scholarly journals Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

2009 ◽  
Vol 75 (24) ◽  
pp. 7807-7813 ◽  
Author(s):  
X. Zhang ◽  
K. Jantama ◽  
K. T. Shanmugam ◽  
L. O. Ingram
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Phosphotransferase System ◽  
Acid Fermentation ◽  
Mineral Salts ◽  
Gene Encoding ◽  
Succinate Production ◽  
Atp Production ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
Genes Encoding

ABSTRACT The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an E scherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.

scholarly journals Genetic and Functional Investigation of Zn 2 Cys 6 Transcription Factors RSE2 and RSE3 in Podospora anserina

2013 ◽  
Vol 13 (1) ◽  
pp. 53-65 ◽  
Author(s):  
Elodie Bovier ◽  
Carole H. Sellem ◽  
Adeline Humbert ◽  
Annie Sainsard-Chanet
Keyword(s):  
Transcription Factors ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Constitutive Expression ◽  
Podospora Anserina ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Content Type ◽  
Zinc Cluster ◽  
Wide Range ◽  
Genes Encoding

ABSTRACT In Podospora anserina , the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase ( aox ) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase ( pck ) and fructose-1,6-biphosphatase ( fbp ). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3 . Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3 . It showed that in addition to aox , fbp , and pck , RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn 2 Cys 6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools.

scholarly journals Pyruvate Formate Lyase Is Required for Pneumococcal Fermentative Metabolism and Virulence

2009 ◽  
Vol 77 (12) ◽  
pp. 5418-5427 ◽  
Author(s):  
Hasan Yesilkaya ◽  
Francesca Spissu ◽  
Sandra M. Carvalho ◽  
Vanessa S. Terra ◽  
Karen A. Homer ◽  
...  
Keyword(s):  
Wild Type ◽  
Product Analysis ◽  
Acid Fermentation ◽  
Pyruvate Formate Lyase ◽  
Growth And Survival ◽  
Mixed Acid Fermentation ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
In The Wild

ABSTRACT Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pfl A genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

scholarly journals Effects of Eliminating Pyruvate Node Pathways and of Coexpression of Heterogeneous Carboxylation Enzymes on Succinate Production by Enterobacter aerogenes

2014 ◽  
Vol 81 (3) ◽  
pp. 929-937 ◽  
Author(s):  
Yoshinori Tajima ◽  
Yoko Yamamoto ◽  
Keita Fukui ◽  
Yousuke Nishio ◽  
Kenichi Hashiguchi ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Enterobacter Aerogenes ◽  
Major Product ◽  
Production Costs ◽  
Total Production ◽  
Acid Fermentation ◽  
Succinate Production ◽  
Content Type ◽  
Acidic Conditions ◽  
The Impact

ABSTRACTLowering the pH in bacterium-based succinate fermentation is considered a feasible approach to reduce total production costs. Newly isolatedEnterobacter aerogenesstrain AJ110637, a rapid carbon source assimilator under weakly acidic (pH 5.0) conditions, was selected as a platform for succinate production. Our previous work showed that the ΔadhE/PCK strain, developed from AJ110637 with inactivated ethanol dehydrogenase and introducedActinobacillus succinogenesphosphoenolpyruvate carboxykinase (PCK), generated succinate as a major product of anaerobic mixed-acid fermentation from glucose under weakly acidic conditions (pH <6.2). To further improve the production of succinate by the ΔadhE/PCK strain, metabolically engineered strains were designed based on the elimination of pathways that produced undesirable products and the introduction of two carboxylation pathways from phosphoenolpyruvate and pyruvate to oxaloacetate. The highest production of succinate was observed with strain ES04/PCK+PYC, which had inactivated ethanol, lactate, acetate, and 2,3-butanediol pathways and coexpressed PCK andCorynebacterium glutamicumpyruvate carboxylase (PYC). This strain produced succinate from glucose with over 70% yield (gram per gram) without any measurable formation of ethanol, lactate, or 2,3-butanediol under weakly acidic conditions. The impact of lowering the pH from 7.0 to 5.5 on succinate production in this strain was evaluated under pH-controlled batch culture conditions and showed that the lower pH decreased the succinate titer but increased its yield. These findings can be applied to identify additional engineering targets to increase succinate production.

scholarly journals Expression of Genes Encoding F1-ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

