Production of Ophthalmic Acid Using Engineered Escherichia coli

Ito, Tomokazu; Tokoro, Maiko; Hori, Ran; Hemmi, Hisashi; Yoshimura, Teizo

doi:10.1128/aem.02806-17

Cited by 10 publications

(8 citation statements)

References 39 publications

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“…The yggS mutant accumulates some metabolites in the Thr/Ile/Val metabolic pathway (Val, ketoisovalerate (KIV), Ile, 2‐AB, and OA) and exhibits decreased level of coenzyme A (Ito et al , , , ). Some of these changes correlate with increased levels of PNP, but the mechanism was unknown (Ito et al , ).…”

Section: Resultsmentioning

confidence: 99%

“…These solutions (180 µl) were deproteinized with HClO 4 (8 M, 20 µl) and neutralized with 100 µl of the 0.8 M K 2 CO 3 . After centrifugation, samples were diluted and used for the HPLC analyses as previously described (Ito et al , , ). Previous results reported that 1 mg of wet cell weight corresponds to 0.23 mg of dry cell weight, and the amount of cytoplasmic water in cells grown in the M9‐glucose is 2.0 µl/mg dry cell weight (Cayley et al ., 1991; Glazyrina et al , ).…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of glycine cleavage system by pyridoxine 5′‐phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli

Ito

Hori

Hemmi

et al. 2019

Molecular Microbiology

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The YggS/Ybl036c/PLPBP family includes conserved pyridoxal 5′-phosphate (PLP)-binding proteins that play a critical role in the homeostasis of vitamin B 6 and amino acids. Disruption of members of this family causes pleiotropic effects in many organisms by unknown mechanisms. In Escherichia coli, conditional lethality of the yggS and glyA (encoding serine hydroxymethyltransferase) has been described, but the mechanism of lethality was not determined. Strains lacking yggS and serA (3-phosphoglycerate dehydrogenase) were conditionally lethality in the M9-glucose medium supplemented with Gly. Analyses of vitamin B 6 pools found the high-levels of pyridoxine 5′-phosphate (PNP) in the two yggS mutants. Growth defects of the double mutants could be eliminated by overexpressing PNP/PMP oxidase (PdxH) to decrease the PNP levels. Further, a serA pdxH strain, which accumulates PNP in the presence of yggS, exhibited similar phenotype to serA yggS mutant. Together these data suggested the inhibition of the glycine cleavage (GCV) system caused the synthetic lethality. Biochemical assays confirmed that PNP disrupts the GCV system by competing with PLP in GcvP protein. Our data are consistent with a model in which PNP-dependent inhibition of the GCV system causes the conditional lethality observed in the glyA yggS or serA yggS mutants.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Inhibition of glycine cleavage system by pyridoxine 5′‐phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli

Ito

Hori

Hemmi

et al. 2019

Molecular Microbiology

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“…Amino acid analysis. Cells were grown in the M9 medium at 30°C to a final OD 600 of 0.5 or on M9 plates at 37°C for 24 h. The cell pellet was washed once with ice-cold PBS and resuspended in 5% (wt/vol) trichloroacetic acid (TCA) solution (100 l for 10 mg wet cells) as described previously (33). After vortex mixing and incubation at 4°C for 30 min, cell debris was removed by centrifugation at 20,000 ϫ g for 20 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…After vortex mixing and incubation at 4°C for 30 min, cell debris was removed by centrifugation at 20,000 ϫ g for 20 min at 4°C. Amino acids were derivatized as described previously (33). Briefly, 25 l of the diluted pCA24N empty vector ASKA clone pCA24N-thrC pCA24N expressing ThrC ASKA clone solution for 10 mg E. coli wet cells).…”

