1-脱氧木酮糖-5-磷酸合成酶(DXS)及其编码基因

萜类物质是广泛分布于生物界的一类天然产物,也是重要生命物质。萜类物质通过甲羟戊酸(MVA)途径和2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途径合成,古细菌、真菌和动物及人的萜类物质主要通过MVA途径合成,而多数真细菌(即通常而言的细菌)则利用MEP途径。植物同时拥有两种途径但分别定位于细胞质和质体。1-脱氧木酮糖-5-磷酸合成酶(DXS)是MEP途径的第一个酶,也是该途径的关键调控位点。现从DXS在MEP途径中的作用、DXS结构、亚细胞定位和酶活性、编码基因及突变体等方面对DXS进行全面阐述。拟南芥DXS基因插入突变体cla1-1发生白化,DXS基因表达与类胡萝卜素等萜类物质积累密切相关,在转基因生物体中过度表达DXS可促进萜类物质合成。植物DXS具有典型的质体转运肽序列,决定了DXS的质体定位。完备的DXS活性分析体系为DXS抑制剂开发筛选等研究奠定良好基础。DXS由一至多个基因编码,随生物种类而异,根据同源性,植物DXS基因可分成两类。DXS基因家族不同成员具有不同的表达模式,但通常有一个成员在多种组织中广泛表达。     

UV-B辐射对神农香菊萜类物质合成 及其相关基因表达的影响

该研究以神农香菊为材料,用强度为400μw·cm~(-2)的紫外光UV-B对其进行辐射处理,辐射时间分别为0、0.5、1、2、4 h,探讨了UV-B辐射对神农香菊萜类物质合成及其相关基因表达的影响。结果表明:(1)较短时间的UV-B辐射对神农香菊萜类物质合成相关基因表达量有明显的促进作用。与对照相比,0.5、1、2、4 h处理对相关基因表达量均有不同程度的提高;在2 h处理下HMGR、DXR、TPS、GPS基因的相对表达量达到最大值,在4 h处理下FPS和DXS的相对表达量达到最大值,其中FPS基因表达量变化最显著,为对照的69倍。(2) MVA途径中,去氢白菖烯、杜松萜烯的含量与FPS基因表达量4 h内持续上升的变化趋势保持一致,1-石竹烯与HMGR的变化趋势保持一致,表现为先升高后降低。(3) MEP合成途径中,α-侧柏酮、崖柏酮、β-侧柏酮的含量呈现与DXR、GPS、TPS基因表达量相同的变化趋势,桉树脑在UV-B辐射4 h内持续上升,与DXS基因的变化一致。由此可以推断,UV-B辐射通过影响各自途径中一些关键基因的表达量,进而影响了神农香菊萜类物质的合成量。     

不同花期‘西伯利亚’百合花瓣单萜合成途径转录组分析

以‘西伯利亚’百合（Lilium ‘Siberia’）花蕾期、半开期、盛开期、衰败期的花瓣为材料,利用RNA-seq技术对其转录组进行高通量测序,分析单萜合成途径中差异表达的基因并阐明其分子机制。结果显示,‘西伯利亚’百合通过转录组测序分析共得到56.28 Gb clean base,223.40 Mb clean reads和124 233个unigene,其中35 749个基因得到注释。萜骨架合成途径中的基因表达水平在不同花期表现出显著差异。其中,甲基赤藓糖醇磷酸（MEP）中的1-脱氧-D-木酮糖-5-磷酸合成酶（DXS）、1-脱氧-D-木酮糖-5-磷酸还原异构酶（DXR）、4-羟基-3-甲丁-2-烯基二磷酸合成酶（HDS）、4-羟基-3-甲丁-2烯基二磷酸还原酶（HDR）、牻牛儿基二磷酸合成酶（GPS）基因的表达水平随花期变化呈先升高后降低的趋势。罗勒烯合成酶（OCS）基因表现出相似变化规律,在盛开期表达量最高。甲羟戊酸（MVA）途径中的3-羟基-3-甲基戊二酸单酰辅酶A还原酶（HMGR）的基因表达同样出现先升高后降低的趋势。单萜合成下游的分支途径中,茄尼基二磷酸合成酶（SDS）、牻牛儿基牻牛儿基二磷酸合成酶（GGDR）基因的表达则出现相反的趋势,在盛开期的表达量最低。研究结果表明MEP途径中的关键基因可随花期变化规律性的表达,以调控单萜的生物合成,在盛开期有较高释放量,且盛开期MVA途径的活化以及泛醌和萜醌代谢支路基因的低表达也促进了单萜的生物合成。     

