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.

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.

scholarly journals The Phosphohistidine Phosphatase SixA Targets a Phosphotransferase System

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Jane E. Schulte ◽  
Mark Goulian
Keyword(s):  
Escherichia Coli ◽  
Protein Phosphatases ◽  
Growth Defect ◽  
Phosphotransferase System ◽  
Protein Activity ◽  
Content Type ◽  
Bacterial Physiology ◽  
Phosphoryl Groups ◽  
Regulate Protein ◽  
Protein Components

ABSTRACTSixA, a well-conserved protein found in proteobacteria, actinobacteria, and cyanobacteria, is the only reported example of a bacterial phosphohistidine phosphatase. A single protein target of SixA has been reported to date: theEscherichia colihistidine kinase ArcB. The present work analyzes an ArcB-independent growth defect of asixAdeletion inE. coli. A screen for suppressors, analysis of various mutants, and phosphorylation assays indicate that SixA modulates phosphorylation of the nitrogen-related phosphotransferase system (PTSNtr). The PTSNtris a widely conserved bacterial pathway that regulates diverse metabolic processes through the phosphorylation states of its protein components, EINtr, NPr, and EIIANtr, which receive phosphoryl groups on histidine residues. However, a mechanism for dephosphorylating this system has not been reported. The results presented here suggest a model in which SixA removes phosphoryl groups from the PTSNtrby acting on NPr. This work uncovers a new role for the phosphohistidine phosphatase SixA and, through factors that affect SixA expression or activity, may point to additional inputs that regulate the PTSNtr.IMPORTANCEOne common means to regulate protein activity is through phosphorylation. Protein phosphatases exist to reverse this process, returning the protein to the unphosphorylated form. The vast majority of protein phosphatases that have been identified target phosphoserine, phosphotheronine, and phosphotyrosine. A widely conserved phosphohistidine phosphatase was identified inEscherichia coli20 years ago but remains relatively understudied. The present work shows that this phosphatase modulates the nitrogen-related phosphotransferase system, a pathway that is regulated by nitrogen and carbon metabolism and affects diverse aspects of bacterial physiology. Until now, there was no known mechanism for removing phosphoryl groups from this pathway.

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 Glucose Uptake in Clostridium beijerinckii NCIMB 8052 and the Solvent-Hyperproducing Mutant BA101

2001 ◽  
Vol 67 (11) ◽  
pp. 5025-5031 ◽  
Author(s):  
Jieun Lee ◽  
H. P. Blaschek
Keyword(s):  
Glucose Uptake ◽  
Clostridium Beijerinckii ◽  
Phosphotransferase System ◽  
Stages Of Growth ◽  
Cell Extracts ◽  
Glucose Phosphorylation ◽  
Phosphorylation Rate

ABSTRACT Glucose uptake and accumulation by Clostridium beijerinckii BA101, a butanol hyperproducing mutant, were examined during various stages of growth. Glucose uptake in C. beijerinckii BA101 was repressed 20% by 2-deoxyglucose and 25% by mannose, while glucose uptake in C. beijerinckii8052 was repressed 52 and 28% by these sugars, respectively. We confirmed the presence of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) associated with cell extracts ofC. beijerinckii BA101 by glucose phosphorylation by PEP. The PTS activity associated with C. beijerinckii BA101 was 50% of that observed for C. beijerinckii 8052.C. beijerinckii BA101 also demonstrated lower PTS activity for fructose and glucitol. Glucose phosphorylation by cell extracts derived from both C. beijerinckii BA101 and 8052 was also dependent on the presence of ATP, a finding consistent with the presence of glucokinase activity in C. beijerinckii extracts. ATP-dependent glucose phosphorylation was predominant during the solventogenic stage, when PEP-dependent glucose phosphorylation was dramatically repressed. A nearly twofold-greater ATP-dependent phosphorylation rate was observed for solventogenic stage C. beijerinckii BA101 than for solventogenic stage C. beijerinckii 8052. These results suggest that C. beijerinckii BA101 is defective in PTS activity and that C. beijerinckii BA101 compensates for this defect with enhanced glucokinase activity, resulting in an ability to transport and utilize glucose during the solventogenic stage.

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 Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

2015 ◽  
Vol 81 (6) ◽  
pp. 1996-2005 ◽  
Author(s):  
Gajendar Komati Reddy ◽  
Steffen N. Lindner ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Point Mutation ◽  
Single Point ◽  
Proton Pumping ◽  
Nadh:Ubiquinone Oxidoreductase ◽  
Single Point Mutation ◽  
Content Type ◽  
Suppressor Mutations ◽  
Substrate Level ◽  
Suppressor Mutants

ABSTRACTCorynebacterium glutamicumuses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GapN) fromClostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154–7160, 2010,http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth ofCorynebacterium glutamicumwith glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression ofudhAencoding soluble transhydrogenase fromEscherichia colipartly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants ofC. glutamicumNDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-IID213Gand thus by coupling to electron transport phosphorylation (ETP).

scholarly journals Increased Glucose Utilization in Corynebacterium glutamicum by Use of Maltose, and Its Application for the Improvement of l-Valine Productivity

2009 ◽  
Vol 76 (1) ◽  
pp. 370-374 ◽  
Author(s):  
Felix S. Krause ◽  
Alexander Henrich ◽  
Bastian Blombach ◽  
Reinhard Krämer ◽  
Bernhard J. Eikmanns ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Glucose Utilization ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphotransferase System ◽  
Dehydrogenase Complex

ABSTRACT Corynebacterium glutamicum efficiently utilizes maltose as a substrate. We show here that the presence of maltose increases glucose utilization by raising the expression of ptsG, which encodes the glucose-specific EII permease of the phosphotransferase system. Consequently, the l-valine productivity of a pyruvate dehydrogenase complex-deficient C. glutamicum strain was improved by the presence of maltose.

scholarly journals Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

2013 ◽  
Vol 80 (4) ◽  
pp. 1388-1393 ◽  
Author(s):  
Zhen Chen ◽  
Rajesh Reddy Bommareddy ◽  
Doinita Frank ◽  
Sugima Rappert ◽  
An-Ping Zeng
Keyword(s):  
Corynebacterium Glutamicum ◽  
Mutant Strain ◽  
Phosphoenolpyruvate Carboxylase ◽  
Metabolic Flux ◽  
Feedback Inhibition ◽  
Allosteric Regulation ◽  
Flux Analysis ◽  
Lysine Production ◽  
Content Type ◽  
Metabolic Flux Distribution

ABSTRACTAllosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition ofCorynebacterium glutamicumPEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into theppcgene, encoding PEPC of the lysine-producing strainC. glutamicumLC298, resulted in ∼37% improved lysine production.In vitroenzyme assays and13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.

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