scholarly journals Rewiring of glycerol metabolism in Escherichia coli for effective production of recombinant proteins

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Chung-Jen Chiang ◽  
Yi-Jing Ho ◽  
Mu-Chen Hu ◽  
Yun-Peng Chao
Keyword(s):  
Escherichia Coli ◽  
Crude Glycerol ◽  
Metabolic Flux ◽  
Raw Materials ◽  
Tricarboxylic Acid Cycle ◽  
Cost Effective ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Glycerol Metabolism ◽  
Related Field

Abstract Background The economic viability of a protein-production process relies highly on the production titer and the price of raw materials. Crude glycerol coming from the production of biodiesel is a renewable and cost-effective resource. However, glycerol is inefficiently utilized by Escherichia coli. Results This issue was addressed by rewiring glycerol metabolism for redistribution of the metabolic flux. Key steps in central metabolism involving the glycerol dissimilation pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle were pinpointed and manipulated to provide precursor metabolites and energy. As a result, the engineered E. coli strain displayed a 9- and 30-fold increase in utilization of crude glycerol and production of the target protein, respectively. Conclusions The result indicates that the present method of metabolic engineering is useful and straightforward for efficient adjustment of the flux distribution in glycerol metabolism. The practical application of this methodology in biorefinery and the related field would be acknowledged.

scholarly journals Responses of theCentral Metabolism in Escherichia coli to PhosphoglucoseIsomerase and Glucose-6-Phosphate DehydrogenaseKnockouts

2003 ◽  
Vol 185 (24) ◽  
pp. 7053-7067 ◽  
Author(s):  
Qiang Hua ◽  
Chen Yang ◽  
Tomoya Baba ◽  
Hirotada Mori ◽  
Kazuyuki Shimizu
Keyword(s):  
Escherichia Coli ◽  
Pentose Phosphate Pathway ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Ratio ◽  
Pentose Phosphate ◽  
Phosphoglucose Isomerase ◽  
Overflow Metabolism ◽  
Flux Analysis ◽  
Glucose Catabolism

ABSTRACT The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-13C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.

scholarly journals Solid State Fermentation of Brewers’ Spent Grains for Improved Nutritional Profile Using Bacillus subtilis WX-17

Fermentation ◽  
2019 ◽  
Vol 5 (3) ◽  
pp. 52 ◽  
Author(s):  
Yong Xing Tan ◽  
Wai Kit Mok ◽  
Jaslyn Lee ◽  
Jaejung Kim ◽  
Wei Ning Chen
Keyword(s):  
Bacillus Subtilis ◽  
Amino Acid ◽  
Solid State ◽  
Food Waste ◽  
Solid State Fermentation ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Fold Increase ◽  
Spent Grains ◽  
Significant Difference

Brewers’ spent grains (BSG) are underutilized food waste materials produced in large quantities from the brewing industry. In this study, solid state fermentation of BSG using Bacillus subtilis WX-17 was carried out to improve the nutritional value of BSG. Fermenting BSG with the strain WX-17, isolated from commercial natto, significantly enhanced the nutritional content in BSG compared to unfermented BSG, as determined by the marked difference in the level of metabolites. In total, 35 metabolites showed significant difference, which could be categorized into amino acids, fatty acids, carbohydrates, and tricarboxylic acid cycle intermediates. Pathway analysis revealed that glycolysis was upregulated, as indicated by the drop in the level of carbohydrate compounds. This shifted the metabolic flux particularly towards the amino acid pathway, leading to a 2-fold increase in the total amount of amino acid from 0.859 ± 0.05 to 1.894 ± 0.1 mg per g of BSG after fermentation. Also, the total amount of unsaturated fatty acid increased by 1.7 times and the total antioxidant quantity remarkably increased by 5.8 times after fermentation. This study demonstrates that novel fermentation processes can value-add food by-products, and valorized food waste could potentially be used for food-related applications. In addition, the study revealed the metabolic changes and mechanisms behind the microbial solid state fermentation of BSG.