2002 ◽  
Vol 68 (9) ◽  
pp. 4274-4282 ◽  
Author(s):  
Brian J. Koebmann ◽  
Christian Solem ◽  
Martin B. Pedersen ◽  
Dan Nilsson ◽  
Peter R. Jensen
Keyword(s):  
Atpase Activity ◽  
Lactococcus Lactis ◽  
Biomass Production ◽  
Energy State ◽  
Sugar Transport ◽  
Growth Conditions ◽  
Glycolytic Flux ◽  
Acid Fermentation ◽  
Mixed Acid ◽  
Genes Encoding

ABSTRACT We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F1 domain of the membrane-bound (F1F0) H+-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F1-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F1-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.

scholarly journals Carbon source-dependent reprogramming of anaerobic metabolism in Staphylococcus aureus

2021 ◽  
Author(s):  
Anne Troitzsch ◽  
Vu Van Loi ◽  
Karen Methling ◽  
Daniela Zühlke ◽  
Michael Lalk ◽  
...  
Keyword(s):  
Carbon Source ◽  
Carbon Sources ◽  
Acid Fermentation ◽  
Glucose Limitation ◽  
Metabolic Switch ◽  
Mixed Acid Fermentation ◽  
Atp Production ◽  
Mixed Acid ◽  
Substrate Level Phosphorylation ◽  
Substrate Level

To be a successful pathogen, S. aureus has to adapt its metabolism to the typically oxygen- and glucose-limited environment of the host. Under fermenting conditions and in the presence of glucose, S. aureus uses glycolysis to generate ATP via substrate level phosphorylation and mainly lactic acid fermentation to maintain the redox balance by re-oxidation of NADH equivalents. However, it is less clear how S. aureus proceeds under anoxic conditions and glucose limitation, likely representing the bona-fide situation in the host. Using a combination of proteomic, transcriptional and metabolomic analyses, we show that in the absence of an abundant glycolysis substrate the available carbon source pyruvate is converted to acetyl-CoA (AcCoA) in a pyruvate formate-lyase (PflB)-dependent reaction to produce ATP and acetate. This process critically depends on de-repression of the catabolite control protein A (CcpA), leading to upregulation of pflB transcription. Under these conditions, ethanol production is repressed to prevent wasteful consumption of AcCoA. In addition, our global and quantitative characterization of the metabolic switch prioritizing acetate over lactate fermentation when glucose is absent illustrates examples of carbon source-dependent control of colonization and pathogenicity factors. Importance: Under infection conditions, S. aureus needs to ensure survival when energy production via oxidative phosphorylation is not possible, e.g. either due to the lack of terminal electron acceptors or by the inactivation of components of the respiratory chain. Under these conditions, S. aureus can switch to mixed acid fermentation to sustain ATP production by substrate-level phosphorylation. The drop in the cellular NAD+/NADH ratio is sensed by the repressor Rex, resulting in de-repression of fermentation genes. Here we show that expression of fermentation pathways is further controlled by CcpA in response to the availability of glucose to ensure optimal resource utilization under growth limiting conditions. We provide evidence for carbon source-dependent control of colonization and virulence factors. These findings add another level to the regulatory network controlling mixed acid fermentation in S. aureus and provide additional evidence for the lifestyle-modulating effect of carbon sources available in S. aureus.

scholarly journals Engineering Lactococcus lactis for Production of Mannitol: High Yields from Food-Grade Strains Deficient in Lactate Dehydrogenase and the Mannitol Transport System

2004 ◽  
Vol 70 (3) ◽  
pp. 1466-1474 ◽  
Author(s):  
Paula Gaspar ◽  
Ana Rute Neves ◽  
Ana Ramos ◽  
Michael J. Gasson ◽  
Claire A. Shearman ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Lactococcus Lactis ◽  
Selection Marker ◽  
Phosphotransferase System ◽  
Acid Fermentation ◽  
Food Grade ◽  
Mixed Acid ◽  
Glucose Depletion ◽  
Mannitol Production

ABSTRACT Mannitol is a sugar polyol claimed to have health-promoting properties. A mannitol-producing strain of Lactococcus lactis was obtained by disruption of two genes of the phosphoenolpyruvate (PEP)-mannitol phosphotransferase system (PTSMtl). Genes mtlA and mtlF were independently deleted by double-crossover recombination in strain L. lactis FI9630 (a food-grade lactate dehydrogenase-deficient strain derived from MG1363), yielding two mutant (ΔldhΔmtlA and ΔldhΔmtlF) strains. The new strains, FI10091 and FI10089, respectively, do not possess any selection marker and are suitable for use in the food industry. The metabolism of glucose in nongrowing cell suspensions of the mutant strains was characterized by in vivo 13C-nuclear magnetic resonance. The intermediate metabolite, mannitol-1-phosphate, accumulated intracellularly to high levels (up to 76 mM). Mannitol was a major end product, one-third of glucose being converted to this hexitol. The double mutants, in contrast to the parent strain, were unable to utilize mannitol even after glucose depletion, showing that mannitol was taken up exclusively by PEP-PTSMtl. Disruption of this system completely blocked mannitol transport in L. lactis, as intended. In addition to mannitol, approximately equimolar amounts of ethanol, 2,3-butanediol, and lactate were produced. A mixed-acid fermentation (formate, ethanol, and acetate) was also observed during growth under controlled conditions of pH and temperature, but mannitol production was low. The reasons for the alteration in the pattern of end products under nongrowing and growing conditions are discussed, and strategies to improve mannitol production during growth are proposed.

