大肠杆菌链长控制基因ddsA原位替换及多基因共表达对辅酶Q10合成能力的影响

目的 强化大肠杆菌合成辅酶Q10(CoQ10)的能力.方法 利用途径工程的基本原理,对大肠杆菌辅酶Q生物合成途径中链长控制基因ispB进行遗传操作,并强化表达该途径中多个功能基因.结果 首次成功用来自Gluconobacter suboxydans的十聚异戊二烯焦磷酸合成酶基因ddsA原位替换大肠杆菌染色体上ispB基因,构建得到原位替换株JKL;同时强化表达CoQ生物合成途径的相关基因,发现在ubiCA和ddsA协同表达的重组菌pDCA/JKL中CoQ的合成能力比JKL提高了2.3倍,CoQ10合成量提高了2.5倍.检测该重组菌中ubiA的转录水平,发现其mRNA相对拷贝数是对照JKL的125倍.结论 成功获得的重组大肠杆菌在降低内源性CoQ8合成能力的同时具备合成较长侧链CoQ10的能力,且通过强化表达相关基因使合成CoQ10的能力得到了提高.     

大肠杆菌链长控制基因ddsA原位替换及多基因共表达对辅酶Q_（10）合成能力的影响

目的强化大肠杆菌合成辅酶Q10（CoQ10）的能力。方法利用途径工程的基本原理,对大肠杆菌辅酶Q生物合成途径中链长控制基因ispB进行遗传操作,并强化表达该途径中多个功能基因。结果首次成功用来自Gluconobacter suboxydans的十聚异戊二烯焦磷酸合成酶基因ddsA原位替换大肠杆菌染色体上ispB基因,构建得到原位替换株JKL;同时强化表达CoQ生物合成途径的相关基因,发现在ubiCA和ddsA协同表达的重组菌pDCA/JKL中CoQ的合成能力比JKL提高了2.3倍,CoQ10合成量提高了2.5倍。检测该重组菌中ubiA的转录水平,发现其mRNA相对拷贝数是对照JKL的125倍。结论成功获得的重组大肠杆菌在降低内源性CoQ8合成能力的同时具备合成较长侧链CoQ10的能力,且通过强化表达相关基因使合成CoQ10的能力得到了提高。     

三孢布拉氏霉合成辅酶Q10及其代谢调控

对三孢布拉氏霉代谢产物的分析表明，该菌能够合成辅酶Q10，且正株合成辅酶Q10的能力高于负株。当正负菌株以5：1混合培养时，辅酶Q10产量最高。三孢布拉氏霉合成辅酶Q10是遵循经典的辅酶Q10生物合成途径的，本实验选育到具有L-甲硫氨酸和D-酪氨酸或对氨基苯丙氨酸双重抗性标记的突变菌各一株，其辅酶Q10产量较出发菌株分别提高了76．7％及94．2％。     

烟草茄尼醇生物合成基因

茄尼醇不仅是合成辅酶Q10和维生素K2等泛醌类药物的重要中间体,而且是卷烟烟气多环芳烃的重要前体物,对卷烟安全性有较大影响。综述了烟草茄尼醇生物合成途径及其关键酶基因的研究进展,展望了其在烟草医药价值开发和低危害烟草品种培育中的应用潜力。     

大肠杆菌ubiA过表达对CoQ产量的影响

旨在建立CoQ高产菌株。通过ubiA基因和pkk223-3质粒构建重组质粒,双酶切和测序验证;发酵ubiA基因过表达菌株,分析CoQ产量和种类变化;通过间歇补加PHBA次数、发酵时间、温度和溶氧量因素优化发酵工艺。结果表明,menA基因敲除菌株基础上,成功获得ubiA基因过表达菌株,CoQ产量增加约为95%,种类不变;补加pHBA3次,摇床175r/min,34℃发酵15h,CoQ合成达2.08mg/g,较原始菌株提高216%。首次获得ubiA过表达的menA敲除菌,结合发酵条件优化,改良株CoQ产量大幅提高,达到预期目标。     

苯丙氨酸生物合成的整体代谢网络调控基因的敲除

csrA基因产物是大肠杆茵芳香族氨基酸生物合成途径中碳中心代谢有关的一种全局性调控蛋白质.采用Red敲除系统介导的同源重组的方法定位缺失大肠杆茵染色体csrA基因,经PCR、DNA测序等多种方法证实了基因剔除的可靠性.csrA基因剔除后,大肠杆菌芳香族氨基酸生物合成中受csrA表达产物负调控的关键酶的酶活力有所提高,而正调控的关键酶基因活性降低,突变菌株发酵生产苯丙氨酸的能力提高近13％.     

