Lysine-producing Corynebacterium Glutamicum Research Articles

Expression of the NADPH+-Dependent Formate-Dehydrogenase Gene from Pseudomonas Increases Lysine Production in Corynebacterium glutamicum

The psefdh_D221Q gene encodes a mutant formate dehydrogenase from Pseudomonas (P-seFDG_D221Q), which catalyzes formate oxidation with the simultaneous formation of NADPH. It is expressed in cells of lysine-producing Corynebacterium glutamicum strains. The psefdh_D221Q gene was introduced into C. glutamicum strains as part of an autonomous plasmid or was integrated into the chromosome with the simultaneous inactivation of the host formate-dehydrogenase genes. It was shown that the C. glutamicum strains with NADP+-dependent formate dehydrogenase have an increased level of L-lysine synthesis in the presence of formate if their own formate dehydrogenase is inactivated.

Metabolic Engineering of Lysine Producing Corynebacterium glutamicum Strains

The review is devoted to the analysis of the current achievements of Corynebacterium glutamicum metabolic engineering for the production of lysine. Key genes of lysine biosynthesis in C. glutamicum and ways of creating new genetically modified strains are considered. The role of different plasmids, vector cassettes, and promoter types for the regulation of gene expression in C. glutamicum is described. Information is provided on the use of carbon-containing substrates (hexose, pentose, lactic acid, mannitol) for the production of lysine. Possibilities of using CRISPR technology in genetic engineering of C. glutamicum are considered. Genetic changes in C. glutamicum allowed the use of alternative substrates and contributed to the increase of lysine accumulation in the culture fluid. The data that may be used for the creation of new lysine overproduction strains are summarized.

Экспрессия гена НАДФ+-зависимой формиатдегидрогеназы из Рseudomonas повышает продукцию лизина в Сorynebacterium glutamicum

Thepsefdh_D221Q gene coding for a mutant formate dehydrogenase (PseFDG_D221Q) from Pseudomonas, which catalyzes the formate oxidation with the simultaneous formation of NADPH, has been expressed in the cells of lysine-producing Corynebacterium glutamicum strains. The psefdh_D221Q gene was introduced into С. glutamicum strains as part of an autonomous plasmid or was integrated into the chromosome with simultaneous inactivation of host formate dehydrogenase genes. It was shown that the С. glutamicum strains with NADP+ -dependent formate dehydrogenase have an increased level of L-lysine synthesis in the presence of formate, if their own formate dehydrogenase is inactivated. L-lysine, formate dehydrogenase, NADPH, Corynebacterium glutamicum The work was carried out using the equipment of the Multipurpose Scientific This work was carried out on the equipment of the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms», National Bio-Resource Center, NRC «Kurchatov Institute»- GosNIIgenetika. This work was financially supported by the Ministry of Education and Science of Russia (Unique Project Identifier - RFMEFI61017X0011).

The small ribosomal protein S12P gene rpsL as an efficient positive selection marker in allelic exchange mutation systems for Corynebacterium glutamicum

We report that the mutant rpsL K43R in streptomycin-resistant and lysine-producing Corynebacterium glutamicum is responsible for streptomycin resistance. In addition, we describe its effective application in gene modification in C. glutamicum.

Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

BackgroundPyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS) and phosphoenolpyruvate carboxylase (PEPC), whereby a reduced amount of carbon may be lost as CO2 due to reduced flux into the tricarboxylic acid (TCA) cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood.ResultsIn this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysCfbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield) which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC) to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose.ConclusionThe metabolic flux analysis performed illustrates the high flexibility of the metabolic network of C. glutamicum to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol, in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering.

