Increase in furfural tolerance by combinatorial overexpression of NAD salvage pathway enzymes in engineered isobutanol-producing E. coli

2017 ◽  
Vol 245 ◽  
pp. 1430-1435 ◽  
Author(s):  
Hun-Suk Song ◽  
Jong-Min Jeon ◽  
Hyun-Joong Kim ◽  
Shashi Kant Bhatia ◽  
Ganesan Sathiyanarayanan ◽  
...  
Keyword(s):  
Salvage Pathway ◽  
E Coli ◽  
Furfural Tolerance

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Increase in Furfural Tolerance in Ethanologenic Escherichia coli LY180 by Plasmid-Based Expression ofthyA

2012 ◽  
Vol 78 (12) ◽  
pp. 4346-4352 ◽  
Author(s):  
Huabao Zheng ◽  
Xuan Wang ◽  
Lorraine P. Yomano ◽  
Keelnatham T. Shanmugam ◽  
Lonnie O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cellular Response ◽  
De Novo ◽  
Content Type ◽  
Genomic Libraries ◽  
Methyl Donor ◽  
E Coli ◽  
Furfural Tolerance ◽  
Small Benefit ◽  
Ethanologenic Escherichia Coli

ABSTRACTFurfural is an inhibitory side product formed during the depolymerization of hemicellulose by mineral acids. Genomic libraries from three different bacteria (Bacillus subtilisYB886,Escherichia coliNC3, andZymomonas mobilisCP4) were screened for genes that conferred furfural resistance on plates. Beneficial plasmids containing thethyAgene (coding for thymidylate synthase) were recovered from all three organisms. Expression of this key gene in thede novopathway for dTMP biosynthesis improved furfural resistance on plates and during fermentation. A similar benefit was observed by supplementation with thymine, thymidine, or the combination of tetrahydrofolate and serine (precursors for 5,10-methylenetetrahydrofolate, the methyl donor for ThyA). Supplementation with deoxyuridine provided a small benefit, and deoxyribose was of no benefit for furfural tolerance. A combination of thymidine and plasmid expression ofthyAwas no more effective than either alone. Together, these results demonstrate that furfural tolerance is increased by approaches that increase the supply of pyrimidine deoxyribonucleotides. However, ThyA activity was not directly affected by the addition of furfural. Furfural has been previously shown to damage DNA inE. coliand to activate a cellular response to oxidative damage in yeast. The added burden of repairing furfural-damaged DNA inE. coliwould be expected to increase the cellular requirement for dTMP. Increased expression ofthyA(E. coli,B. subtilis, orZ. mobilis), supplementation of cultures with thymidine, and supplementation with precursors for 5,10-methylenetetrahydrofolate (methyl donor) are each proposed to increase furfural tolerance by increasing the availability of dTMP for DNA repair.

scholarly journals RNase I regulates Escherichia coli 2′,3′-cyclic nucleotide monophosphate levels and biofilm formation

2018 ◽  
Vol 475 (8) ◽  
pp. 1491-1506 ◽  
Author(s):  
Benjamin M. Fontaine ◽  
Kevin S. Martin ◽  
Jennifer M. Garcia-Rodriguez ◽  
Claire Jung ◽  
Laura Briggs ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Cyclic Nucleotide ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Rna Degradation ◽  
Bacterial Biofilm ◽  
Biological Functions ◽  
Salvage Pathway ◽  
E Coli

Regulation of nucleotide and nucleoside concentrations is critical for faithful DNA replication, transcription, and translation in all organisms, and has been linked to bacterial biofilm formation. Unusual 2′,3′-cyclic nucleotide monophosphates (2′,3′-cNMPs) recently were quantified in mammalian systems, and previous reports have linked these nucleotides to cellular stress and damage in eukaryotes, suggesting an intriguing connection with nucleotide/nucleoside pools and/or cyclic nucleotide signaling. This work reports the first quantification of 2′,3′-cNMPs in Escherichia coli and demonstrates that 2′,3′-cNMP levels in E. coli are generated specifically from RNase I-catalyzed RNA degradation, presumably as part of a previously unidentified nucleotide salvage pathway. Furthermore, RNase I and 2′,3′-cNMP levels are demonstrated to play an important role in controlling biofilm formation. This work identifies a physiological role for cytoplasmic RNase I and constitutes the first progress toward elucidating the biological functions of bacterial 2′,3′-cNMPs.