脱羧酶基因ARO10的过量表达及其对Saccharomy cescerevisiae的β-苯乙醇合成代谢影响

为了验证脱羧酶在S.cerevisiaeβ-苯乙醇合成途径中的调控作用,本文从S.cerevisiae S288C中克隆脱羧酶基因ARO10,并构建由3-磷酸甘油酸激酶基因PGK1组成型强启动子控制的ARO10基因表达载体pYES2-Ppgk-ARO10,将重组载体导入S.cerevisiae S288C,研究ARO10基因过量表达对重组菌株中β-苯乙醇合成的影响。经摇瓶实验测定,携带pYES2-Ppgk-ARO10的转化子SP10在发酵60h时β-苯乙醇产量达到最大量1.0g/L,较野生型的对照菌提高16.3%。研究结果表明,脱羧酶是S.cerevisiae S288C中β-苯乙醇生物合成途径的关键酶之一,增加ARO10基因表达量有利于提高β-苯乙醇产量,研究为构建β-苯乙醇高产工程菌株奠定了重要基础。     

脱羧酶基因ARO10的过量表达及其对Saccharomy cescerevisiae的β-苯乙醇合成代谢影响

为了验证脱羧酶在S.cerevisiaeβ-苯乙醇合成途径中的调控作用,本文从S.cerevisiae S288C中克隆脱羧酶基因ARO10,并构建由3-磷酸甘油酸激酶基因PGK1组成型强启动子控制的ARO10基因表达载体pYES2-Ppgk-ARO10,将重组载体导入S.cerevisiae S288C,研究ARO10基因过量表达对重组菌株中β-苯乙醇合成的影响。经摇瓶实验测定,携带pYES2-Ppgk-ARO10的转化子SP10在发酵60h时β-苯乙醇产量达到最大量1.0g/L,较野生型的对照菌提高16.3%。研究结果表明,脱羧酶是S.cerevisiae S288C中β-苯乙醇生物合成途径的关键酶之一,增加ARO10基因表达量有利于提高β-苯乙醇产量,研究为构建β-苯乙醇高产工程菌株奠定了重要基础。      

噬菌体基因Ⅴ蛋白基因的合成、重组表达及功能分析

目的:研究丝状噬菌体基因V蛋白(gene V protein,GVP)基因的合成、重组表达及其功能。方法:根据GVP的基因序列,选择大肠杆菌偏爱的密码子,设计合成了8个寡核苷酸片段,利用重叠延伸PCR合成GVP基因序列,将其与原核表达载体pET-28a-c(+)质粒重组,转化大肠杆菌,获得GVP蛋白阳性表达菌株,异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,产物经Ni+-NTA琼脂糖凝胶层析纯化,获得目的蛋白GVP,DNA结合实验检测其功能。结果:成功合成出GVP基因,重组体在大肠杆菌BL21(DE3)中诱导获得高效表达,DNA结合实验表明GVP与单链DNA间解离平衡常数Kd=7.27×10-5mol/L。结论:重组构建并高效表达的GVP蛋白具有较高单链DNA结合能力,可用于食品病原微生物特定单链DNA分子的浓缩和分离。     

大肠杆菌铁相关基因的调控对血红素合成的影响

血红素(heme)是一种金属卟啉化合物，在细胞中具有重要的生理功能，实际生产中应用广泛，目前对大肠杆菌(Escherichia coli)血红素合成途径调控的研究主要集中在上游，而下游部分较少。为了研究下游途径的改造对大肠杆菌血红素合成的影响，对合成途径最后一步的相关基因进行了不同的表达调控。研究结果表明，在铁摄取调节子(ferric uptake regulator, Fur)基因fur缺失菌中过表达亚铁鳌合酶(ferrochelatase)基因hemH会抑制血红素的合成；而过表达染料脱色过氧化物酶(dye-decolorizing peroxidase)基因efeB可以缓解因fur缺失而造成的血红素含量下降；efeB和hemH基因的共表达则可以有效提高血红素的含量，达到出发菌株E.coli BL21的1.72倍。研究结果为提升大肠杆菌血红素含量的研究提供了一定的策略和理论依据。     