Phytate utilization by genetically engineered lysine-producing Corynebacterium glutamicum

Heterologous expression of a phytase gene ( phyC) from Bacillus amyloliquefaciens DSM 7 enabled the growth of Corynebacterium glutamicum with phytate ( myo-inositol-1,2,3,4,5,6-hexakisphosphate) as a new, sole source of phosphorus. Phytate was not used as a carbon source. During growth of the phyC-expressing amino acid ( l-lysine)-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) with phytate as the source of phosphorus, merely a small, transient accumulation of inorganic phosphate was observed in the fermentation broth. At the later stages of fermentation, free inorganic phosphate from phytate degradation was no longer detectable. Growth and l-lysine production by the phytase-producing strain C. glutamicum ATCC 21253 (pWLQ2::phyC) in phytate medium did not differ significantly from control experiments with strain C. glutamicum ATCC 21253 (pWLQ2) conducted with an excess of inorganic phosphate, demonstrating that there was no phosphate limitation when phytate was used as the phosphorus source. Under the expression conditions employed, only part of PhyC was secreted to the culture broth by C. glutamicum, but this did not significantly affect growth or lysine production.

The effect of variations in intracellular pyruvate availability on acetohydroxy acid synthase activity and l-valine overproduction in Corynebacterium glutamicum

A methionine-producing strain was derived from a lysine-producing Corynebacterium glutamicum through a process of genetic manipulation in order to assess its potential to synthesize and accumulate methionine during growth. The strain carries a deregulated hom gene (homFBR) to abolish feedback inhibition of homoserine dehydrogenase by threonine and a deletion of the thrB gene (ΔthrB) to abolish threonine synthesis. The constructed C. glutamicum MH20-22B/homFBR/ΔthrB strain accumulated 2.9 g/l of methionine by batch fermentation and showed resistance to methionine analogue ethionine at concentrations up to 30 mM. The growth of the strain was apparently impaired as a result of the accumulation of methionine biosynthetic intermediate, homocysteine. Production assays also revealed that the accumulation of methionine in the growth medium was transient and declined as the carbon source was depleted. During the period of methionine disappearance, the methionine biosynthetic genes were completely repressed in the engineered strains but not in the parental strain. After all, we have not only successfully constructed a methionine-producing C. glutamicum strain by genetic manipulation, but also revealed cellular constraints in attaining high yield and productivity.

Characteristics of methionine production by an engineered Corynebacterium glutamicum strain

A methionine-producing strain was derived from a lysine-producing Corynebacterium glutamicum through a process of genetic manipulation in order to assess its potential to synthesize and accumulate methionine during growth. The strain carries a deregulated hom gene ( hom FBR) to abolish feedback inhibition of homoserine dehydrogenase by threonine and a deletion of the thrB gene (Δ thrB) to abolish threonine synthesis. The constructed C. glutamicum MH20-22B/ hom FBR/Δ thrB strain accumulated 2.9 g/l of methionine by batch fermentation and showed resistance to methionine analogue ethionine at concentrations up to 30 mM. The growth of the strain was apparently impaired as a result of the accumulation of methionine biosynthetic intermediate, homocysteine. Production assays also revealed that the accumulation of methionine in the growth medium was transient and declined as the carbon source was depleted. During the period of methionine disappearance, the methionine biosynthetic genes were completely repressed in the engineered strains but not in the parental strain. After all, we have not only successfully constructed a methionine-producing C. glutamicum strain by genetic manipulation, but also revealed cellular constraints in attaining high yield and productivity.

A Multi-Scale Modeling Concept and Computational Tools for the Integrative Analysis of Stationary Metabolic Data

SummaryMetabolic Engineering aims at the systematic analysis and targeted manipulation of the metabolism in biotechnologically utilized microorganisms [1,2]. Recently, consistent stationary in vivo data sets of intracellular metabolite concentrations, fluxes and specific enzyme activities have become available for this purpose [3,4]. For the integrative analysis of the metabolic data at hand, a novel multi-scale modeling concept, computeralgebraic methods and efficient numerical algorithms are proposed. The available metabolic data are typically afflicted with comparatively large measurement errors. Therefore, reliable comprehensive error estimations are essential for the reasonable interpretation of consecutive outcomes, such as simulation results. The concepts, methods and algorithms are first presented as universal methods and subsequently applied to the anaplerotic regulation in lysine-producing Corynebacterium glutamicum. A multi-scale model is set up, fitted to the available experimental data and validated by the prediction of further experiments. This model is capable of forecasting the quantitative effect of changes in the specific activity of anaplerotic enzymes, namely phosphoenolpyruvate carboxylase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase, on lysine productivity and yield.

Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale.

We present a straightforward approach comprising (13)C tracer experiments at 200-microL volume in 96-well microtiter plates with on-line measurement of dissolved oxygen for quantitative high-throughput metabolic network analysis at a miniaturized scale. This method was successfully applied for cultivation and (13)C metabolic flux analysis of two mutants of lysine producing Corynebacterium glutamicum (ATCC 13287 and ATCC 21543). Microtiter-plate cultivations showed excellent accordance in kinetics and stoichiometry of growth and product formation as well as in intracellular flux distributions as compared with parallel shake-flask experiments. These cultivations further allowed clear identification of strain-specific flux differences such as increased flux toward lysine, increased flux through the pentose phosphate pathway (PPP), decreased flux through the tricarboxylic (TCA) cycle, and increased dihydroxyacetone formation in C. glutamicum ATCC 21543 compared with ATCC 13287. The present approach has strong potential for broad quantitative screening of metabolic network activities, especially those involving high-cost tracer substrates.

Membrane Inlet Mass Spectrometry for the on‐line Measurement of Metabolic Fluxes: Case Lysine‐Production by Corynebacterium glutamicum

AbstractA miniaturized reactor system with on‐line measurement of respiration rates by membrane inlet mass spectrometry was applied for the on‐line metabolic flux analysis at different phases of a 1.2 L batch culture of lysine‐producing Corynebacterium glutamicum. For this purpose, cells taken from the batch culture were transferred into the 12 mL mini reactor, and incubated for 15 min with [1‐18O]glucose. Quantification of oxygen uptake rate and CO2 mass isotopomer production rates in combination with a simple metabolic model allowed the estimation of the flux partitioning ratio between the pentose phosphate pathway and glycolysis during the process. The relative flux into the pentose pathway increased during growth, and reached maxima at 11 and 17 h cultivation time coinciding with maxima of the differential lysine yield. The developed system is a promising tool for determination of metabolic flux dynamics in industrially relevant batch and fed‐batch cultures.

Metabolic network simulation using logical loop algorithm and Jacobian matrix

A novel method to accomplish efficient numerical simulation of metabolic networks for flux analysis was developed. The only inputs required are the set of stoichiometric balances and the atom mapping matrices of all components of the reaction network. The latter are used to automatically calculate isotopomer mapping matrices. Using the symbolic toolbox of MATLAB the analytical solution of the stoichiometric balance equation system, isotopomer balances and the analytical Jacobian matrix of the total set of stoichiometric and isotopomer balances are created automatically. The number of variables in the isotopomer distribution equation system is significantly reduced applying modified isotopomer mapping matrices. These allow lumping of several consecutive isotopomer reactions into a single one. The solution of the complete system of equations is improved by implementing an iterative logical loop algorithm and using the analytical Jacobian matrix. This new method provided quick and robust convergence to the root of such equation systems in all cases tested. The method was applied to a network of lysine producing Corynebacterium glutamicum. The resulting equation system with the dimension of 546×546 was directly derived from 12 isotopomer balance equations. The results obtained yielded identical labeling patterns for metabolites as compared to the relaxation method.

Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum: a novel approach for metabolic flux analysis.

In the present work, a novel comprehensive approach of (13)C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 microL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-(13)C glucose or with mixtures of (13)C6/(12)C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.

Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis

Metabolic carbon labelling experiments enable a large amount of extracellular fluxes and intracellular carbon isotope enrichments to be measured. Since the relation between the measured quantities and the unknown intracellular metabolic fluxes is given by bilinear balance equations, flux determination from this data set requires the numerical solution of a nonlinear inverse problem. To this end, a general algorithm for flux estimation from metabolic carbon labelling experiments based on the least squares approach is developed in this contribution and complemented by appropriate tools for statistical analysis. The linearization technique usually applied for the computation of nonlinear confidence regions is shown to be inappropriate in the case of large exchange fluxes. For this reason a sophisticated compactification transformation technique for nonlinear statistical analysis is developed. Statistical analysis is then performed by computing appropriate statistical quality measures like output sensitivities, parameter sensitivities and the parameter covariance matrix. This allows one to determine the order of magnitude of exchange fluxes in most practical situations. An application study with a large data set from lysine-producing Corynebacterium glutamicum demonstrates the power and limitations of the carbon-labelling technique. It is shown that all intracellular fluxes in central metabolism can be quantitated without assumptions on intracellular energy yields. At the same time several exchange fluxes are determined which is invaluable information for metabolic engineering.

