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.     

Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with <Emphasis Type="Italic">Escherichia coli</Emphasis>

Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8–4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.     

筛选地中海拟无枝酸菌无痕基因敲除菌株的简便方法

将抗生素抗性基因作为标记筛选无痕基因敲除菌株比较费时,因而建立筛选无痕基因敲除菌株的简便方法。通过敲除茄红素生物合成途径中第一个反应的酶编码基因dxs(1-脱氧-D-木酮糖-5-磷酸合酶基因),获得白色地中海拟无枝酸菌突变菌株,以此菌株为受体菌,对S-丙二酰转移酶基因(mtf)进行无痕敲除。针对菌落本身携带颜色的地中海拟无枝酸菌(橘红色),利用茄红素合成酶基因dxs无痕敲除获得了白色菌株,在此基础上进行mtf的无痕敲除。以茄红素生物合成途径中任意一个反应的酶编码基因作为标记,很容易筛选得到无痕基因敲除的突变菌株。     

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     

Characterization of the Escherichia coli gene for 1-acyl-sn-glycerol-3-phosphate acyltransferase (pIsC)

Summary An Escherichia coli strain deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity has previously been isolated, and the gene (plsC) has been shown to map near min 65 on the chromosome. I precisely mapped the location of plsC on the chromosome, and determined its DNA sequence. plsC is located between parC and sufI, and is separated from sufI by 74 bp. Upstream of plsC is parC, separated by 233 bp, which includes an active promoter. parC, plsC, and sufI are all transcribed in the counterclockwise direction on the chromosome, possibly in an operon with multiple promoters. The amino-terminal sequence of the partially purified protein, combined with the DNA sequence, reveal 1-acyl-sn-glycerol-3-phosphate acyltransferase to be a 27.5 kDa highly basic protein. The plsC gene product, 1-acyl-sn-glycerol-3-phosphate acyltransferase, is localized to the cytoplasmic membrane of the cell. The amino-terminal sequence of the purified protein reveals the first amino acid to be a blocked methionine residue, most probably a formyl-methionine. The amino acid sequence of 1-acyl-sn-glycerol-3-phosphate acyltransferase has a short region of homology to two other E. coli acyltransferases that utilize acyl-acyl carrier protein as the acyl donor, sn-glycerol-3-phosphate acyltransferase and UDP-N-acetyl-glucosamine acyltransferase (involved in lipid A biosynthesis).     

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

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.     

冬凌草1-脱氧-D-木酮糖-5-磷酸合成酶基因克隆与表达分析

1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose 5-phosphate synthase,DXS)是植物萜类代谢通路中2-C-甲基-D-赤藓糖醇-4-磷酸(MEP)途径的第一个关键酶,在植物萜类物质的生物合成中发挥重要的作用.为了研究该基因在冬凌草二萜类成分合成中的作用,该研究在冬凌草转录组测序结果的基础上设计一对特异性引物,采用RT-PCR方法得到冬凌草IrDXS基因cDNA全长序列,并对其蛋白进行理化性质分析、信号肽预测、亚细胞定位预测、蛋白质二级结构、三级结构预测分析及跨膜域分析等生物信息学分析,同时利用实时荧光定量PCR的方法检测IrDXS基因在冬凌草不同部位中的表达情况.结果表明:从冬凌草叶片中分离得到了一条编码DXS的全长基因,通过生物信息学软件分析发现,该基因编码全长2169 bp,编码722个氨基酸,分子量为77.7 kD.多序列比对发现该基因编码的蛋白和其他植物中已知的DXS蛋白序列具有较高的同源性,N端均包含了一段质体转运肽序列,并均具有一个保守的焦磷酸硫胺素结构域和与吡啶结合相关的DRAG结构域.序列进化树分析显示,IrDXS基因属于植物DXS2家族.DXS基因在冬凌草根中表达量最高、愈伤组织中最低.该研究首次获得了IrDXS基因的全长cDNA序列,并揭示了其在不同组织中的表达差异,为后续的深入研究IrDXS基因在冬凌草二萜类成分合成途径中的功能奠定了基础.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

An exception among diatoms: unique organization of genes involved in isoprenoid biosynthesis in Rhizosolenia setigera CCMP 1694

The marine diatom Rhizosolenia setigera is unique among this group of microalgae given that it is only one of a handful of diatom species that can produce highly branched isoprenoid (HBI) hydrocarbons. In our efforts to determine distinguishing molecular characteristics in R. setigera CCMP 1694 that could help elucidate the underlying mechanisms for its ability to biosynthesize HBIs, we discovered the occurrence of independent genes encoding for two isopentenyl diphosphate isomerases (RsIDI1 and RsIDI2) and one squalene synthase (RsSQS), enzymes that catalyze non‐consecutive steps in isoprenoid biosynthesis. These genes are peculiarly fused in all other genome‐sequenced diatoms to date, making their organization in R. setigera CCMP 1694 a clear distinguishing molecular feature. Phylogenetic and sequence analysis of RsIDI1, RsIDI2, and RsSQS revealed that such an arrangement of individually transcribed genes involved in isoprenoid biosynthesis could have arisen through a secondary gene fission event. We further demonstrate that inhibition of squalene synthase (SQS) shifts the flux of exogenous isoprenoid precursors towards HBI biosynthesis suggesting the competition for isoprenoid substrates in the form of farnesyl diphosphate between the sterol and HBI biosynthetic pathways in this diatom.     

