RESUMO
Strains of the bacterium, Corynebacterium glutamicum, are widely used for the industrial production of L-glutamic acid and various other substances. C. glutamicum ssp. lactofermentum AJ 1511, formerly classified as Brevibacterium lactofermentum, and the closely related C. glutamicum ATCC 13032 have been used as industrial strains for more than 50 years. We determined the whole genome sequence of C. glutamicum AJ 1511 and performed genome-wide comparative analysis with C. glutamicum ATCC 13032 to determine strain-specific genetic differences. This analysis revealed that the genomes of the two industrial strains are highly similar despite the phenotypic differences between the two strains. Both strains harbored unique genes but gene transpositions or inversions were not observed. The largest unique region, a 220-kb AT-rich region located between 1.78 and 2.00 Mb position in C. glutamicum ATCC 13032 genome, was missing in the genome of C. glutamicum AJ 1511. The next two largest unique regions were present in C. glutamicum AJ 1511. The first region (413-484 kb position) contains several predicted transport proteins, enzymes involved in sugar metabolism, and transposases. The second region (1.47-1.50 Mb position) encodes restriction modification systems. A gene predicted to encode NADH-dependent glutamate dehydrogenase, which is involved in L-glutamate biosynthesis, is present in C. glutamicum AJ 1511. Strain-specific genes identified in this study are likely to govern phenotypes unique to each strain.
Assuntos
Brevibacterium/genética , Corynebacterium glutamicum/genética , Genoma Bacteriano , Ácido Glutâmico/biossíntese , Análise de Sequência de DNA , Corynebacterium glutamicum/enzimologia , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA Bacteriano , Glutamato Desidrogenase/genética , Glutamato Desidrogenase/metabolismo , Fenótipo , Especificidade da Espécie , Transposases/genética , Transposases/metabolismoRESUMO
Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5% yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.
Assuntos
Enterobacter aerogenes/metabolismo , Ácido Succínico/metabolismo , Anaerobiose , Fermentação/fisiologia , Succinatos/metabolismoRESUMO
Corynebacterium glutamicum produces succinate from glucose via the reductive tricarboxylic acid cycle under microaerobic and anaerobic conditions. We identified a NCgl2130 gene of C. glutamicum as a novel succinate exporter that functions in succinate production, and designated sucE1. sucE1 expression levels were higher under microaerobic conditions than aerobic conditions, and overexpression or disruption of sucE1 respectively increased or decreased succinate productivity during fermentation. Under microaerobic conditions, the sucE1 disruptant sucE1Δ showed 30% less succinate productivity and a lower sugar-consumption rate than the parental strain. Under anaerobic conditions, succinate production by sucE1Δ ceased. The intracellular succinate and fructose-1,6-bisphosphate levels of sucE1Δ under microaerobic conditions were respectively 1.7-fold and 1.6-fold higher than those of the parental strain, suggesting that loss of SucE1 function caused a failure of succinate removal from the cells, leading to intracellular accumulation that inhibited upstream sugar metabolism. Homology and transmembrane helix searches identified SucE1 as a membrane protein belonging to the aspartate:alanine exchanger (AAE) family. Partially purified 6x-histidine-tagged SucE1 (SucE1-[His](6)) reconstituted in succinate-loaded liposomes clearly demonstrated counterflow and self-exchange activities for succinate. Together, these findings suggest that sucE1 encodes a novel succinate exporter that is induced under microaerobic conditions, and is important for succinate production under both microaerobic and anaerobic conditions.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Ácido Succínico/metabolismo , Aerobiose , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Bioensaio , Transporte Biológico , Reatores Biológicos/microbiologia , Corynebacterium glutamicum/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Interações Hidrofóbicas e Hidrofílicas , Espaço Intracelular/metabolismo , Metaboloma , Filogenia , Proteolipídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low-pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis-based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO(3). Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L(-1) in shaking flasks with buffering and more than 17 g L(-1) without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed.