Potential metabolic limitations in lysine production by Corynebacterium glutamicum as revealed by metabolic network analysis

Analysis of the metabolic network of lysine-producing Corynebacterium glutamicum showed that lysine yields are limited by the excess energy production in lysine biosynthesis. The most probable maximum yield is 0.47 mol/mol on glucose, when phosphoenolpyruvate carboxylase functions in an anaplerotic rection. When this function is fulfilled by the glyoxylate pathway, a maximum yield of 0.38 mol/mol is obtained.

Effects of phosphoenol pyruvate carboxylase deficiency on metabolism and lysine production in Corynebacterium glutamicum

The phosphoenol pyruvate carboxylase gene (ppc) of lysine-producing Corynebacterium glutamicum and C. lactofermentum strains was inactivated by marker exchange mutagenesis. The mutants lacked completely phosphoenol pyruvate carboxylase (PEP carboxylase) activity, but grew in minimal medium containing glucose as the sole carbon source. In addition, the ppc− strains produced equivalent titers of lysine in shake flasks and in 10-l fermentation experiments as their parent strains. To address the question of how ppc−Corynebacterium strains generate oxaloacetate (OAA) for their own metabolism as well as for high-level lysine production, we measured the activities of enzymes leading to OAA synthesis. Whereas pyruvate carboxylase activity was not detected in any of the strains, phosphoenol pyruvate carboxykinase (PEP carboxykinase) activity was found to be significantly higher in C. glutamicum ppc mutants compared to the parent strains. On the other hand, PEP carboxykinase activity in C. lactofermentum was essentially absent. As glyxylate cycle enzymes are strongly repressed by glucose, they are not likely to compensate for the lack of PEP carboxylase activity. PEP carboxykinase, among several candidates, could play this role.

Aspartokinase genes lysCα and lysCβ overlap and are adjacent to the aspartate β-semialdehyde dehydrogenase gene asd in Corynebacterium glutamicum

A 2.1 kb DNA fragment of the recombinant plasmid pCS2, isolated from an aminoethyl cysteine (AEC)-resistant and lysine-producing Corynebacterium glutamicum mutant strain, and which confers AEC resistance and lysine production on the wild-type G. glutamicum ATCC 13032 was analysed. DNA sequence analysis of this fragment revealed three large open reading frames (ORFs). The incomplete ORF1 does not contain the 5' end of the coding region. ORF2, which uses the same reading frame as ORF1, is identical to the 3' end of ORF1 and encodes a putative protein of 172 amino acids (aa) and of Mr 18,584. ORF3 encodes a putative protein of 344 aa and of Mr 36,275. The amino acid sequences deduced from ORF1 and ORF2 display strong homologies to those of the alpha- and beta-subunits of the Bacillus subtilis aspartokinase II. It is therefore proposed that the incomplete ORF1, termed lysC alpha, encodes part of the alpha-subunit of the C. glutamicum aspartokinase whereas the complete ORF2, termed lysC beta, encodes the beta-subunit of the same enzyme. ORF2 is responsible for AEC resistance and lysine production due to a feedback-resistant aspartokinase. The amino acid sequence deduced from ORF3, termed asd, is highly homologous to that of the Streptococcus mutans aspartate beta-semialdehyde dehydrogenase (ASD). Plasmids carrying the C. glutamicum asd gene complemented Escherichia coli asd mutants. Increase in ASD activity by a factor of 30-60 was measured for C. glutamicum cells harbouring high copy-number plasmids with the C. glutamicum asd gene.