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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.

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.

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.

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, μ, 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 13C 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 ΔsucA). 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.

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 .

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.

Metabolic flux analysis using 13C-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 13C 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.

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.

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.

Understanding the regulation and control of the expression of genes encoding metabolic enzymes is crucial for production using microbes. To overcome technical difficulties involved in identifying regulatory network systems, we designed a DNA motif finding procedure combining transcriptome and genome sequence data. Here, we used the ArcAB two-component system of Escherichia coli, which controls genes involved in the TCA cycle and energy metabolism, as a model to identify DNA motifs involved in gene-expression regulation. DNA-array data were used to extract up-regulated genes from ΔarcA and ΔarcB E. coli strains, and the upstream sequences were subjected to DNA-motif finding. Sequence similarity and conserved residues identified the known ArcA-binding motif and a novel DNA-motif candidate that was estimated to be related to YdcI, a putative LysR-type transcriptional regulator. A hypothetical YdcI-binding motif was found upstream of the gltA gene, suggesting that YdcI might control the carbon flux into the TCA cycle. To verify this, l-glutamic-acid production and citrate synthase activity in the ydcI gene-amplified strain were investigated. Our findings suggested that YdcI is a transcription factor that regulates the expression of gltA and other genes, and controls the carbon flux into the TCA cycle.

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.

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.

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.

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.

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°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.

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.

Two kinds of 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase genes were cloned from an L-Phe-producing mutant of Brevibacterium lactofermentum, AJ11957, which was resistant to m-fluoro-D, L-phenylalanine (mFP) and p-fluoro-D, L-phenylalanine (pFP) and which had DAHP synthase free from feedback inhibition. Both genes were cloned using a vector plasmid, pAJ1844, and the resulting recombinant plasmids were named pARl and pAR2. They had different structures on the restriction maps. Both plasmids, pARl and pAR2, conferred mFP and pFP resistance and L-Phe and L-Tyr productivity on a wild-type strain of B. lactofermentum, AJ 12036. The degrees of L-Phe-analogue resistance and aromatic amino acid productivity of the pARl-transformant were larger than those of the pAR2-transformant. The introduction of pARl to AJ12036 resulted in the alteration of DAHP synthase activity from a feedback-sensitive mechanism to a feedback-insensitive one accompanied by an elevated level of specific activity. A DAHP synthase deficient mutant of Escherichia coli was complemented by pAR2, but not by pARl. Characteristics of the two kinds of DAHP synthase genes are discussed.

The aroL gene, encoding shikimate kinase of Brevibacterium lactofermentum, a coryneform glutamic acid-producing bacterium, was cloned. Recombinant plasmids containing the aroL gene caused elevated levels of shikimate kinase synthesis in B. lactofermentum. It was found that in addition to the aroL gene, the aroB and aroE genes, encoding dehydroquinate synthase and shikimate dehydrogenase, respectively, also existed on these recombinant plasmids, in complementation tests with various Escherichia coli and B. lactofermentum aromatic amino acid auxotrophs. The aroL, aroB and aroE genes of B. lactofermentum are located closely on the cloned DNA fragment, in that order. It was shown that at least these three aro genes form a cluster on the chromosome of B. lactofermentum.

Five genes for tryptophan biosynthesis, trpE, trpD, trpC, trpB, and trpA of Brevibacterium lactofermentum, a coryne form glutamic acid-producing bacterium, were cloned as a 9.6 kb BamHl DNA fragment by colony hybridization. A previously cloned 1.2 kb Pst I DNA fragment containing a major part of the trpE gene was used as a probe. By complementation tests using the subclones of this 9.6 kb BamHl fragment and various tryptophan auxotrophs of B. lactofermentum and Escherichia coli, this fragment was found to contain a gene cluster composed of trpE, trpD, trpC, trpB, and trpA in this order. It suggests that genes for tryptophan biosynthesis in B. lactofermentum may be an operon.

The trp genes of Brevibacterium lactofermentum lie within a 7.72-kb HapII-BamHI fragment whose sequence has been determined (Matsui et al., 1986). The 5'- and the 3'-flanking regions of this gene cluster were subcloned as a 1.8-kb PstI-PstI and a 0.6-kb Xhol-BamHI fragment, respectively. The 5'-flanking region encodes two open reading frames (ORFs); one corresponds to trpL, while the other corresponds to the N-terminal half of the trpE gene. Within the 17 amino acid residues of the predicted leader peptide encoded by trpL are found three contiguous tryptophan residues. By subcloning parts of the 1.8-kb PstI-PstI fragment into promoter probe vectors, a promoter situated 32 bp upstream from the presumptive trpL gene was identified. The -35 and the -10 region of this promoter, TACACA and AATAAT, respectively, are very similar to the Escherichia coli trp promoter. The trp promoter of B. lactofermentum functions in E. coli. A 14-bp imperfect palindrome that overlaps the -10 region apparently functions in B. lactofermentum but not in E. coli as a trp operator. Upstream of trpE, attenuator-like sequences are found that resemble the corresponding E. coli sequences. The 0.6-kb Xhol-BamHI 3'-flanking fragment contains one ORF that encodes the C-terminal part of trpA. Downstream from trpA lies a Rho-independent terminator that resembles E. coli sequences that are situated downstream from the E. coli trp operon. Thus the trp control regions of the Gram-positive B. lactofermentum are more closely related in structure to the corresponding regions of the Gram-negative E. coli than to those of the Gram-positive Bacillus subtilis.