The commercial production of chemicals using pathway engineering.
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Integration of metabolic pathway engineering and fermentation production technologies is necessary for the successful commercial production of chemicals. The 'toolbox' to do pathway engineering is ever expanding to enable mining of biodiversity, to maximize productivity, enhance carbon efficiency, improve product purity, expand product lines, and broaden markets. Functional genomics, proteomics, fluxomics, and physiomics are complementary to pathway engineering, and their successful applications are bound to multiply product turnover per cell, channel carbon efficiently, shrink the size of factories (i.e., reduce steel in the ground), and minimize product development cycle times to bring products to market. (+info)
The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.
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The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides). Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments. (+info)
Ascorbate-synthesizing system in rat liver microsomes. II. A peptide-bound flavin as the prosthetic group of L-gulono-gamma-lactone oxidase.
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L-Gulono-gamma-lactone oxidase [EC 1.1.3.8] was purified 80-fold from rat liver microsomes. In confirmation of our previous finding with a cruder preparation, the purified enzyme was shown to contain an L-gulono-gamma-lactone-reducible pigment as a prosthetic group. This pigment was not liberated from the protein by acid ammonium sulfate, 10% trichloroacetic acid or 2 M area, but was effectively released by proteolytic digestion. The pigment thus released showed a reduced-minus-oxidized difference spectrum characteristic of a flavin compound. The pigment was liberated from a trichloroacetic acid-treated preparation of the enzyme by pronase digestion and purified by Florisil column chromatography and paper chromatography. The absorption spectrum as well as the fluorescence emission and excitation spectra of the purified pigment indicated that it was actually a flavin peptide. It was, however, different not only from FMN but also from flavin peptides isolated from other sources such as succinate dehydrogenase [EC 1.3.99.1] and monoamine oxidase [EC 1.4.3.4] as regards the pH dependence of fluorescence intensity and the Rf value on thin-layer chromatography. A preliminary analysis showed that the purified flavin compound contained several amino acid residues. Alkaline photolysis of the purified flavin peptide suggested that the isoalloxazine ring of the flavin is involved in its binding to the peptide. The hypsochromic shift of the absorption peak in the near-ultraviolet region suggested further that the linkage between the flavin and the peptide may be mediated by the 8-methyl group of the isoalloxazine nucleus. It can be concluded that the prosthetic group of gulonolactone oxidase is a flavin which is covalently bound to the enzyme protein. (+info)
Control of exuT activity for galacturonate transport by the negative regulator ExuR in Erwinia chrysanthemi EC16.
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The negative regulatory protein ExuR in Erwinia chrysanthemi regulates expression of the galacturonate uptake (exuT) and utilization (uxaA, uxaB, uxaC) genes. We cloned and determined the nucleotide sequence of the exuR gene from E. chrysanthemi EC16. Analysis of the deduced amino acid sequence indicates that this protein possesses a helix-turn-helix motif and belongs to the GntR family of transcriptional repressors. Northern blot analysis and studies with transcriptional fusions of exuT in wild-type and exuR mutant backgrounds indicate that exuT transcription is deregulated in the exuR strain in vivo and in planta. [14C]-galacturonic acid uptake was constitutively high under inducing and noninducing conditions in the exuR mutant. Maximal exuT transcription activity was observed within 8 h of bacterial inoculation into potato tubers, well before any visible symptoms of disease were detected. This suggests that ExuT transport activity in E. chrysanthemi is important in the early stages of disease development. (+info)
Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid.
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Four bacterial strains that oxidize L-sorbose to 2-keto-L-gulonic acid, a key intermediate in the synthesis of vitamin C, were isolated from soils of geographically distinct locations. All were Gram-negative, facultatively anaerobic, chemoheterotrophic rods. Comparative analysis revealed nearly identical 16S rDNA sequences amongst them (99.7-100% identical) and identified them as members of the alpha-subclass of the Proteobacteria. Phylogenetic analysis identified the closest taxonomically defined genus as Roseobacter (92.1-92.8% identical). On the basis of phylogenetic, phenotypic and genotypic analyses, a new genus is proposed, Ketogulonigenium gen. nov. Based upon these analyses, we also propose the reclassification of strain DSM 4025TP, originally identified as Gluconobacter oxydans, to the genus Ketogulonigenium. Two species are proposed: the type species Ketogulonigenium vulgare gen. nov., sp. nov., consisting of strains 62A-12APP, 266-13BPP and the type strain K. vulgare DSM 4025TP, and Ketogulonigenium robustum gen. nov., sp. nov., consisting of the type strain K. robustum X6LTP (= NRRL B-21627 = KCTC 0858BP). The species affiliation of the fifth strain (291-19PP) remains unresolved. (+info)
Nutritional consequences of the Maillard reaction. The absorption of fructose-L-tryptophan in the large intestine of the rat.
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The absorption of fructose-L-tryptophan, an early Maillard reaction product formed from tryptophan and glucose, in the large intestine of the rat was examined. The possible involvement of cecal microflora of the rat in the absorption of fructose-L-tryptophan was investigated. Fructose-L-tryptophan was degraded by the cecal microflora (in vitro) after a 12-hour incubation at 37 degrees. Experiments with rat colonic rings and everted sacs indicated that a passive diffusion of this compound occurs in vitro in the colon of the rat. In vivo absorption was determined by introducing fructose-L-[3-14C]tryptophan directly into the cecum. Twenty percent of the total radioactivity injected was recovered in the urine after 24 hours. Fructose-L-tryptophan was detected in the urine, which suggested that this compound was absorbed by the large intestine but excreted without being metabolized. Approximately 1% of the total radioactivity was recovered in the expired air within 24 hours after injection. The small recovery of fructose-L-tryptophan in feces might indicate that this compound was degraded by the action of microorganisms in the large intestine of the rat. (+info)
Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion.
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A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone. KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli. (+info)
Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives.
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The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids. (+info)