Gnd Gene Research Articles

Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens in Escherichia coli, and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of the gnd gene in E. coli K-12 MG1655. We show that GndA pulls down components of respiratory complex I (RCI) and is required for proper localization of a RCI subunit during heat shock. At high temperature GndA deletion (ΔGndA) cells exhibit perturbations in cell growth, NADH+/NAD ratio, and expression of a number of genes including several associated with oxidative stress. These findings suggest that GndA may function in maintenance of homeostasis during heat shock. Characterization of GndA therefore supports the nascent but growing consensus that functional, overlapping genes occur in genomes from viruses to humans.

Optimizing Hexose Utilization Pathways of Cupriavidus necator for Improving Growth and L-Alanine Production under Heterotrophic and Autotrophic Conditions.

Cupriavidus necator is a versatile microbial chassis to produce high-value products. Blocking the poly-β-hydroxybutyrate synthesis pathway (encoded by the phaC1AB1 operon) can effectively enhance the production of C. necator, but usually decreases cell density in the stationary phase. To address this problem, we modified the hexose utilization pathways of C. necator in this study by implementing strategies such as blocking the Entner-Doudoroff pathway, completing the phosphopentose pathway by expressing the gnd gene (encoding 6-phosphogluconate dehydrogenase), and completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene (encoding 6-phosphofructokinase). During heterotrophic fermentation, the OD600 of the phaC1AB1-knockout strain increased by 44.8% with pfkA gene expression alone, and by 93.1% with gnd and pfkA genes expressing simultaneously. During autotrophic fermentation, gnd and pfkA genes raised the OD600 of phaC1AB1-knockout strains by 19.4% and 12.0%, respectively. To explore the effect of the pfkA gene on the production of C. necator, an alanine-producing C. necator was constructed by expressing the NADPH-dependent L-alanine dehydrogenase, alanine exporter, and knocking out the phaC1AB1 operon. The alanine-producing strain had maximum alanine titer and yield of 784 mg/L and 11.0%, respectively. And these values were significantly improved to 998 mg/L and 13.4% by expressing the pfkA gene. The results indicate that completing the Embden-Meyerhof-Parnas pathway by expressing the pfkA gene is an effective method to improve the growth and production of C. necator.

Real-Time PCR based Rapid and Culture-Independent Detection of Citrobacter: ‘An Alarming State for Environment and Human Health’

The changing global scenario of environmental conditions and altered microbial community has severely deteriorated the quality of water which enforced an elevated health risks on human beings due to unavailability of safe and pristine water. Citrobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. It is an opportunistic pathogen formerly used as an indicator bacterium along with other fecal coliforms namely Escherichia coli, Klebsiella and Enterobacter, but is now known to cause several diseases in human beings. In present study we collected 135 samples from potable water and riverine-systems and confirmed the existence of Citrobacter using the conserved sequence of gnd (6-phosphogluconate dehydrogenate) gene with the application of SYBR green real-time PCR, which demonstrated extremely high specificity, fidelity and stringency in detection strategy. The Citrobacter counts in potable water were in the range of zero to 1.54 x104 ± 572.88 cfu/100ml. The Citrobacter levels in surface water were 2.18 x104 ±848, 1.54 x104 ±462, 3.53 x104 ±1299, 2.57 x104 ±912, 8.78 x103 ±201, 1.28 x104 ±370, CFU/100ml; associated with hydrophytes were 6.24 x104 ±1535, 8.42 x104 ±2711, 1.19 x105 ±4792, 1.12 x105 ±4177, 8.32 x104 ±2329, 8.08 x104 ±2149 CFU/10g and in the sediments the counts were 2.50 x104 ±647, 5.02 x104 ±1852, 3.26 x104 ±1186, 7.65 x104 ±2884, 1.50 x104 ±355, and 2.56 x104 ±737 CFU/10g at sampling site # 1, site # 2, site # 3, site # 4, site # 5 and site # 6, respectively. The present assay could be applied in the detection and regular monitoring of potable water and other environmental samples to check waterborne diseases and outbreaks caused by Citrobacter.

