scholarly journals Facile synthesis of bioactive Allitol from D-psicose by coexpression of ribitol dehydrogenase and formate dehydrogenase in Escherichia Coli

2018 ◽  
Vol 4 ◽  
Author(s):  
Hinawi A.M. Hassanin ◽  
Mohammed A.A. Eassa ◽  
Bo Jiang
Keyword(s):  
Escherichia Coli ◽  
Substrate Concentration ◽  
Formate Dehydrogenase ◽  
Maximum Yield ◽  
Optimum Conditions ◽  
Facile Synthesis ◽  
Nadh Regeneration ◽  
E Coli ◽  
Ribitol Dehydrogenase

Coexpression of formate dehydrogenase (FDH) and ribitol dehydrogenase (RDH) in Escherichia coli was used for the synthesis of Allitol from D-psicose. FDH was coexpressed with RDH for continuous NADH regeneration. The results revealed that the optimum conditions for allitol production occurred at pH 7.0 and 30 °C. Allitol reached the maximum yield of 19.2 mg at 2.0% substrate concentration after 48 hours. Using D-psicose as a substrate, allitol was successfully produced using an engineered E. coli coexpressed with RDH and FDH.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

scholarly journals Chemobiosynthesis of Novel 6-Deoxyerythronolide B Analogues by Mutation of the Loading Module of 6-Deoxyerythronolide B Synthase 1

2005 ◽  
Vol 71 (8) ◽  
pp. 4503-4509 ◽  
Author(s):  
Sumati Murli ◽  
Karen S. MacMillan ◽  
Zhihao Hu ◽  
Gary W. Ashley ◽  
Steven D. Dong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptomyces Coelicolor ◽  
Cost Effective ◽  
Effective Alternative ◽  
Facile Synthesis ◽  
E Coli ◽  
Cost Effective Alternative ◽  
Deoxyerythronolide B Synthase

ABSTRACT Chemobiosynthesis (J. R. Jacobsen, C. R. Hutchinson, D. E. Cane, and C. Khosla, Science 277:367-369, 1997) is an important route for the production of polyketide analogues and has been used extensively for the production of analogues of 6-deoxyerythronolide B (6-dEB). Here we describe a new route for chemobiosynthesis using a version of 6-deoxyerythronolide B synthase (DEBS) that lacks the loading module. When the engineered DEBS was expressed in both Escherichia coli and Streptomyces coelicolor and fed a variety of acyl-thioesters, several novel 15-R-6-dEB analogues were produced. The simpler “monoketide” acyl-thioester substrates required for this route of 15-R-6-dEB chemobiosynthesis allow greater flexibility and provide a cost-effective alternative to diketide-thioester feeding to DEBS KS1o for the production of 15-R-6-dEB analogues. Moreover, the facile synthesis of the monoketide acyl-thioesters allowed investigation of alternative thioester carriers. Several alternatives to N-acetyl cysteamine were found to work efficiently, and one of these, methyl thioglycolate, was verified as a productive thioester carrier for mono- and diketide feeding in both E. coli and S. coelicolor.

scholarly journals The broad amine scope of pantothenate synthetase enables the synthesis of pharmaceutically relevant amides

2021 ◽  
Author(s):  
Mohammad Z. Abidin ◽  
Thangavelu Saravanan ◽  
Erick Strauss ◽  
Gerrit J. Poelarends
Keyword(s):  
Escherichia Coli ◽  
Facile Synthesis ◽  
E Coli ◽  
Pantothenate Synthetase ◽  
Vitamin B5

Pantothenate synthetase from Escherichia coli (PSE. coli) has a broad substrate scope, accepting diverse amines in the amidation of (R)-pantoate, enabling the facile synthesis of pharmaceutically relevant vitamin B5 antimetabolites.

scholarly journals Purification and properties of Klebsiella aerogenesd-arabitol dehydrogenase

1979 ◽  
Vol 183 (1) ◽  
pp. 31-42 ◽  
Author(s):  
M S Neuberger ◽  
R A Patterson ◽  
B S Hartley
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Activity Ratio ◽  
Klebsiella Aerogenes ◽  
Wild Type ◽  
E Coli ◽  
Type K ◽  
Purification And Properties ◽  
Immunological Cross Reaction ◽  
Ribitol Dehydrogenase

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.

scholarly journals Selenium Is Mobilized In Vivo from Free Selenocysteine and Is Incorporated Specifically into Formate Dehydrogenase H and tRNA Nucleosides

2002 ◽  
Vol 184 (7) ◽  
pp. 1940-1946 ◽  
Author(s):  
Gerard M. Lacourciere
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Formate Dehydrogenase ◽  
E Coli ◽  
Selenium Metabolism ◽  
Concomitant Decrease ◽  
Selenophosphate Synthetase

ABSTRACT Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP. It was recently demonstrated that selenium delivered from selenocysteine by an E. coli NifS-like protein could replace free selenide in the in vitro SPS assay for selenophosphate formation (G. M. Lacourciere, H. Mihara, T. Kurihara, N. Esaki, and T. C. Stadtman, J. Biol. Chem. 275:23769-23773, 2000). During growth of E. coli in the presence of 0.1 μM 75SeO3 2− and increasing amounts of l-selenocysteine, a concomitant decrease in 75Se incorporation into formate dehydrogenase H and nucleosides of bulk tRNA was observed. This is consistent with the mobilization of selenium from l-selenocysteine in vivo and its use in selenophosphate formation. The ability of E. coli to utilize selenocysteine as a selenium source for selenophosphate biosynthesis in vivo supports the participation of the NifS-like proteins in selenium metabolism.

