scholarly journals A large decrease in heat-shock-induced proteolysis after tryptophan starvation leads to increased expression of phage λ lysozyme cloned in Escherichia coli

1992 ◽  
Vol 286 (1) ◽  
pp. 187-191 ◽  
Author(s):  
P Soumillion ◽  
J Fastrez
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Stationary Phase ◽  
Coli Strain ◽  
Present Knowledge ◽  
R Gene ◽  
Gene Coding ◽  
Heat Shock Responses ◽  
Multicopy Vector ◽  
Thermal Induction

The R gene coding for phage lambda lysozyme (lambda L), cloned under the control of the PL promoter on a multicopy vector, is expressed in an Escherichia coli strain auxotrophic for tryptophan. Induction by a thermal shift after tryptophan supplementation in a culture initially brought into stationary phase by tryptophan starvation leads to highly increased expression. A thermally unstable mutant protein, difficult to obtain under standard conditions, can be easily produced by post-stationary-phase expression. It is shown that this is due to a drastic decrease in the heat-shock-induced proteolysis normally observed on thermal induction. These data are discussed in relation to our present knowledge of stringent and heat-shock responses.

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12

1982 ◽  
Vol 152 (3) ◽  
pp. 1138-1146
Author(s):  
L J Lee ◽  
J B Hansen ◽  
E K Jagusztyn-Krynicka ◽  
B M Chassy
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Protein Product ◽  
Coli Strain ◽  
Cloning And Expression ◽  
E Coli ◽  
Lactose Metabolism ◽  
Gene Coding ◽  
K 12

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

scholarly journals Construction and Characterization of anEscherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation

2000 ◽  
Vol 66 (12) ◽  
pp. 5383-5386 ◽  
Author(s):  
Rahul Krishnaswamy ◽  
David B. Wilson
Keyword(s):  
Escherichia Coli ◽  
Energy Source ◽  
Helicobacter Pylori ◽  
Fusion Protein ◽  
Dry Weight ◽  
Coli Strain ◽  
Genetically Engineered ◽  
Gene Coding ◽  
Ph Range

ABSTRACT An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase–pea metallothionein fusion protein. The resulting strain accumulated 15 μmol of Ni(II) per g (dry weight) from a 10 μM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.

Study of the Potential of Reversal of the Fatty Acid Beta-Oxidation Pathway for Stereoselective Biosynthesis of (S)-1,3-Butanediol from Glucose by Recombinant Escherichia coli Strains

2019 ◽  
Vol 35 (5) ◽  
pp. 12-19
Author(s):  
A.Yu. Skorokhodova ◽  
V.G. Debabov
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Essential Gene ◽  
Coli Strain ◽  
Recombinant Escherichia Coli ◽  
Beta Oxidation ◽  
Oxidation Pathway ◽  
Gene Coding ◽  
Fundamental Research ◽  
Fatty Acid Beta Oxidation

A possible contribution of collateral enzymes to the formation of key precursor metabolite, 3-hydroxybutyryl-CoA, in a recombinant Escherichia coli strain engineered for 1,3-butanediol biosynthesis from glucose through the inverted fatty acid beta-oxidation pathway has been evaluated. The inactivation of the 3-hydroxyadipyl-CoA dehydrogenase gene, paaH, did not prevent the 1,3-butanol biosynthesis during anaerobic glucose utilization by the strain with the intact essential gene fabG coding for 3-ketoacyl-ACP reductase, which can catalyze the conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. The subsequent inactivation in the strain of fadB gene coding for (S)-stereospecific 3-hydroxyacyl-CoA dehydrogenase of the fatty acid beta-oxidation led to the abolishment of the 1,3-butanediol synthesis. The respective diol was also not found among the products secreted by the strain possessing the intact fabG and paaH genes upon an individual deletion of fadB gene. It was established that the collateral enzymes did not participate in the formation of 3-hydroxybutyryl-CoA in the studied strains and the respective CoA-derivative was synthesized solely by the (S)-specific enzyme of the fatty acid beta-oxidation pathway. The obtained results indicate that the reversal of the fatty acid beta-oxidation pathway can ensure the enantioselective biosynthesis of the (S)-stereoisomer of 1,3-butanediol in engineered E. coli strains. 1,3-butanediol, fatty acid beta-oxidation, Escherichia coli, glucose, metabolic engineering, stereoisomer. The work was carried out with financial support Russian Foundation for Fundamental Research (No. 18-29-08059).

