Functional Expression of Bacterial Zymobacter palmae Pyruvate Decarboxylase Gene in Lactococcus lactis

A pyruvate decarboxylase (PDC) gene from bacterial Zymobacter palmae (Zymopdc) was cloned, characterized, and introduced into Lactococcus lactis via a shuttle vector pAK80 as part of a research strategy to develop an efficient ethanol-producing lactic acid bacteria (LAB). The expression levels of Zymopdc gene in the host, as measured by a colorimetric assay based on PDC catalyzed formation of (R)-phenylacetylcarbinol ((R)-PAC), appeared to be dependent on the strength of corresponding Gram-positive promoters. A constitutive, highly expressed promoter conferred the greatest PDC activity, and an acid-inducible promoter demonstrated acid-inducible expression. The metabolic production of ethanol and other products was examined in flask fermentations. More than eightfold increases in acetaldehyde concentrations were detected in two recombinant strains. However, no detectable differences for ethanol fermentation in these engineered strains were observed compared with that of the strain carrying lacZ reporter.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

巴斯德毕赤酵母表达系统研究进展

巴斯德毕赤酵母外源基因表达系统是近年来发展的一种优秀的真核表达系统 ,与传统的大肠杆菌和酿酒酵母表达体系相比 ,其具有的诸多优势使之研究价值及应用价值不断体现。综述了其在生物学特性、表达载体、基因整合、外源蛋白的分泌、诱导表达及发酵等方面的基础研究及新近研究进展。     

Characterization and Heterologous Gene Expression of a Novel Esterase from Lactobacillus casei CL96

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P_mxaF), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S·tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37°C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C₈), with K_m and k_cat values of 14 ± 1.08 μM and 1,245 ± 42.3 S⁻¹, respectively.     

结核分枝杆菌FtsZ的异源表达及酶学性质研究

作为细菌分裂所必需的微管蛋白类似物,丝状温度敏感蛋白Z(filamentous temperature-sensitive protein Z,FtsZ)被认为是一个具有潜力的药物作用新靶点。为了构建高纯度的FtsZ重组蛋白分离纯化体系,探讨其酶学性质,该研究利用大肠杆菌BL21异源表达结核分枝杆菌FtsZ重组蛋白,通过Ni亲和层析柱和G-50层析柱纯化目的蛋白,采用孔雀石绿法和90°光散射法测定FtsZ重组蛋白的GTP酶活和蛋白聚集。研究结果表明:成功获得具有生物学活性的结核分枝杆菌FtsZ重组蛋白,其相对分子质量约为49kD;该酶最适反应温度为37℃,最适pH为6.8,金属离子Mg~(2+)和K~+对FtsZ重组蛋白酶活具有促进作用,有机溶剂DMSO和TritonX-100的体积分数分别高于0.1%和0.005%时对酶活有显著抑制作用(P0.05)。此外,FtsZ重组蛋白在加入底物GTP诱导后,快速聚集。本实验利用基因工程技术成功获得具有生物活性的FtsZ重组蛋白,并明确了该蛋白质的酶学性质,为其进一步的研究和应用奠定了基础。     

Inactivation of the Glutamate Decarboxylase Gene in Lactococcus lactis subsp. cremoris

Lactococcus lactis subsp. lactis strains show glutamate decarboxylase activity, whereas L. lactis subsp. cremoris strains do not. The gadB gene encoding glutamate decarboxylase was detected in the L. lactis subsp. cremoris genome but was poorly expressed. Sequence analysis showed that the gene is inactivated by the frameshift mutation and encoded in a nonfunctional protein.     

罗中链霉菌中衣霉素基因簇的克隆及异源表达

衣霉素属于核苷类抗生素,具有抑制蛋白质N-糖基化的活性,是潜在的药物先导化合物.罗中链霉菌(Streptomyces luozzhongensis)TRM49605是一株产衣霉素的链霉菌属(Streptomyces)的新物种.本研究旨在探索TRM49605中衣霉素生物合成基因簇的生物学功能,为新型药物开发提供理论依据....     