不同花期‘西伯利亚’百合花瓣单萜合成途径转录组分析（英文）

以‘西伯利亚’百合(Lilium‘Siberia’)花蕾期、半开期、盛开期、衰败期的花瓣为材料,利用RNA-seq技术对其转录组进行高通量测序,分析单萜合成途径中差异表达的基因并阐明其分子机制。结果显示,‘西伯利亚’百合通过转录组测序分析共得到56.28 Gb clean base,223.40 Mb clean reads和124 233个unigene,其中35 749个基因得到注释。萜骨架合成途径中的基因表达水平在不同花期表现出显著差异。其中,甲基赤藓糖醇磷酸(MEP)中的1-脱氧-D-木酮糖-5-磷酸合成酶(DXS)、1-脱氧-D-木酮糖-5-磷酸还原异构酶(DXR)、4-羟基-3-甲丁-2-烯基二磷酸合成酶(HDS)、4-羟基-3-甲丁-2烯基二磷酸还原酶(HDR)、牻牛儿基二磷酸合成酶(GPS)基因的表达水平随花期变化呈先升高后降低的趋势。罗勒烯合成酶(OCS)基因表现出相似变化规律,在盛开期表达量最高。甲羟戊酸(MVA)途径中的3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR)的基因表达同样出现先升高后降低的趋势。单萜合成下游的分支途径中,茄尼基二磷酸合成酶(SDS)、牻牛儿基牻牛儿基二磷酸合成酶(GGDR)基因的表达则出现相反的趋势,在盛开期的表达量最低。研究结果表明MEP途径中的关键基因可随花期变化规律性的表达,以调控单萜的生物合成,在盛开期有较高释放量,且盛开期MVA途径的活化以及泛醌和萜醌代谢支路基因的低表达也促进了单萜的生物合成。     

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。     

甘草过表达HMGR、SQS1、β-AS基因再生植株的诱导

目的:诱导过表达3-羟基-3-甲基戊二酰CoA还原酶(HMGR)、鲨烯合成酶1(SQS1)和β-香树脂醇合成酶(β-AS)基因的甘草再生植株。方法:以植物双元表达载体pCAMBIA1305.1为载体骨架,HMGR、SQS1、β-AS基因为目的基因,利用基因重组试剂盒eFusion CloningKit分别构建含有3个外源基因的重组载体,并将其转入根癌农杆菌EHA105,采用根癌农杆菌介导法将目的基因转入甘草外植体。结果与结论:获得了过表达HMGR、SQS1和β-AS基因的甘草愈伤组织及试管苗,为进一步研究这3个功能基因过表达对甘草酸合成的影响奠定了基础。     

