scholarly journals Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli

2021 ◽  
Vol 22 (5) ◽  
pp. 2266
Author(s):  
Zamira E. Soto-Varela ◽  
Gema Cabrera ◽  
Agustin Romero ◽  
Domingo Cantero ◽  
Antonio Valle ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Anaerobic Fermentation ◽  
Waste Product ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Tca Cycle ◽  
Biotechnological Production ◽  
Phosphoenol Pyruvate ◽  
Valuable Compounds ◽  
C4 Production

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.

scholarly journals Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

2016 ◽  
Vol 82 (19) ◽  
pp. 5960-5968 ◽  
Author(s):  
Tomohiro Shimada ◽  
Kan Tanaka
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Monitoring System ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Bacterial Cells ◽  
Bacterial Luciferase ◽  
Content Type ◽  
Time Dynamics ◽  
The Tca Cycle

ABSTRACTRegulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employingVibrio fischeriluciferase to probe the intracellular metabolic condition inEscherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used aVibrio fischeriluciferase monitoring system to probe the intracellular metabolic condition inEscherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.

Regulation of catalase level in Escherichia coli K12

1977 ◽  
Vol 23 (1) ◽  
pp. 84-91 ◽  
Author(s):  
Yona Yoshpe-Purer ◽  
Yigal Henis ◽  
Jacob Yashphe
Keyword(s):  
Escherichia Coli ◽  
Catabolite Repression ◽  
Nitrogen Starvation ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Enzyme Synthesis ◽  
Anaerobic Growth ◽  
Nutrient Agar ◽  
Tca Cycle Intermediates ◽  
Transient Repression

The synthesis of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) in Escherichia coli K12 was regulated by repression–induction and catabolite repression. Transient repression by glucose was also demonstrated. Under certain conditions, preferential synthesis was observed. Enzyme synthesis was induced in glycerol-grown cells by 0.15 to 0.2 μmol H2O2 (5–6 μg) per millilitre of culture, added at 10 to 15-min intervals, which were shortened progressively as the level of catalase in the cells rose. Catabolite repression of catalase synthesis was demonstrated with glucose, glycerol, maltose, lactose, xylose, mannitol, sorbitol, and trehalose, and to a lesser extent with arabinose, galactose, sucrose, rhamnose, and dulcitol as sole carbon sources in mineral medium. Catabolite repression was prevented by anaerobic growth and nitrogen starvation. All TCA-cycle intermediates examined afforded high catalase levels when serving as sole carbon sources. High catalase levels were also obtained when cells were grown on nutrient broth, nutrient agar, casitone, and peptone.

Levels of coenzyme A – glutathione mixed disulfide in Escherichia coli

1978 ◽  
Vol 56 (7) ◽  
pp. 753-759 ◽  
Author(s):  
Peter C. Loewen
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Anaerobic Fermentation ◽  
Carbon Sources ◽  
Lower Proportion ◽  
Fermentable Sugars ◽  
Glucose Starvation ◽  
Aerobic Growth ◽  
Mixed Disulfide ◽  
Oxygen Starvation

The pool of coenzyme A – glutathione mixed disulfide (CoASSG) rapidly increased 2.0 times in response to oxygen starvation and 1.5 times in response to glucose starvation but did not change following ammonia starvation. The increase in the CoASSG pool resulted from an increase in the CoASSG fraction of the CoA pool from 42 to 66–93%. Fluoride, cyanide, chloramphenicol, and rifampicin all caused similar increases. Aerobic growth on fermentable sugars resulted in CoASSG making up 40–55% of the CoA pool while growth on nonfermentable carbon sources or anaerobic fermentation resulted in CoASSG replacing acetyl CoA and free CoA to make up 85–95% of the CoA pool. The CoASSG:ATP ratio varied inversely with the growth rate in two groupings of carbon sources made up of either fermentable or nonfermentable molecules. Cultures grown aerobically on fermentable sugars exhibited a lower CoASSG:ATP ratio reflecting the lower proportion of CoASSG in the CoA pool.

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 Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti

Genetics ◽  
1997 ◽  
Vol 147 (4) ◽  
pp. 1521-1531 ◽  
Author(s):  
Magne Østerås ◽  
Shelley A P O'Brien ◽  
Turlough M Finan
Keyword(s):  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Root Nodules ◽  
Genetic Regulation ◽  
Carbon Sources ◽  
Dicarboxylic Acids ◽  
Phenotypic Analysis ◽  
Gene Encoding ◽  
Type Locus ◽  
Rapid Induction

Abstract The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium (now Sinorhizobium) meliloti. This bacterium forms N2-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C4-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the Ga1R/LacI family of transcriptional regulators that are involved in metabolism.

scholarly journals Sporulation in solventogenic and acetogenic clostridia

2021 ◽  
Author(s):  
Mamou Diallo ◽  
Servé W. M. Kengen ◽  
Ana M. López-Contreras
Keyword(s):  
Current Knowledge ◽  
Carbon Sources ◽  
Cost Effective ◽  
Biotechnological Production ◽  
Key Points ◽  
Wide Range ◽  
Potential Applications ◽  
A Cell ◽  
Sporulation Initiation ◽  
Solventogenic Clostridia

AbstractThe Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

scholarly journals Conditional Essentiality of the csrA Gene in Escherichia coli

2008 ◽  
Vol 191 (5) ◽  
pp. 1722-1724 ◽  
Author(s):  
Johan Timmermans ◽  
Laurence Van Melderen
Keyword(s):  
Escherichia Coli ◽  
Growth Inhibition ◽  
Deletion Mutant ◽  
Glycogen Synthesis ◽  
Synthetic Medium ◽  
Carbon Sources ◽  
Krebs Cycle ◽  
Physiological Processes

ABSTRACT CsrA is a global posttranscriptional regulator of numerous physiological processes, such as glycogenesis and glycolysis. Here, we show that the csrA gene of Escherichia coli is essential for growth on LB and on synthetic medium containing glycolytic carbon sources. However, csrA is not necessary for growth on synthetic medium containing pyruvate, showing that the Krebs cycle is functional in the csrA::cat deletion mutant. Deletion of the glgCAP operon in the csrA::cat mutant restored the ability to grow on LB and on synthetic medium containing glycolytic carbon sources, showing that growth inhibition is due to an excess of glycogen synthesis.

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