Section: Methodsmentioning

confidence: 99%

Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli

Ito

Yamamoto

Hori

et al. 2019

Appl Environ Microbiol

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Escherichia coli YggS (COG0325) is a member of the highly conserved pyridoxal 5=-phosphate (PLP)-binding protein (PLPBP) family. Recent studies suggested a role for this protein family in the homeostasis of vitamin B 6 and amino acids. The deletion or mutation of a member of this protein family causes pleiotropic effects in many organisms and is causative of vitamin B 6 -dependent epilepsy in humans. To date, little has been known about the mechanism by which lack of YggS results in these diverse phenotypes. In this study, we determined that the pyridoxine (PN) sensitivity observed in yggS-deficient E. coli was caused by the pyridoxine 5=phosphate (PNP)-dependent overproduction of Val, which is toxic to E. coli. The data suggest that the yggS mutation impacts Val accumulation by perturbing the biosynthetic of Thr from homoserine (Hse). Exogenous Hse inhibited the growth of the yggS mutant, caused further accumulation of PNP, and increased the levels of some intermediates in the Thr-Ile-Val metabolic pathways. Blocking the Thr biosynthetic pathway or decreasing the intracellular PNP levels abolished the perturbations of amino acid metabolism caused by the exogenous PN and Hse. Our data showed that a high concentration of intracellular PNP is the root cause of at least some of the pleiotropic phenotypes described for a yggS mutant of E. coli. IMPORTANCE Recent studies showed that deletion or mutation of members of the YggS protein family causes pleiotropic effects in many organisms. Little is known about the causes, mechanisms, and consequences of these diverse phenotypes. It was previously shown that yggS mutations in E. coli result in the accumulation of PNP and some metabolites in the Ile/Val biosynthetic pathway. This work revealed that some exogenous stresses increase the aberrant accumulation of PNP in the yggS mutant. In addition, the current report provides evidence indicating that some, but not all, of the phenotypes of the yggS mutant in E. coli are due to the elevated PNP level. These results will contribute to continuing efforts to determine the molecular functions of the members of the YggS protein family.

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“…OA is a tripeptide GSH analog with L-2-aminobutyrate (2-AB) in place of the cysteine group. The physiological role of ophthalmate is not entirely clear, but it is assumed that OA possesses antioxidant properties and exerts a protective function due to its similarity to GSH (40). OA can be synthesized from γ-glutamyl-2-aminobutyric acid (γ-Glu-2AB), using the same enzymes as GSH biosynthesis.…”

Section: Dynamics Of Adenylates: Optimal Energy Storage For Rapidmentioning

confidence: 99%

Combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect

Ryabova

Cornette

Cherkasov

et al. 2020

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

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Some organisms have evolved a survival strategy to withstand severe dehydration in an ametabolic state, called anhydrobiosis. The only known example of anhydrobiosis among insects is observed in larvae of the chironomid Polypedilum vanderplanki. Recent studies have led to a better understanding of the molecular mechanisms underlying anhydrobiosis and the action of specific protective proteins. However, gene regulation alone cannot explain the rapid biochemical reactions and independent metabolic changes that are expected to sustain anhydrobiosis. For this reason, we conducted a comprehensive comparative metabolome–transcriptome analysis in the larvae. We showed that anhydrobiotic larvae adopt a unique metabolic strategy to cope with complete desiccation and, in particular, to allow recovery after rehydration. We argue that trehalose, previously known for its anhydroprotective properties, plays additional vital roles, providing both the principal source of energy and also the restoration of antioxidant potential via the pentose phosphate pathway during the early stages of rehydration. Thus, larval viability might be directly dependent on the total amount of carbohydrate (glycogen and trehalose). Furthermore, in the anhydrobiotic state, energy is stored as accumulated citrate and adenosine monophosphate, allowing rapid reactivation of the citric acid cycle and mitochondrial activity immediately after rehydration, before glycolysis is fully functional. Other specific adaptations to desiccation include potential antioxidants (e.g., ophthalmic acid) and measures to avoid the accumulation of toxic waste metabolites by converting these to stable and inert counterparts (e.g., xanthurenic acid and allantoin). Finally, we confirmed that these metabolic adaptations correlate with unique organization and expression of the corresponding enzyme genes.

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Production of Ophthalmic Acid Using Engineered Escherichia coli

Cited by 10 publications

References 39 publications

Inhibition of glycine cleavage system by pyridoxine 5′‐phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli

Inhibition of glycine cleavage system by pyridoxine 5′‐phosphate causes synthetic lethality in glyA yggS and serA yggS in Escherichia coli

Conserved Pyridoxal 5'-Phosphate-Binding Protein YggS Impacts Amino Acid Metabolism through Pyridoxine 5'-Phosphate in Escherichia coli

Combined metabolome and transcriptome analysis reveals key components of complete desiccation tolerance in an anhydrobiotic insect

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