油橄榄HMGR基因家族的克隆表达及功能验证

3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR),催化3-羟基-3-甲基戊二酸单酰辅酶A(HMG-Co A)生成甲羟戊酸(MVA),是MVA途径中第一个关键酶。油橄榄是一种重要的经济油料作物,其含有的萜类物质具有重要营养价值,但关于调控萜类物质代谢途径的关键基因研究较少。为研究萜类合成途径关键酶基因HMGR,本研究采用RT-PCR法克隆油橄榄HMGR基因,进行生物信息学分析及构建原核表达载体,对表达蛋白生物活性进行功能验证,并采用荧光定量PCR分析HMGR在油橄榄不同生长阶段的表达量。结果表明:克隆出油橄榄HMGR基因家族的三个基因,分别命名为Oe HMGR1、Oe HMGR2、Oe HMGR3,m RNA ORF的长度分别为1 713 bp、1 773 bp、1 737 bp,编码570、590、578个氨基酸;基因编码蛋白均有2个跨膜区及HMGR催化活性的结构域,不含信号肽;构建了原核表达载体p ET30b(+)-Oe HMGR,转入到大肠杆菌BL21中成功表达,3个重组酶蛋白分子量大小均在66.2~68.0 k D之间,分离纯化重组蛋白进行功能验证,GC-MS检测表明该蛋白具有HMGR催化活性功能;荧光定量PCR分析表明该家族基因在果实和叶片中表达量较高,而在根、茎、花中表达量较低,在开花后45 d、90 d、120 d表达量较低,但在花后165 d表达量上升至较高水平。该研究为进一步鉴定Oe HMGR的功能及油橄榄萜类物质的合成生物学研究奠定基础。     

杜仲MVA和MEP途径相关基因的亚细胞定位与表达分析

通过对杜仲基因组分析,筛选并克隆出MVA途径和MEP途径的相关基因全长（EuDXR,EuMCT,EuCMK,EuMDS,EuACOT,EuHMGS和EuHMGR）,并通过生物信息学方法分析其结构特征,结果表明上述基因与其他已知物种相应基因的相似度达73%~85%。通过构建亚细胞定位表达载体,并瞬时转化烟草下表皮细胞后激光共聚焦显微镜下观察显示,EuDXR,EuMCT,EuCMK,EuMDS基因编码蛋白定位于叶绿体,EuACOT和EuHMGR基因编码蛋白定位于内质网,EuHMGS基因编码蛋白定位于细胞质膜。利用转录组测序技术分析上述基因的时空表达特性表明,MEP途径相关基因在杜仲叶片中大量表达,而MVA途径相关基因在杜仲幼果中大量表达,且杜仲幼果比叶片中的橡胶含量高,因此,推断MVA途径在杜仲橡胶合成中占主导作用。     

钙离子和水杨酸诱导灵芝多糖和三萜的合成

灵芝多糖和三萜是灵芝主要的药理活性物质。如何提高液体发酵中灵芝多糖和三萜的含量是目前研究的热点。本研究以J‐7/AL‐2菌株为例,在灵芝的液体发酵中加入10mmol/L钙离子诱导,多糖含量比对照提高34%,三萜含量提高64%。在发酵液中加入150μmol/L水杨酸(SA)诱导,多糖含量提高57.54%,三萜含量提高37.19%。在灵芝的液体发酵第12天加入10mmol/L钙离子和150μmol/L水杨酸,三萜含量提高46.9%。在不同诱导条件下,三萜的高效液相图谱(HPLC)显示钙离子与对照的差异性比较大。灵芝三萜关键酶基因的表达也有所不同。水杨酸提高法尼基焦磷酸合酶(FPS)、鲨烯合酶(SQS)和羊毛兹醇合酶(LS)基因的表达量,而钙离子和钙离子与水杨酸共同处理提高3‐羟基‐3‐甲基戊二酰辅酶A合酶(HMGS)、3‐羟基‐3‐甲基戊二酰辅酶A转录水平还原酶(HMGR)、焦磷酸甲羟戊酸脱羧酶(MVD)、法尼基焦磷酸合酶(FPS)、鲨烯合酶(SQS)和羊毛兹醇合酶(LS)6个三萜关键酶基因的表达量。在3种处理下,都是SQS的基因表达量提高最多,推测SQS基因是灵芝三萜合成过程中重要的基因。     

