Dataset Information

¹³C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in Escherichia coli.

ABSTRACT: Phenol is an important chemical product that can be used in a wide variety of applications, and it is currently produced from fossil resources. Fermentation production of phenol from renewable biomass resources by microorganisms is highly desirable for sustainable development. However, phenol toxicity hampers phenol production in industrial microorganisms such as Escherichia coli. In the present study, it was revealed that culturing E. coli in the presence of phenol not only decreased growth rate, but also biomass yield. This suggests that phenol affects the carbon flow of the metabolism, but the mechanism is unknown. To investigate the effect of phenol on the flux distribution of central carbon metabolism, ¹³C-metabolic flux analysis (¹³C-MFA) was performed on cells grown under different phenol concentrations (0, 0.1, and 0.15%). ¹³C-MFA revealed that the TCA cycle flux reduced by 25% increased acetate production from acetyl-CoA by 30% in the presence of 0.1% phenol. This trend of flux changes was emphasized at a phenol concentration of 0.15%. Although the expression level of citrate synthase, which catalyzes the first reaction of the TCA cycle, does not change regardless of phenol concentrations, the in vitro enzyme activity assay shows that the reaction was inhibited by phenol. These results suggest that the TCA cycle flux decreased due to phenol inhibition of citrate synthase; therefore, ATP could not be sufficiently produced by respiration, and growth rate decreased. Furthermore, since carbon was lost as acetate due to overflow metabolism, the biomass yield became low in the presence of phenol.

SUBMITTER: Kitamura S

PROVIDER: S-EPMC6514248 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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13C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in Escherichia coli.

Kitamura Sayaka S Toya Yoshihiro Y Shimizu Hiroshi H

Frontiers in microbiology 20190507

Phenol is an important chemical product that can be used in a wide variety of applications, and it is currently produced from fossil resources. Fermentation production of phenol from renewable biomass resources by microorganisms is highly desirable for sustainable development. However, phenol toxicity hampers phenol production in industrial microorganisms such as Escherichia coli. In the present study, it was revealed that culturing E. coli in the presence of phenol not only decrea ...[more]

PMID: 31134035

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Project description:BACKGROUND: It has long been recognized that analyzing the behaviour of the complex intracellular biological networks is important for breeding industrially useful microorganisms. However, because of the complexity of these biological networks, it is currently not possible to obtain all the desired microorganisms. In this study, we constructed a system for analyzing the effect of gene expression perturbations on the behavior of biological networks in Escherichia coli. Specifically, we utilized (13)C metabolic flux analysis ((13)C-MFA) to analyze the effect of perturbations to the expression levels of pgi and eno genes encoding phosphoglucose isomerase and enolase, respectively on metabolic fluxes. RESULTS: We constructed gene expression-controllable E. coli strains using a single-copy mini F plasmid. Using the pgi expression-controllable strain, we found that the specific growth rate correlated with the pgi expression level. (13)C-MFA of this strain revealed that the fluxes for the pentose phosphate pathway and Entner-Doudoroff pathway decreased, as the pgi expression lelvel increased. In addition, the glyoxylate shunt became active when the pgi expression level was almost zero. Moreover, the flux for the glyoxylate shunt increased when the pgi expression level decreased, but was significantly reduced in the pgi-knockout cells. Comparatively, eno expression could not be decreased compared to the parent strain, but we found that increased eno expression resulted in a decreased specific growth rate. (13)C-MFA revealed that the metabolic flux distribution was not altered by an increased eno expression level, but the overall metabolic activity of the central metabolism decreased. Furthermore, to evaluate the impact of perturbed expression of pgi and eno genes on changes in metabolic fluxes in E. coli quantitatively, metabolic sensitivity analysis was performed. As a result, the perturbed expression of pgi gene had a great impact to the metabolic flux changes in the branch point between the glycolysis and pentose phosphate pathway, isocitrate dehydrogenase reaction, anaplerotic pathways and Entner-Doudoroff pathway. In contrast, the impact of perturbed eno expression to the flux changes in E. coli metabolic network was small. CONCLUSIONS: Our results indicate that the response of metabolic fluxes to perturbation to pgi expression was different from that to eno expression; perturbations to pgi expression affect the reaction related to the Pgi protein function, the isocitrate dehydrogenase reaction, anaplerotic reactions and Entner-Doudoroff pathway. Meanwhile, eno expression seems to affect the overall metabolic activity, and the impact of perturbed eno expression on metabolic flux change is small. Using the gene expression control system reported here, it is expected that we can analyze the response and adaptation process of complex biological networks to gene expression perturbations.

