大肠杆菌中表达关键基因产异丁醇的研究

目的:改造大肠杆菌的代谢途径,使非生产菌株大肠杆菌具备产异丁醇的能力.方法:将乳脂乳球菌NIZO B1157的2-酮酸脱羧酶基因kdcA克隆到大肠杆菌中,使大肠杆菌产异丁醇;另外,采取两种方法过量表达alsS、ilvC、ilvD基因,增加前体物质酮酸的供应,以提高异丁醇的产量:一是与kdcA串联表达;二是在另一个相容质粒中表达.结果:大肠杆菌工程菌具备产异丁醇的能力,其中相关基因在一个质粒中串联表达的产量比其在两个相容质粒中共表达的产量高30倍,达到3g/L.结论:导入的酮酸合成途径与醇类生产途径结合,能使非生产菌株大肠杆菌生产异丁醇,并且单质粒表达代谢途径相关基因的效果优于双质粒表达.     

产异丁醇大肠杆菌工程菌的构建

摘要:酮酸脱羧酶是异丁醇生物合成的关键酶,而大肠杆菌中没有此基因.将乳脂乳球菌NIZO B1157的2-酮酸脱羧酶基因kdcA克隆到非生产菌株大肠杆菌中,同时串联表达与前体物质酮酸相关的alsS、ilvC和ilvD基因,构建了串联表达质粒pSTV29-alsS-ilvC-ilvD-kdcA.大肠杆茵工程茵成功表达了4个基因,并能利用葡萄糖发酵产异丁醇,发酵24h后最高产量为3 g/L.     

短芽孢杆菌a-乙酰乳酸脱羧酶基因在大肠杆菌中的克隆和表达

用PCR方法扩增短芽孢杆菌α—乙酰乳酸脱羧酶基因,引入原核表达载体pBV220中,得到重组质粒pALDl。pALDl中的ALDC基因在大肠杆菌中高效表达,每毫升发酵液产酶80单位以下,比原始菌株提高200余倍。SDS-PAGE蛋白质分析表明,大肠杆菌DH5α(pALDl)表达的ALDC占细胞总蛋白量的40％以上。研究了重组质粒稳定性,大肠杆菌DH5α(PALDl)和HB101(pALDl)分别在无选择压力下30℃连续培养50代以上,41℃诱导不同时间,DH5α(pALDl)在热诱导5h后,未发现质粒不稳定性,HB101(pALDl)在热诱导3h后,质粒基本稳定,但热诱导5h后,丢失质粒的细胞约占70％。     

α-乙酰乳酸脱羧酶在大肠杆菌中的克隆表达

根据已知的α-乙酰乳酸脱羧酶（ALDC）的基因序列,通过PCR获得了枯草芽孢杆菌168的ALDC基因,将该基因克隆到克隆载体pUC18和表达载体pQE60中,构建了pUC18-ALDC和pQE60-ALDC,经DNA测序证明序列正确。重组大肠杆菌DH5a／pQE60-ALDC经IPTG诱导能够高表达ALDC。对该酶进行了活力测定,结果显示工程菌产酶活力为0．11U／mL。     

基因敲除提高大肠杆菌工程菌生产异丁醇产量的研究

探究pflB、frdAB、fnr和AdhE四基因缺失突变株对大肠杆菌工程菌发酵生产异丁醇的影响。运用Red重组系统敲除大肠杆菌BW25113的pflB、frdAB、fnr和AdhE基因,构建pflB、frdAB、fnr和AdhE四基因缺失突变株E.coliBW25113H,结合本实验室已经构建的表达质粒pSTV29-alsS-ilvC-ilvD-kdcA,并检测该工程菌在1L发酵罐的发酵过程中的生物量、突变菌株的稳定性、异丁醇产量及有机酸含量的变化情况。成功获得pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H。发酵结果表明,该工程菌能以较长时间,较高比生长速率保持对数生长期,其稳定性较好,异丁醇产量增加了40%。成功构建pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H,结合非自身发酵途径使异丁醇的产量由3 g/L提升至4.2 g/L。     

大肠杆菌DH5α菌株感受态制备及转化率变化研究

通过氯化钙法制备大肠杆菌DH5α菌株感受态,讨论了不同保存温度和保存时间对感受态转化率的影响。结果表明,在4℃下保存,8h达到最高转化率;在-20℃和-70℃下保存,均为48h达到最高转化率。通过氯化钙法制备的DH5α菌株感受态细胞,在-20℃条件下简单保存,20d内完全可以满足一般转化研究的要求,不需要复杂的甘油、液氮处理及超低温要求。     

