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At least 199 records · Page 11Linked to original sources

PubMed2006

Fogacin, a novel cyclic octaketide produced by Streptomyces strain Tü 6319.

A new octaketide named fogacin (1) was isolated from Streptomyces sp. (strain Tü 6319). Furthermore two shunt metabolites, SEK4b (2) and anhydroSEK4b (3), were detected and identified as non-enzymatically cyclized products of polyketide intermediates built during the biosynthesis of actinorhodin. SEK4b (2) as well as anhydroSEK4b (3) were previously described as metabolites of genetically engineered strains.

Anti-Bacterial Agents↗

PubMed2006

Enhancement and selective production of oligomycin through inactivation of avermectin's starter unit in Streptomyces avermitilis.

Oligomycin and its analogues, produced by Streptomyces avermitilis and other actinomycetes, are of interest for their potent and selective biological activities. PCR-mediated gene replacement, targeting bkdF, one of avermectin's starter unit encoding genes in S. avermitilis, was performed to yield an oligomycin producer, BIB0423. The engineered strain produced oligomycin A at 2.3 mg/ml compared to the wild type strain at 0.1 mg/ml. This resulting mutant was genetically stable and should be useful for the industrial production of oligomycin.

Gene Expression Regulation, Bacterial↗

PubMed2006

Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli.

The Saccharomyces cerevisiae glycerol pathway (GPD1 and GPP2) was evolved in vivo in Escherichia coli. The central metabolism of E. coli was engineered to link glucose consumption and glycerol production. The engineered strain was evolved in a chemostat culture and a high glycerol producer was rapidly obtained. The evolution of the strain was associated to a deletion between GPD1 and GPP2, resulting in the production of a fusion protein with both glycerol-3-P dehydrogenase and glycerol-3-P phosphatase activities. The higher efficiency of the fusion protein was due to partial glycerol-3-P channeling between the two active sites. The evolved strain produces glycerol from glucose at high yield, concentration and productivity.

Adaptation, Physiological↗

PubMedDate not supplied

Improved degradation of organophosphorus nerve agents and p-nitrophenol by Pseudomonas putida JS444 with surface-expressed organophosphorus hydrolase.

Pseudomonas putida JS444, isolated from p-nitrophenol (PNP) contaminated waste sites, was genetically engineered to simultaneously degrade organophosphorus pesticides (OP) and PNP. A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringae was used to target the organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida JS444, reducing the potential substrate uptake limitation. Engineered cells were capable of targeting OPH onto the cell surface as demonstrated by western blotting, cell fractionation, and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without instability problems associated with the engineered Moraxella sp. The initial hydrolysis rate was 7.90, 3.54, and 1.53 micromol/h/mg dry weight for paraoxon, parathion, and methyl parathion, respectively. The excellent stability in combination with the rapid degradation rate for organophosphates and PNP make this engineered strain an ideal biocatalyst for complete mineralization of organophosphates.

Aryldialkylphosphatase↗

PubMed1988

Dicarboxylic acid transport in Bradyrhizobium japonicum: use of Rhizobium meliloti dct gene(s) to enhance nitrogen fixation.

A recombinant plasmid encoding Rhizobium meliloti sequences involved in dicarboxylic acid transport (plasmid pRK290:4:46) (E. Bolton, B. Higgisson, A. Harrington, and F. O'Gara, Arch. Microbiol. 144:142-146, 1986) was used to study the relationship between dicarboxylic acid transport and nitrogen fixation in Bradyrhizobium japonicum. The expression of the dct sequences on plasmid pRK290:4:46 in B. japonicum CJ1 resulted in increased growth rates in media containing dicarboxylic acids as the sole source of carbon. In addition, strain CJ1(pRK290:4:46) exhibited enhanced succinate uptake activity when grown on dicarboxylic acids under aerobic conditions. Under free-living nitrogen-fixing conditions, strain CJ1(pRK290:4:46) exhibited higher nitrogenase (acetylene reduction) activity compared with that of the wild-type strain. This increase in nitrogenase activity also correlated with an enhanced dicarboxylic acid uptake rate under these microaerobic conditions. The regulation of dicarboxylic acid transport by factors such as metabolic inhibitors and the presence of additional carbon sources was similar in both the wild-type and the engineered strains. The implications of increasing nitrogenase activity through alterations in the dicarboxylic acid transport system are discussed.

Biological Transport, Active↗

PubMed2000

Genomic determinants of cardiovirulence in coxsackievirus B3 clinical isolates: localization to the 5' nontranslated region.

