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

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%.

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

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 %.

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

2004 ◽  
Vol 70 (12) ◽  
pp. 7277-7287 ◽  
Author(s):  
Christoph Wittmann ◽  
Patrick Kiefer ◽  
Oskar Zelder
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Flux Analysis ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Metabolic Fluxes ◽  
Fructose 1,6 Bisphosphate

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.

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

2013 ◽  
Vol 79 (8) ◽  
pp. 2588-2595 ◽  
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.

Increased glucose utilization and cell growth of Corynebacterium glutamicum by modifying the glucose-specific phosphotransferase system (PTSGlc) genes

2016 ◽  
Vol 62 (12) ◽  
pp. 983-992 ◽  
Author(s):  
Jianzhong Xu ◽  
Junlan Zhang ◽  
Dongdong Liu ◽  
Weiguo Zhang
Keyword(s):  
Growth Rate ◽  
Cell Growth ◽  
Corynebacterium Glutamicum ◽  
Production Rate ◽  
Glucose Consumption ◽  
Chemical Mutagenesis ◽  
Transport Systems ◽  
Phosphotransferase System ◽  
Lysine Production ◽  
Consumption Growth

The phosphoenolpyruvate:glucose phosphotransferase system (PTSGlc) is the major pathway of glucose uptake in Corynebacterium glutamicum. This study investigated glucose consumption rate, cell growth, and metabolite changes resulting from modification of PTSGlc. The classical l-lysine producer C. glutamicum XQ-8 exhibited low glucose consumption, cell growth, and l-lysine production rates, whereas these parameters were significantly increased during cultivating on glucose plus maltose, through inactivation of SugR, or by overexpression of PTSGlc genes. XQ-8sugR::cat/pDXW-8-ptsI exhibited the highest increase in glucose consumption, growth rate, and l-lysine production, followed by XQ-8sugR::cat/pDXW-8-ptsG. However, overexpression of ptsH had little effect on the above-mentioned factors. Although co-overexpression of ptsGHI led to the highest glucose consumption, growth rate, and final l-lysine production; the l-lysine production rate was lower than that of XQ-8sugR::cat/pDXW-8-ptsIH. In fed-batch fermentation, XQ-8sugR::cat/pDXW-8-ptsIH had a higher growth rate of 0.54 h−1 to a dry cell mass of 66 g·L−1 after 16 h, and had a higher l-lysine production rate of 159.2 g·L−1 after 36 h. These results indicate that modification of the sugar transport systems improves amino acid production, especially for mutants obtained by repeated physical and (or) chemical mutagenesis. However, modification of these systems needs to be performed on a case-by-case basis.

scholarly journals 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

2005 ◽  
Vol 71 (12) ◽  
pp. 8587-8596 ◽  
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.

scholarly journals Regulation of ldh expression during biotin-limited growth of Corynebacterium glutamicum

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1360-1375 ◽  
Author(s):  
Christiane Dietrich ◽  
Aimé Nato ◽  
Bruno Bost ◽  
Pierre Le Maréchal ◽  
Armel Guyonvarch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Time Course ◽  
Site Directed Mutagenesis ◽  
Transcriptional Level ◽  
Phosphotransferase System ◽  
Limited Growth ◽  
Mobility Shift ◽  
Batch Cultures ◽  
Fructose 1,6 Bisphosphate ◽  
Electrophoretic Mobility Shift Assays

Corynebacterium glutamicum is a biotin-auxotrophic bacterium and some strains efficiently produce glutamic acid under biotin-limiting conditions. In an effort to understand C. glutamicum metabolism under biotin limitation, growth of the type strain ATCC 13032 was investigated in batch cultures and a time-course analysis was performed. A transient excretion of organic acids was observed and we focused our attention on lactate synthesis. Lactate synthesis was due to the ldh-encoded l-lactate dehydrogenase (Ldh). Features of Ldh activity and ldh transcription were analysed. The ldh gene was shown to be regulated at the transcriptional level by SugR, a pleiotropic transcriptional repressor also acting on most phosphotransferase system (PTS) genes. Electrophoretic mobility shift assays (EMSAs) and site-directed mutagenesis allowed the identification of the SugR-binding site. Effector studies using EMSAs and analysis of ldh expression in a ptsF mutant revealed fructose 1-phosphate as a highly efficient negative effector of SugR. Fructose 1,6-bisphosphate also affected SugR binding.

