Spatial and developmental expression of key genes of terpene biosynthesis in Tanacetum parthenium

Feverfew (Tanacetum parthenium) is a medicinal plant belonging to the Asteraceae family. To improve understanding terpene metabolism in feverfew, the relative gene expression of four key genes coding 3-hydroxy-3-methylglutarylcoenzyme A reductase (HMGR) and germacrene A synthase (GAS) from the mevalonic acid pathway (MVA), as well as 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) and hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) from the methyl erythritol phosphate pathway (MEP), were examined. Target organs and tissues included young leaves (not fully expanded), mature leaves (fully expanded), flowers, stems, roots, and glandular trichomes. HMGR, DXR, and HDR were isolated and sequenced for the first time in feverfew. Real-time quantitative PCR analysis revealed differential expression of these genes in feverfew tissues and developmental stages.     

Combinational biosynthesis of isoprene by engineering the MEP pathway in Escherichia coli

As an important feedstock in petrochemistry, isoprene is used in a wide range of industrial applications. It is produced almost entirely from petrochemical sources; however, these sources are being progressively depleted. A reliable biological process for isoprene production utilizing renewable feedstocks would be an industry-redefining development. There are two biosynthetic pathways producing isoprene: the mevalonate (MVA) pathway and the methyl erythritol 1-phosphate (MEP) pathway. In this study, the MEP pathway was modified in Escherichia coli BL21 (DE3) to produce isoprene. The isoprene synthase (IspS) gene chemically synthesized from Populus alba after codon optimization for expression in E. coli was heterologously expressed. The endogenous genes of 1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) were over-expressed. The isopentenyl pyrophosphate isomerase (Idi) gene from Streptococcus pneumoniae was exogenously over-expressed, and farnesyl diphosphate synthase (ispA) was weakened to enhance the yield. The control strain harboring empty plasmids did not emit any isoprene. The over-expression of the DXR gene only had little impact on the yield of isoprene. Idi from S. pneumoniae played a significant role in the improvement of isoprene production. The highest yield was achieved by an ispA-weakened DXS-IDI-IspS recombinant with 19.9 mg/l isoprene, which resulted in a 33-fold enhancement of the isoprene yield from the IspS recombinant.     

Crystal structure of Brucella abortus deoxyxylulose-5-phosphate reductoisomerase-like (DRL) enzyme involved in isoprenoid biosynthesis

Most bacteria use the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for the synthesis of their essential isoprenoid precursors. The absence of the MEP pathway in humans makes it a promising new target for the development of much needed new and safe antimicrobial drugs. However, bacteria show a remarkable metabolic plasticity for isoprenoid production. For example, the NADPH-dependent production of MEP from 1-deoxy-d-xylulose 5-phosphate in the first committed step of the MEP pathway is catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) in most bacteria, whereas an unrelated DXR-like (DRL) protein was recently found to catalyze the same reaction in some organisms, including the emerging human and animal pathogens Bartonella and Brucella. Here, we report the x-ray crystal structures of the Brucella abortus DRL enzyme in its apo form and in complex with the broad-spectrum antibiotic fosmidomycin solved to 1.5 and 1.8 Å resolution, respectively. DRL is a dimer, with each polypeptide folding into three distinct domains starting with the NADPH-binding domain, in resemblance to the structure of bacterial DXR enzymes. Other than that, DRL and DXR show a low structural relationship, with a different disposition of the domains and a topologically unrelated C-terminal domain. In particular, the active site of DRL presents a unique arrangement, suggesting that the design of drugs that would selectively inhibit DRL-harboring pathogens without affecting beneficial or innocuous bacteria harboring DXR should be feasible. As a proof of concept, we identified two strong DXR inhibitors that have virtually no effect on DRL activity.     

Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts.     

Effect of down-regulating 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose-5-phosphate reductoisomerase by RNAi on growth and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), an important enzyme in the 2-c-methyl-d-erythritol-4-phosphate (MEP) pathway in plant plastids, provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the roles of the MEP pathway in regulating growth, development and artemisinin biosynthesis of Artemisia annua L., we used RNA interference technology to generate transgenic plants with suppressed expression of DXR in A. annua (AaDXR). Suppression of AaDXR resulted in shorter stems, decreased branch numbers and leaf area, lower density of leaf trichomes. Although AaDXR-RNAi plants had no significant changes on the stomatal conductance, the net photosynthesis rate was decreased by 20.0–31.4% due to the marked decline in the contents of chlorophyll. Decreased levels of endogenous gibberellic acid (GA₃) and abscisic acid were also detected in the transgenic lines. The artemisinin contents in leaves of all tested transgenic lines declined by 41.8–73.4% at the vegetative stage and 61.5–63.6% at the stages of flowering. The enhancement of artemisinin contents by methyl jasmonate at 300 µM has been abolished at seedling and vegetative stages in AaDXR-RNAi plants. These results demonstrate that AaDXR play import roles in the control of plan vegetative growth and artemisinin biosynthesis in A. annua.     

