Metabolic Fluxes in Corynebacterium glutamicum during Lysine Production with Sucrose as Carbon Source

ABSTRACT Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-13CFru]sucrose, [1-13CGlc]sucrose, and [13C6 Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTSMan or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.

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Evolving a New Efficient Mode of Fructose Utilization for Improved Bioproduction in Corynebacterium glutamicum

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.669093 ◽

2021 ◽

Vol 9 ◽

Author(s):

Irene Krahn ◽

Daniel Bonder ◽

Lucía Torregrosa-Barragán ◽

Dominik Stoppel ◽

Jens P. Krause ◽

...

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Pentose Phosphate ◽

Phosphotransferase System ◽

Lysine Production ◽

Depth Analysis ◽

Mutant Strains ◽

Phosphofructokinase Activity ◽

Fructose 1,6 Bisphosphate ◽

Product Accumulation

Fructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP-dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (∼10%) compared to glucose (∼60%). Consequently, the biosynthesis of NADPH demanding products, e.g., L-lysine, by C. glutamicum is largely decreased when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose-specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and, consequently, NADPH availability. Here, we generated deletion strains lacking either the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose-specific PTS as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose-specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, and P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased ODs, reduced side-product accumulation, and increased L-lysine production by 50%.

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Evolving a new efficient mode of fructose utilization for improved bioproduction in Corynebacterium glutamicum

10.1101/2021.02.18.431779 ◽

2021 ◽

Author(s):

Irene Krahn ◽

Daniel Bonder ◽

Lucia Torregrosa ◽

Dominik Stoppel ◽

Jens P. Krause ◽

...

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Pentose Phosphate ◽

Phosphotransferase System ◽

Lysine Production ◽

Depth Analysis ◽

Mutant Strains ◽

Phosphofructokinase Activity ◽

Fructose 1,6 Bisphosphate ◽

Product Accumulation

AbstractFructose utilization in Corynebacterium glutamicum starts with its uptake and concomitant phosphorylation via the phosphotransferase system (PTS) to yield intracellular fructose 1-phosphate, which enters glycolysis upon ATP dependent phosphorylation to fructose 1,6-bisphosphate by 1-phosphofructokinase. This is known to result in a significantly reduced oxidative pentose phosphate pathway (oxPPP) flux on fructose (~10 %) compared to glucose (~60 %). Consequently, the biosynthesis of NADPH demanding products, e.g. L-lysine, by C. glutamicum is largely decreased, when fructose is the only carbon source. Previous works reported that fructose is partially utilized via the glucose specific PTS presumably generating fructose 6-phosphate. This closer proximity to the entry point of the oxPPP might increase oxPPP flux and consequently NADPH availability. Here, we generated deletion strains either lacking in the fructose-specific PTS or 1-phosphofructokinase activity. We used these strains in short-term evolution experiments on fructose minimal medium and isolated mutant strains, which regained the ability of fast growth on fructose as a sole carbon source. In these fructose mutants, the deletion of the glucose specific PTS, as well as the 6-phosphofructokinase gene, abolished growth, unequivocally showing fructose phosphorylation via glucose specific PTS to fructose 6-phosphate. Gene sequencing revealed three independent amino acid substitutions in PtsG (M260V, M260T, P318S). These three PtsG variants mediated faster fructose uptake and utilization compared to native PtsG. In-depth analysis of the effects of fructose utilization via these PtsG variants revealed significantly increased biomass formation, reduced side-product accumulation, and increased L-lysine production by 50 %.

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Amplified Expression of Fructose 1,6-Bisphosphatase in Corynebacterium glutamicum Increases In Vivo Flux through the Pentose Phosphate Pathway and Lysine Production on Different Carbon Sources

Applied and Environmental Microbiology ◽

10.1128/aem.71.12.8587-8596.2005 ◽

2005 ◽

Vol 71 (12) ◽

pp. 8587-8596 ◽

Cited By ~ 140

Author(s):

Judith Becker ◽

Corinna Klopprogge ◽

Oskar Zelder ◽

Elmar Heinzle ◽

Christoph Wittmann

Keyword(s):

Corynebacterium Glutamicum ◽

Pentose Phosphate Pathway ◽

Metabolic Flux ◽

Carbon Sources ◽

Pentose Phosphate ◽

Metabolic Effects ◽

Flux Analysis ◽

Lysine Production ◽

Fructose 1,6 Bisphosphatase

ABSTRACT The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfb r . The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.