Nutrition promotion of brewer's spent grain by symbiotic fermentation adding Bacillus velezensis and Levilactobacillus brevis

Brewer's spent grain (BSG) is the main byproduct of the beer industry and is commonly used as a low-value animal feed or food ingredient. This study investigated the role of symbiotic fermentation with Bacillus velezensis and Levilactobacillus brevis in improving the nutritional value of BSG. The addition of B. velezensis increased the cell growth of L. brevis by over thirty-fold higher than that in the single-strain system. Simultaneously, cellulase and protease activities were substantially increased, and more soluble sugars were produced. Total amino acid, glutamic acid, and γ-aminobutyric acid (GABA) levels increased by 52.2%, 155.1%, and 144.1%, respectively. Conversely, lactic acid production decreased. Genetic analysis showed that the glycolysis-related gene pykA, pentose phosphate pathway gene gnd, and GABA-related gene gad were upregulated, while the heterolactin-related gene xpkA was downregulated, explaining that B. velezensis might inhibit heterolactin fermentation while enhancing GABA biosynthesis. This study provides a potential strategy for the high-value utilization of BSG in the food or feed industry.

Metabolomics for the design of new metabolic engineering strategies for improving aerobic succinic acid production in Escherichia coli

IntroductionGlycerol is a byproduct from the biodiesel industry that can be biotransformed by Escherichia coli to high added-value products such as succinate under aerobic conditions. The main genetic engineering strategies to achieve this aim involve the mutation of succinate dehydrogenase (sdhA) gene and also those responsible for acetate synthesis including acetate kinase, phosphate acetyl transferase and pyruvate oxidase encoded by ackA, pta and pox genes respectively in the ΔsdhAΔack-ptaΔpox (M4) mutant. Other genetic manipulations to rewire the metabolism toward succinate consist on the activation of the glyoxylate shunt or blockage the pentose phosphate pathway (PPP) by deletion of isocitrate lyase repressor (iclR) or gluconate dehydrogenase (gnd) genes on M4-ΔiclR and M4-Δgnd mutants respectively.ObjectiveTo deeply understand the effect of the blocking of the pentose phosphate pathway (PPP) or the activation of the glyoxylate shunt, metabolite profiles were analyzed on M4-Δgnd, M4-ΔiclR and M4 mutants.MethodsMetabolomics was performed by FT-IR and GC–MS for metabolite fingerprinting and HPLC for quantification of succinate and glycerol.ResultsMost of the 65 identified metabolites showed lower relative levels in the M4-ΔiclR and M4-Δgnd mutants than those of the M4. However, fructose 1,6-biphosphate, trehalose, isovaleric acid and mannitol relative concentrations were increased in M4-ΔiclR and M4-Δgnd mutants. To further improve succinate production, the synthesis of mannitol was suppressed by deletion of mannitol dehydrogenase (mtlD) on M4-ΔgndΔmtlD mutant that increase ~ 20% respect to M4-Δgnd.ConclusionMetabolomics can serve as a holistic tool to identify bottlenecks in metabolic pathways by a non-rational design. Genetic manipulation to release these restrictions could increase the production of succinate.

Increased NADPH Supply Enhances Glycolysis Metabolic Flux and L-methionine Production in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important strain for the industrial production of amino acids, but the fermentation of L-methionine has not been realized. The purpose of this study is to clarify the effect of reducing power NADPH on L-methionine synthesis. Site-directed mutagenesis of zwf and gnd genes in pentose phosphate pathway relieved feedback inhibition, increased NADPH supply by 151.8%, and increased L-methionine production by 28.3%; Heterologous expression of gapC gene to introduce NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase increased NADPH supply by 75.0% and L-methionine production by 48.7%; Heterologous expression of pntAB gene to introduce membrane-integral nicotinamide nucleotide transhydrogenase increased NADPH by 89.2% and L-methionine production by 35.9%. Finally, the engineering strain YM6 with a high NADPH supply was constructed, which increased the NADPH supply by 348.2% and the L-methionine production by 64.1%. The analysis of metabolic flux showed that YM6 significantly increased the glycolytic flux, including the metabolic flux of metabolites such as glycosyldehyde-3-phosphate, dihydroxyacetate phosphate, 3-phosphoglycate and pyruvate, and the significant increase of L-methionine flux also confirmed the increase of its synthesis. This study provides a research basis for the systematic metabolic engineering construction of L-methionine high-yield engineering strains.