A study on biosynthesis of “citral” in lemongrass (<Emphasis Type="Italic">C. flexuosus</Emphasis>) cv. Suvarna

Incorporation of [1-¹³C]-glucose and fosmidomycin was achieved in young and rapidly expanding (aged 15 days) leaves of lemongrass (C. flexuosus) cv. suvarna to elucidate biosynthetic origin of citral (3,7-dimethyl-2,6-octadienal). Analyses of the resultant ¹³C-labeling patterns of citral by quantitative ¹³C-NMR spectroscopy revealed significant %¹³C enrichment at carbons C-3, C-5, C-7 and C-9 in citral. This labeling pattern of the citral is in accordance with their biosynthesis via 2C-methyl-d-erythritol-4-phosphate (MEP) pathway. However, incorporation of [1-¹³C]-glucose achieved in the presence of fosmidomycin resulted in a ¹³C-labeling pattern of citral which did not match with labeling pattern characteristic of the MEP pathway. In addition, we studied the activity pattern of the DXR enzyme following fosmidomycin (25, 50, 75 and 100 μM concentrations) treatment of the young (aged 15 days) leaves for 48 h. The results revealed that fosmidomycin (100 μM) caused drastic inhibition (>50 %) of the DXR enzyme activity. The levels of the citral measured in the fosmidomycin treated leaves were also found to be reduced with decrease the activity of DXR enzyme. In conclusion, the results of the present work revealed the presence of the MEP pathway and its role in the biosynthesis of citral in lemongrass. In addition, the critical role of the DXR enzyme in the citral biosynthesis is highlighted. This is the first report on elucidation of the MEP pathway in lemongrass and may help in deeper understanding of the monoterpene biosynthesis and regulation in the genus Cymbopogon of high industrial significance.     

Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera

Withania somnifera (L.) is one of the most valuable medicinal plants used in Ayurvedic and other indigenous medicines. Pharmaceutical activities of this herb are associated with presence of secondary metabolites known as withanolides, a class of phytosteroids synthesized via mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate pathways. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, not much is known about the genes responsible for biosynthesis of these compounds. In this study, we have characterized two genes encoding 1-deoxy-d-xylulose-5-phosphate synthase (DXS; EC 2.2.1.7) and 1-deoxy-d-xylulose-5-phosphate reductase (DXR; EC 1.1.1.267) enzymes involved in the biosynthesis of isoprenoids. The full-length cDNAs of W. somnifera DXS (WsDXS) and DXR (WsDXR) of 2,154 and 1,428 bps encode polypeptides of 717 and 475 amino acids residues, respectively. The expression analysis suggests that WsDXS and WsDXR are differentially expressed in different tissues (with maximal expression in flower and young leaf), chemotypes of Withania, and in response to salicylic acid, methyl jasmonate, as well as in mechanical injury. Analysis of genomic organization of WsDXS shows close similarity with tomato DXS in terms of exon–intron arrangements. This is the first report on characterization of isoprenoid biosynthesis pathway genes from Withania.     

苏云金芽胞杆菌dxr1基因的转录受SigH控制

【目的】萜类化合物广泛分布在生物界,是重要的生命物质。目前发现有两条萜类化合物的生物合成途径,即甲羟戊酸(MVA)途径和2-甲基-D-赤藓糖醇-4-磷酸(MEP)途径。MEP代谢途径中的关键酶1-脱氧-D-木酮糖-5-磷酸还原异构化酶(DXR,EC1.1.1.267)催化1-脱氧-D-木酮糖-5-磷酸生成MEP。枯草芽胞杆菌中dxr基因编码DXR酶,而在苏云金芽胞杆菌(Bacillusthuringiensis,Bt)中有2个基因(dxr1和dxr2)编码DXR酶。通过分析BtHD73菌株的dxr1基因的转录活性和dxr1突变体表型,明确dxr1基因的转录调控机制和功能。【方法】通过5?RACE分析dxr1的转录起始位点;β-半乳糖苷酶活性测定分析dxr1基因启动子(Pdxr1)的转录活性;采用同源重组技术分别敲除BtHD73菌株的dxr1和dxr2基因;利用总蛋白定量确定Cry1Ac蛋白产量;利用DXR检测试剂盒检测Bt菌株的DXR活性。【结果】dxr1基因的转录起始位点位于起始密码子上游39 bp处的G碱基;与出发菌株HD73相比,Pdxr1在sig H突变体中的转录活性明显降低;dxr1或dxr2基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无显著影响,但使DXR活性下降。【结论】Bt中dxr1基因的转录受Sig H控制,dxr1基因的缺失影响DXR的活性。     

Molecular characterization of the Oncidium orchid HDR gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, the last step of the methylerythritol phosphate pathway

