丙酮酸脱羧酶基因和乙醇脱氢酶基因表达载体的构建

以运动发酵单胞菌(Zymomonas mobilis)的总DNA为模板,PCR扩增运动发酵单胞菌中的丙酮酸脱羧酶( Pyruvate decarboxylase,PDC)基因和乙醇脱氢酶Ⅱ(Alcohol dehydrogenaseⅡ,ADHⅡ)基因.将丙酮酸脱羧酶基因和含核糖体结合位点(RBS)的乙醇脱氢酶基因串联起来置于T7启动子控制下,构成多顺反子表达质粒pQR-PRA.经酶切和PCR验证,表达载体构建成功.将pQR-PRA转入大肠埃希菌(Escherichia coli)BL21中,转化子的定性检测表明有酶表达,并且初步测定了2种酶的表达量.     

运动发酵单胞菌乙醇脱氢酶基因的克隆及在大肠杆菌中的表达

采用PCR技术,以基因组DNA为模板克隆得到运动发酵单胞菌(Zyrnomonas mobilis)乙醇脱氢酶(alcohol dehydrogenaseⅡ)基因adhB,连接到表达载体pSE380上,得到重组质粒pSE-adhB。将此重组质粒转化到大肠杆菌菌株DH5α中,重组菌株经IPTG诱导后,在乙醛指示平板检测到乙醇脱氢酶活性。SDS-PAGE电泳结果显示出明显的40KD特异性蛋白质条带。重组菌株经诱导培养,每毫升发酵液酶活力为5u。     

产L-乳酸的运动发酵单胞菌代谢工程菌株的构建

以运动发酵单胞菌(Zymomonas mobilis)CP4基因组DNA为模板,采用PCR技术克隆得到其丙酮酸脱氢酶基因(pdc)同源下游p3片段,并连接到广谱宿主载体pBBR1MCS3-Ppdc-ldhL中构建了重组质粒pBBR1MCS3-Ppdc-ldhL-p3,将此重组质粒转化到受体菌Z.mobilis CP4中,分别以Ppdc和p3片段作为同源上游和下游片段,利用同源双交换重组技术将重组质粒中的ldhL基因置换了Z.mobilis染色体中的pdc基因,得到重组菌株Z.mobilis CP4(△pdc∷ldhL).测得重组菌株乳酸产量为10.8g/L,明显高于出发菌株,说明初步成功构建了产L-乳酸的运动发酵单胞菌代谢工程菌株.     

产乙醇运动发酵单胞菌的研究进展

运动发酵单胞菌作为天然生产乙醇的主要微生物之一,具有特殊的Entner Doudoroff途径和其他一些特殊的糖代谢和能量代谢方式,因此具有乙醇产率高和乙醇耐受力强的显著特点。通过简述运动发酵单胞菌的糖代谢和能量代谢、乙醇和高渗透压等耐性及其遗传改造三方面的研究进展,阐明其应用于燃料乙醇生产的巨大潜力     

运动发酵单孢菌ZM4 adh Ⅱ基因的克隆及在大肠杆菌TOP10中的表达

利用高保真聚合酶从运动发酵单胞菌中克隆出乙醇脱氢酶基因,加A后克隆到pGM-T载体,测序验证无误。经酶切、酶连到表达载体pUC-18。形成重组质粒pUC-18-adhⅡ,转化到大肠杆菌TOP10中,经定性定量分析乙醇脱氢酶酶基因在大肠杆菌中高效表达,成功构建出乙醇脱氢酶基因的表达载体。     

热休克蛋白增加大肠杆菌抗逆性和乙醇产量的研究

目的：研究热休克蛋白对增加大肠杆菌抗逆性和乙醇产量的影响。方法：运用基因工程技术,用大肠杆菌的Lac启动子、运动发酵单胞菌的丙酮酸脱羧酶基因（pdc）和乙醇脱氢酶基因（adhB）,构建可以在大肠杆菌中表达的Lac-AP操纵子。Lac-AP操纵子导入大肠杆菌,可使大肠杆菌发酵糖生产乙醇。再用来自超嗜热菌强烈火球菌（Pyrococcus furiosus）的小分子热休克蛋基因（sHsp）,构建在大肠杆菌中表达的Lac-APH操纵子。结果：成功地构建了耐高温产生乙醇的大肠杆菌LAPH和LAP,它们发酵后乙醇的产量分别为11.5g/L、7.9g/L,而对照菌LH的产量只有为0.5g/L。与对照菌LH相比,LAPH和LAP的产量分别提高了23倍和15.8倍。结果证明：与对照LAP相比,热休克蛋白使菌种LAPH的45℃温度耐受性提高15.75倍、50℃温度耐受性提高40.7倍,乙醇的产量增高高达4.74倍。结论：研究表明,小分子热休克蛋白的表达,可使细菌在致死温度下的存活率显著提高,耐受温度明显增强,乙醇产量显著提高。     