Sulfide oxidation by gene expressions of sulfide-quinone oxidoreductase and ubiquinone-8 biosynthase in Escherichia coli

Sulfides (S2),SH-) such as hydrogen sulfide belong to a class of sulfur compounds with unpleasant odors. In order to confer sulfide-oxidizing ability on the intestine-inhabiting bacteria, the sulfide-quinone oxidoreductase gene (sqr) in Rhodobacter capsulatus DSM-155 and genes for quinone biosynthesis (ubiC, ubiA and ispB) in Escherichia coli XL1 Blue-MRF' were transduced into E. coli BL21(DE3). Plasmids pT7-7 and pSTV were used as vectors of sqr, and ubiCA and ispB, respectively. The recombinants sqr-BL21(DE3) and ubiCA,ispB-sqr-BL21(DE3) were successfully constructed. The maximal sulfide-removing activities of the whole cells and membrane fractions of sqr-BL21(DE3) attained at pH 8.0 and 7.8, were 267 nmol/mg cells (dry weight)/min and 1250 nmol/mg membrane fraction (protein)/min, respectively. The molecular ratio of sulfide (S2-) oxidized and oxygen (O2) consumed was 2:1. SQR activity in the recombinant cells was positively restricted under anaerobic conditions and also by the addition of electron transfer inhibitors. Ubiquinone-8 (UQ-8) biosynthesis in the cells of ubiCA,ispB-sqr-BL21(DE3) increased as much as 2.2-fold compared with that of (pSTV)-sqr-BL21(DE3) during the 12-16 h incubation period. The maximal sulfide removal in the quinone-raised E. coli was attained slightly earlier, however, SQR activities thereafter were lower than those in (pSTV)-sqr-BL21(DE3).     

fps基因过量表达对烟叶类胡萝卜素生物合成的影响

为探索法呢基焦磷酸合酶(FPS)基因过量表达对烤烟叶片类胡萝卜素生物合成的影响,利用RT-PCR技术,比较了4个fps转基因株系(K4、K-6、K-17、K-35)及其非转基因对照中参与类胡萝卜素生物合成的关键酶基因的表达强度,并利用LC/MS技术,分析了叶片发育过程中类胡萝卜素及其各组分含量的变化规律.结果表明:外源fps在烟草中的过量表达,对烟叶类胡萝卜素合成相关基因Psy、Lycb皆有正向调节作用,但对Zds的表达影响不大;化学分析表明fps转基因烤烟株系中,总类胡萝卜素及其各组分的含量在叶片不同发育期均高于未转基因K326对照株系.说明外源fps基因在烟草中的过表达,对类胡萝卜素生物合成具有促进作用.     

Purification, characterization and gene cloning of thermostable O-acetyl-L-homoserine sulfhydrylase forming gamma-cyano-alpha-aminobutyric acid

A thermophilic and cyanide ion-tolerant bacterium, Bacillus stearothermophilus CN3 isolated from a hot spring in Japan, was found to produce thermostable gamma-cyano-alpha-aminobutyric acid synthase. The enzyme was purified and characterized. The purified enzyme has a molecular mass of approximately 180 kDa and consists of four identical subunits. It was stable in the pH range of 6.0 to 10.5 and up to 60 degrees C. The enzyme catalyzed the gamma-replacement reaction of O-acetyl-L-homo-serine with cyanide ions. The gene encoding the gamma-cyano-alpha-aminobutyric acid synthase was isolated from B. stearothermophilus CN3. Sequence homology analysis revealed that the gamma-cyano-alpha-aminobutyric acid synthase from the bacterium is O-acetyl-L-homoserine sulfhydrylase. A recombinant plasmid, constructed by ligation of the cloned gene and an expression vector, was introduced into Escherichia coli JM109. The transformed E. coli cells overexpressed gamma-cyano-alpha-aminobutyric acid synthase. The heat stable gamma-cyano-alpha-aminobutyric acid synthase can be applied to the synthesis of [5-11C]L-glutamic acid used as a tracer for positron emission tomography.     

