scholarly journals Improved Yield of Recombinant Protein via Flagella Regulator Deletion in Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Jae-Ho Han ◽  
Sang Taek Jung ◽  
Min-Kyu Oh
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Production ◽  
Metabolic Flux ◽  
Tricarboxylic Acid Cycle ◽  
Glucose Consumption ◽  
High Yield ◽  
Carbon Substrate ◽  
Flux Analysis ◽  
Flagella Assembly

Protein production requires a significant amount of intracellular energy. Eliminating the flagella has been proposed to help Escherichia coli improve protein production by reducing energy consumption. In this study, the gene encoding a subunit of FlhC, a master regulator of flagella assembly, was deleted to reduce the expression of flagella-related genes. FlhC knockout in the ptsG-deleted strain triggered significant growth retardation with increased ATP levels and a higher NADPH/NADP+ ratio. Metabolic flux analysis using a 13C-labeled carbon substrate showed increased fluxes toward the pentose phosphate and tricarboxylic acid cycle pathways in the flhC- and ptsG-deleted strains. Introduction of a high copy number plasmid or overexpression of the recombinant protein in this strain restored growth rate without increasing glucose consumption. These results suggest that the metabolic burden caused by flhC deletion was resolved by recombinant protein production. The recombinant enhanced green fluorescent protein yield per glucose consumption increased 1.81-fold in the flhC mutant strain. Thus, our study demonstrates that high-yield production of the recombinant protein was achieved with reduced flagella formation.

scholarly journals Metabolic Characterization of Escherichia coli Strains Adapted to Growth on Lactate

2007 ◽  
Vol 73 (14) ◽  
pp. 4639-4647 ◽  
Author(s):  
Qiang Hua ◽  
Andrew R. Joyce ◽  
Bernhard Ø. Palsson ◽  
Stephen S. Fong
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Carbon Sources ◽  
Carbon Substrate ◽  
Flux Analysis ◽  
Average Growth ◽  
Laboratory Evolution ◽  
Average Biomass ◽  
Evolutionary Trajectories ◽  
Genetic Mechanisms

ABSTRACT In comparison with intensive studies of genetic mechanisms related to biological evolutionary systems, much less analysis has been conducted on metabolic network responses to adaptive evolution that are directly associated with evolved metabolic phenotypes. Metabolic mechanisms involved in laboratory evolution of Escherichia coli on gluconeogenic carbon sources, such as lactate, were studied based on intracellular flux states determined from 13C tracer experiments and 13C-constrained flux analysis. At the end point of laboratory evolution, strains exhibited a more than doubling of the average growth rate and a 50% increase in the average biomass yield. Despite different evolutionary trajectories among parallel evolved populations, most improvements were obtained within the first 250 generations of evolution and were generally characterized by a significant increase in pathway capacity. Partitioning between gluconeogenic and pyruvate catabolic flux at the pyruvate node remained almost unchanged, while flux distributions around the key metabolites phosphoenolpyruvate, oxaloacetate, and acetyl-coenzyme A were relatively flexible over the course of evolution on lactate to meet energetic and anabolic demands during rapid growth on this gluconeogenic carbon substrate. There were no clear qualitative correlations between most transcriptional expression and metabolic flux changes, suggesting complex regulatory mechanisms at multiple levels of genetics and molecular biology. Moreover, higher fitness gains for cell growth on both evolutionary and alternative carbon sources were found for strains that adaptively evolved on gluconeogenic carbon sources compared to those that evolved on glucose. These results provide a novel systematic view of the mechanisms underlying microbial adaptation to growth on a gluconeogenic substrate.

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.

Metabolic flux analysis of recombinant protein overproduction in Escherichia coli

2005 ◽  
Vol 22 (2) ◽  
pp. 167-195 ◽  
Author(s):  
Pınar Özkan ◽  
Berna Sariyar ◽  
F. Özde Ütkür ◽  
Uğur Akman ◽  
Amable Hortaçsu
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Flux Analysis ◽  
Protein Overproduction

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.

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