RESUMO
Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B ⢠Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control ⢠Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.
Assuntos
Corynebacterium glutamicum , Aminoácidos , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , AlemanhaRESUMO
Corynebacterium glutamicum, a Gram-positive soil bacterium employed in the industrial production of various amino acids, is able to use a number of different nitrogen sources, such as ammonium, urea or creatinine. This study shows that l-glutamine serves as an excellent nitrogen source for C. glutamicum and allows similar growth rates in glucose minimal medium to those in ammonium. A transcriptome comparison revealed that the nitrogen starvation response was elicited when glutamine served as the sole nitrogen source, meaning that the target genes of the global nitrogen regulator AmtR were derepressed. Subsequent growth experiments with a variety of mutants defective in nitrogen metabolism showed that glutamate synthase is crucial for glutamine utilization, while a putative glutaminase is dispensable under the experimental conditions used. The gltBD operon encoding the glutamate synthase is a member of the AmtR regulon. The observation that the nitrogen starvation response was elicited at high intracellular l-glutamine levels has implications for nitrogen sensing. In contrast with other Gram-positive and Gram-negative bacteria such as Bacillus subtilis, Salmonella enterica serovar Typhimurium and Klebsiella pneumoniae, a drop in glutamine concentration obviously does not serve as a nitrogen starvation signal in C. glutamicum.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Glutamina/metabolismo , Nitrogênio/metabolismo , Regulon , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Meios de Cultura , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Glutamato Sintase/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Compostos de Amônio Quaternário/metabolismo , RNA Bacteriano/genética , Proteínas Repressoras/genéticaRESUMO
Corynebacterium glutamicum can utilize acetic acid and propionic acid for growth and amino acid production. Growth on acetate as sole carbon source requires acetate activation by acetate kinase (AK) and phosphotransacetylase (PTA), encoded in the pta-ack operon. Genetic and enzymatic studies showed that these enzymes also catalyze propionate activation and were required for growth on propionate as sole carbon source. However, when glucose was present as a co-substrate strain lacking the AK-PTA pathway was still able to utilize acetate or propionate for growth indicating that an alternative activation pathway exists. As shown by (13)C-labelling experiments, the carbon skeleton of acetate is conserved during activation to acetyl-CoA in this pathway. Metabolic flux analysis during growth on an acetate-glucose mixture revealed that in the absence of the AK-PTA pathway carbon fluxes in glycolysis, the tricarboxylic acid (TCA) cycle and anaplerosis via PEP carboxylase and/or pyruvate carboxylase were increased, while the glyoxylate cycle flux was decreased. DNA microarray experiments identified cg2840 as a constitutively and highly expressed gene putatively encoding a CoA transferase. Purified His-tagged Cg2840 protein was active as CoA transferase interconverting acetyl-, propionyl- and succinyl-moieties as CoA acceptors and donors. Strains lacking both the CoA transferase and the AK-PTA pathway could neither activate acetate nor propionate in the presence or absence of glucose. Thus, when these short-chain fatty acids are co-metabolized with other carbon sources, CoA transferase and the AK-PTA pathway constitute a redundant system for activation of acetate and propionate.
Assuntos
Acetato Quinase/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Fosfato Acetiltransferase/metabolismo , Acetato Quinase/genética , Acetatos/metabolismo , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Isótopos de Carbono/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Genômica , Glucose/metabolismo , Marcação por Isótopo , Mutação , Fosfato Acetiltransferase/genética , Propionatos/metabolismoRESUMO
Corynebacterium glutamicum can grow on a variety of carbohydrates from which glucose, fructose and sucrose are taken up and phosphorylated by the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Here, we show that cg2927 (scrB) encodes sucrose-6-phosphate hydrolase. The purified His-tagged protein hydrolyzed sucrose-6-phosphate and sucrose, but not sucrose-6'-phosphate. The Km value for sucrose was 190 mM while the Km for sucrose-6-phosphate was much lower, 0.04 mM. Sucrose-6-phosphate hydrolase activity was stimulated by MgSO4 and fructose-6-phosphate and was inhibited by MnCl2, CaCl2, CuSO4 and ZnSO4. A scrB deletion mutant could not grow on sucrose as the sole carbon source. In addition, growth in the absence of scrB was severely decreased when sucrose was present in addition to glucose, fructose or acetate, suggesting that higher intracellular concentrations of sucrose-6-phosphate are toxic. Transcriptional start sites in the cg2929-cg2928-scrB-ptsS locus could be revealed upstream of cg2929 and upstream of the sucrose-specific PTS gene ptsS. Of these, only ptsS showed increased expression when grown in the presence of sucrose, which was due to control by the transcriptional regulator SugR. The sucrose-6-phosphate hydrolase activity, however, was increased two- to threefold during growth in fructose- or sucrose-containing media, regardless of the presence or absence of SugR.