Amplification of the NADPH-related genes zwf and gnd for the oddball biosynthesis of PHB in an E. coli transformant harboring a cloned phbCAB operon

NADPH, a major reducing power in microorganisms, is mostly generated from the pentose phosphate (PP) pathway by glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) expressed by the zwf and gnd genes, respectively. The characteristics of these two genes in Escherichia coli were compared after their re-introduction into the parent strain for over-expression. zwf encoding G6PDH increased the level of NADPH 3 folds compared to gnd encoding 6PGDH. An excess of NADPH depressed cell growth mainly due to the inhibition of citrate synthase in the TCA cycle. Recombinant plasmids containing zwf or gnd co-integrated with the phbCAB operon from Ralstonia eutropha were constructed, and introduced into E. coli for the oddball biosynthesis of PHB. The amount of PHB increased after enforcing the genes; especially the zwf gene an increase of around 41%, due to the rise in NADPH and the depressed TCA cycle, leading to the metabolic flux of intermediates to the pathway for the biosynthesis of PHB.     

Modulation of talA gene in pentose phosphate pathway for overproduction of poly-beta-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon

The talA gene encoding transaldolase, the key enzyme in the nonoxidative pentose phosphate pathway, was amplified in a transformant Escherichia coli harboring the phbCAB operon to shift the metabolic flux of the hexose mono-phosphate shunt to the odd-ball biosynthesis pathway for poly-beta-hydroxybutyrate overproduction. The PHB content in the transformant E. coli coharboring the phbCAB operon and talA gene increased from 28.2% to 52.3%, and the retarded cell growth was overcome. This increase seems to be mainly due to the concomitant supplies of the intermediates NADPH and acetyl-CoA, which are from the activated pentose phosphate pathway through the modulation of the talA gene and from the Embden-Meyerhof pathway.     

保加利亚乳杆菌D-乳酸脱氢酶同工酶基因在大肠杆菌中的表达

通过对保加利亚乳杆菌ATCC11842的D-乳酸脱氢酶基因的克隆,分离获得了ldb0101、ldb0813、ldb1010、ldb1147和ldb2021五种同工酶基因,并分别构建重组质粒转化大肠杆菌JM109,实现D-LDH同工酶基因的表达。结合重组大肠杆菌的摇瓶发酵实验和D-乳酸的合成,初步确定D-乳酸脱氢酶Ldb0101和Ldb1010在保加利亚乳杆菌中对D-乳酸的合成起着重要的作用。      

Role of tktA gene in pentose phosphate pathway on odd-ball biosynthesis of poly-beta-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon

A cloned tktA gene encoding transketolase (TK), the most critical enzyme in the nonoxidative pentose phosphate (PP) pathway, was reinforced into the parent Escherichia coli for metabolic flux control of carbohydrate. It was also transformed into the transformant E. coli harboring phbCAB operon for the odd-ball biosynthesis of PHB. The biosynthesis of PHB significantly increased up to 1.7-fold after the cotransformation of the phbCAB operon and tktA gene, due to the active supplementation of the precursor molecules, acetyl-CoA and NADPH.     

Biosynthesis, bioproduction and novel roles of ubiquinone

Ubiquinone (coenzyme Q) is a well-known component of the electron transfer system in living organisms. It is known that ubiquinone transfers electrons from Complex I (or Complex II) to Complex III in the respiratory chain. However, recent evidence indicates that an involvement in respiration is not the sole role of ubiquinone, and various novel roles have been elucidated. A role as a lipid soluble antioxidant is now widely accepted. The relationship between lifespan and ubiquinone has attracted much interest based on the study of a Caenorhabditis elegans clk-1 mutant. The connection between disulfide bond formation and ubiquinone (or menaquinone) in Escherichia coli has been well studied. The production of hydrogen sulfide in a ubiquinone-deficient fission yeast is an interesting phenotype recently observed. These are some examples of the novel roles of ubiquinone and this review summarizes the recent findings relating to the biosynthesis, bioproduction and novel roles of ubiquinone.     

Genetic manipulation system in propionibacteria

Members of the genus Propionibacterium are widely used in the production of vitamin B12, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propionibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibacterium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in P. freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin B12 biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in P. freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probiotics and the vitamin B12 biosynthetic pathway.