大肠杆菌K-12转醛醇酶基因talB的克隆及在运动发酵单胞菌CP4中的表达

研究了E.coliK-12转醛醇酶基因(talB)在自身启动子和在Z.mobilisCP4eno基因启动子的启动下在E.coliDH5α和Z.mobilisCP4中的表达情况。首先克隆了E.coliK-12talB基因,并连接到穿梭载体pZB1上构建成pZB1-talB;然后利用PCR重叠延伸技术将E.coliK-12talB自身的启动子换成Z.mobilisCP4eno的启动子,构建得到pZB1-Peno-talB。将这两个质粒分别转化E.coliDH5α和Z.mobilisCP4。对转化子粗酶液进行的转醛醇酶酶活力测定结果表明,E.coli talB自身启动子和Z.mobilis eno启动子能以基本相同的效率启动talB基因在E.coli和Z.mobilis中的表达。     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

Overexpression of extracellular sucrase (SacC) of Zymomonas mobilis in Escherichia coli

Abstract The extracellular sucrase (SacC) gene of Zymomonas mobilis was overexpressed in Escherichia coli BL21 using the T7 polymerase expression system. A low cell density induction method was designed to have maximum expression, and the conditions (IPTG concentration, ampicillin addition) were optimised to overexpress to the level of more than 60% of the total cellular protein representing SacC protein.     

Disruption of the Zymomonas mobilis extracellular sucrase gene (sacC) improves levan production

AIMS: Disruption of the extracellular Zymomonas mobilis sucrase gene (sacC) to improve levan production. METHODS AND RESULTS: A PCR-amplified tetracycline resistance cassette was inserted within the cloned sacC gene in pZS2811. The recombinant construct was transferred to Z. mobilis by electroporation. The Z. mobilis sacC gene, encoding an efficient extracellular sucrase, was inactivated. A sacC defective mutant of Z. mobilis, which resulted from homologous recombination, was selected and the sacC gene disruption was confirmed by PCR. Fermentation trials with this mutant were conducted, and levansucrase activity and levan production were measured. In sucrose medium, the sacC mutant strain produced threefold higher levansucrase (SacB) than the parent strain. This resulted in higher levels of levan production, whilst ethanol production was considerably decreased. CONCLUSIONS: Zymomonas mobilis sacC gene encoding an extracellular sucrase was inactivated by gene disruption. This sacC mutant strain produced higher level of levan in sucrose medium because of the improved levansucrase (SacB) than the parent strain. SIGNIFICANCE AND IMPACT OF THE STUDY: The Z. mobilis CT2, sacC mutant produces high level of levansucrase (SacB) and can be used for the production of levan.     

重组运动发酵单胞菌的构建及木糖利用特性研究

将大肠杆菌(Escherichia coli)木糖代谢的关键酶基因.引入到运动发酵单胞菌中,获得能利用木糖发酵生产乙醇的重组工程菌株PZM.混合糖发酵过程中,重组菌利用葡萄糖和木糖生成乙醇的效率分别达到理论值的81.2%和63.1%.     

Over-expression of xylulokinase in a xylose-metabolising recombinant strain of Zymomonas mobilis

The broad host range vector pBBR1MCS-2 has been evaluated as an expression vector for Zymomonas mobilis. The transformation efficiency of this vector was 2 x 10(3) CFU per mug of DNA in a recombinant strain of Z. mobilis ZM4/AcR containing the plasmid pZB5. Stable replication for this expression vector was demonstrated for 50 generations. This vector was used to study xylose metabolism in acetate resistant Z. mobilis ZM4/AcR (pZB5) by over-expression of xylulokinase (XK), as previous studies had suggested that XK could be the rate-limiting enzyme for such strains. Based on the above vector, a recombinant plasmid pJX1 harboring xylB (expressing XK) under control of a native Z. mobilis promotor Ppdc was constructed. When this plasmid was introduced into ZM4/AcR (pZB5) a 3-fold higher XK expression was found compared to the control strain. However, fermentation studies with ZM4/AcR (pZB5, pJX1) on xylose medium did not result in any increase in rate of growth or xylose metabolism, suggesting that XK expression was not rate-limiting for ZM4/AcR (pZB5) and related strains.     