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Regulatory Tasks of the Phosphoenolpyruvate-Phosphotransferase System of Pseudomonas putida in Central Carbon Metabolism

mBio ◽

10.1128/mbio.00028-12 ◽

2012 ◽

Vol 3 (2) ◽

Cited By ~ 58

Author(s):

Max Chavarría ◽

Roelco J. Kleijn ◽

Uwe Sauer ◽

Katharina Pflüger-Grau ◽

Víctor de Lorenzo

Keyword(s):

Pseudomonas Putida ◽

Metabolic Networks ◽

Tca Cycle ◽

High Energy ◽

Pentose Phosphate ◽

Cell Physiology ◽

Phosphotransferase System ◽

Content Type ◽

Metabolic Fluxes ◽

Central Carbon

ABSTRACTTwo branches of the phosphoenolpyruvate-phosphotransferase system (PTS) operate in the soil bacteriumPseudomonas putidaKT2440. One branch encompasses a complete set of enzymes for fructose intake (PTSFru), while the other (N-related PTS, or PTSNtr) controls various cellular functions unrelated to the transport of carbohydrates. The potential of these two systems for regulating central carbon catabolism has been investigated by measuring the metabolic fluxes of isogenic strains bearing nonpolar mutations in PTSFruor PTSNtrgenes and grown on either fructose (a PTS substrate) or glucose, the transport of which is not governed by the PTS in this bacterium. The flow of carbon from each sugar was distinctly split between the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways in a ratio that was maintained in each of the PTS mutants examined. However, strains lacking PtsN (EIIANtr) displayed significantly higher fluxes in the reactions of the pyruvate shunt, which bypasses malate dehydrogenase in the TCA cycle. This was consistent with the increased activity of the malic enzyme and the pyruvate carboxylase found in the corresponding PTS mutants. Genetic evidence suggested that such a metabolic effect of PtsN required the transfer of high-energy phosphate through the system. The EIIANtrprotein of the PTSNtrthus helps adjust central metabolic fluxes to satisfy the anabolic and energetic demands of the overall cell physiology.IMPORTANCEThis study demonstrates that EIIANtrinfluences the biochemical reactions that deliver carbon between the upper and lower central metabolic domains for the consumption of sugars byP. putida. These findings indicate that the EIIANtrprotein is a key player for orchestrating the fate of carbon in various physiological destinations in this bacterium. Additionally, these results highlight the importance of the posttranslational regulation of extant enzymatic complexes for increasing the robustness of the corresponding metabolic networks.

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Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.70.7.4222-4229.2004 ◽

2004 ◽

Vol 70 (7) ◽

pp. 4222-4229 ◽

Cited By ~ 25

Author(s):

Cristian A. Varela ◽

Mauricio E. Baez ◽

Eduardo Agosin

Keyword(s):

Osmotic Stress ◽

Corynebacterium Glutamicum ◽

Metabolic Flux ◽

Cell Metabolism ◽

Compatible Solutes ◽

Flux Analysis ◽

High Dilution ◽

Metabolic Fluxes ◽

Cell Productivity ◽

Osmotic Stress Response

ABSTRACT Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.

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Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

10.1101/2021.01.29.428863 ◽

2021 ◽

Author(s):

Khushboo Borah ◽

Tom A. Mendum ◽

Nathaniel D. Hawkins ◽

Jane L. Ward ◽

Michael H. Beale ◽

...

Keyword(s):

Metabolic Network ◽

Metabolic Flux ◽

Carbon Sources ◽

Tca Cycle ◽

Flux Analysis ◽

Glyoxylate Shunt ◽

Citrate Cycle ◽

Metabolic Fluxes ◽

The Tca Cycle ◽

Carbon Substrates

AbstractThe utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

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The metabolic origins of non-photorespiratory CO2 release during photosynthesis: A metabolic flux analysis

Plant Physiology ◽

10.1093/plphys/kiab076 ◽

2021 ◽

Author(s):

Yuan Xu ◽

Xinyu Fu ◽

Thomas D Sharkey ◽

Yair Shachar-Hill ◽

Berkley J Walker

Keyword(s):