小麦几丁质酶基因的异种表达及其功能鉴定

几丁质酶参与植物的发育及防卫反应,并与人类疾病发生有关.文章研究了小麦几丁质酶基因Wch2经根癌农杆菌介导的烟草瞬间表达和转基因拟南芥的稳定表达,Western杂交及酶活测定证实,瞬间表达的小麦几丁质酶分子量约30 kD,具有降解几丁质多聚物的功能;Wch2在转入拟南芥后表达量高,尖孢镰刀菌接种的鉴定表明,表达Wch2的转基因植株的抗病性显著高于表达绿色荧光蛋白的对照植株.这些结果说明Wch2的异种表达,可用于植物抗病基因工程,以增强植物的抗病性.     

Heterologous Expression and Functional Characterization of CAP18 from Oryctolagus cuniculus

Background:Antimicrobial peptides belong to the innate defence system of creatures. These peptides attach to the bacterial membrane in order to die microorganisms by penetrating them. Hence, biotechnology researchers pay more attention to produce antimicrobial peptides for use in various fields. The studies showed that rabbit tissue with inflammation and skin ulcers would be producing CAP18 peptide, which belongs to the cathelicidin group.Methods:In this study, the optimized sequence of the cap18 gene was placed into the pPICZAα plasmid after the alpha-factor signal and transformed into Pichia pastoris (X-33 strain). Purification of the recombinant peptide was done based on its histidine tail at C-terminal, and western blotting method was used to demonstrate the purification of rCAP18. The antibacterial activity of the purified and desalted rCAP18 was investigated at different concentrations against pathogenic bacteria.Results:The maximum expression level of rCAP18 (17.5 kDa) was seen 90 h after induction of alcohol oxidase I (AOX1) promoter with methanol. The concentration of rCAP18 was 33 mg/L after purification with Ni-NTA Sepharose column. The function of rCAP18 (4.3, 5.7, 7 µg/ml) was investigated against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Results showed that %CFU/cm² reached 28% after P. aeruginosa cells treatment with 7 μg/ml of rCAP18.Conclusion:This study presented the findings related to heterologous expression of cap18 gene, and evaluation of rCAP18 antibacterial effects. Our results showed that rCAP18 plays a significant role in inhibiting bacterial growth, especially Gram-negative bacteria.Key Words: Antibacterial activity, Cathelicidin, Recombinant CAP18, Heterologous expression      

Novel Type of ADP-Forming Acetyl Coenzyme A Synthetase in Hyperthermophilic Archaea: Heterologous Expression and Characterization of Isoenzymes from the Sulfate Reducer Archaeoglobus fulgidus and the Methanogen Methanococcus jannaschii

Acetyl coenzyme A (CoA) synthetase (ADP forming) (ACD) represents a novel enzyme of acetate formation and energy conservation (acetyl-CoA + ADP + P(i) right harpoon over left harpoon acetate + ATP + CoA) in Archaea and eukaryotic protists. The only characterized ACD in archaea, two isoenzymes from the hyperthermophile Pyrococcus furiosus, constitute 145-kDa heterotetramers (alpha(2), beta(2)). The coding genes for the alpha and beta subunits are located at different sites in the P. furiosus chromosome. Based on significant sequence similarity of the P. furiosus genes, five open reading frames (ORFs) encoding putative ACD were identified in the genome of the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus and one ORF was identified in the hyperthermophilic methanogen Methanococcus jannaschii. The ORFs constitute fusions of the homologous P. furiosus genes encoding the alpha and beta subunits. Two ORFs, AF1211 and AF1938, of A. fulgidus and ORF MJ0590 of M. jannaschii were cloned and functionally overexpressed in Escherichia coli. The purified recombinant proteins were characterized as distinctive isoenzymes of ACD with different substrate specificities. In contrast to the Pyrococcus ACD, the ACDs of Archaeoglobus and Methanococcus constitute homodimers of about 140 kDa composed of two identical 70-kDa subunits, which represent fusions of the homologous P. furiosus alpha and beta subunits in an alphabeta (AF1211 and MJ0590) or betaalpha (AF1938) orientation. The data indicate that A. fulgidus and M. jannaschii contains a novel type of ADP-forming acetyl-CoA synthetase in Archaea, in which the subunit polypeptides and their coding genes are fused.     

