Yoshihiro Usuda

Altered Metabolic Flux due to Deletion of odhA causes l-Glutamate Overproduction in Corynebacterium glutamicum

ABSTRACT 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 in biotechnology processes

Metabolic flux analysis (MFA) has become a fundamental tool of metabolic engineering to elucidate the metabolic state of the cell and has been applied to various biotechnological processes. In recent years, considerable technical advances have been made. Developments of analytical instruments allow us to determine 13C labeling distribution of intracellular metabolites with high accuracy and sensitivity. Moreover, kinetic information of intracellular label distribution during isotopic instationary enables us to calculate metabolic fluxes with shortened experimental time and decreased amount of labeled substrate. The 13C MFA may be one of the most promising approaches for the target estimation to improve strain performances and production processes.

Dynamic modeling of Escherichia coli metabolic and regulatory systems for amino-acid production.

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.

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.

Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions.

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.

Effects of Deregulation of Methionine Biosynthesis on Methionine Excretion in Escherichia coli

ABSTRACT Several regulators of methionine biosynthesis have been reported in Escherichia coli, which might represent barriers to the production of excess l-methionine (Met). In order to examine the effects of these factors on Met biosynthesis and metabolism, deletion mutations of the methionine repressor (metJ) and threonine biosynthetic (thrBC) genes were introduced into the W3110 wild-type strain of E. coli. Mutations of the metK gene encoding S-adenosylmethionine synthetase, which is involved in Met metabolism, were detected in 12 norleucine-resistant mutants. Three of the mutations in the metK structural gene were then introduced into metJ and thrBC double-mutant strains; one of the resultant strains was found to accumulate 0.13 g/liter Met. Mutations of the metA gene encoding homoserine succinyltransferase were detected in α-methylmethionine-resistant mutants, and these mutations were found to encode feedback-resistant enzymes in a 14C-labeled homoserine assay. Three metA mutations were introduced, using expression plasmids, into an E. coli strain that was shown to accumulate 0.24 g/liter Met. Combining mutations that affect the deregulation of Met biosynthesis and metabolism is therefore an effective approach for the production of Met-excreting strains.

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.

Structural basis for amino acid export by DMT superfamily transporter YddG

The drug/metabolite transporter (DMT) superfamily is a large group of membrane transporters ubiquitously found in eukaryotes, bacteria and archaea, and includes exporters for a remarkably wide range of substrates, such as toxic compounds and metabolites. YddG is a bacterial DMT protein that expels aromatic amino acids and exogenous toxic compounds, thereby contributing to cellular homeostasis. Here we present structural and functional analyses of YddG. Using liposome-based analyses, we show that Escherichia coli and Starkeya novella YddG export various amino acids. The crystal structure of S. novella YddG at 2.4 Å resolution reveals a new membrane transporter topology, with ten transmembrane segments in an outward-facing state. The overall structure is basket-shaped, with a large substrate-binding cavity at the centre of the molecule, and is composed of inverted structural repeats related by two-fold pseudo-symmetry. On the basis of this intramolecular symmetry, we propose a structural model for the inward-facing state and a mechanism of the conformational change for substrate transport, which we confirmed by biochemical analyses. These findings provide a structural basis for the mechanism of transport of DMT superfamily proteins.

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for l-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H2O2) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsAD210Y identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and l-lysine production from fatty acid. Introduction of this rpsAD210Y mutation into a wild-type strain resulted in lower H2O2 concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H2O2 concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H2O2 concentrations and promoted E. coli fatty acid utilization and l-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted l-lysine production from fatty acids and decreased intracellular H2O2 concentrations. Among the ROS generated by fatty-acid β-oxidation, H2O2 critically affected E. coli growth and l-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.

Vibrio algivorus sp. nov., an alginate and agarose assimilating bacterium isolated from the gut flora of a turban shell marine snail.

An agarose- and alginate-assimilating, Gram-reaction-negative, non-motile, rod-shaped bacterium, designated strain SA2T, was isolated from the gut of a turban shell sea snail (Turbo cornutus) collected near Noto Peninsula, Ishikawa Prefecture, Japan. The 16S rRNA gene sequence of strain SA2T was 99.59 % identical to that of Vibrio rumoiensis DSM 19141T and 98.19 % identical to that of Vibrio litoralis DSM 17657T. This suggested that strain SA2T could be a subspecies of V. rumoiensis or V. litoralis. However, DNA-DNA hybridization results showed only 37.5 % relatedness to DSM 19141T and 44.7 % relatedness to DSM 17657T, which was far lower than the 70 % widely accepted to define common species. Strain SA2T could assimilate agarose as a sole carbon source, whereas strains DSM 19141T and DSM 17657T could not assimilate it at all. Furthermore, results using API 20NE and API ZYM kits indicated that their enzymic and physiological phenotypes were also different. These results suggested that strain SA2T represented a novel species within the genus Vibrio. The major isoprenoid quinone in SA2T was Q-8, and its major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major fatty acids were summed feature 3, (comprising C16 : 1ω6c and/or C16 : 1ω7c), C16 : 0, and summed feature 8 (comprising C18 : 1ω6c and/or C18 : 1ω7c). The DNA G+C content of SA2T was 40.7 mol%. The name proposed for this novel species of the genus Vibrio is Vibrio algivorus sp. nov., with the type strain designated SA2T (=DSM 29824T=NBRC 111146T).