scholarly journals One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

2015 ◽  
Vol 81 (11) ◽  
pp. 3648-3654 ◽  
Author(s):  
Ryotaro Hara ◽  
Masashi Nakano ◽  
Kuniki Kino
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Maximum Yield ◽  
Test Tube ◽  
Efficient Production ◽  
Scale Production ◽  
Deficient Mutant ◽  
One Pot ◽  
Content Type ◽  
E Coli

ABSTRACTWe developed a novel process for efficient synthesis ofl-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation ofl-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesizel-THA. By using these recombinant enzymes,l-THA was obtained byl-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube.l-THA was obtained in a small amount with a molar yield of 0.076% by using intactEscherichia coliexpressing theasnOgene, and thus, two asparaginase-deficient mutants ofE. coliwere investigated. A remarkably increasedl-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of theasnOgene was enhanced by using the T7 promoter inE. coliinstead of thelacpromoter, thel-THA yield was significantly increased to 92%. By using a combination of theE. coliasparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently,l-THA was successfully obtained froml-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production ofl-THA.

scholarly journals Identification of a Formate-Dependent Uric Acid Degradation Pathway inEscherichia coli

2019 ◽  
Vol 201 (11) ◽  
Author(s):  
Yumi Iwadate ◽  
Jun-ichi Kato
Keyword(s):  
Escherichia Coli ◽  
Uric Acid ◽  
Formate Dehydrogenase ◽  
Sole Source ◽  
Degradation Pathway ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Purine Catabolism ◽  
E Coli ◽  
Acid Degradation

ABSTRACTPurine is a nitrogen-containing compound that is abundant in nature. In organisms that utilize purine as a nitrogen source, purine is converted to uric acid, which is then converted to allantoin. Allantoin is then converted to ammonia. InEscherichia coli, neither urate-degrading activity nor a gene encoding an enzyme homologous to the known urate-degrading enzymes had previously been found. Here, we demonstrate urate-degrading activity inE. coli. We first identifiedaegAas anE. coligene involved in oxidative stress tolerance. An examination of gene expression revealed that bothaegAand its paralogygfTare expressed under both microaerobic and anaerobic conditions. TheygfTgene is localized within a chromosomal gene cluster presumably involved in purine catabolism. Accordingly, the expression ofygfTincreased in the presence of exogenous uric acid, suggesting thatygfTis involved in urate degradation. Examination of the change of uric acid levels in the growth medium with time revealed urate-degrading activity under microaerobic and anaerobic conditions in the wild-type strain but not in theaegA ygfTdouble-deletion mutant. Furthermore, AegA- and YgfT-dependent urate-degrading activity was detected only in the presence of formate and formate dehydrogenase H. Collectively, these observations indicate the presence of urate-degrading activity inE. colithat is operational under microaerobic and anaerobic conditions. The activity requires formate, formate dehydrogenase H, and eitheraegAorygfT. We also identified other putative genes which are involved not only in formate-dependent but also in formate-independent urate degradation and may function in the regulation or cofactor synthesis in purine catabolism.IMPORTANCEThe metabolic pathway of uric acid degradation to date has been elucidated only in aerobic environments and is not understood in anaerobic and microaerobic environments. In the current study, we showed thatEscherichia coli, a facultative anaerobic organism, uses uric acid as a sole source of nitrogen under anaerobic and microaerobic conditions. We also showed that formate, formate dehydrogenase H, and either AegA or YgfT are involved in uric acid degradation. We propose that formate may act as an electron donor for a uric acid-degrading enzyme in this bacterium.

scholarly journals Conservation and Variation between Rhodobacter capsulatus and Escherichia coli Tat Systems

2006 ◽  
Vol 188 (22) ◽  
pp. 7807-7814 ◽  
Author(s):  
Ute Lindenstrauß ◽  
Thomas Brüser
Keyword(s):  
Escherichia Coli ◽  
Dimethyl Sulfoxide ◽  
Formate Dehydrogenase ◽  
Rhodobacter Capsulatus ◽  
Translational Coupling ◽  
E Coli ◽  
Biochemical Assays ◽  
Tat System ◽  
Respiration Deficiency ◽  
Physiological And Biochemical

ABSTRACT The Tat system allows the translocation of folded and often cofactor-containing proteins across biological membranes. Here, we show by an interspecies transfer of a complete Tat translocon that Tat systems are largely, but not fully, interchangeable even between different classes of proteobacteria. The Tat apparatus from the α-proteobacterium Rhodobacter capsulatus was transferred to a Tat-deficient Escherichia coli strain, which is a γ-proteobacterium. Similar to that of E. coli, the R. capsulatus Tat system consists of three components, rc-TatA, rc-TatB, and rc-TatC. A fourth gene (rc-tatF) is present in the rc-tatABCF operon which has no apparent relevance for translocation. The translational starts of rc-tatC and rc-tatF overlap in four nucleotides (ATGA) with the preceding tat genes, pointing to efficient translational coupling of rc-tatB, rc-tatC, and rc-tatF. We show by a variety of physiological and biochemical assays that the R. capsulatus Tat system functionally targets the E. coli Tat substrates TorA, AmiA, AmiC, and formate dehydrogenase. Even a Tat substrate from a third organism is accepted, demonstrating that usually Tat systems and Tat substrates from different proteobacteria are compatible with each other. Only one exceptional Tat substrate of E. coli, a membrane-anchored dimethyl sulfoxide (DMSO) reductase, was not targeted by the R. capsulatus Tat system, resulting in a DMSO respiration deficiency. Although the general features of Tat substrates and translocons are similar between species, the data indicate that details in the targeting pathways can vary considerably.

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