Cloning and Heterologous Expression of the Thioviridamide Biosynthesis Gene Cluster from Streptomyces olivoviridis

Thioviridamide is a unique peptide antibiotic containing five thioamide bonds from Streptomyces olivoviridis. Draft genome sequencing revealed a gene (the tvaA gene) encoding the thioviridamide precursor peptide. The thioviridamide biosynthesis gene cluster was identified by heterologous production of thioviridamide in Streptomyces lividans.     

Identification and Heterologous Expression of the Chaxamycin Biosynthesis Gene Cluster from Streptomyces leeuwenhoekii

Streptomyces leeuwenhoekii, isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate S. leeuwenhoekii and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in Streptomyces coelicolor A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing ΔcxmK mutant (cxmK encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.     

金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究

透明质酸酶能够将透明质酸聚糖降解成具有抗氧化等生物活性的低分子量寡糖.微生物来源透明质酸酶具有酶学性质多样和易于重组表达等特点,是开发透明质酸酶的研究热点.通过基因组测序获得一个潜在的金黄色葡萄球菌来源透明质酸裂解酶基因hylS,将其进行了大肠杆菌BL21(DE3)异源重组表达,并对重组酶进行了酶学特性和酶解产物抗氧化...     

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

The first step of amino acid degradation in lactococci is a transamination, which requires an α-keto acid as the amino group acceptor. We have previously shown that the level of available α-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding α-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce α-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added α-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added α-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding α-ketoglutarate to improve aroma development in cheese.     

Cloning, Heterologous Expression, and Functional Characterization of a Chitinase Gene, Lbchi32, from Limonium bicolor

In the present study, an endochitinase gene, Lbchi32, was cloned from Limonium bicolor. The cDNA sequence of Lbchi32 was 1,443 bp in length and encoded 319 amino acid residues. The DNA sequence of Lbchi32 was 2,512 bp in length and contained three exons and two introns. The Lbchi32 gene was inserted into a pPIC9 vector and transferred into Pichia pastoris strains GS115 and KM71 for heterologous expression. SDS-PAGE analyses indicated that LbCHI32 was expressed in both GS115 and KM71 and that it was secreted extracellularly. The optimal reaction conditions for LbCHI32 activity are 45°C, pH 5.0, and 5 mM Ba²⁺. The LbCHI32 enzyme can efficiently degrade chitin, chitin derivatives, and the cell walls of different pathogenic fungi, including phytopathogenic Rhizoctonia solani, Fusarium oxysporum, Sclerotinia sclerotiorum, Valsa sordida, Septoria tritici, and Phytophthora sojae. These findings suggest that Lbchi32 has potential use in the degradation of chitin and chitin derivatives.     

Hydrolysis of oligofructoses by the recombinant beta-fructofuranosidase from Bifidobacterium lactis

The ability of the beta-fructofuranosidase (EC 3.2.1.26) from Bifidobacterium lactis DSM 10140T to cleave a variety of fructooligosaccharides was characterised. We identified its gene on a cloned chromosomal DNA fragment by sequence similarity (69% identity) to the putative CscA protein encoded in the Bifidobacterium longum genome. The deduced amino acid sequence of 532 residues (59.4 kDa) appeared to be identical to the beta-fructofuranosidase from the same strain recently described by Ehrmann et al. (Curr. Microbiol. 2003, 46, 391-397). However, the characterisation of the heterologously expressed enzyme showed several discrepancies to the referred study. First, the B. lactis beta-fructofuranosidase gene was found to have 41% identity with CscA from E. coli in contrast to the 16% reported, therefore it was assigned to as CscA protein instead of BfrA. Second, we observed only low activity of the enzyme towards sucrose (6%) instead of the 100% previously reported. Instead, we measured highest activity (100%) of the enzyme with the oligofructose Raftilose as a substrate compared with the inulin of low degree of polymerisation Raftiline LS (29%) and the highly polymerised Raftiline HP (10%). Altogether, the enzyme showed high affinity to terminal beta(2-1) glycosyl linkages between fructose moieties. The Km values obtained for Raftilose, Raftiline LS and sucrose were 0.12, 7.08 and 8.37 mM, respectively, and V(max) values for the conversion to fructose were calculated to be 5, 21 and 17 micromol/min per mg of protein, respectively. Growth of B. lactis was supported by fructans of low degree of polymerisation (Raftilose and Raftiline LS), whereas we observed no growth with highly polymerised inulin (Raftiline HP).     

Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1β, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.