Two pathways are used by higher plants for the biosynthesis of isoprenoid precursors: the mevalonate pathway in the cytosol and a 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway in the plastids, with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) catalyzing the last step in the MEP pathway. In order to understand the contribution of MEP pathway in isoprenoid biosynthesis of Oncidium orchid, a full-length cDNA corresponding to HDR from the flower tissues of Oncidium Gower Ramsey was cloned. The deduced OncHDR amino acid sequence contains a plastid signal peptide at the N-terminus and four conserved cysteine residues. RT-PCR analysis of HDR in Oncidium flowering plants revealed ubiquitous expression in organs and tissues, with preferential expression in the floral organs. Phylogenetic analysis revealed evolutionary conservation of the encoding HDR protein sequence. The genomic sequence of the HDR in Oncidium is similar to that in Arabidopsis, grape, and rice in structure. Successful complementation by OncHDR of an E. coli hdr ⁻ mutant confirmed its function. Transgenic tobacco carrying the OncHDR promoter-GUS gene fusion showed expression in most tissues, as well as in reproductive organs, as revealed by histochemical staining. Light induced strong GUS expression driven by the OncHDR promoter in transgenic tobacco seedlings. Taken together, our data suggest a role for OncHDR as a light-activated gene.     

Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxyxylulose 5-phosphate reductoisomerase

The methylerythritol 4-phosphate (MEP) pathway synthesizes the precursors for an astonishing diversity of plastid isoprenoids, including the major photosynthetic pigments chlorophylls and carotenoids. Since the identification of the first two enzymes of the pathway, deoxyxylulose 5-phoshate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), they both were proposed as potential control points. Increased DXS activity has been shown to up-regulate the production of plastid isoprenoids in all systems tested, but the relative contribution of DXR to the supply of isoprenoid precursors is less clear. In this work, we have generated transgenic Arabidopsis thaliana plants with altered DXS and DXR enzyme levels, as estimated from their resistance to clomazone and fosmidomycin, respectively. The down-regulation of DXR resulted in variegation, reduced pigmentation and defects in chloroplast development, whereas DXR-overexpressing lines showed an increased accumulation of MEP- derived plastid isoprenoids such as chlorophylls, carotenoids, and taxadiene in transgenic plants engineered to produce this non-native isoprenoid. Changes in DXR levels in transgenic plants did not result in changes in␣DXS gene expression or enzyme accumulation, confirming that the observed effects on plastid isoprenoid levels in DXR-overexpressing lines were not an indirect consequence of altering DXS levels. The results indicate that the biosynthesis of MEP (the first committed intermediate of the pathway) limits the production of downstream isoprenoids in Arabidopsis chloroplasts, supporting a role for DXR in the control of the metabolic flux through the MEP pathway.     

Molecular characterization and expression of 1-deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-xylulose 5-phosphate reductoisomerase (DXR) gene from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes the first committed step of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plants. The present study describes the cloning and characterization of a cDNA encoding DXR from Salvia miltiorrhiza (designated as SmDXR, GenBank Accession No. FJ476255). Comparative and bioinformatic analyses revealed that SmDXR showed extensive homology with DXRs from other plant species. Phylogenetic tree analysis indicated that SmDXR belongs to the plant DXR superfamily and has the closest relationship with DXR from Lycopersicon esculentum. Tissue expression pattern analysis revealed that SmDXR expressed strongly in leaves, followed by roots and stems, implying that SmDXR was a constitutively expressed gene. This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinone biosynthetic pathway in Salvia plants. The expression profiles revealed by RT-PCR under different elicitor treatments such as methyl jasmonate (MJ) and salicylic acid (SA) were compared for the first time, and the results revealed that SmDXR was an elicitor-responsive gene, which could be induced by SA in leaves and inhibited by exogenous MJ in three tested tissues. The functional color assay in Escherichia coli showed that SmDXR could accelerate the biosynthesis of lycopene, indicating that SmDXR encoded a functional protein. The characterization, expression profile and functional analysis of SmDXR gene will be helpful for further study in the role of SmDXR in tanshinones biosynthetic pathway and metabolic engineering to increase tanshinones production in S. miltiorrhiza.     

Methylerythritol phosphate pathway to isoprenoids: Kinetic modeling and in silico enzyme inhibitions in Plasmodium falciparum

The methylerythritol phosphate (MEP) pathway of Plasmodium falciparum (P. falciparum) has become an attractive target for anti-malarial drug discovery. This study describes a kinetic model of this pathway, its use in validating 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) as drug target from the systemic perspective, and additional target identification, using metabolic control analysis and in silico inhibition studies. In addition to DXR, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) can be targeted because it is the first enzyme of the pathway and has the highest flux control coefficient followed by that of DXR. In silico inhibition of both enzymes caused large decrement in the pathway flux. An added advantage of targeting DXS is its influence on vitamin B1 and B6 biosynthesis. Two more potential targets, 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase, were also identified. Their inhibition caused large accumulation of their substrates causing instability of the system.