Coxsackievirus B3 (CVB3) infections can cause myocarditis in humans and are implicated in the pathogenesis of dilated cardiomyopathy. The natural genetic determinants of cardiovirulence for CVB3 have not been identified, although using strains engineered in the laboratory, cardiovirulence determinants have been identified in the CVB3 5' nontranslated region (5'NTR) and capsid. The myocarditic phenotypes of two CVB3 clinical isolates were determined using an established murine model of inflammatory heart disease. The 5'NTRs and capsid proteins of the noncardiovirulent CVB3/CO strain and cardiovirulent CVB3/AS strain were examined to determine their influence on the cardiovirulence phenotype. Six intratypic chimeric viruses were constructed in which 5'NTR and capsid sequences of the infectious cDNA copy of the cardiovirulent CVB3/20 genome were replaced by homologous sequences from CVB3/CO or CVB3/AS. Chimeric strains were tested for cardiovirulence by inoculation of C3H/HeJ mice. Sections of hearts removed at 10 days postinoculation were examined for evidence of myocarditis by light microscopy and assayed for the presence of virus. Replacement of the CVB3/20 capsid coding region by that from the homologous region of CVB3/CO resulted in no change in the cardiovirulent CVB3/20 phenotype, with virus recoverable from the heart at 10 days postinoculation. However, recombinant virus containing the CVB3/CO 5'NTR alone or the 5'NTR and capsid sequences together were not myocarditic, and infectious virus was not recovered from the myocardium. Chimeric viruses containing the CVB3/AS 5'NTR alone, capsid sequence alone, or both together preserved the myocarditic phenotype. These data support the 5'NTR as the primary site in the determination of the natural cardiovirulence phenotype of CVB3.

5' Untranslated Regions↗

PubMed1999

[Expression of genes aroG and pheA in phenylalanine biosynthesis].

aroG and pheA genes, encoding 3-Deoxy-D-arabinoheptulonate-7-phosphate synthase(DS) and Chorismate mutase (CM)-prephenate dehydratase(PD) in the pathway of phenylalanine biosynthesis respectively, were amplified by polymerase chain reaction(PCR). The genes were assembled on the multicopy vectors and expressed in both Escherichia coli and Brevibacterium. The products of two gene were detected by SDS-PAGE. The activities of relevant enzymes were measured in the crude extract of the host strain. When aroG-pheA genes were introduced into E. coli p2392, the activities of DS, CM and PD were increased by 4.3-fold, 4.4-fold and 2.2-fold respectively. Whereas in the case of Brevibacterium flavum 2732, the activities of DS, CM and PD were increased by 12.3-fold, 2.3-fold and 5.6-fold, respectively. As the results, the overproduction of phenylalanine was brought about by using the genetic engineering strain of B. flavum.

3-Deoxy-7-Phosphoheptulonate Synthase↗

PubMed2000

Metabolic engineering applications to renewable resource utilization.

Lignocellulosic materials containing cellulose, hemicellulose, and lignin are the most abundant renewable organic resource on earth. The utilization of renewable resources for energy and chemicals is expected to increase in the near future. The conversion of both cellulose (glucose) and hemicellulose (hexose and pentose) for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction (xylose and arabinose, the main pentose sugars obtained on hydrolysis of hemicellulose), presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds, such as ethanol. Metabolic strategies seek to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulosic sugars to products that can be derived from the primary metabolism, such as ethanol. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra.

Bacteria↗

PubMed2004

Bioactivation of the heterocyclic aromatic amine 2-amino-3-methyl-9H-pyrido [2,3-b]indole (MeAalphaC) in recombinant test systems expressing human xenobiotic-metabolizing enzymes.