Assuntos
Ácido Succínico/metabolismo , Yarrowia/enzimologia , Yarrowia/fisiologia , Aerobiose , Carbonato de Cálcio/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Glucose/metabolismo , Glicerol/metabolismo , Concentração de Íons de Hidrogênio , Viabilidade Microbiana , Mutagênese , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Yarrowia/genética , Yarrowia/metabolismoRESUMO
Our aim is to construct a practical dynamic-simulation system that can model the metabolic and regulatory processes involved in the production of primary metabolites, such as amino acids. We have simulated the production of glutamate by transient batch-cultivation using a model of Escherichia coli central metabolism. Kinetic data were used to produce both the metabolic parts of the model, including the phosphotransferase system, glycolysis, the pentose-phosphate pathway, the tricarboxylic acid cycle, the glyoxylate shunt, and the anaplerotic pathways, and the regulatory parts of the model, including regulation by transcription factors, cyclic AMP receptor protein (CRP), making large colonies protein (Mlc), catabolite repressor/activator (Cra), pyruvate dehydrogenase complex repressor (PdhR), and acetate operon repressor (IclR). RNA polymerase and ribosome concentrations were expressed as a function of the specific growth rate, mu, corresponding to the changes in the growth rate during batch cultivation. Parameter fitting was performed using both extracellular concentration measurements and in vivo enzyme activities determined by (13)C flux analysis. By manual adjustment of the parameters, we simulated the batch fermentation of glucose or fructose by a wild-type strain (MG1655) and a glutamate-producing strain (MG1655 Delta sucA). The differences caused by the carbon source, and by wild-type and glutamate-producing strains, were clearly shown by the simulation. A sensitivity analysis revealed the factors that could be altered to improve the production process. Furthermore, an in silico deletion experiments could suggested the existence of uncharacterized regulation. We concluded that our simulation model could function as a new tool for the rational improvement and design of metabolic and regulatory networks.
Assuntos
Escherichia coli/metabolismo , Ácido Glutâmico/biossíntese , Modelos Biológicos , Carbono/farmacologia , Simulação por Computador , Escherichia coli/efeitos dos fármacos , Frutose/farmacologia , Malato Desidrogenase/genética , Malatos/metabolismo , Reprodutibilidade dos Testes , Ativação Transcricional/efeitos dos fármacosRESUMO
The twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum has been described previously. The minimal functional Tat system in C. glutamicum required TatA and TatC but did not require TatB, although this component was required for maximal efficiency of Tat-dependent secretion. We previously demonstrated that Chryseobacterium proteolyticum pro-protein glutaminase (pro-PG) and Streptomyces mobaraensis pro-transglutaminase (pro-TG) could be secreted via the Tat pathway in C. glutamicum. Here we report that the amounts of pro-PG secreted were more than threefold larger when TatC or TatAC was overexpressed, and there was a further threefold increase when TatABC was overexpressed. These results show that the amount of TatC protein is the first bottleneck and the amount of TatB protein is the second bottleneck in Tat-dependent protein secretion in C. glutamicum. In addition, the amount of pro-TG that accumulated via the Tat pathway when TatABC was overexpressed with the TorA signal peptide in C. glutamicum was larger than the amount that accumulated via the Sec pathway. We concluded that TatABC overexpression improves Tat-dependent pro-PG and pro-TG secretion in C. glutamicum.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Dosagem de Genes , Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Chryseobacterium/enzimologia , Chryseobacterium/genética , Proteínas Recombinantes/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Transglutaminases/genética , Transglutaminases/metabolismoRESUMO
The phosphotransferase system (PTS) is the sugar transportation machinery that is widely distributed in prokaryotes and is critical for enhanced production of useful metabolites. To increase the glucose uptake rate, we propose a rational strategy for designing the molecular architecture of the Escherichia coli glucose PTS by using a computer-aided design (CAD) system and verified the simulated results with biological experiments. CAD supports construction of a biochemical map, mathematical modeling, simulation, and system analysis. Assuming that the PTS aims at controlling the glucose uptake rate, the PTS was decomposed into hierarchical modules, functional and flux modules, and the effect of changes in gene expression on the glucose uptake rate was simulated to make a rational strategy of how the gene regulatory network is engineered. Such design and analysis predicted that the mlc knockout mutant with ptsI gene overexpression would greatly increase the specific glucose uptake rate. By using biological experiments, we validated the prediction and the presented strategy, thereby enhancing the specific glucose uptake rate.