Mechanism of pyridoxine 5'-phosphate accumulation in PLPBP protein-deficiency

2022 ◽  
Author(s):  
Tomokazu Ito ◽  
Honoka Ogawa ◽  
Hisashi Hemmi ◽  
Diana M. Downs ◽  
Tohru Yoshimura
Keyword(s):  
De Novo ◽  
Binding Protein ◽  
Vitamin B ◽  
Wild Type ◽  
Salvage Pathway ◽  
Intracellular Accumulation ◽  
Pyridoxal Kinase ◽  
Genetic Studies ◽  
E Coli ◽  
Metabolic Systems

The pyridoxal 5'-phosphate (PLP)-binding protein (PLPBP) plays an important role in vitamin B 6 homeostasis. Loss of this protein in organisms such as Escherichia coli and humans disrupts the vitamin B 6 pool and induces intracellular accumulation of pyridoxine 5'-phosphate (PNP), which is normally undetectable in wild-type cells. The accumulated PNP could affect diverse metabolic systems through inhibition of some PLP-dependent enzymes. In this study, we investigated the as yet unclear mechanism of intracellular accumulation of PNP by the loss of PLPBP protein encoded by yggS in E. coli . Genetic studies using several PLPBP-deficient strains of E. coli lacking known enzyme(s) in the de novo or salvage pathway of vitamin B 6 , which includes pyridoxine (amine) 5'-phosphate oxidase (PNPO), PNP synthase, pyridoxal kinase, and pyridoxal reductase, demonstrated that neither the flux from the de novo pathway nor the salvage pathway solely contributed to the PNP accumulation caused by the PLPBP mutation. Studies with the strains lacking both PLPBP and PNPO suggested that PNP shares the same pool with PMP, and showed that PNP levels are impacted by PMP levels and vice versa . We show that disruption of PLPBP lead to perturb PMP homeostasis, which may result in PNP accumulation in the PLPBP-deficient strains. Importance A PLP-binding protein PLPBP from the conserved COG0325 family has recently been recognized as a key player in vitamin B 6 homeostasis in various organisms. Loss of PLPBP disrupts vitamin B 6 homeostasis and perturbs diverse metabolisms, including amino acid and α-keto acid metabolism. Accumulation of PNP is a characteristic phenotype of the PLPBP deficiency and is suggested to be a potential cause of the pleiotropic effects, but the mechanism of the PNP accumulation was poorly understood. In this study, we show that fluxes for PNP synthesis/metabolism are not responsible for the accumulation of PNP. Our results indicate that PLPBP is involved in the homeostasis of pyridoxamine 5'-phosphate, and its disruption may lead to the accumulation of PNP in PLPBP-deficiency.

Bifunctional cytosolic UDP-glucose 4-epimerases catalyse the interconversion between UDP-D-xylose and UDP-L-arabinose in plants

2009 ◽  
Vol 424 (2) ◽  
pp. 169-177 ◽  
Author(s):  
Toshihisa Kotake ◽  
Ryohei Takata ◽  
Rajeev Verma ◽  
Masato Takaba ◽  
Daisuke Yamaguchi ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
De Novo ◽  
Salvage Pathway ◽  
Cell Wall Polysaccharides ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Pisum Sativum L ◽  
Net Flux ◽  
Terminal Amino ◽  
Apparent Equilibrium

UDP-sugars serve as substrates in the synthesis of cell wall polysaccharides and are themselves generated through sequential interconversion reactions from UDP-Glc (UDP-glucose) as the starting substrate in the cytosol and the Golgi apparatus. For the present study, a soluble enzyme with UDP-Xyl (UDP-xylose) 4-epimerase activity was purified approx. 300-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The N-terminal amino acid sequence of the enzyme revealed that it is encoded by a predicted UDP-Glc 4-epimerase gene, PsUGE1, and is distinct from the UDP-Xyl 4-epimerase localized in the Golgi apparatus. rPsUGE1 (recombinant P. sativum UGE1) expressed in Escherichia coli exhibited both UDP-Xyl 4-epimerase and UDP-Glc 4-epimerase activities with apparent Km values of 0.31, 0.29, 0.16 and 0.15 mM for UDP-Glc, UDP-Gal (UDP-galactose), UDP-Ara (UDP-L-arabinose) and UDP-Xyl respectively. The apparent equilibrium constant for UDP-Ara formation from UDP-Xyl was 0.89, whereas that for UDP-Gal formation from UDP-Glc was 0.24. Phylogenetic analysis revealed that PsUGE1 forms a group with Arabidopsis UDP-Glc 4-epimerases, AtUGE1 and AtUGE3, apart from a group including AtUGE2, AtUGE4 and AtUGE5. Similar to rPsUGE1, recombinant AtUGE1 and AtUGE3 expressed in E. coli showed high UDP-Xyl 4-epimerase activity in addition to their UDP-Glc 4-epimerase activity. Our results suggest that PsUGE1 and its close homologues catalyse the interconversion between UDP-Xyl and UDP-Ara as the last step in the cytosolic de novo pathway for UDP-Ara generation. Alternatively, the net flux of metabolites may be from UDP-Ara to UDP-Xyl as part of the salvage pathway for Ara.