2-Amino-3-methyl-9H-pyrido[2,3-b]indole (MeAalphaC) and some metabolites were investigated for mutagenicity in mammalian cell lines and bacterial strains engineered for the expression of human enzymes. MeAalphaC induced gene mutations (studied at the hprt locus) in Chinese hamster V79-derived cells co-expressing cytochrome (CYP) 1A2 and sulphotransferase (SULT) 1A1 even at a concentration of 30 nM, but was inactive in cells co-expressing CYP1A2 and N-acetyltransferase (NAT) 1 or 2. MeAalphaC, tested in the presence of rat liver post-mitochondrial fraction, showed strongly enhanced mutagenicity in a Salmonella typhimurium strain expressing human SULT1A1 compared with the control (recipient) strain TA1538/1,8-DNP (deficient in endogenous acetyltransferase). Mutagenicity was also enhanced, although to a lesser extent, when NAT2 was expressed in the latter strain. The metabolite, 2-hydroxylamino-3-methyl-9H-pyrido[2,3-b]indole (N-OH-MeAalphaC) was a direct mutagen to strains TA1538 and TA1538/ 1,8-DNP. This mutagenicity was strongly enhanced in corresponding strains expressing SULT1A1. A moderate enhancement was observed when SULT1A2, SULT1B1, SULT1C2 or NAT2 were expressed in strain TA1538. The remaining enzymes studied (SULT1A3, 1C1, 1E1, 2A1, 2B1a, 2B1b, 4A1 and NAT1) did not indicate any activation of N-OH-MeAalphaC. Preliminary mutagenicity experiments in SULT-expressing S.typhimurium strains were conducted with other hydroxylated metabolites of MeAalphaC. The phenols, 6- and 7-hydroxy-MeAalphaC, were inactive under the conditions studied. The benzylic alcohol, 2-amino-3-hydroxymethyl-9H-pyrido[2,3-b]indole, was mutagenic in a strain expressing SULT1A1, but its activity was much weaker than that of N-OH-MeAalphaC. Thus, N-hydroxylation (e.g. mediated by CYP1A2) and sulpho conjugation (primarily mediated by SULT1A1) was the dominating activation pathway of MeAalphaC in model systems engineered for human enzymes. Some other SULT forms as well as NAT2 were also capable of activating N-OH-MeAalphaC, although with much lower efficiency than SULT1A1. Another minor activation pathway involved benzylic hydroxylation followed by sulpho conjugation by SULT1A1.

Animals↗

PubMed1996

Protection of non-murine mammals against encephalomyocarditis virus using a genetically engineered Mengo virus.

Genetically engineered Mengo viruses with artificial deletions in the 5' noncoding poly(C) tracts are highly attenuated for pathogenicity when introduced as live vaccines into the natural murine host. Inoculation produces lifelong protective immunity without disease or viral persistence. This report extends the vaccination studies to non-murine hosts, including baboons, macaques and domestic pigs, all of which are susceptible to severe cardiovirus epizootics. All animals of these species that were inoculated with vMC24, an engineered strain of Mengo, seroconverted. When the immunized animals were challenged, they were protected against lethal doses of encephalomyocarditis virus (EMCV) derived from currently circulating epizootic strains. In baboons, the neutralizing antibody titers induced by vMC24 were significantly higher than from an inactivated EMCV vaccine. Moreover, terminal histopathology on baboons (inoculated intramuscularly), macaques (inoculated intracerebrally), and pigs (inoculated intramuscularly) showed few, if any, gross lesions characteristic of EMCV-like disease, in the vMC24 vaccinates. We suggest that genetically engineered, short poly(C) Mengo viruses may be universally potent attenuated vaccines for many types of animals and can possibly provide safe, efficacious protection against all cardioviruses of the EMCV serotype.

Animals↗

PubMed2000

Generation of new hydrogen-recycling Rhizobiaceae strains by introduction of a novel hup minitransposon.

Hydrogen evolution by nitrogenase is a source of inefficiency for the nitrogen fixation process by the Rhizobium-legume symbiosis. To develop a strategy to generate rhizobial strains with H(2)-recycling ability, we have constructed a Tn5 derivative minitransposon (TnHB100) that contains the ca. 18-kb H(2) uptake (hup) gene cluster from Rhizobium leguminosarum bv. viciae UPM791. Bacteroids from TnHB100-containing strains of R. leguminosarum bv. viciae PRE, Bradyrhizobium japonicum, R. etli, and Mesorhizobium loti expressed high levels of hydrogenase activity that resulted in full recycling of the hydrogen evolved by nitrogenase in nodules. Efficient processing of the hydrogenase large subunit (HupL) in these strains was shown by immunoblot analysis of bacteroid extracts. In contrast, Sinorhizobium meliloti, M. ciceri, and R. leguminosarum bv. viciae UML2 strains showed poor expression of the hup system that resulted in H(2)-evolving nodules. For the latter group of strains, no immunoreactive material was detected in bacteroid extracts using anti-HupL antiserum, suggesting a low level of transcription of hup genes or HupL instability. A general procedure for the characterization of the minitransposon insertion site and removal of antibiotic resistance gene included in TnHB100 has been developed and used to generate engineered strains suitable for field release.

Bacterial Proteins↗

PubMed2004

Microarray-based resequencing of multiple Bacillus anthracis isolates.

We used custom-designed resequencing arrays to generate 3.1 Mb of genomic sequence from a panel of 56 Bacillus anthracis strains. Sequence quality was shown to be very high by replication (discrepancy rate of 7.4 x 10(-7)) and by comparison to independently generated shotgun sequence (discrepancy rate < 2.5 x 10(-6)). Population genomics studies of microbial pathogens using rapid resequencing technologies such as resequencing arrays are critical for recognizing newly emerging or genetically engineered strains.

