Comparative studies of glycolytic pathways and channeling under <i>in vitro</i> and <i>in vivo</i> modes

Abernathy, Mary H.; Zhang, Yuchen; Hollinshead, Whitney D.; Wang, George; Baidoo, Edward E. K.; Liu, Tiangang; Tang, Yinjie J.

doi:10.1002/aic.16367

Cited by 13 publications

(12 citation statements)

References 48 publications

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“…Proximity channeling could be another explanation for the difficulty in redirecting flux from one glycolytic pathway to another. This has been observed previously, where overexpression of homologous ED enzymes in E. coli did not capture significant flux from EMP pathway unless PTS was knocked out (Abernathy et al, 2019). Although there is no evidence of similar channels in Z. mobilis, it is possible that difficulties in redirecting glucose flux from ED pathway to EMP may be partially due to existence of ED proximity channels.…”

Section: Discussionsupporting

confidence: 76%

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4

et al. 2019

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Zymomonas mobilis is a bacterium that produces ethanol from glucose at up to 97% of theoretical efficiency on a carbon basis. One factor contributing to the high efficiency of ethanol production is that Z. mobilis has a low biomass yield. The low biomass yield may be caused partly by the low ATP yield of the Entner-Doudoroff (ED) glycolytic pathway used by Z. mobilis, which produces only one ATP per glucose consumed. To test the hypothesis that ATP yield limits biomass yield in Z. mobilis, we attempted to introduce the Embden-Meyerhof-Parnas (EMP) glycolytic pathway (with double the ATP yield) by expressing phosphofructokinase (Pfk I) from Escherichia coli. Expression of Pfk I caused growth inhibition and resulted in accumulation of mutations in the pfkA gene. Co-expression of additional EMP enzymes, fructose bisphosphate aldolase (Fba) and triose phosphate isomerase (Tpi), with Pfk I did not enable EMP flux, and resulted in production of glycerol as a side product. Further analysis indicated that heterologous reactions may have operated in the reverse direction because of native metabolite concentrations. This study reveals how the metabolomic context of a chassis organism influences the range of pathways that can be added by heterologous expression.

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Section: Discussionsupporting

confidence: 76%

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4

et al. 2019

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“…In contrast, model microorganisms that utilize the EMP pathway, like E. coli, often experience catabolic repression. Directing glycolytic flux through the ED pathway rather than the EMP pathway may be a key strategy for avoiding glucose catabolic repression (44). The ED pathway also requires fewer enzymes (40), saving cellular resources by reducing the amount of protein needed for catabolism.…”

Section: Discussionmentioning

confidence: 99%

“…Analysis of Engineered ED Pathway in E. coli. The ΔptsG and ΔptsG + ED E. coli mutants were generated as reported previously (44). Briefly, the ΔptsG strain was obtained from the Keio Collection (Coli Genetic Stock Center) and transformed with the plasmid carrying the ED pathway genes (edd and eda).…”

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confidence: 99%

Model metabolic strategy for heterotrophic bacteria in the cold ocean based on Colwellia psychrerythraea 34H

Czajka

Abernathy

Benites

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Colwellia psychrerythraea 34H is a model psychrophilic bacterium found in the cold ocean—polar sediments, sea ice, and the deep sea. Although the genomes of such psychrophiles have been sequenced, their metabolic strategies at low temperature have not been quantified. We measured the metabolic fluxes and gene expression of 34H at 4 °C (the mean global-ocean temperature and a normal-growth temperature for 34H), making comparative analyses at room temperature (above its upper-growth temperature of 18 °C) and with mesophilic Escherichia coli. When grown at 4 °C, 34H utilized multiple carbon substrates without catabolite repression or overflow byproducts; its anaplerotic pathways increased flux network flexibility and enabled CO2 fixation. In glucose-only medium, the Entner–Doudoroff (ED) pathway was the primary glycolytic route; in lactate-only medium, gluconeogenesis and the glyoxylate shunt became active. In comparison, E. coli, cold stressed at 4 °C, had rapid glycolytic fluxes but no biomass synthesis. At their respective normal-growth temperatures, intracellular concentrations of TCA cycle metabolites (α-ketoglutarate, succinate, malate) were 4–17 times higher in 34H than in E. coli, while levels of energy molecules (ATP, NADH, NADPH) were 10- to 100-fold lower. Experiments with E. coli mutants supported the thermodynamic advantage of the ED pathway at cold temperature. Heat-stressed 34H at room temperature (2 hours) revealed significant down-regulation of genes associated with glycolytic enzymes and flagella, while 24 hours at room temperature caused irreversible cellular damage. We suggest that marine heterotrophic bacteria in general may rely upon simplified metabolic strategies to overcome thermodynamic constraints and thrive in the cold ocean.

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“…In eukaryotes, the formation of metabolons are associated with cellular organelles and membrane compartments, such as the mitochondria or peroxisomes (Graham et al, 2007). In prokaryotes, evidence suggests that enzyme proximity channeling may be widely present in central pathways (Abernathy et al, 2019). Cyanobacterial RuBisCO is the starting point for reduced carbon metabolism in the cell, and the encapsulating carboxysome may offer an anchoring point for cascade enzymes.…”

Section: Introductionmentioning

confidence: 99%

Cyanobacterial carboxysome mutant analysis reveals the influence of enzyme compartmentalization on cellular metabolism and metabolic network rigidity

Abernathy

Czajka

Allen

et al. 2019

Metabolic Engineering

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Cyanobacterial carboxysomes encapsulate carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Genetic deletion of the major structural proteins encoded within the ccm operon in Synechococcus sp. PCC 7002 (ΔccmKLMN) disrupts carboxysome formation and significantly affects cellular physiology. Here we employed both metabolite pool size analysis and isotopically nonstationary metabolic flux analysis (INST-MFA) to examine metabolic regulation in cells lacking carboxysomes. Under high CO2 environments (1%), the ΔccmKLMN mutant could recover growth and had a similar central flux distribution as the control strain, with the exceptions of moderately decreased photosynthesis and elevated biomass protein content and photorespiration activity. Metabolite analyses indicated that the ΔccmKLMN strain had significantly larger pool sizes of pyruvate (> 18 folds), UDPG (uridine diphosphate glucose), and aspartate as well as higher levels of secreted organic acids (e.g., malate and succinate). These results suggest that the ΔccmKLMN mutant is able to largely maintain a fluxome similar to the control strain by changing in intracellular metabolite concentrations and metabolite overflows under optimal growth conditions. When CO2 was insufficient (0.2%), provision of acetate moderately promoted mutant growth. Interestingly, the removal of microcompartments may loosen the flux network and promote RuBisCO side-reactions, facilitating redirection of central metabolites to competing pathways (i.e., pyruvate to heterologous lactate production). This study provides important insights into metabolic regulation via enzyme compartmentation and cyanobacterial compensatory responses.

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Comparative studies of glycolytic pathways and channeling under in vitro and in vivo modes

Cited by 13 publications

References 48 publications

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4

Expression of Phosphofructokinase Is Not Sufficient to Enable Embden-Meyerhof-Parnas Glycolysis in Zymomonas mobilis ZM4

Model metabolic strategy for heterotrophic bacteria in the cold ocean based on Colwellia psychrerythraea 34H

Cyanobacterial carboxysome mutant analysis reveals the influence of enzyme compartmentalization on cellular metabolism and metabolic network rigidity

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