酮型广藿香MVA途径基因表达分析及与主要成分合成相关性研究

广藿香药材以广藿香酮含量较高的酮型广藿香为最优质。而广藿香酮为一种萜类成分,其生物合成途径尚未明确。MVA(甲羟戊酸)途径是萜类化合物生物合成的重要途径。为了分析MVA途经基因表达与化学成分的相关性从而获得促进广藿香酮合成的潜在基因,该文以2种酮型广藿香栽培品种(石牌广藿香、高要广藿香)为材料,通过实时定量PCR分析基因表达和主要成分含量测定,并研究了供试材料不同时期的茎、叶中与甲羟戊酸代谢途径相关的HMGR、MK、MDD基因表达及化学成分。结果表明：(1)HMGR基因在石牌广藿香嫩叶中表达更明显;MK基因在石牌广藿香和高要广藿香中表达模式相似,主要在老茎中表达;MDD基因在石牌广藿香叶中比高要广藿香表达量更高,在两种广藿香的茎中表达模式相似。(2)同属于酮型广藿香,石牌广藿香与高要广藿香的化学成分相似,老叶广藿香醇含量最高,老茎的广藿香酮含量更高。(3)MDD和MK基因与广藿香酮的合成正相关。综上结果所述,酮型广藿香两个栽培种MVA途径的基因表达模式相似,MDD和MK基因可能为酮型广藿香萜类代谢途径的关键基因。     

Cloning and characterization of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway genes of a natural-rubber producing plant, Hevea brasiliensis

Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-(13)C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.     

Reconstruction and Evaluation of the Synthetic Bacterial MEP Pathway in Saccharomyces cerevisiae

Isoprenoids, which are a large group of natural and chemical compounds with a variety of applications as e.g. fragrances, pharmaceuticals and potential biofuels, are produced via two different metabolic pathways, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we attempted to replace the endogenous MVA pathway in Saccharomyces cerevisiae by a synthetic bacterial MEP pathway integrated into the genome to benefit from its superior properties in terms of energy consumption and productivity at defined growth conditions. It was shown that the growth of a MVA pathway deficient S. cerevisiae strain could not be restored by the heterologous MEP pathway even when accompanied by the co-expression of genes erpA, hISCA1 and CpIscA involved in the Fe-S trafficking routes leading to maturation of IspG and IspH and E. coli genes fldA and fpr encoding flavodoxin and flavodoxin reductase believed to be responsible for electron transfer to IspG and IspH.     

Early Steps in Isoprenoid Biosynthesis: Multilevel Regulation of the Supply of Common Precursors in Plant Cells

Isoprenoids are produced in all organisms but are especially abundant and diverse in plants. Two separate pathways operate in plant cells to synthesize prenyl diphosphate precursors common to all isoprenoids. Cytosolic and mitochondrial precursors are produced by the mevalonic acid (MVA) pathway whereas the recently discovered methylerythritol phosphate (MEP) pathway is located in plastids. However, both pathways may participate in the synthesis of at least some isoprenoids under certain circumstances. Although genes encoding all the enzymes from both pathways have already been cloned, little is known about the regulatory mechanisms that control the supply of isoprenoid precursors. Genetic approaches are providing valuable information on the regulation of both pathways. Thus, recent data from overexpression experiments in transgenic plants show that several enzymes share control over the metabolic flux through the MEP pathway, whereas a single regulatory step has been proposed for the MVA pathway. Identification of Arabidopsis thaliana mutants that are resistant to the inhibition of the MVA and the MEP pathways is a promising approach to uncover mechanisms involved in the crosstalk between pathways. The characterization of some of these mutants impaired in light perception and signaling has recently provided genetic evidence for a role of light as a key factor to modulate the availability of isoprenoid precursors in Arabidopsis seedlings. The picture emerging from recent data supports that a complex regulatory network appears to be at work in plant cells to ensure the supply of isoprenoid precursors when needed.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Evidence of Isoprenoid Precursor Toxicity in Bacillus subtilis

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.