PUMA publications for /tag/glutamicum,%20Bacterial%20gene%20proteins,Fri Feb 09 13:18:17 CET 2018Appl. Environ. Microbiol.jan2419--427Acetohydroxyacid synthase, a novel target for improvement of {L}-lysine production by {Corynebacterium} glutamicum752009Acetolactate Acid, Bacterial Butyrates Corynebacterium Deletion, Enzyme Expression Gene Inhibitors, Isoleucine, Kinetics, Leucine, Lysine, Profiling, Proteins, Pyruvic Sequence Synthase, Valine, glutamicum, myown The influence of acetohydroxy acid synthase (AHAS) on L-lysine production by Corynebacterium glutamicum was investigated. An AHAS with a deleted C-terminal domain in the regulatory subunit IlvN was engineered by truncating the ilvN gene. Compared to the wild-type AHAS, the newly constructed enzyme showed altered kinetic properties, i.e., (i) an about twofold-lower K(m) for the substrate pyruvate and an about fourfold-lower V(max); (ii) a slightly increased K(m) for the substrate alpha-ketobutyrate with an about twofold-lower V(max); and (iii) insensitivity against the inhibitors L-valine, L-isoleucine, and L-leucine (10 mM each). Introduction of the modified AHAS into the L-lysine producers C. glutamicum DM1729 and DM1933 increased L-lysine formation by 43\% (30 mM versus 21 mM) and 36\% (51 mM versus 37 mM), respectively, suggesting that decreased AHAS activity is linked to increased L-lysine formation. Complete inactivation of the AHAS in C. glutamicum DM1729 and DM1933 by deletion of the ilvB gene, encoding the catalytic subunit of AHAS, led to L-valine, L-isoleucine, and L-leucine auxotrophy and to further-improved L-lysine production. In batch fermentations, C. glutamicum DM1729 Delta ilvB produced about 85\% more L-lysine (70 mM versus 38 mM) and showed an 85\%-higher substrate-specific product yield (0.180 versus 0.098 mol C/mol C) than C. glutamicum DM1729. Comparative transcriptome analysis of C. glutamicum DM1729 and C. glutamicum DM1729 Delta ilvB indicated transcriptional differences for about 50 genes, although not for those encoding enzymes involved in the L-lysine biosynthetic pathway.Fri Feb 09 13:18:17 CET 2018Appl. Microbiol. Biotechnol.jun3471--479Corynebacterium glutamicum tailored for high-yield {L}-valine production792008Bacterial Biomass, Biosynthetic Complex, Corynebacterium Dehydrogenase Engineering, Expression, Fermentation Gene Genetic Ketol-Acid Oxidoreductases, Pathways, Proteins, Pyruvate Reductoisomerase, Transaminases, Valine, glutamicum, myown We recently engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of L: -valine from glucose by inactivation of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. Based on the first generation of pyruvate-dehydrogenase-complex-deficient C. glutamicum strains, a second generation of high-yield L-valine producers was constructed by successive deletion of the genes encoding pyruvate:quinone oxidoreductase, phosphoglucose isomerase, and pyruvate carboxylase and overexpression of ilvBNCE. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 410 mM (48 g/l) L-valine, showed a maximum yield of 0.75 to 0.86 mol/mol (0.49 to 0.56 g/g) of glucose in the production phase and, in contrast to the first generation strains, excreted neither pyruvate nor any other by-product tested.Fri Feb 09 13:18:17 CET 2018J. Biotechnol.dec4331--340Impact of different {CO}2/{HCO}3- levels on metabolism and regulation in {Corynebacterium} glutamicum1682013Alanine, Bacterial Bacterial, Biomass, CO(2)/HCO(3)(−), Carbon Corynebacterium DNA-Binding Dehydrogenase, Dioxide, Diphtheria Dissolved DtxR, Expression Gene Glucose, Glucosephosphate Proteins, Regulation, Thiamin Thiamine, Toxin, Valine, biosynthesis carbon dioxide, glutamicum, myown repressor toxin We investigated the growth kinetics and transcriptional responses of Corynebacterium glutamicum in environments with low (pCO2{\textless}40 mbar) and high (pCO2 ≥ 300 mbar) CO2/HCO3(-) levels compared to standard conditions. When cultivated at high CO2/HCO3(-)-levels, C. glutamicum showed increased (63\%) biomass to substrate yields during the initial growth phase. Other kinetic parameters such as growth rate (μ), specific glucose consumption rate (qS), and selected enzymatic activities of anaplerotic reactions, the pentose phosphate pathway and the tricarboxylic acid cycle were similar to standard conditions. However, microarray hybridization disclosed a complex transcriptional response involving 117 differentially expressed genes. Among those, 60 genes were assigned to the complete DtxR/RipA regulon controlling iron homeostasis in C. glutamicum. Impaired growth of a ΔdtxR mutant at high CO2/HCO3(-) levels validated the relevance of this master regulator to cope with excessive CO2/HCO3(-) availability. At low CO2/HCO3(-) levels, C. glutamicum grew in a bi-level manner with three distinct growth phases. Differential analyses revealed approximately doubled activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase accompanied by the formation of L-alanine and L-valine during the lowest μ occurring in mid-phase of the cultivation. DNA microarray analysis revealed more than 100 differentially expressed genes in growth phase II compared to phase I including almost all thiamin pyrophosphate (TPP) biosynthesis genes, which were significantly up regulated. Concluding, we hypothesize that C. glutamicum counteracts the lack of CO2/HCO3(-) by triggering TPP biosynthesis for increasing the activities of TPP-dependent enzymes involved in CO2 formation.Fri Feb 09 13:18:17 CET 2018J. Mol. Microbiol. Biotechnol.3-4236--239{RamB} is an activator of the pyruvate dehydrogenase complex subunit {E}1p gene in {Corynebacterium} glutamicum162009(Lipoamide) Acetates, Bacterial Bacterial, Base Corynebacterium Data, Dehydrogenase Expression Gene Molecular Proteins, Pyruvate Regulation, Sequence Sequence, glutamicum, myown In Corynebacterium glutamicum, the transcriptional regulator RamB negatively controls the expression of genes involved in acetate metabolism. Here we show that during growth in media containing glucose and in complex medium without glucose RamB activates expression of the aceE gene, encoding the E1p subunit of the pyruvate dehydrogenase complex. Thus, RamB functions both as repressor and as activator in C. glutamicum.glutamicum, Bacterial gene proteins,Community for tag(s) glutamicum, Bacterial gene proteins,