene along with the gnd gene. The presence of the his operon in the constructed plasmids was confirmed by hybridization to S. typhimurium his RNA. The location of the gnd gene in the CBHAFIE fragment of the his gene was confirmed genetically: after transfection with the plasmid bearing the gnd gene, a gnd recipient gained the capacity to utilize gluconate as a sole carbon source. The DNAs of the three hybrid plasmids were analyzed by gel electrophoresis. By comparing the EcoRI endonuclease cleavage pattern of these three hybrid plasmids with the DNA cleavage pattern of phi 80 his imm lambda, phi 80 imm lambda and lambda phages, the EcoRI cleavage map of phi 80 his imm lambda was obtained.     

基于2-C-甲基-D-赤藓糖醇-4磷酸途径的产紫槐二烯大肠杆菌构建及其补糖策略优化

2-C-甲基-D-赤藻糖醇-4-磷酸(2-methyl-D-erythritol-4-phosphate, MEP) 途径是大肠杆菌Escherichiacoli 唯一的萜类前体合成途径,研究表明它比甲羟戊酸(Mevalonate, MVA)途径具有更高的理论产率。但目前有关MEP 途径的调控所知非常有限,故单独强化MEP 途径对萜类异源合成产量的提高效果并不理想。研究中通过引入外源MEP 途径基因强化E. coli 萜类合成的遗传改造策略和发酵过程补糖控制优化,尝试更有效地释放MEP 途径的潜力,建立青蒿素前体——紫槐二烯的高密度发酵过程。研究结果表明共表达阿维链霉菌Streptomyces avermitilis dxs2 基因和枯草芽胞杆菌Bacillus subtilis idi 基因可使紫槐二烯的摇瓶发酵产量比野生菌株提高12.2 倍。随后针对该菌株建立了高密度发酵过程,发现稳定期的中前期(24?72 h) 是产物合成的关键期,通过稳定期补糖速率的调整,明显改善了产物合成速度,使紫槐二烯的产量从2.5 g/L 提高到了4.85 g/L,但不影响产物积累的周期。考虑到72 h 后菌体老化可能会影响产物合成,进一步采取了调整对数期的补糖速率控制菌体生长的策略,使紫槐二烯的产量达到6.1 g/L。研究结果为基于MEP 途径的萜类异源合成工程菌构建及其发酵工艺的建立奠定了基础。     

Metabolic engineering of carotenoid accumulation in Escherichia coli by modulation of the isoprenoid precursor pool with expression of deoxyxylulose phosphate synthase

The recently discovered non-mevalonate pathway to isoprenoids, which uses glycolytic intermediates, has been modulated by overexpression of Escherichia coli d-1-deoxyxylulose 5-phosphate synthase (DXS) to increase deoxyxylulose 5-phosphate and, consequently, increase the isoprenoid precursor pool in E. coli. Carotenoids are a large class of biologically important compounds synthesized from isoprenoid precursors and of interest for metabolic engineering. However, carotenoids are not ordinarily present in E. coli. Co-overexpression of E. coli dxs with Erwinia uredovora gene clusters encoding carotenoid biosynthetic enzymes led to an increased accumulation of the carotenoids lycopene or zeaxanthin over controls not expressing DXS. Thus, rate-controlling enzymes encoded by the carotenogenic gene clusters are responsive to an increase in isoprenoid precursor pools. Levels of accumulated carotenoids were increased up to 10.8 times the levels of controls not overexpressing DXS. Lycopene accumulated to a level as high as 1333 μg/g dw and zeaxanthin accumulated to a level as high as 592 μg/g dw, when pigments were extracted from colonies. Zeaxanthin-producing colonies grew about twice as fast as lycopene-producing colonies throughout a time course of 11 days. Metabolic engineering of carbon flow from simple glucose metabolites to representatives of the largest class of natural products was demonstrated in this model system. Received: 6 August 1999 / Received revision: 25 October 1999 / Accepted: 5 November 1999     

Cloning and analysis of the spc ribosomal protein operon of Bacillus subtilis: comparison with the spc operon of Escherichia coli.

A segment of Bacillus subtilis chromosomal DNA homologous to the Escherichia coli spc ribosomal protein operon was isolated using cloned E. coli rplE (L5) DNA as a hybridization probe. DNA sequence analysis of the B. subtilis cloned DNA indicated a high degree of conservation of spc operon ribosomal protein genes between B. subtilis and E. coli. This fragment contains DNA homologous to the promoter-proximal region of the spc operon, including coding sequences for ribosomal proteins L14, L24, L5, S14, and part of S8; the organization of B. subtilis genes in this region is identical to that found in E. coli. A region homologous to the E. coli L16, L29 and S17 genes, the last genes of the S10 operon, was located upstream from the gene for L14, the first gene in the spc operon. Although the ribosomal protein coding sequences showed 40-60% amino acid identity with E. coli sequences, we failed to find sequences which would form a structure resembling the E. coli target site for the S8 translational repressor, located near the beginning of the L5 coding region in E. coli, in this region or elsewhere in the B. subtilis spc DNA.     

The methylerythritol phosphate pathway is functionally active in all intraerythrocytic stages of Plasmodium falciparum

Two genes encoding the enzymes 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase have been recently identified, suggesting that isoprenoid biosynthesis in Plasmodium falciparum depends on the methylerythritol phosphate (MEP) pathway, and that fosmidomycin could inhibit the activity of 1-deoxy-D-xylulose-5-phosphate reductoisomerase. The metabolite 1-deoxy-D-xylulose-5-phosphate is not only an intermediate of the MEP pathway for the biosynthesis of isopentenyl diphosphate but is also involved in the biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) in plants and many microorganisms. Herein we report the first isolation and characterization of most downstream intermediates of the MEP pathway in the three intraerythrocytic stages of P. falciparum. These include, 1-deoxy-D-xylulose-5-phosphate, 2-C-methyl-D-erythritol-4-phosphate, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate, and 2-C-methyl-D-erythritol-2,4-cyclodiphosphate. These intermediates were purified by HPLC and structurally characterized via biochemical and electrospray mass spectrometric analyses. We have also investigated the effect of fosmidomycin on the biosynthesis of each intermediate of this pathway and isoprenoid biosynthesis (dolichols and ubiquinones). For the first time, therefore, it is demonstrated that the MEP pathway is functionally active in all intraerythrocytic forms of P. falciparum, and de novo biosynthesis of pyridoxal in a protozoan is reported. Its absence in the human host makes both pathways very attractive as potential new targets for antimalarial drug development.