An In Vivo Metabolic Approach for Deciphering the Product Specificity of Glycerate Kinase Proves that Both E. coli’s Glycerate Kinases Generate 2-Phosphoglycerate

Zelcbuch, Lior; Razo-Mejia, Manuel; Herz, Elad; Yahav, Sagit; Antonovsky, Niv; Kroytoro, Hagar; Milo, Ron; Bar‐Even, Arren

doi:10.1371/journal.pone.0122957

Cited by 20 publications

(13 citation statements)

References 28 publications

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“…We suspect that the conversion of d -glyceryl-CoA to d -glycerate is genetically redundant and therefore cannot be identified by assaying mutant strains with only one transposon insertion. The glycerate kinase of P. simiae (PS417_13970) is 51% identical to glycerate kinase 1 from Escherichia coli ( garK ), which forms 2-phosphoglycerate (14, 15). 2-Phosphoglycerate is an intermediate in glycolysis and thus links the proposed pathway to central metabolism.…”

Section: Resultsmentioning

confidence: 99%

Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism

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Iavarone

et al. 2019

mSystems

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Deoxyribose is one of the building blocks of DNA and is released when cells die and their DNA degrades. We identified a bacterium that can grow with deoxyribose as its sole source of carbon even though its genome does not contain any of the known genes for breaking down deoxyribose. By growing many mutants of this bacterium together on deoxyribose and using DNA sequencing to measure the change in the mutants’ abundance, we identified multiple protein-coding genes that are required for growth on deoxyribose. Based on the similarity of these proteins to enzymes of known function, we propose a 6-step pathway in which deoxyribose is oxidized and then cleaved. Diverse bacteria use a portion of this pathway to break down a related compound, deoxyribonate, which is a waste product of metabolism. Our study illustrates the utility of large-scale bacterial genetics to identify previously unknown metabolic pathways.

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Section: Resultsmentioning

confidence: 99%

Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism

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Iavarone

et al. 2019

mSystems

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“…Pyruvate can enter the central metabolism either towards the generation of biosynthetic intermediates or via the pyruvate dehydrogenase reaction (thus producing acetyl-CoA). The glycolaldehyde is first converted to glycolate by the aldehyde dehydrogenase, aldA [ 39–41 ], which can be metabolized via either the glyoxylate shunt or the glyoxylate degradation pathway, which converts glyoxylate to tartronate semialdehyde, d -glycerate and then 2-phosphoglycerate [ 42 ]. Therefore, the Dahms pathway would provide intermediates for sugar biosynthesis without the requirement to produce phosphoenolpyruvate from TCA cycle intermediates.…”

Section: Discussionmentioning

confidence: 99%

Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway

et al. 2018

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Bio-production of fuels and chemicals from lignocellulosic C5 sugars usually requires the use of the pentose phosphate pathway (PPP) to produce pyruvate. Unfortunately, the oxidation of pyruvate to acetyl-coenzyme A results in the loss of 33 % of the carbon as CO2, to the detriment of sustainability and process economics. To improve atom efficiency, we engineered Escherichia coli to utilize d-xylose constitutively using the Weimberg pathway, to allow direct production of 2-oxoglutarate without CO2 loss. After confirming enzyme expression in vitro, the pathway expression was optimized in vivo using a combinatorial approach, by screening a range of constitutive promoters whilst systematically varying the gene order. A PPP-deficient (ΔxylAB), 2-oxoglutarate auxotroph (Δicd) was used as the host strain, so that growth on d-xylose depended on the expression of the Weimberg pathway, and variants expressing Caulobacter crescentus xylXAB could be selected on minimal agar plates. The strains were isolated and high-throughput measurement of the growth rates on d-xylose was used to identify the fastest growing variant. This strain contained the pL promoter, with C. crescentus xylA at the first position in the synthetic operon, and grew at 42 % of the rate on d-xylose compared to wild-type E. coli using the PPP. Remarkably, the biomass yield was improved by 53.5 % compared with the wild-type upon restoration of icd activity. Therefore, the strain grows efficiently and constitutively on d-xylose, and offers great potential for use as a new host strain to engineer carbon-efficient production of fuels and chemicals via the Weimberg pathway.

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“…We suspect that the conversion of D-glyceryl-CoA to D-glycerate is genetically redundant and therefore cannot be identified by assaying mutant strains with only one transposon insertion. The glycerate kinase of P. simiae (PS417_13970) is 51% identical to glycerate kinase 1 from Escherichia coli (garK) , which forms 2-phosphoglycerate (Bartsch et al 2008;Zelcbuch et al 2015) . 2-phosphoglycerate is an intermediate in glycolysis and thus links the proposed pathway to central metabolism.…”

Section: Resultsmentioning

confidence: 99%

Oxidative pathways of deoxyribose and deoxyribonate catabolism

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Iavarone

et al. 2017

Preprint

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High-throughput genetics is a powerful approach to identify new genes involved in bacterial carbon catabolism. Here, we used genome-wide fitness assays to identify a novel pathway for 2-deoxy-D-ribose catabolism in Pseudomonas simiae WCS417. The genes that were important for deoxyribose utilization but not in other conditions included two putative dehydrogenases, a lactonase, a β-keto acid cleavage enzyme, and a glycerate kinase. We propose that deoxyribose is oxidized twice to 2-deoxy-3-keto-D-ribonoate (a β-keto acid) before cleavage to D-glycerate, which is phosphorylated and enters lower glycolysis. We purified the two dehydrogenases and reconstituted the enzymatic pathway for the conversion of deoxyribose to 2-deoxy-3-keto-D-ribonoate in vitro .

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An In Vivo Metabolic Approach for Deciphering the Product Specificity of Glycerate Kinase Proves that Both E. coli’s Glycerate Kinases Generate 2-Phosphoglycerate

Cited by 20 publications

References 28 publications

Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism

Oxidative Pathways of Deoxyribose and Deoxyribonate Catabolism

Engineering Escherichia coli to grow constitutively on D-xylose using the carbon-efficient Weimberg pathway

Oxidative pathways of deoxyribose and deoxyribonate catabolism

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