Characterization of the str operon genes from Spirulina platensis and their evolutionary relationship to those of other prokaryotes

Summary A 5.3 kb DNA segment containing the str operon (ca. 4.5 kb) of the cyanobacterium Spirulina platensis has been sequenced. The str operon includes the structural genes rpsL (ribosomal protein S12), rpsG (ribosomal protein S7), fus (translation elngation factor EF-G) and tuf (translation elongation factor EF-Tu). From the nucleotide sequence of this operon, the primary structures of the four gene products have been derived and compared with the available corresponding structures from eubacteria, archaebacteria and chloroplasts. Extensive homologies were found in almost all cases and in the order S12>EF-Tu>EF-G>S7; the largest homologies were generally found between the cyanobacterial proteins and the corresponding chloroplast gene products. Overall codon usage in S. platensis was found to be rather unbiased.     

Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids

The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.     

Characterisation of actI-homologous DNA encoding polyketide synthase genes from the monensin producer Streptomyces cinnamonensis

Summary Cloned DNA encoding polyketide synthase (PKS) genes from one Streptomyces species was previously shown to serve as a useful hybridisation probe for the isolation of other PKS gene clusters from the same or different species. In this work, the actI and actIII genes, encoding components of the actinorhodin PKS of Streptomyces coelicolor, were used to identify and clone a region of homologous DNA from the monensin-producing organism S. cinnamonensis. A 4799 by fragment containing the S. cinnamonensis act-homologous DNA was sequenced. Five open reading frames (ORFs 1–5) were identified on one strand of this DNA. The five ORFs show high sequence similarities to ORFs that were previously identified in the granaticin, actinorhodin, tetracenomycin and whiE PKS gene clusters. This allowed the assignment of the following putative functions to these five ORFS : a heterodimeric -ketoacyl synthase (ORF1 and ORF2), an acyl carrier protein (ORF3), a -ketoacyl reductase (ORF5), and a bifunctional cyclase/dehydrase (ORF4). The ORFs are encoded in the order ORFl-ORF2-ORF3-ORF5-ORF4, and ORFs-1 and -2 show evidence for translational coupling. This act-homologous region therefore appears to encode a PKS gene cluster. A gene disruption experiment using the vector pGM 160, and other evidence, suggests that this cluster is not essential for monensin biosynthesis but rather is involved in the biosynthesis of a cryptic aromatic polyketide in S. cinnamonensis. An efficient plasmid transformation system for S. cinnamonensis has been established, using the multicopy plasmids pWOR120 and pWOR125.     

罗汉果色氨酸转氨酶基因SgTAR2的克隆及表达分析

色氨酸转氨酶基因家族,是直接参与植物生长素生物合成途径的关键酶基因。该研究在罗汉果转录组测序的基础上,结合RACE技术克隆罗汉果色氨酸转氨酶基因SgTAR2的全长cDNA序列和DNA序列;并对其进行生物信息学分析和时空表达分析。结果表明:克隆所得SgTAR2的cDNA全长序列2078bp,最长ORF为1332bp,编码443个氨基酸,Gen Bank登录号为KU949381,其编码蛋白具有2个蒜氨酸酶保守结构域和多个5'-PLP结合位点,推测其可能参与催化色氨酸转氨基作用、化学防御作用、生长素生物合成等生物学过程;SgTAR2基因DNA长为4103bp,含有4个内含子和5个外显子,其内含子具有多个高水平转录调控因子和多个与激素、环境等胁迫响应相关的作用元件,暗示SgTAR2基因内含子协同调控罗汉果生长素合成、抗胁迫反应、形态发育等生物学过程。实时荧光定量结果显示,SgTAR2基因在罗汉果各组织器官均有表达,在雌蕊和15d幼果期表达量较高,暗示该基因参与罗汉果果实早期发育。该研究结果表明SgTAR2参与了生长素介导的罗汉果不同生长发育过程,特别对幼果及花的起始发育和器官形态建成等具有重要意义。     

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.     