The exceptional photofermentative hydrogen metabolism of the green alga Chlamydomonas reinhardtii

2005 ◽  
Vol 33 (1) ◽  
pp. 39-41 ◽  
Author(s):  
A. Hemschemeier ◽  
T. Happe
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Alga ◽  
Photosynthetic Electron Transport ◽  
H2 Production ◽  
Hydrogen Metabolism ◽  
Acid Fermentation ◽  
Mixed Acid ◽  
Fermentative Metabolism ◽  
Transport Chain ◽  
Important Enzyme

The photosynthetic green alga Chlamydomonas reinhardtii is capable of performing a complex fermentative metabolism which is related to the mixed acid fermentation of bacteria such as Escherichia coli. The fermentative pattern includes the products formate, ethanol, acetate, glycerol, lactate, carbon dioxide and molecular hydrogen (H2). H2 production is catalysed by an active [Fe]-hydrogenase (HydA) which is coupled with the photosynthetic electron-transport chain. The most important enzyme of the classic fermentation pathway is pyruvate formate-lyase, which is common in bacteria but seldom found in eukaryotes. An interaction between fermentation, photosynthesis and H2 evolution allows the algae to overcome long periods of anaerobiosis. In the absence of sulphur, the cells establish a photofermentative metabolism and accumulate large amounts of H2.

Creation of Plasmidless and Markerless Escherichia coli Succinate Producing Strain and Evaluation of its Biosynthetic Potential during Utilization of Lignocellulosic Sugars

2020 ◽  
Vol 36 (2) ◽  
pp. 3-11
Author(s):  
O.A. Zhuravliova ◽  
Т.А. Voeikova ◽  
A.Yu. Gulevich ◽  
V.G. Debabov
Keyword(s):  
Escherichia Coli ◽  
Succinic Acid ◽  
Plant Biomass ◽  
Anaerobic Fermentation ◽  
Basic Research ◽  
Acid Fermentation ◽  
Mixed Acid ◽  
Gene Coding ◽  
Lignocellulosic Sugars ◽  
Basic Research Project

The plasmidless and markerless Escherichia coli succinate producing strain SGM2.0Pyc-int has been engineered and characterized. The strain has the inactivated main mixed-acid fermentation pathways due to the deletions of ldhA,poxB, ackA,pta, and adhE genes, constitutively expresses the genes of the aceEF-lpdA operon encoding components of pyravate dehydrogenase complex, and possesses the chromosomally integrated Bacillus subtilis pycA gene coding for pyruvate carboxylase. The capacity of the strain to synthesize succinic acid in course of dual-phase aerobic-anaerobic fermentation with lignocellulosic sugars as substrates was studied. The SGM2.0Pyc-int strain synthesized succinic acid from glucose, xylose, and arabinose with a molar yields of 1.41 mol/mol, 1.18 mol/mol, and 1.18 mol/mol, respectively, during the anaerobic production stage. The constructed strain has great potential for developing efficient processes for the succinic acid production from plant biomass-derived sugars. Escherichia coli, fermentation, arabinose, glucose, xylose, succinic acid. The work was supported by a Grant from the Russian Foundation for Basic Research (Project no. 18-29-14005).

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Structure, expression, chromosomal location and product of the gene encoding Adh2 in Petunia.

Genetics ◽  
1993 ◽  
Vol 133 (4) ◽  
pp. 999-1007
Author(s):  
R G Gregerson ◽  
L Cameron ◽  
M McLean ◽  
P Dennis ◽  
J Strommer
Keyword(s):  
Chromosomal Location ◽  
Higher Plants ◽  
Gene Encoding ◽  
Genomic Libraries ◽  
Regulatory Strategy ◽  
Adh1 Gene ◽  
Adh Genes ◽  
Genes Encoding ◽  
Adh Gene ◽  
Polymerase Chain

Abstract In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

Sign in / Sign up

Export Citation Format

Share Document