Cloning and expression in Escherichia coli of the dnaK gene of Zymomonas mobilis.

The DnaK protein of Zymomonas mobilis (DnaKz) was identified and found to be 80% identical to the DnaK protein of Escherichia coli on the basis of the sequence of the N-terminal 21 amino acids. The dnaKz gene was cloned and found to be expressed in a thermosensitive dnaK mutant of Escherichia coli. Expression of the foreign gene restored a thermoresistant phenotype but failed to modulate the heat shock response in E. coli.     

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD’s SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD’s SP were also higher than the other two strains containing PhoC or ZMO0331’s SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.     

Towards the characterization of squalene synthase activity in extracts of Zymomonas mobilis

Abstract Extracts of Zymomonas mobilis in the presence of NADPH converted tritium-labelled farnesyl diphosphate (FPP) into squalene, resulting from the activity of squalene synthase, as well as diploptene and diplopterol, derived from further squalene cyclisation. An unidentified isoprenoid representing up to 70% of the conversion products of FPP and different from presqualene alcohol was also formed, even in the absence of NADPH. Addition of squalestatin 1, an inhibitor of squalene synthase, blocked biosynthesis from FPP of the three former triterpenes, in accordance with the role of squalene synthase in their formation, as well as that of the unknown compound.     

Localisation of the glucose-fructose oxidoreductase in wild type and overproducing strains of Zymomonas mobilis

Abstract The enzyme glucose-fructose oxidoreductase (GFOR) from the Gram-negative ethanologenic bacterium Zymomonas mobilis was purified to homogeneity and was shown to be a tetrameric protein with a subunit size of M _r 42 500. Using immunogold-labelling in combination with electron microscopy, ultrathin sections of Z. mobilis wild type cells showed that the enzyme GFOR is located in the periplasm off the bacterial cells. Z. mobilis strains which carried the cloned gfo gene on plasmid pSUP104, had 5–6-fold increased GFOR enzyme activities. Moreover, these cells accumulated large amounts of a presumable unprocessed pre-GFOR protein ( M _r 48 000).     

Cloning of the Acetobacter xylinum cellulase gene and its expression in Escherichia coli and Zymomonas mobilis

A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201–3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.     

A new biosensor for specific determination of sucrose using an oxidoreductase of Zymomonas mobilis and invertase

A new biosensor for specific determination of sucrose was developed using an oxidoreductase of Zymomonas mobilis and invertase. Cells of Z. mobilis were permeabilized with toluene in order to utilize the enzymes of glucose-fructose oxidoreductase and gluconolactonase inside the intact cells. Permeabilized cells and invertase were coimmobilized in a gelatin membrane, and a whole cell enzyme electrode was constructed by fixing the membrane on a pH electrode. The production of hydrogen ion was detected using the biosensor-connected microcomputer, and the concentration of sucrose was determined by using both the initial rate and the steady-state methods. Optimum conditions for biosensor response were pH 6.2 and temperature 35 degrees C. The effect of interfering compounds on the electrode response was investigated, and the interference by various sugars was eliminated by determining sucrose concentration using the steady-state method. The biosensor developed is simple and reproducible, and the calibration curve for sucrose is linear up to 70 g/L.     

Isolation of the dxr gene of Zymomonas mobilis and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase

The gene encoding the second enzyme of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway for isopentenyl diphosphate biosynthesis, 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, was cloned and sequenced from Zymomonas mobilis. The deduced amino acid sequence showed the highest identity (48.2%) to the DXP reductoisomerase of Escherichia coli. Biochemical characterization of the purified DXP reductoisomerase showed a strict dependence of the enzyme on NADPH and divalent cations (Mn(2+), Co(2+) or Mg(2+)). The enzyme is a dimer with a molecular mass of 39 kDa per subunit and has a specific activity of 19.5 U mg protein(-1). Catalysis of the intramolecular rearrangement and reduction of DXP to MEP is competitively inhibited by the antibiotic fosmidomycin with a K(i) of 0.6 microM.     

