Yousuke Nishio

The genome stability in Corynebacterium species due to lack of the recombinational repair system.

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.

Computer-aided rational design of the phosphotransferase system for enhanced glucose uptake in Escherichia coli

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.

Analysis of L-glutamic acid fermentation by using a dynamic metabolic simulation model of Escherichia coli.

BackgroundUnderstanding the process of amino acid fermentation as a comprehensive system is a challenging task. Previously, we developed a literature-based dynamic simulation model, which included transcriptional regulation, transcription, translation, and enzymatic reactions related to glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and the anaplerotic pathway of Escherichia coli. During simulation, cell growth was defined such as to reproduce the experimental cell growth profile of fed-batch cultivation in jar fermenters. However, to confirm the biological appropriateness of our model, sensitivity analysis and experimental validation were required.ResultsWe constructed an l-glutamic acid fermentation simulation model by removing sucAB, a gene encoding α-ketoglutarate dehydrogenase. We then performed systematic sensitivity analysis for l-glutamic acid production; the results of this process corresponded with previous experimental data regarding l-glutamic acid fermentation. Furthermore, it allowed us to predicted the possibility that accumulation of 3-phosphoglycerate in the cell would regulate the carbon flux into the TCA cycle and lead to an increase in the yield of l-glutamic acid via fermentation. We validated this hypothesis through a fermentation experiment involving a model l-glutamic acid-production strain, E. coli MG1655 ΔsucA in which the phosphoglycerate kinase gene had been amplified to cause accumulation of 3-phosphoglycerate. The observed increase in l-glutamic acid production verified the biologically meaningful predictive power of our dynamic metabolic simulation model.ConclusionsIn this study, dynamic simulation using a literature-based model was shown to be useful for elucidating the precise mechanisms involved in fermentation processes inside the cell. Further exhaustive sensitivity analysis will facilitate identification of novel factors involved in the metabolic regulation of amino acid fermentation.

Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

ABSTRACT Lowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolated Enterobacter aerogenes strain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introduced Actinobacillus succinogenes phosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK and Corynebacterium glutamicum pyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.

Metabolic Control of the TCA cycle by the YdcI Transcriptional Regulatorin Escherichia coli

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.

Characterization of feedback-resistant mevalonate kinases from the methanogenic archaeons Methanosaeta concilii and Methanocella paludicola

The inhibition of mevalonate kinase (MVK) by downstream metabolites is an important mechanism in the regulation of isoprenoid production in a broad range of organisms. The first feedback-resistant MVK was previously discovered in the methanogenic archaeon Methanosarcinamazei. Here, we report the cloning, expression, purification, kinetic characterization and inhibition analysis of MVKs from two other methanogens, Methanosaetaconcilii and Methanocellapaludicola. Similar to the M. mazei MVK, these enzymes were not inhibited by diphosphomevalonate (DPM), dimethylallyl diphosphate (DMAPP), isopentenyldiphosphate (IPP), geranylpyrophosphate (GPP) or farnesylpyrophosphate (FPP). However, they exhibited significantly higher affinity to mevalonate and higher catalytic efficiency than the previously characterized enzyme.

Analysis of strain-specific genes in glutamic acid-producing Corynebacterium glutamicum ssp. lactofermentum AJ 1511

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.

Comparative Whole Genome Sequence Analysis of Corynebacteria

Complete genome sequences are available for three corynebacterial species: Corynebacterium glutamicum, which is widely used for industrial amino acid production by fermentation; Corynebacterium efficiens, which produces glutamic acid at a higher temperature than C. glutamicum; and Corynebacterium diphtheriae, which is a well-known pathogenic bacterium. Comparative genomic studies highlighted the evolutionary mechanisms underlying various aspects of the functional differentiation of these species, such as the unique metabolic features and thermostability of C. efficiens. The GC content of the C. efficiens genome amounted to 63.1 %, which was approximately 10 % higher than those of C. glutamicum and C. diphtheriae were. This difference was reflected in codon usage and nucleotide substitutions. Analyzing orthologous gene pairs with 60–95 % amino acid sequence identity between C. efficiens and C. glutamicum revealed a significant bias in amino acid substitutions. In particular, accumulations of three asymmetrical amino acid substitutions (lysine to arginine, serine to alanine, and serine to threonine) were associated with the thermostability and increased GC content of C. efficiens. A phylogenetic tree constructed using Mycobacterium and Streptomyces as outgroups indicated that the common ancestor of the corynebacteria was likely to have possessed most of the gene sets necessary for amino acid production. C. diphtheriae appeared to have lost the genes responsible for amino acid production. Glutamate overproduction in C. glutamicum was induced by a shortage of biotin, and this bacterium showed an incomplete biotin biosynthesis pathway. By contrast, C. diphtheriae might have acquired the complete biotin biosynthesis pathway by horizontal gene transfer. This process could have affected metabolic regulation in the corynebacteria following the loss of the glutamate overproduction mechanism in C. diphtheriae. Our findings suggest that dynamic genome evolution has been a motivational force for functional differentiation among the corynebacteria.