scholarly journals Truncation of poly-N-acetylglucosamine (PNAG) polymerization with N-acetylglucosamine analogues

2021 ◽  
Author(s):  
Zachary Morrison ◽  
Alexander Eddenden ◽  
Adithya S Subramanian ◽  
P. Lynne Howell ◽  
mark nitz
Keyword(s):  
Biofilm Formation ◽  
Chain Termination ◽  
Salvage Pathway ◽  
Biofilm Inhibition ◽  
E Coli ◽  
Substrate Analogues

Bacteria require polysaccharides for structure, survival, and virulence. Despite the central role these structures play in microbiology few tools are available to manipulate their production. In E. coli the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro the inhibitors cause PNAG chain termination; consistent with the mechanism of PNAG polymerization from the non-reducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

scholarly journals Crystal Structure of Pyridoxal Kinase from the Escherichia coli pdxK Gene: Implications for the Classification of Pyridoxal Kinases

2006 ◽  
Vol 188 (12) ◽  
pp. 4542-4552 ◽  
Author(s):  
Martin K. Safo ◽  
Faik N. Musayev ◽  
Martino L. di Salvo ◽  
Sharyn Hunt ◽  
Jean-Baptiste Claude ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Active Site ◽  
Pyridoxal Phosphate ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
E Coli ◽  
Binary Complexes ◽  
Significant Conformational Change

ABSTRACT The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-Å resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4′ substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4′ of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

scholarly journals Pyridoxal Reductase, PdxI, Is Critical for Salvage of Pyridoxal in Escherichia coli

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Tomokazu Ito ◽  
Diana M. Downs
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Active Form ◽  
Biologically Active ◽  
Vitamin B ◽  
Limited Information ◽  
Salvage Pathway ◽  
Content Type ◽  
E Coli ◽  
Wide Range

ABSTRACT Pyridoxal 5′-phosphate (PLP) is the biologically active form of vitamin B6 and an essential cofactor in all organisms. In Escherichia coli, PLP is synthesized via the deoxyxylulose 5-phosphate (DXP)-dependent pathway that includes seven enzymatic steps and generates pyridoxine 5′-phosphate as an intermediate. Additionally, E. coli is able to salvage pyridoxal, pyridoxine, and pyridoxamine B6 vitamers to produce PLP using kinases PdxK/PdxY and pyridox(am)ine phosphate oxidase (PdxH). We found that E. coli strains blocked in PLP synthesis prior to the formation of pyridoxine 5′-phosphate (PNP) required significantly less exogenous pyridoxal (PL) than strains lacking pdxH and identified the conversion of PL to pyridoxine (PN) during cultivation to be the cause. Our data showed that PdxI, shown to have PL reductase activity in vitro, was required for the efficient salvage of PL in E. coli. The pdxI+ E. coli strains converted exogenous PL to PN during growth, while pdxI mutants did not. In total, the data herein demonstrated that PdxI is a critical enzyme in the salvage of PL by E. coli. IMPORTANCE The biosynthetic pathway of pyridoxal 5′-phosphate (PLP) has extensively been studied in Escherichia coli, yet limited information is available about the vitamin B6 salvage pathway. We show that the protein PdxI (YdbC) is the primary pyridoxal (PL) reductase in E. coli and is involved in the salvage of PL. The orthologs of PdxI occur in a wide range of bacteria and plants, suggesting that PL reductase in the B6 salvage pathway is more widely distributed than previously expected.

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