Bacillus anthracis↗

PubMed2006

Genetically engineered underdominance for manipulation of pest populations: a deterministic model.

We theoretically investigate the potential for introgressing a desired engineered gene into a pest population by linking the desired gene to DNA constructs that exhibit underdominance properties. Our deterministic model includes two independently segregating engineered constructs that both carry a lethal gene, but suppress each other. Only genotypes containing both or neither construct are viable. Both constructs also carry the desired gene with an independent regulatory mechanism. We examine the minimal number of individuals of an engineered strain that must be released into a natural population to successfully introgress the desired gene. We compare results for strains carrying single and multiple insertions of the constructs. When there are no fitness costs associated with the inserted constructs (when the lethal sequences are not expressed), the number of individuals that must be released decreases as the number of insertions in the genome of the released strain increases. As fitness costs increase, the number of individuals that must be released increases at a greater rate for release strains with more insertions. Under specific conditions this results in the strain with only a single insertion of each construct being the most efficient for introgressing the desired gene. We discuss practical implications of our findings.

Animals↗

PubMed2004

Genetic engineering in the mouse: tuning TNF/TNFR expression.

With the exponential increase in the number of genes identified by various genome projects, it has become imperative that efficient methods be developed for deciphering gene function. Genetically engineered strains of mice are now critical research tools for basic biomedical research and for genomic approaches for the development of new therapeutic treatments for human disease. Over the past ten years it has become possible to make essentially any mutation in the mouse by transgenesis and homologous recombination in embryonic stem cells. Current advances in the "genetic engineering of the mouse," including the tissue-specific activation or inactivation of gene expression combined with developing technologies for switching gene expression on and off at will, provide experimental settings unprecedented in their potential to offer answers to long-standing questions.

Alleles↗

PubMed2005

Combinatorial biosynthesis of lipopeptide antibiotics in Streptomyces roseosporus.

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. Cubicin (daptomycin-for-injection) was approved in 2003 by the FDA to treat skin and skin structure infections caused by Gram-positive pathogens. Daptomycin is particularly significant in that it represents the first new natural product antibacterial structural class approved for clinical use in three decades. The daptomycin gene cluster contains three very large genes (dptA, dptBC, and dptD) that encode the nonribosomal peptide synthetase (NRPS). The related cyclic lipopeptide A54145 has four NRPS genes (lptA, lptB, lptC, and lptD), and calcium dependent antibiotic (CDA) has three (cdaPS1, cdaPS2, and cdaPS3). Mutants of S. roseosporus containing deletions of one or more of the NRPS genes have been trans-complemented with dptA, dptBC, and dptD by inserting these genes under the control of the ermEp* promoter into separate conjugal cloning vectors containing phiC31 or IS117 attachment (attP int) sites; delivering the plasmids into S. roseosporus by conjugation from Escherichia coli; and inserting the plasmids site-specifically into the chromosome at the corresponding attB sites. This trans-complementation system was used to generate subunit exchanges with lptD and cdaPS3 and the recombinants produced novel hybrid molecules. Module exchanges at positions D: -Ala(8) and D: -Ser(11) in the peptide have produced additional novel derivatives of daptomycin. The approaches of subunit exchanges and module exchanges were combined with amino acid modifications of Glu at position 12 and natural variations in lipid side chain starter units to generate a combinatorial library of antibiotics related to daptomycin. Many of the engineered strains produced levels of novel molecules amenable to isolation and antimicrobial testing, and most of the compounds displayed antibacterial activities.

Anti-Bacterial Agents↗

PubMed1999

Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli.

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.

Alkyl and Aryl Transferases↗

PubMed1995

Production of fully human antibodies by transgenic mice.

The ability to produce a diverse repertoire of fully human monoclonal antibodies may have significant applications to human therapy. One of the most promising approaches to the production of therapeutic human monoclonal antibodies is the creation of a mouse strain engineered to produce a large repertoire of human antibodies in the absence of mouse antibodies. Recently, such mice have been generated by introducing segments of human immunoglobulin loci into the germlines of mice deficient in mouse antibody production as a result of gene targeting. These mice produce significant levels of fully human antibodies with a diverse adult-like repertoire and, upon immunization with antigens, generate antigen-specific fully human monoclonal antibodies. Such strains of mice may provide the optimal source for producing human monoclonal antibodies with high affinity and specificity against a broad spectrum of antigens, including human antigens.

Animals↗

PubMed2005

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana.

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

Arabidopsis Proteins↗