Creation of Plasmidless and Markerless Escherichia coli Succinate Producing Strain and Evaluation of its Biosynthetic Potential during Utilization of Lignocellulosic Sugars

2020 ◽  
Vol 36 (2) ◽  
pp. 3-11
Author(s):  
O.A. Zhuravliova ◽  
Т.А. Voeikova ◽  
A.Yu. Gulevich ◽  
V.G. Debabov
Keyword(s):  
Escherichia Coli ◽  
Succinic Acid ◽  
Plant Biomass ◽  
Anaerobic Fermentation ◽  
Basic Research ◽  
Acid Fermentation ◽  
Mixed Acid ◽  
Gene Coding ◽  
Lignocellulosic Sugars ◽  
Basic Research Project

The plasmidless and markerless Escherichia coli succinate producing strain SGM2.0Pyc-int has been engineered and characterized. The strain has the inactivated main mixed-acid fermentation pathways due to the deletions of ldhA,poxB, ackA,pta, and adhE genes, constitutively expresses the genes of the aceEF-lpdA operon encoding components of pyravate dehydrogenase complex, and possesses the chromosomally integrated Bacillus subtilis pycA gene coding for pyruvate carboxylase. The capacity of the strain to synthesize succinic acid in course of dual-phase aerobic-anaerobic fermentation with lignocellulosic sugars as substrates was studied. The SGM2.0Pyc-int strain synthesized succinic acid from glucose, xylose, and arabinose with a molar yields of 1.41 mol/mol, 1.18 mol/mol, and 1.18 mol/mol, respectively, during the anaerobic production stage. The constructed strain has great potential for developing efficient processes for the succinic acid production from plant biomass-derived sugars. Escherichia coli, fermentation, arabinose, glucose, xylose, succinic acid. The work was supported by a Grant from the Russian Foundation for Basic Research (Project no. 18-29-14005).

Expression and characterization of an α-glucosidase from Thermoanaerobacter ethanolicus JW200 with potential for industrial application

Biologia ◽  
2009 ◽  
Vol 64 (6) ◽  
Author(s):  
Yue-Hong Wang ◽  
Yu Jiang ◽  
Zuo-Ying Duan ◽  
Wei-Lan Shao ◽  
Hua-Zhong Li
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Optimal Condition ◽  
Industrial Application ◽  
Conversion Rate ◽  
Hydrolytic Activity ◽  
Thermoanaerobacter Ethanolicus ◽  
Transglycosylation Activity

AbstractIn this study, a new α-glucosidase gene from Thermoanaerobacter ethanolicus JW200 was cloned and expressed in Escherichia coli by a novel heat-shock vector pHsh. The recombinant α-glucosidase exhibited its maximum hydrolytic activity at 70°C and pH 5.0∼5.5. With p-nitrophenyl-α-D-glucoside as a substrate and under the optimal condition (70°C, pH 5.5), K m and V max of the enzyme was 1.72 mM and 39 U/mg, respectively. The purified α-glucosidase could hydrolyze oligosaccharides with both α-1,4 and α-1,6 linkages. The enzyme also had strong transglycosylation activity when maltose was used as sugar donor. The transglucosylation products towards maltose are isomaltose, maltotriose, panose, isomaltotriose and tetrasaccharides. The enzyme could convert 400 g/L maltose to oligosaccharides with a conversion rate of 52%, and 83% of the oligosaccharides formed were prebiotic isomaltooligosaccharides (containing isomaltose, panose and isomaltotriose).

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