scholarly journals Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

2013 ◽  
Vol 79 (9) ◽  
pp. 3091-3100 ◽  
Author(s):  
Francesco Falcioni ◽  
Lars M. Blank ◽  
Oliver Frick ◽  
Andreas Karau ◽  
Bruno Bühler ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Analysis ◽  
Substrate Uptake ◽  
Mrna Levels ◽  
Microbial Physiology ◽  
Whole Cell ◽  
Content Type ◽  
Proline Hydroxylation

ABSTRACTMicrobial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinantEscherichia coliexpressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alterp4hmRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription ofputAandputP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.

scholarly journals Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

2002 ◽  
Vol 184 (1) ◽  
pp. 152-164 ◽  
Author(s):  
Marcel Emmerling ◽  
Michael Dauner ◽  
Aaron Ponti ◽  
Jocelyne Fiaux ◽  
Michel Hochuli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Glucose Uptake ◽  
Dilution Rate ◽  
Pyruvate Kinase ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Flux Analysis ◽  
E Coli ◽  
General Response

ABSTRACT The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

scholarly journals Nonlinear Dependency of Intracellular Fluxes on Growth Rate in Miniaturized Continuous Cultures of Escherichia coli

2006 ◽  
Vol 72 (2) ◽  
pp. 1164-1172 ◽  
Author(s):  
Annik Nanchen ◽  
Alexander Schicker ◽  
Uwe Sauer
Keyword(s):  
Escherichia Coli ◽  
Dilution Rate ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Pentose Phosphate ◽  
Glyoxylate Shunt ◽  
Parallel Operation ◽  
Physiological Variables

ABSTRACT A novel mini-scale chemostat system was developed for the physiological characterization of 10-ml cultures. The parallel operation of eight such mini-scale chemostats was exploited for systematic 13C analysis of intracellular fluxes over a broad range of growth rates in glucose-limited Escherichia coli. As expected, physiological variables changed monotonously with the dilution rate, allowing for the assessment of maintenance metabolism. Despite the linear dependence of total cellular carbon influx on dilution rate, the distribution of almost all major fluxes varied nonlinearly with dilution rate. Most prominent were the distinct maximum of glyoxylate shunt activity and the concomitant minimum of tricarboxylic acid cycle activity at low to intermediate dilution rates of 0.05 to 0.2 h−1. During growth on glucose, this glyoxylate shunt activity is best understood from a network perspective as the recently described phosphoenolpyruvate (PEP)-glyoxylate cycle that oxidizes PEP (or pyruvate) to CO2. At higher or extremely low dilution rates, in vivo PEP-glyoxylate cycle activity was low or absent. The step increase in pentose phosphate pathway activity at around 0.2 h−1 was not related to the cellular demand for the reduction equivalent NADPH, since NADPH formation was 20 to 50% in excess of the anabolic demand at all dilution rates. The results demonstrate that mini-scale continuous cultivation enables quantitative and parallel characterization of intra- and extracellular phenotypes in steady state, thereby greatly reducing workload and costs for stable-isotope experiments.

scholarly journals Pyruvate production by Escherichia coli using pyruvate dehydrogenase variants

2021 ◽  
Author(s):  
W. Chris Moxley ◽  
Mark A. Eiteman
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Carbon Flux ◽  
Metabolic Flux ◽  
Pyruvate Dehydrogenase Complex ◽  
Cost Effective ◽  
Specific Productivity ◽  
Synthesis Methods ◽  
Additional Tool ◽  
Dehydrogenase Complex

Altering metabolic flux at a key branchpoint in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 – 0.28 h−1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59-0.66 g pyruvate/g glucose with a specific productivity of 0.78 – 0.92 g pyruvate/g cells·h. These results provide proof-of-concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. Importance Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.

scholarly journals Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G

2015 ◽  
Vol 112 (32) ◽  
pp. 9855-9859 ◽  
Author(s):  
Baixue Lin ◽  
Keqiang Fan ◽  
Jian Zhao ◽  
Junjie Ji ◽  
Linjun Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Fold Increase ◽  
Tca Cycle ◽  
Penicillin G ◽  
Whole Cell ◽  
Knock Out ◽  
Engineering Strategy ◽  
Acetate Accumulation ◽  
Biocatalytic Process

Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate–dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host β-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

scholarly journals Enzymes of the intermediary carbohydrate metabolism of Polyangium cellulosum

1985 ◽  
Vol 31 (12) ◽  
pp. 1142-1146 ◽  
Author(s):  
Renu Sarao ◽  
Howard D. McCurdy ◽  
Luciano Passador
Keyword(s):  
Carbohydrate Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Fold Increase ◽  
Pentose Phosphate ◽  
Acid Cycle ◽  
The Family ◽  
The Glyoxylate Cycle ◽  
Pentose Phosphate Shunt

Crude extracts of vegetative cells of the cellulolytic myxobacter Polyangium cellulosum contained significant levels of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle. Key enzymes of glycolysis and the pentose phosphate shunt were also detected. Specific activities of hexokinase and fructose- 1,6-diphosphate aldolase exhibited a 10-fold increase when the cells were grown in complex medium containing glucose. Cytochromes of a, b, and c type were demonstrated. By the use of a dispersly growing strain of P. cellulosum, its generation time was determined to be 22–24 h. This study suggests that the organism probably uses glycolysis and citric acid cycle for complete oxidation of glucose. The exact role of the glyoxylate cycle and pentose phosphate shunt cannot be deduced from this study. This is the first report on the study of intermediary carbohydrate metabolism in any member of the family Polyangiaceae.

scholarly journals Legionella pneumophila CsrA regulates a metabolic switch from amino acid to glycerolipid metabolism

Open Biology ◽  
2017 ◽  
Vol 7 (11) ◽  
pp. 170149 ◽  
Author(s):  
Ina Häuslein ◽  
Tobias Sahr ◽  
Pedro Escoll ◽  
Nadine Klausner ◽  
Wolfgang Eisenreich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Legionella Pneumophila ◽  
Tricarboxylic Acid Cycle ◽  
Life Stage ◽  
Pentose Phosphate ◽  
Glycerol Metabolism ◽  
Specific Expression ◽  
Metabolic Switch ◽  
Metabolic Functions ◽  
Glycerol Utilization

Legionella pneumophila CsrA plays a crucial role in the life-stage-specific expression of virulence phenotypes and metabolic activity. However, its exact role is only partly known. To elucidate how CsrA impacts L. pneumophila metabolism we analysed the CsrA depended regulation of metabolic functions by comparative 13 C-isotopologue profiling and oxygen consumption experiments of a L. pneumophila wild-type (wt) strain and its isogenic csrA − mutant. We show that a csrA − mutant has significantly lower respiration rates when serine, alanine, pyruvate, α-ketoglutarate or palmitate is the sole carbon source. By contrast, when grown in glucose or glycerol, no differences in respiration were detected. Isotopologue profiling uncovered that the transfer of label from [U- 13 C 3 ]serine via pyruvate into the citrate cycle and gluconeogenesis was lower in the mutant as judged from the labelling patterns of protein-derived amino acids, cell-wall-derived diaminopimelate, sugars and amino sugars and 3-hydroxybutyrate derived from polyhydroxybutyrate (PHB). Similarly, the incorporation of [U- 13 C 6 ]glucose via the glycolysis/Entner–Doudoroff (ED) pathway but not via the pentose phosphate pathway was repressed in the csrA − mutant. On the other hand, fluxes due to [U- 13 C 3 ]glycerol utilization were increased in the csrA − mutant. In addition, we showed that exogenous [1,2,3,4- 13 C 4 ]palmitic acid is efficiently used for PHB synthesis via 13 C 2 -acetyl-CoA. Taken together, CsrA induces serine catabolism via the tricarboxylic acid cycle and glucose degradation via the ED pathway, but represses glycerol metabolism, fatty acid degradation and PHB biosynthesis, in particular during exponential growth. Thus, CsrA has a determining role in substrate usage and carbon partitioning during the L. pneumophila life cycle and regulates a switch from amino acid usage in replicative phase to glycerolipid usage during transmissive growth.

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