一种新的DyP-type过氧化物酶在大肠杆菌中的重组表达、纯化及鉴定

染料脱色过氧化物酶(DyP-type过氧化物酶)是含有亚铁血红素,能降解各种有毒染料的一类蛋白.为了研究运动发酵单胞菌Zymomonas mobilis ZM4 (ATCC 31821)中一种新的DyP-type过氧化物酶的特点和功能,以Z.mobilis基因组DNA为模板,通过PCR扩增目的基因,克隆到大肠杆菌表达载体pET-21b(+)中.通过ZmDyP与其他DyP-type过氧化物酶的比对,发现它们存在着共同保守氨基酸D149、R239、T254、F256和GXXDG结构基序,说明ZmDyP是Dyp-type过氧化物酶家族的一个新成员.经IPTG诱导大肠杆菌中pET21 b(+)-ZmDyP表达,并将表达的酶进行金属螯合层析纯化.SDS-PAGE分析表明,纯酶分子量为36 kDa,而活性染色显示分子量为108 kDa,表明该酶在活性状态下可能是一个三聚体.光谱扫描显示ZmDyP有一个典型的亚铁血红素吸收峰,说明它是含有亚铁血红素的蛋白.对ZmDyP性质进行了研究,发现以2,2-二氨-双(3-乙基苯并噻唑-6-磺酸)ABTS为底物,ZmDyP表现出更高的转化效率.这些研究结果丰富了DyP-type 过氧化物酶家族信息,并且为ZmDyP的结构功能和反应机制研究奠定了基础.     

Cloning,sequencing and characterization of the alkaline phosphatase gene (phoD) from Zymomonas mobilis

The phoD gene encoding the membrane-bound alkaline phosphatase (ALPI) from Zymomonas mobilis CP4 was cloned and sequenced. Both the translated sequence and the properties of the recombinant enzyme were unusual. Z. mobilis ALPI was monomeric (_r 62926) and hydrolysed nucleotides more effectively than sugar phosphates. The translated sequence contained a single hydrophobic segment near the N-terminus which may serve as a membrane-anchor in Z. mobilis, although the recombinant enzyme was recovered in the cytoplasmic fraction of Escherichia coli. The predicted amino acid sequence for ALPI did not align well with other ALPs or other known genes. However, some similarity to E. coli ALP was noted in the metal-binding and phosphate-binding regions. Two other regions were identified with similarity to the active sites of pyruvate kinase and mammalian 5′-nucleotide phosphodiesterase (also membrane-bound), respectively. It is likely that Z. mobilis phoD represents a new class of alkaline phosphatase genes which has not been described previously.     

Cloning of the Zymomonas mobilis structural gene encoding alcohol dehydrogenase I (adhA): sequence comparison and expression in Escherichia coli.

Zymomonas mobilis ferments sugars to produce ethanol with two biochemically distinct isoenzymes of alcohol dehydrogenase. The adhA gene encoding alcohol dehydrogenase I has now been sequenced and compared with the adhB gene, which encodes the second isoenzyme. The deduced amino acid sequences for these gene products exhibited no apparent homology. Alcohol dehydrogenase I contained 337 amino acids, with a subunit molecular weight of 36,096. Based on comparisons of primary amino acid sequences, this enzyme belongs to the family of zinc alcohol dehydrogenases which have been described primarily in eucaryotes. Nearly all of the 22 strictly conserved amino acids in this group were also conserved in Z. mobilis alcohol dehydrogenase I. Alcohol dehydrogenase I is an abundant protein, although adhA lacked many of the features previously reported in four other highly expressed genes from Z. mobilis. Codon usage in adhA is not highly biased and includes many codons which were unused by pdc, adhB, gap, and pgk. The ribosomal binding region of adhA lacked the canonical Shine-Dalgarno sequence found in the other highly expressed genes from Z. mobilis. Although these features may facilitate the expression of high enzyme levels, they do not appear to be essential for the expression of Z. mobilis adhA.