Assuntos
Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/crescimento & desenvolvimento , Sacarose/metabolismo , beta-Frutofuranosidase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Corynebacterium glutamicum/genética , Meios de Cultura , Frutose/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Sacarose/análogos & derivados , Fosfatos Açúcares/metabolismo , beta-Frutofuranosidase/genéticaRESUMO
Corynebacterium glutamicum can grow on L-lactate as a sole carbon and energy source. The NCgl2816-lldD operon encoding a putative transporter (NCgl2816) and a quinone-dependent L-lactate dehydrogenase (LldD) is required for L-lactate utilization. DNA affinity chromatography revealed that the FadR-type regulator LldR (encoded by NCgl2814) binds to the upstream region of NCgl2816-lldD. Overexpression of lldR resulted in strongly reduced NCgl2816-lldD mRNA levels and strongly reduced LldD activity, and as a consequence, a severe growth defect was observed in cells grown on L-lactate as the sole carbon and energy source, but not in cells grown on glucose, ribose, or acetate. Deletion of lldR had no effect on growth on these carbon sources but resulted in high NCgl2816-lldD mRNA levels and high LldD activity in the presence and absence of L-lactate. Purified His-tagged LldR bound to a 54-bp fragment of the NCgl2816-lldD promoter, which overlaps with the transcriptional start site determined by random amplification of cDNA ends-PCR and contains a putative operator motif typical of FadR-type regulators, which is -1TNGTNNNACNA10. Mutational analysis revealed that this motif with hyphenated dyad symmetry is essential for binding of LldD to the NCgl2816-lldD promoter. L-Lactate, but not D-lactate, interfered with binding of LldRHis to the NCgl2816-lldD promoter. Thus, during growth on media lacking L-lactate, LldR represses expression of NCgl2816-lldD. In the presence of L-lactate in the growth medium or under conditions leading to intracellular L-lactate accumulation, the L-lactate utilization operon is induced.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Regulação Bacteriana da Expressão Gênica , L-Lactato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Proteínas de Bactérias/genética , Benzoquinonas/metabolismo , Corynebacterium glutamicum/crescimento & desenvolvimento , Corynebacterium glutamicum/metabolismo , Deleção de Genes , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteoma , Transcrição GênicaRESUMO
A critical factor in the biotechnological production of L: -lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP(+) via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysine-producing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by approximately 10% on glucose, approximately 70% on fructose, approximately 50% on glucose plus fructose, and even by approximately 300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.
Assuntos
Biotecnologia/métodos , Corynebacterium glutamicum/enzimologia , Escherichia coli/genética , Lisina/biossíntese , NADP Trans-Hidrogenases/genética , NADP Trans-Hidrogenases/metabolismo , Membrana Celular/enzimologia , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/crescimento & desenvolvimento , Meios de Cultura , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , NADP/metabolismoRESUMO
In the biotechnological production of L-lysine and L-glutamate by Corynebacterium glutamicum media based on glucose, fructose or sucrose are typically used. Glutamate production by C. glutamicum was very similar on glucose, fructose, glucose plus fructose and sucrose. In contrast, lysine production of genetically defined C. glutamicum strains was significantly higher on glucose than on the other carbon sources. To test whether malic enzyme or fructose-1,6-bisphosphatase might limit growth and lysine on fructose, glucose plus fructose or sucrose, strains overexpressing either malE which encodes the NADPH-dependent malic enzyme or the fructose-1,6-bisphosphatase gene fbp were generated. Overexpression of malE did not improve lysine production on any of the tested carbon sources. Upon overexpression of fbp lysine yields on glucose and/or fructose were unchanged, but the lysine yield on sucrose increased twofold. Thus, fructose-1,6-bisphosphatase was identified as a limiting factor for lysine production by C. glutamicum with sucrose as the carbon source.