氯化镁对运动发酵单胞菌乙醇产率及耗糖的影响

研究了不同氯化镁浓度的合成培养基和复合培养基中,运动发酵单胞菌的乙醇产量及培养基中的残糖量。结果表明,在复合培养基和合成培养基中,乙醇产量最高、残糖量最低的最佳氯化镁浓度分别为0.005mol/L和0.01mol/L。     

一株葡萄糖和木糖共发酵高效产乙醇重组运动发酵单胞菌的性能评价

木糖是木质纤维素原料水解液中的第二大组分,木糖和葡萄糖的充分利用是有经济性地生产纤维素乙醇的关键。通过基因克隆手段构建了一株可以高效利用木糖产乙醇的重组运动发酵单胞菌Zymomonas mobilis TSH01,并进行了利用单糖溶液、混合糖溶液及玉米秸秆水解液发酵产乙醇效率的研究。结果表明,利用单一葡萄糖或单一木糖溶液发酵时,当糖浓度为8%、发酵72 h后,糖利用率分别为100%和98.9%,乙醇代谢收率分别为87.8%和78.3%;利用8%葡萄糖和8%木糖的混合溶液发酵时,72 h后,葡萄糖和木糖的利用率分别为98.5%和97.4%,乙醇代谢收率为94.9%。利用含3.2%葡萄糖和3.5%木糖的玉米秸秆水解液发酵72 h后,葡萄糖和木糖的利用率分别为100%和92.3%,乙醇代谢收率为91.5%。此外,磷酸二氢钾对发酵过程中木糖利用率以及乙醇收率的提高有明显促进作用。     

代谢工程改造运动发酵单胞菌用于提高乙醇产量

目的:采用可以在运动发酵单胞菌中表达的操纵子构建重组运动发酵单胞菌,用于提高该细菌对高温高糖的耐受性和提高乙醇产量.方法:用外来的YfdZ、MetB和Hsp构建的多顺反子质粒,转化运动发酵单胞菌而使其获得新的代谢途径.在玉米水解液中,验证了该多顺反子质粒对运动发酵单胞菌产生乙醇的影响.结果:与对照菌相比,在37℃和糖浓度为28%的培养条件下,该基因工程菌的乙醇产量提高到183.2%.在37℃,糖浓度为28%并添加氮源的条件下,该基因工程菌的乙醇产量提高到148.0%.结论:YfdZ、MetB及Hsp三种基因的共同作用能显著提高运动发酵单胞菌的乙醇产量和发酵温度.     

Establishment of a Highly Efficient Ammonia Secreting Mutant of Synechococcus sp. PCC 7942 and the Glutamine Synthetase Activity,Photosynthesis and Growth in Its Immobilized Cells

A recombinate plasmid pDC-ATGS was constructed, which contained the antisense fragment of glnA gene from Anabaena sp. PCC 7120 and transformed the unicellular cyanobactefium Synechococcus sp. PCC 7942. The foreign DNA was inserted into the site of glnA locus of the chromosome through the homologous recombination. By using neomyisin, a highly efficient ammonia secretion mutant was selected. After immobilized, the cells of the mutant within polyurethane (PU) foams, glutamine synthetase (GS) and NIt4+ secretory activity of GS, and its growth and photosynthesis were measured. It was shown that NH4+ secretion of the immobilized mutant was enhanced 156 folds which was much higher than that of free-living cells of the wild type. The activity of GS was decreased by 73.6%. Growth of the mutant was the same as that of the wild type. The activity of photosystem Ⅱ in the immobilized mutant cells increased by 44% with 77 K fluorescence spectrum measurement.     

Structure and Function of the Catalytic Domain of the Dihydrolipoyl Acetyltransferase Component in Escherichia coli Pyruvate Dehydrogenase Complex

The Escherichia coli pyruvate dehydrogenase complex (PDHc) catalyzing conversion of pyruvate to acetyl-CoA comprises three components: E1p, E2p, and E3. The E2p is the five-domain core component, consisting of three tandem lipoyl domains (LDs), a peripheral subunit binding domain (PSBD), and a catalytic domain (E2pCD). Herein are reported the following. 1) The x-ray structure of E2pCD revealed both intra- and intertrimer interactions, similar to those reported for other E2pCDs. 2) Reconstitution of recombinant LD and E2pCD with E1p and E3p into PDHc could maintain at least 6.4% activity (NADH production), confirming the functional competence of the E2pCD and active center coupling among E1p, LD, E2pCD, and E3 even in the absence of PSBD and of a covalent link between domains within E2p. 3) Direct acetyl transfer between LD and coenzyme A catalyzed by E2pCD was observed with a rate constant of 199 s⁻¹, comparable with the rate of NADH production in the PDHc reaction. Hence, neither reductive acetylation of E2p nor acetyl transfer within E2p is rate-limiting. 4) An unprecedented finding is that although no interaction could be detected between E1p and E2pCD by itself, a domain-induced interaction was identified on E1p active centers upon assembly with E2p and C-terminally truncated E2p proteins by hydrogen/deuterium exchange mass spectrometry. The inclusion of each additional domain of E2p strengthened the interaction with E1p, and the interaction was strongest with intact E2p. E2p domain-induced changes at the E1p active site were also manifested by the appearance of a circular dichroism band characteristic of the canonical 4′-aminopyrimidine tautomer of bound thiamin diphosphate (AP).     