Assuntos
Desenho Assistido por Computador , Escherichia coli/metabolismo , Glucose/metabolismo , Fosfotransferases/metabolismo , Adenilil Ciclases/metabolismo , Algoritmos , Simulação por Computador , AMP Cíclico/metabolismo , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Modelos Biológicos , Mutação , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems.
Assuntos
Corynebacterium glutamicum/enzimologia , Glutaminase/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Glutaminase/genética , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Proteoma/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.
Assuntos
Aminoácidos/biossíntese , Aminoácidos/metabolismo , Modelos Biológicos , Reatores Biológicos , Vias Biossintéticas , Isótopos de Carbono , Cromatografia Líquida , Escherichia coli/metabolismo , Proteínas/metabolismo , Espectrometria de Massas em TandemRESUMO
L-glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and L-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces L-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that L-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the L-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the L-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on L-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to L-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to L-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.
Assuntos
Corynebacterium glutamicum/metabolismo , Glutamatos/biossíntese , Complexo Cetoglutarato Desidrogenase/deficiência , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Complexo Cetoglutarato Desidrogenase/genética , Complexo Cetoglutarato Desidrogenase/metabolismoRESUMO
Compared to those of other gram-positive bacteria, the genetic structure of the Corynebacterium glutamicum Tat system is unique in that it contains the tatE gene in addition to tatA, tatB, and tatC. The tatE homologue has been detected only in the genomes of gram-negative enterobacteria. To assess the function of the C. glutamicum Tat pathway, we cloned the tatA, tatB, tatC, and tatE genes from C. glutamicum ATCC 13869 and constructed mutants carrying deletions of each tat gene or of both the tatA and tatE genes. Using green fluorescent protein (GFP) fused with the twin-arginine signal peptide of the Escherichia coli TorA protein, we demonstrated that the minimal functional Tat system required TatA and TatC. TatA and TatE provide overlapping function. Unlike the TatB proteins from gram-negative bacteria, C. glutamicum TatB was dispensable for Tat function, although it was required for maximal efficiency of secretion. The signal peptide sequence of the isomaltodextranase (IMD) of Arthrobacter globiformis contains a twin-arginine motif. We showed that both IMD and GFP fused with the signal peptide of IMD were secreted via the C. glutamicum Tat pathway. These observations indicate that IMD is a bona fide Tat substrate and imply great potential of the C. glutamicum Tat system for industrial production of heterologous folded proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Arthrobacter/enzimologia , Arthrobacter/metabolismo , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Dextranase/metabolismo , Proteínas de Escherichia coli , Deleção de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microbiologia Industrial/métodos , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Dobramento de Proteína , Análise de Sequência de DNARESUMO
This work demonstrates a novel computational approach combining flux balance modeling with statistical methods to identify correlations among fluxes in a metabolic network, providing insight as to how the fluxes should be redirected to achieve maximum product yield. The procedure is demonstrated using the example of amino acid production from an industrial Escherichia coli production strain and a hypothetical engineered strain overexpressing two heterologous genes. Regression analysis based on a random sampling of 5,000 points within the feasible solution space of the E. coli stoichiometric network suggested that increased activity of the glyoxylate cycle or PEP carboxylase and elimination of malic enzyme will improve lysine and arginine synthesis.
Assuntos
Aminoácidos/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Modelos Biológicos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Aminoácidos/genética , Simulação por Computador , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Lineares , Modelos Estatísticos , Análise Multivariada , Análise de Componente Principal , Análise de RegressãoRESUMO
We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.
Assuntos
Corynebacterium glutamicum/genética , Replicação do DNA/genética , Vetores Genéticos/genética , Temperatura , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Componentes do Gene , Hidroxilamina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Análise de Sequência de DNARESUMO
Although the enhancement of amino-acid synthesis by guanosine-3',5'-tetraphosphate (ppGpp) is well known, the effect of intracellular ppGpp levels on amino-acid overproduction in Escherichia coli has not been investigated. In this study, we demonstrate that overexpression of the relA gene, encoding ppGpp synthetase, increases the accumulation of amino acids, such as glutamate and lysine, in amino-acid-overproducing strains of E. coli. Elevation of intracellular ppGpp levels due to depletion of required amino acids also enhances glutamate overproduction. Moreover, the extent of overproduction is highly dependent on the intracellular ppGpp level. These results demonstrate that amino-acid overproduction in E. coli is closely connected to amino-acid auxotrophy via the accumulation of ppGpp.