基于大肠杆菌受体菌DH5α △asd缺失株的构建及作为基因转化、表达操作系统的评估

基于大肠杆菌(E.coli)染色体上asd基因的已知序列,利用λ噬菌体的Red同源重组系统一步法构建E.coliDH5α的asd基因缺失突变株DH5α△asd::cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20介导二次同源重组,以去除上述缺失突变株中氯霉素抗性筛选基因。结合PCR扩增和测序结果,证明DH5α△asd缺失突变株的正确构建。该缺失突变株失去了在普通LB培养基上生长的能力,只有添加DAP或导入表达asd基因的质粒(asd基因互补试验)才能在LB培养基上生长,与原型DH5α比较,其生长速度和生长对数期、接受不同拷贝数质粒的转化效率几乎相一致。基于该缺失突变株构建出以asd营养基因为标志的大肠杆菌染色体-质粒平衡致死系统。体外培养连续传代50代次,pnirBMisL-fedF-asd质粒不丢失,并功能性表达F18大肠杆菌黏附素FedF。     

大肠杆菌astE、rph基因敲除突变株的构建

目的:分别构建大肠杆菌astE、rph基因敲除突变株,并检测其异丁醇耐受性的变化。方法:利用Red重组系统分别敲除大肠杆菌的astE和rph基因,并对所获得的突变株进行异丁醇耐受性相关实验研究。结果:成功构建了astE基因缺失突变株△astE和rph基因缺失突变株Δrph,发现两种突变株的异丁醇耐受性均有所提高。结论:通过缺陷菌株的构建,为未来进一步代谢改造生产异丁醇和研究异丁醇耐受机制奠定了基础。     

在大肠杆菌中表达酮酸脱羧酶产异戊醇

酮酸脱羧酶作为异戊醇生物合成的关键酶,不存在于大肠杆菌中。以乳酸乳球菌的基因组DNA为模板,经过PCR扩增得到酮酸脱羧酶基因kivD(rbs),插入到大肠杆菌高效表达载体pET-28a(+)上形成pET-kivD(rbs),重组质粒热击转化进大肠杆菌BL21(DE3)中,其成功表达了酮酸脱羧酶。对发酵产物进行分析,检测到了微量的目标产物—异戊醇。     

An evolutionary strategy for isobutanol production strain development in Escherichia coli

Higher alcohols such as isobutanol possess several physical characteristics that make them attractive as biofuels such as higher energy densities and infrastructure compatibility. Here we have developed a rapid evolutionary strategy for isolating strains of Escherichia coli that effectively produce isobutanol from glucose utilizing random mutagenesis and a growth selection scheme. By selecting for mutants with the ability to grow in the presence of the valine analog norvaline, we obtained E. coli NV3; a strain with improved 24-h isobutanol production (8.0 g/L) in comparison with a productivity of 5.3 g/L isobutanol obtained with the parental wild type strain. Genomic sequencing of NV3 identified the insertion of a stop codon in the C-terminus of the RNA polymerase σ^s-factor, RpoS. Upon repair of this inhibitory mutation (strain NV3r1), a final isobutanol titer of 21.2 g/L isobutanol was achieved in 99 h with a yield of 0.31 g isobutanol/g glucose or 76% of theoretical maximum. Furthermore, a mutation in ldhA, encoding d-lactate dehydrogenase, was identified in NV3; however, repair of LdhA in NV3r1 had no affect on LdhA activity detected from cell extracts or on isobutanol productivity. Further study of NV3r1 may identify novel genotypes that confer improved isobutanol production.     