Gas Exchange ◽

Goodness Of Fit ◽

Time Course ◽

Metabolic Flux ◽

Carbon Fixation ◽

Tca Cycle ◽

Isotopic Labeling ◽

Pentose Phosphate ◽

Flux Analysis ◽

Oilseed Crop

Abstract Respiration in the light (RL) releases CO2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since RL lowers net carbon fixation, understanding RL could help improve plant carbon-use efficiency and models of crop photosynthesis. Although RL was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to RL, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13CO2 labeling time course, gas exchange and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N2; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that RL releases 5.2 μmol CO2 g−1 FW hr−1 of CO2, which is relatively consistent with a value of 9.3 μmol CO2 g−1 FW hr−1 measured by CO2 gas exchange. The results indicated that ≤10% of RL results from TCA cycle reactions, which are widely considered to dominate RL. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO2 released by RL.

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Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.01942-09 ◽

2009 ◽

Vol 75 (24) ◽

pp. 7866-7869 ◽

Cited By ~ 85

Author(s):

Judith Becker ◽

Corinna Klopprogge ◽

Hartwig Schröder ◽

Christoph Wittmann

Keyword(s):

Metabolic Engineering ◽

Corynebacterium Glutamicum ◽

Tricarboxylic Acid Cycle ◽

Tca Cycle ◽

Tricarboxylic Acid ◽

Start Codon ◽

Flux Analysis ◽

Lysine Production ◽

Acid Cycle ◽

The Tca Cycle

ABSTRACT In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.

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A Cyclic Metabolic Network inPseudomonas protegensPf-5 Prioritizes the Entner-Doudoroff Pathway and Exhibits Substrate Hierarchy during Carbohydrate Co-Utilization

Applied and Environmental Microbiology ◽

10.1128/aem.02084-18 ◽

2018 ◽

Vol 85 (1) ◽

Cited By ~ 7

Author(s):

Rebecca A. Wilkes ◽

Caroll M. Mendonca ◽

Ludmilla Aristilde

Keyword(s):

Metabolic Network ◽

Carbohydrate Metabolism ◽

Metabolic Flux ◽

Gene Annotation ◽

Pentose Phosphate ◽

Flux Analysis ◽

Environmental Matrices ◽

Content Type ◽

Phosphofructokinase Activity ◽

Fructose 1,6 Bisphosphate

ABSTRACTThe genetic characterization ofPseudomonas protegensPf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed13C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes inP. protegensPf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates inP. protegensPf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways.IMPORTANCESpecies of thePseudomonasgenus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism inPseudomonas protegensPf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network inP. protegensis important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untappedPseudomonasspecies.

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Phosphotransferase System-Mediated Glucose Uptake Is Repressed in Phosphoglucoisomerase-Deficient Corynebacterium glutamicum Strains

Applied and Environmental Microbiology ◽

10.1128/aem.03231-12 ◽

2013 ◽

Vol 79 (8) ◽

pp. 2588-2595 ◽

Cited By ~ 27

Author(s):

Steffen N. Lindner ◽

Dimitar P. Petrov ◽

Christian T. Hagmann ◽

Alexander Henrich ◽

Reinhard Krämer ◽

...

Keyword(s):

Amino Acid ◽

Glucose Uptake ◽

Corynebacterium Glutamicum ◽

Pentose Phosphate ◽

Phosphotransferase System ◽

Negative Effects ◽

Gene Encoding ◽

Lysine Production ◽

Content Type ◽

Model Strain

ABSTRACTCorynebacterium glutamicumis particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of thepgigene, encoding the phosphoglucoisomerase, has been applied for the improvement ofC. glutamicumamino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted thepgigene in the type strainC. glutamicumATCC 13032. The resulting strain,C. glutamicumΔpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake inC. glutamicumΔpgi. Furthermore, Northern blot analyses revealed that expression ofptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimizedl-lysine production in the model strainC. glutamicumDM1729 by deletion ofpgiand overexpression of plasmid-encodedptsG.l-Lysine yields and productivity withC. glutamicumΔpgi(pBB1-ptsG) were significantly higher than those withC. glutamicumΔpgi(pBB1). These results show thatptsGoverexpression is required to overcome the repressed activity of PTS-mediated glucose uptake inpgi-deficientC. glutamicumstrains, thus enabling efficient as well as fastl-lysine production.

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