Gene content and organization of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii

Spiroplasma kunkelii, the causative agent of corn stunt disease in maize ( Zea mays L.), is a helical, cell wall-less prokaryote assigned to the class Mollicutes. As part of a project to sequence the entire S. kunkelii genome, we analyzed an 85-kb DNA segment from the pathogenic strain CR2-3x. This genome segment contains 101 ORFs and two tRNA genes. The majority of the ORFs code for predicted proteins that can be assigned to respective clusters of orthologous groups (COGs). These COGs cover diverse functional categories including genetic information storage and processing, cellular processes, and metabolism. The most notable gene cluster in this genome segment is a super-operon capable of encoding 24 ribosomal proteins. The organization of genes in this operon reflects the unique evolutionary position of the spiroplasma. Gene duplications, domain rearrangements, and frameshift mutations in the segment are interpreted as indicators of phase variation in the spiroplasma. To our knowledge, this is the first analysis of a large genome segment from a plant pathogenic spiroplasma.Communicated by W. Goebel     

The Arabidopsis thaliana FPP synthase isozymes have overlapping and specific functions in isoprenoid biosynthesis,and complete loss of FPP synthase activity causes early developmental arrest

Farnesyl diphosphate (FPP) synthase (FPS) catalyses the synthesis of FPP, the major substrate used by cytosolic and mitochondrial branches of the isoprenoid pathway. Arabidopsis contains two farnesyl diphosphate synthase genes, FPS1 and FPS2, that encode isozymes FPS1L (mitochondrial), FPS1S and FPS2 (both cytosolic). Here we show that simultaneous knockout of both FPS genes is lethal for Arabidopsis, and embryo development is arrested at the pre‐globular stage, demonstrating that FPP‐derived isoprenoid metabolism is essential. In addition, lack of FPS enzyme activity severely impairs male genetic transmission. In contrast, no major developmental and metabolic defects were observed in fps1 and fps2 single knockout mutants, demonstrating the redundancy of the genes. The levels of sterols and ubiquinone, the major mitochondrial isoprenoid, are only slightly reduced in the single mutants. Although one functional FPS gene is sufficient to support isoprenoid biosynthesis for normal growth and development, the functions of FPS1 and FPS2 during development are not completely redundant. FPS1 activity has a predominant role during most of the plant life cycle, and FPS2 appears to have a major role in seeds and during the early stages of seedling development. Lack of FPS2 activity in seeds, but not of FPS1 activity, is associated with a marked reduction in sitosterol content and positive feedback regulation of 3‐hydroxy‐3‐methylglutaryl CoA reductase activity that renders seeds hypersensitive to the 3‐hydroxy‐3‐methylglutaryl CoA reductase inhibitor mevastatin.     

Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO₂. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C₅ sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.     

Genetics of pentose-phosphate pathway enzymes ofEscherichia coli K-12

The pentose-phosphate pathway ofEscherichia coli K-12, in addition to its role as a route for the breakdown of sugars such as glucose or pentoses, provides the cell with intermediates for the anabolism of amino acids, vitamins, nucleotides, and cell wall constituents. Through its oxidative branch, it is a major source of NADPH. The expression of the gene for NADP-dependent 6-phospho-gluconate dehydrogenase (gnd) is regulated by the growth rate inE. coli. The recently identified gene for ribulose-5-phosphate 3-epimerase (rpe) is part of a large operon that comprises among others genes for the biosynthesis of aromatic amino acids. In recent years, genes for all enzymes of the pathway have been cloned and sequenced. Isoenzymes have been found for transketolase (genestktA andtktB), ribose-5-phosphate isomerase (rpiA andrpiB) and transaldolase (talA andtalB).     

Engineering a functional 1-deoxy-D-xylulose 5-phosphate (DXP) pathway in Saccharomyces cerevisiae

Isoprenoids are used in many commercial applications and much work has gone into engineering microbial hosts for their production. Isoprenoids are produced either from acetyl-CoA via the mevalonate pathway or from pyruvate and glyceraldehyde 3-phosphate via the 1-deoxy-D-xylulose 5-phosphate (DXP) pathway. Saccharomyces cerevisiae exclusively utilizes the mevalonate pathway to synthesize native isoprenoids and in fact the alternative DXP pathway has never been found or successfully reconstructed in the eukaryotic cytosol. There are, however, several advantages to isoprenoid synthesis via the DXP pathway, such as a higher theoretical yield, and it has long been a goal to transplant the pathway into yeast. In this work, we investigate and address barriers to DXP pathway functionality in S. cerevisiae using a combination of synthetic biology, biochemistry and metabolomics. We report, for the first time, functional expression of the DXP pathway in S. cerevisiae. Under low aeration conditions, an engineered strain relying solely on the DXP pathway for isoprenoid biosynthesis achieved an endpoint biomass 80% of that of the same strain using the mevalonate pathway.