猪源大肠杆菌(ETEC、STEC、AEEC)毒力基因及其与O抗原型的关系

[目的]揭示从我国部分地区仔猪腹泻或水肿病病猪体内分离到的300个大肠杆菌分离株所属病原型(pathotype)、毒力基因及其与O血清型的关系.[方法]O血清型采用常规的凝集试验进行测定,毒力基因采用PCR方法检测.[结果]通过对这300个分离株的O血清型及其毒素、紧密素和黏附素基因进行鉴定,结果显示除50株未定型、17株自凝外,测定出233个分离株的血清型,这些分离株覆盖了45个血清型,其中以0149、0107、0139、093和091为主,共133株,占定型菌株的57.1%;拥有est Ⅰ、estⅡ、elt、stx2e和eae A基因的菌株分别为102(34.0%)、190(63.3%)、81(27.0%)、57(19.0%)和54(18.0%)株;分离株中有51株K88基因阳性(其中菌毛表达率为100%),75株F18基因阳性(其中菌毛表达率为50.7%),在K88菌株中,0149血清型与est Ⅰ或estⅡ elt密切相关,在F18菌株中,0107血清型与est Ⅰ或estⅡ、0139血清型与stx2e紧密相关.依其毒力特征可将这些分离株分为以下6种类型:ETEC、STEC、AEEC、ETEC/STEC、AEEC/ETEC和AEEC/ETEC/STEC,分别拥有190、24、36、32、17和1个菌株,占分离株的63.3%、8.0%、12.0%、10.7%、5.7%和0.3%.通过分析这些分离株的O血清型、毒素类型和黏附素型之间的相关性:猪源ETEC以0149、0107、093和098等血清型为主,0149:K88菌株主要与estⅡ或estⅡ elt肠毒素相关,0107:F18菌株主要与estⅡ相关,093和098血清型菌株主要与estⅡ肠毒素相关;STEC菌株以0139:F18血清型为主,拥有stx2e;AEEC菌株拥有紧密素,无明显优势血清型;ETEC/STEC菌株以0107:F18和0116:F18血清型为主,主要与est Ⅰ stx2e或estⅡ stx2e密切相关,ETEC/AEEC菌株以091和0107血清型为主,全部拥有肠毒素est Ⅰ和紧密素基因.[结论]我国至少存在6种病原型的猪肠道致病性大肠杆菌,其中ETEC为我国部分地区猪大肠杆菌病的主要病原,同时其病原型日益复杂.     

以蔗糖为底物利用重组大肠杆菌合成甘露醇

【目的】异型发酵乳酸菌可利用胞内产生的甘露醇脱氢酶将果糖高效转化为甘露醇,但果糖作为底物相对昂贵,不利于工业化生产。为了降低生产成本,必须选择廉价的底物。蔗糖相对便宜,并且大量存在于自然界中,能够被重组大肠杆菌利用产生甘露醇。蔗糖水解酶(Sucrose hydrolase)和甘露醇脱氢酶(Mannitol dehydrogenase)是发酵生产甘露醇中催化蔗糖转化成甘露醇的关键酶,构建蔗糖水解酶和甘露醇脱氢酶共表达菌株并进行相关研究是本文的主旨。【方法】利用PCR方法分别从植物乳杆菌(Lactobacillus plantarum)和布氏乳杆菌(Lactobacillus buchneri)基因组DNA中获得sac A和mdh基因,得到大小分别为1 502 bp和1 032 bp的目的基因,经序列分析后将其连接到表达载体p ET-28a(+)上,得到重组表达载体p ET28a-sac A-mdh。将重组质粒转化到大肠杆菌BL21(DE3)中,并用SDS-PAGE分析目的蛋白的表达情况并测定其酶活。【结果】SDS-PAGE显示表达蛋白的大小亚基分子量分别为55.1 k D和37.8 k D,与预期分子量一致,实现sac A和mdh基因的表达。蔗糖水解酶和甘露醇脱氢酶酶活分别为25.78 U/m L和14.56 U/m L。对重组菌株BL21(DE3)/p ET28a-sac A-mdh进行发酵条件优化,甘露醇质量浓度达到45.19 g/L,总糖转化率为37.66%。【结论】与乳酸菌利用蔗糖发酵生产甘露醇相比,产量提高了6倍,且具有发酵周期短、稳定性高等优点,菌株的成功构建为甘露醇工业化生产奠定了基础。     

多基因在大肠杆菌中的共表达策略

多基因共表达在多领域有重要的应用价值,大肠杆菌共表达系统包括多顺反子系统和双质粒系统,两者各有优缺点。多顺反子系统不需外界2种抗生素的同时存在,但操作较为复杂。通常认为,具有相同复制子的质粒是不相容的,但近来的实验表明,在双抗生素的选择压力下,不相容的双质粒系统也能稳定传代,且操作简单,周期较短。双质粒系统已经应用于生产、医学等各个领域。     

Global proteomic profiling of native outer membrane vesicles derived from Escherichia coli

Gram-negative bacteria constitutively secrete native outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress in this area has revealed that OMVs are essential for bacterial survival and pathogenesis, the mechanism of vesicle formation and the biological roles of OMVs have not been clearly defined. Using a proteomics approach, we identified 141 protein components of Escherichia coli-derived native OMVs with high confidence; two separate analyses yielded identifications of 104 and 117 proteins, respectively, with 80 proteins overlapping between the two trials. In the group of identified proteins, the outer membrane proteins were highly enriched, whereas inner membrane proteins were lacking, suggesting that a specific sorting mechanism for vesicular proteins exists. We also identified proteins involved in vesicle formation, the removal of toxic compounds and attacking phage, and the elimination of competing organisms, as well as those involved in facilitating the transfer of genetic material and protein to other bacteria, targeting host cells, and modulating host immune responses. This study provides a global view of native bacterial OMVs. This information will help us not only to elucidate the biogenesis and functions of OMV from nonpathogenic and pathogenic bacteria but also to develop vaccines and antibiotics effective against pathogenic strains.     

Carboxymethylhydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli

5-Carboxymethyl-2-hydroxymuconic semialdehyde dehydrogenase in the 4-hydroxyphenylacetate meta-cleavage pathway has been purified to 96% homogeneity. The native enzyme, which appears to be a tetramer, has an apparent molecular weight of 210000. The purified enzyme shows a narrow pH optimum at pH 7.8 and does not require ions for its catalytic activity. Under standard assay conditions the enzyme acts preferentially with NAD but reduces NADP at 11% of the rate observed for NAD, primarily because of a difference in K_m. Apparent K_m values are 6.4 μM for 5-carboxymethyl-2-hydroxymuconic semialdehyde and 52.2 μM for NAD.     

过表达氨基葡萄糖脱氨酶对大肠杆菌氨基葡萄糖合成及中心碳代谢的影响

考察过表达氨基葡萄糖脱氨酶对氨基葡萄糖合成及大肠杆菌（Escherichia coli）中心碳代谢的影响。实验结果表明：过表达氨基葡萄糖脱氨酶使得在36 g/L葡萄糖,pH为9.0的发酵条件下,发酵24 h后,重组菌发酵液中氨基葡萄糖、丙酮酸和乙酸的量分别是对照菌Rosetta的2.1、1.48和1.74倍;而乳酸的量为2.53 g/L,对照菌Rosetta发酵液中的乳酸含量未检测到,重组菌发酵液中柠檬酸及α-酮戊二酸的含量分别是Rosetta的2.99和2.73倍。     

代谢工程改造大肠杆菌合成D-1,2,4-丁三醇

【目的】D-1,2,4-丁三醇是一种四碳的多元醇,在军事和医药领域具有广泛的应用。为实现生物法一步转化生产D-1,2,4-丁三醇,对Escherichia coli W3100的木糖代谢途径进行改造。【方法】将来源于柄杆菌的D-木糖脱氢酶基因xylB和恶臭假单胞菌的苯甲酰甲酸脱羧酶基因mdlC克隆至E.coli W3100,得到重组菌E.coli(pEtac-mdlC-tac-xylB)。在此基础上对重组菌代谢木糖合成D-1,2,4-丁三醇的能力进行考察。【结果】在30°C下,以30 g/L D-木糖为底物,重组菌E.coli(pEtac-mdlC-tac-xylB)的D-1,2,4-丁三醇产量达到了0.9 g/L,摩尔转化率为4%。【结论】实现了D-1,2,4-丁三醇的一步法发酵生产,为国内开展相关研究奠定了坚实的基础。     

Escherichia coli dinJ-yafQ genes act as a toxin-antitoxin module

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.     

Effect of finishing diets on Escherichia coli populations and prevalence of enterohaemorrhagic E. coli virulence genes in cattle faeces

AIM: To determine the effect of different carbohydrate-based finishing diets on fermentation characteristics and the shedding of Escherichia coli and enterohaemorrhagic E. coli (EHEC) virulence genes in cattle faeces. METHODS AND RESULTS: The size of faecal E. coli populations and fermentation characteristics were ascertained in three experiments where cattle were maintained on a range of finishing diets including high grain, roughage, and roughage + molasses (50%) diets. Increased E. coli numbers, decreased pH and enhanced butyrate and lactate fermentation pathways were associated with grain diets, whereas roughage and roughage + molasses diets resulted in decreased concentrations of ehxA, eaeA and stx(1) genes, this trend remaining at lairage. In one experiment, faecal E. coli numbers were significantly lower in animals fed roughage and roughage + molasses, than animals fed grain (4.5, 5.2 and 6.3 mean log10 g(-1) digesta respectively). In a second experiment, faecal E. coli numbers were 2 log lower in the roughage and roughage + molasses diets compared with grain-fed animals prior to lairage (5.6, 5.5 and 7.9 mean log10 g(-1) digesta respectively) this difference increasing to 2.5 log at lairage. CONCLUSIONS: The type of dietary carbohydrate has a significant effect on E. coli numbers and concentration of EHEC virulence genes in faeces of cattle. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a better understanding of the impact finishing diet and commercial lairage management practices may have on the shedding of E. coli and EHEC virulence factors, thus reducing the risk of carcass contamination by EHEC.