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

2018 ◽  
Vol 85 (1) ◽  
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.

scholarly journals The DeoR-Type Regulator SugR Represses Expression of ptsG in Corynebacterium glutamicum

2007 ◽  
Vol 189 (8) ◽  
pp. 2955-2966 ◽  
Author(s):  
Verena Engels ◽  
Volker F. Wendisch
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Dna Microarray ◽  
Sole Carbon Source ◽  
Glucose Utilization ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Phosphotransferase System ◽  
High Fructose ◽  
Promoter Dna

ABSTRACT Corynebacterium glutamicum grows on a variety of carbohydrates and organic acids. Uptake of the preferred carbon source glucose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) is reduced during coutilization of glucose with acetate, sucrose, or fructose compared to growth on glucose as the sole carbon source. Here we show that the DeoR-type regulator SugR (NCgl1856) represses expression of ptsG, which encodes the glucose-specific PTS enzyme II. Overexpression of sugR resulted in reduced ptsG mRNA levels, decreased glucose utilization, and perturbed growth on media containing glucose. In mutants lacking sugR, expression of the ptsG′-′cat fusion was increased two- to sevenfold during growth on gluconeogenic carbon sources but remained similar during growth on glucose or other sugars. As shown by DNA microarray analysis, SugR also regulates expression of other genes, including ptsS and the putative NCgl1859-fruK-ptsF operon. Purified SugR bound to DNA regions upstream of ptsG, ptsS, and NCgl1859, and a 75-bp ptsG promoter fragment was sufficient for SugR binding. Fructose-6-phosphate interfered with binding of SugR to the ptsG promoter DNA. Thus, while during growth on gluconeogenic carbon sources SugR represses ptsG, ptsG expression is derepressed during growth on glucose or under other conditions characterized by high fructose-6-phosphate concentrations, representing one mechanism which allows C. glutamicum to adapt glucose uptake to carbon source availability.

scholarly journals Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases

2011 ◽  
Vol 77 (11) ◽  
pp. 3571-3581 ◽  
Author(s):  
Steffen N. Lindner ◽  
Gerd M. Seibold ◽  
Alexander Henrich ◽  
Reinhard Krämer ◽  
Volker F. Wendisch
Keyword(s):  
Glucose Uptake ◽  
Corynebacterium Glutamicum ◽  
Glucose Utilization ◽  
Growth Defect ◽  
Phosphotransferase System ◽  
Glucokinase Gene ◽  
Lysine Production ◽  
Content Type ◽  
Suppressor Mutations ◽  
Glucose Phosphorylation

ABSTRACTPhosphoenolpyruvate-dependent glucose phosphorylation via the phosphotransferase system (PTS) is the major path of glucose uptake inCorynebacterium glutamicum, but some growth from glucose is retained in the absence of the PTS. The growth defect of a deletion mutant lacking the general PTS component HPr in glucose medium could be overcome by suppressor mutations leading to the high expression of inositol utilization genes or by the addition of inositol to the growth medium if a glucokinase is overproduced simultaneously. PTS-independent glucose uptake was shown to require at least one of the inositol transporters IolT1 and IolT2 as a mutant lacking IolT1, IolT2, and the PTS component HPr could not grow with glucose as the sole carbon source. Efficient glucose utilization in the absence of the PTS necessitated the overexpression of a glucokinase gene in addition to eitheriolT1oriolT2. IolT1 and IolT2 are low-affinity glucose permeases withKsvalues of 2.8 and 1.9 mM, respectively. As glucose uptake and phosphorylation via the PTS differs from glucose uptake via IolT1 or IolT2 and phosphorylation via glucokinase by the requirement for phosphoenolpyruvate, the roles of the two pathways forl-lysine production were tested. Thel-lysine yield byC. glutamicumDM1729, a rationally engineeredl-lysine-producing strain, was lower than that by its PTS-deficient derivate DM1729Δhpr, which, however, showed low production rates. The combined overexpression ofiolT1oriolT2withppgK, the gene for PolyP/ATP-dependent glucokinase, in DM1729Δhprenabledl-lysine production as fast as that by the parent strain DM1729 but with 10 to 20% higherl-lysine yield.

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