Novel Binding Motif and New Flexibility Revealed by Structural Analyses of a Pyruvate Dehydrogenase-Dihydrolipoyl Acetyltransferase Subcomplex from the Escherichia coli Pyruvate Dehydrogenase Multienzyme Complex

The Escherichia coli pyruvate dehydrogenase multienzyme complex contains multiple copies of three enzymatic components, E1p, E2p, and E3, that sequentially carry out distinct steps in the overall reaction converting pyruvate to acetyl-CoA. Efficient functioning requires the enzymatic components to assemble into a large complex, the integrity of which is maintained by tethering of the displaced, peripheral E1p and E3 components to the E2p core through non-covalent binding. We here report the crystal structure of a subcomplex between E1p and an E2p didomain containing a hybrid lipoyl domain along with the peripheral subunit-binding domain responsible for tethering to the core. In the structure, a region at the N terminus of each subunit in the E1p homodimer previously unseen due to crystallographic disorder was observed, revealing a new folding motif involved in E1p-E2p didomain interactions, and an additional, unexpected, flexibility was discovered in the E1p-E2p didomain subcomplex, both of which probably have consequences in the overall multienzyme complex assembly. This represents the first structure of an E1p-E2p didomain subcomplex involving a homodimeric E1p, and the results may be applicable to a large range of complexes with homodimeric E1 components. Results of HD exchange mass spectrometric experiments using the intact, wild type 3-lipoyl E2p and E1p are consistent with the crystallographic data obtained from the E1p-E2p didomain subcomplex as well as with other biochemical and NMR data reported from our groups, confirming that our findings are applicable to the entire E1p-E2p assembly.     

Comparative evaluation of the Ridascreen Verotoxin enzyme immunoassay for detection of Shiga-toxin producing strains of Escherichia coli (STEC) from food and other sources

AIMS: To evaluate the suitability of the commercially distributed Ridascreen Verotoxin enzyme immunoassay (EIA) for detection of known genetic types of the Vero (Shiga) toxins 1 (Stx1) and 2 (Stx2) families and to determine its relative sensitivity and specificity. METHODS AND RESULTS: The Ridascreen-EIA was compared with the Vero cell assay, a P(1)-glycoprotein receptor EIA and with stx gene-specific PCs for detection of Stx with 43 Shiga toxin-producing strains of Escherichia coli (STEC) reference strains and with 241 test strains. The Ridascreen-EIA detects strains producing Stx1 and variants Stx1c and Stx1d, as well as Stx2 and variants Stx2d1, Stx2d2, Stx2e, Stx2d, Stx2-O118 (Stx2d-ount), Stx2-NV206, Stx2f and Stx2g. The assay showed a relative sensitivity of 95.7% and a relative specificity of 98.7%. Some of the Stx2-O118-, Stx2e- and Stx2g-producing STEC were not detected with the Ridascreen-EIA probably because of low amount of toxin produced by these strains. CONCLUSIONS: The Ridascreen-EIA is able to detect all known types of Stx and is applicable for routine screening of bacterial isolates owing to its high specificity. It is less applicable for testing samples where low amounts of Stx are expected, such as mixed cultures and certain Stx2 variants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study presents a first comprehensive evaluation of the Ridascreen-EIA, a rapid standardized STEC screening test for routine diagnostic laboratories. Data are presented on the type of the spectrum of Stx that are detected with this immunoassay and its advantages and limits for practical use.     

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes

Aims:  To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains.
Methods and Results:  GeneDiscs for detection of genes encoding Shiga toxins ( stx ), intimins ( eae ), E. coli O157 ( rfbE _O157) and H7 ( fliC _H7) antigens as well as genes specific for EHEC O26 ( wzx _O26), O103 ( wzx _O103), O111 ( wbd1 _O111), O145 ( ihp1 _O145) and O157 ( ihp1 _O157) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae , except stx _2f and eae -rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1 _O157 gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates.
Conclusions:  The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria.
Significance and Impact of the Study:  The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.     

PhoE protein pores in the outer membrane of Escherichia coli K-12 not only have a preference for Pi and Pi-containing solutes but are general anion-preferring channels

Previous studies on the question of whether the PhoE protein pore has a preference for P_i and P_i-containing solutes only or whether it constitutes a general anion-preferring channel, have not given an unequivocal answer either because the presence of the phosphate binding protein was not ascertained or because only arsenate was tested as a non P_i-containing control solute. Permeability properties of PhoE, OmpF and OmpC protein pores for negatively charged solutes were measured in vivo in the presence of phosphate-binding protein. It appeared that the PhoE protein pore is the most efficient channel for the three tested solutes phosphate, succinate and sulphate. Conditions were established to measure the frequency of ethyl methane sulphonate induced mutations as a function of the presence of pore proteins. These results indicate that PhoE protein also forms the most efficient channel for ethyl methane sulphonate. We conclude that the preference of the PhoE protein pore is not restricted to P_i and P_i-containing solutes but also concerns several other negatively charged solutes.