Assuntos
Escherichia coli/metabolismo , Ácido Glutâmico/biossíntese , Guanosina Tetrafosfato/farmacologia , Lisina/biossíntese , Aminoácidos/biossíntese , Escherichia coli/crescimento & desenvolvimento , Guanosina Tetrafosfato/metabolismo , Espaço Intracelular/metabolismo , Fator de Transcrição RelA/genética , Transformação BacterianaRESUMO
Using a DNA macroarray, we investigated the effects of rmf gene (encoding ribosome modulation factor) disruption on gene expression profiles in Escherichia coli. This strain showed a phosphate-starvation-like response in gene expression even under phosphate sufficient conditions; significant upregulation of the Pho regulon genes was observed. Further, the production of alkaline phosphatase, a product of the Pho regulon gene, phoA, increased in the rmf disruptant under a Pi sufficient condition. Furthermore, production of PhoC acid phosphatase/nucleoside pyrophosphate phosphotransferase derived from Morganella morganii also increased significantly in the rmf disruptant. We concluded that host modification by the rmf gene disruption has potential benefit in industrial enzyme production using Escherichia coli.
Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Regiões Promotoras Genéticas/genética , Regulon/genética , Proteínas Ribossômicas/metabolismo , Fosfatase Alcalina/metabolismo , Sequência de Bases , Proliferação de Células/efeitos dos fármacos , Meios de Cultura , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/genética , Dados de Sequência Molecular , Fosfatos/farmacologia , Proteínas Ribossômicas/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Lisina/biossíntese , Proteínas Ribossômicas/fisiologia , Sequência de Bases , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Fosfatos/metabolismo , Proteínas Ribossômicas/genéticaRESUMO
Corynebacterium glutamicum, which is the closest relative of Corynebacterium efficiens, is widely used for the large scale production of many kinds of amino acids, particularly glutamic acid and lysine, by fermentation. Corynebacterium diphtheriae, which is well known as a human pathogen, is also closely related to these two species of Corynebacteria, but it lacks such productivity of amino acids. It is an important and interesting question to ask how those closely related bacterial species have undergone such significant functional differentiation in amino acid biosynthesis. The main purpose of the present study is to clarify the evolutionary process of functional differentiation among the three species of Corynebacteria by conducting a comparative analysis of genome sequences. When Mycobacterium and Streptomyces were used as out groups, our comparative study suggested that the common ancestor of Corynebacteria already possessed almost all of the gene sets necessary for amino acid production. However, C. diphtheriae was found to have lost the genes responsible for amino acid production. Moreover, we found that the common ancestor of C. efficiens and C. glutamicum have acquired some of genes responsible for amino acid production by horizontal gene transfer. Thus, we conclude that the evolutionary events of gene loss and horizontal gene transfer must have been responsible for functional differentiation in amino acid biosynthesis of the three species of Corynebacteria.
Assuntos
Aminoácidos/biossíntese , Corynebacterium/genética , Corynebacterium/metabolismo , Genoma Bacteriano , Composição de Bases , Corynebacterium/patogenicidade , Corynebacterium diphtheriae/genética , Corynebacterium diphtheriae/metabolismo , Corynebacterium diphtheriae/patogenicidade , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , Evolução Molecular , Deleção de Genes , Transferência Genética Horizontal , Genes Bacterianos , Humanos , Modelos Biológicos , Fases de Leitura Aberta , Filogenia , Especificidade da EspécieRESUMO
Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids. C. efficiens but not C. glutamicum can grow above 40 degrees C. We sequenced the complete C. efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs. Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of the C. efficiens proteins: from lysine to arginine, serine to alanine, and serine to threonine. Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content of C. efficiens.