Project description:BackgroundBioprocessing offers a sustainable and green approach to manufacture various chemicals and materials. Development of bioprocesses requires transforming common producer strains to cell factories. 13C metabolic flux analysis (13C-MFA) can be applied to identify relevant targets to accomplish the desired phenotype, which has become one of the major tools to support systems metabolic engineering. In this research, we applied 13C-MFA to identify bottlenecks in the bioconversion of glycerol into acetol by Escherichia coli. Valorization of glycerol, the main by-product of biodiesel, has contributed to the viability of biofuel economy.ResultsWe performed 13C-MFA and measured intracellular pyridine nucleotide pools in a first-generation acetol producer strain (HJ06) and a non-producer strain (HJ06C), and identified that engineering the NADPH regeneration is a promising target. Based on this finding, we overexpressed nadK encoding NAD kinase or pntAB encoding membrane-bound transhydrogenase either individually or in combination with HJ06, obtaining HJ06N, HJ06P and HJ06PN. The step-wise approach resulted in increasing the acetol titer from 0.91 g/L (HJ06) to 2.81 g/L (HJ06PN). To systematically characterize and the effect of mutation(s) on the metabolism, we also examined the metabolomics and transcriptional levels of key genes in four strains. The pool sizes of NADPH, NADP+ and the NADPH/NADP+ ratio were progressively increased from HJ06 to HJ06PN, demonstrating that the sufficient NADPH supply is critical for acetol production. Flux distribution was optimized towards acetol formation from HJ06 to HJ06PN: (1) The carbon partitioning at the DHAP node directed gradually more carbon from the lower glycolytic pathway through the acetol biosynthetic pathway; (2) The transhydrogenation flux was constantly increased. In addition, 13C-MFA showed the rigidity of upper glycolytic pathway, PP pathway and the TCA cycle to support growth. The flux patterns were supported by most metabolomics data and gene expression profiles.ConclusionsThis research demonstrated how 13C-MFA can be applied to drive the cycles of design, build, test and learn implementation for strain development. This succeeding engineering strategy can also be applicable for rational design of other microbial cell factories.

Project description:BACKGROUND:Glycerol, a byproduct of biodiesel, has become a readily available and inexpensive carbon source for the production of high-value products. However, the main drawback of glycerol utilization is the low consumption rate and shortage of NADPH formation, which may limit the production of NADPH-requiring products. To overcome these problems, we constructed a carbon catabolite repression-negative ?ptsGglpK* mutant by both blocking a key glucose PTS transporter and enhancing the glycerol conversion. The mutant can recover normal growth by co-utilization of glycerol and glucose after loss of glucose PTS transporter. To reveal the metabolic potential of the ?ptsGglpK* mutant, this study examined the flux distributions and regulation of the co-metabolism of glycerol and glucose in the mutant. RESULTS:By labeling experiments using [1,3-(13)C]glycerol and [1-(13)C]glucose, (13)C metabolic flux analysis was employed to decipher the metabolisms of both the wild-type strain and the ?ptsGglpK* mutant in chemostat cultures. When cells were maintained at a low dilution rate (0.1 h(-1)), the two strains showed similar fluxome profiles. When the dilution rate was increased, both strains upgraded their pentose phosphate pathway, glycolysis and anaplerotic reactions, while the ?ptsGglpK* mutant was able to catabolize much more glycerol than glucose (more than tenfold higher). Compared with the wild-type strain, the mutant repressed its flux through the TCA cycle, resulting in higher acetate overflow. The regulation of fluxomes was consistent with transcriptional profiling of several key genes relevant to the TCA cycle and transhydrogenase, namely gltA, icdA, sdhA and pntA. In addition, cofactor fluxes and their pool sizes were determined. The ?ptsGglpK* mutant affected the redox NADPH/NADH state and reduced the ATP level. Redox signaling activated the ArcA regulatory system, which was responsible for TCA cycle repression. CONCLUSIONS:This work employs both (13)C-MFA and transcription/metabolite analysis for quantitative investigation of the co-metabolism of glycerol and glucose in the ?ptsGglpK* mutant. The ArcA regulatory system dominates the control of flux redistribution. The ?ptsGglpK* mutant can be used as a platform for microbial cell factories for the production of biofuels and biochemicals, since most of fuel molecule (e.g., alcohols) synthesis requires excess reducing equivalents.

Dataset Information

¹³C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in Escherichia coli.

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<sup>13</sup>C-Metabolic Flux Analysis Reveals Effect of Phenol on Central Carbon Metabolism in <i>Escherichia coli</i>.

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