Genotyping of Salmonella enterica strains from animal and human origin using three molecular techniques

This study aims to characterize different Salmonella enterica subsp molecularly. enterica strains (n=49) were isolated from human gastrointestinal cases in the Tolima region and poultry from Santander and Tolima regions using PCR-RFLP, PCR-ribotyping, and PCR-SSCP. The band patterns obtained with each technique were analyzed by building dendrograms based on the Unweighted Pair Group Method with Arithmetic mean (UPGMA) method and using the Dice coefficient. On the other hand, the discriminatory power of each technique was assessed using Simpson's discriminatory index. The genetic profiles of the gnd gene obtained with AciI restriction enzyme and the PCR-SSCP carried out with groEL gene allowed the inter-and intraserovar differentiation. Finally, the PCR-ribotyping method exhibited the highest discriminatory power (0.8571). In conclusion, we show three PCR-based genotyping methods providing an alternative for identifying similarities and differences within Salmonella enterica strains from different geographic and biological regions.

A new species of Aphis Linnaeus from Guangxi, China (Hemiptera, Aphididae), based on morphological characters and DNA sequences

• A new species of the genus Aphis feeding on Gnetum parvifolium from Guangxi, China. • A key to Aphis species with stridulatory spines on the hind tibiae is provided. • The taxonomic position of this new species is supported by molecular annalysis. A new species of the genus Aphis with stridulatory apparatus, Aphis gnetuma Qiao sp. n. , feeding on Gnetum parvifolium which is a new host plant record for aphids from Guangxi, China is described. The new species is distinguished by a well-developed median frontal tubercle and body bearing white wax powder in life. The taxonomic position of this new species is supported by analysis results of COI and gnd genes. A key to Aphis species with stridulatory spines on the hind tibiae of apterous viviparous females is provided. The type specimens studied are deposited in the National Zoological Museum of China, Institute of Zoology, Chinese Academy of Sciences, Beijing, China. www.zoobank.org/urn:lsid:zoobank.org:act:BBB024DA-8454-4E22-8736-66ED92A3091B .

A single-gene approach for the subspecies classification of Mycobacteroides abscessus.

The subspecies classification of Mycobacteroides abscessus complex into M. abscessus, M. massiliense and M. bolletii requires the amplification and sequencing of multiple genes. The objective of this study was to evaluate the possibility of subspecies classification using a single PCR target. An in silico study was performed to classify 1613 strains deposited in a public database using 9 genes (partial gene sequences of hsp65, rpoB, sodA, argH, cya, glpK, gnd, and murC, and the full gene sequence of MAB_3542c). We found the housekeeping gene gnd to be able to classify the M. abscessus subspecieswith high accuracy (99.94%). A single-gene PCR approach based on gnd would be a suitable replacement for the more expensive, labor-intensive and time-consuming multi-gene PCR analysis currently in use for the subspecies identification of M. abscessus.

The relevance of enzyme specificity for coenzymes and the presence of 6-phosphogluconate dehydrogenase for polyhydroxyalkanoates production in the metabolism of Pseudomonas sp. LFM046

Reconstruction of genome-based metabolic model is a useful approach for the assessment of metabolic pathways, genes and proteins involved in the environmental fitness capabilities or pathogenic potential as well as for biotechnological processes development. Pseudomonas sp. LFM046 was selected as a good polyhydroxyalkanoates (PHA) producer from carbohydrates and plant oils. Its complete genome sequence and metabolic model were obtained. Analysis revealed that the gnd gene, encoding 6-phosphogluconate dehydrogenase, is absent in Pseudomonas sp. LFM046 genome. In order to improve the knowledge about LFM046 metabolism, the coenzyme specificities of different enzymes was evaluated. Furthermore, the heterologous expression of gnd genes from Pseudomonas putida KT2440 (NAD+ dependent) and Escherichia coli MG1655 (NADP+ dependent) in LFM046 was carried out and provoke a delay on cell growth and a reduction in PHA yield, respectively. The results indicate that the adjustment in cyclic Entner-Doudoroff pathway may be an interesting strategy for it and other bacteria to simultaneously meet divergent cell needs during cultivation phases of growth and PHA production.

Shiga-toxigenic Escherichia coli from animal food sources in Mauritius: Prevalence, serogroup diversity and virulence profiles

Shiga-toxigenic Escherichia coli (STEC) are important human pathogens associated with diarrhea and in some cases haemorrhagic colitis. Contaminated food derived from cattle and wildlife species are often associated with disease outbreaks. In this study, we report the prevalence, serogroup diversity and virulence profiles of STEC strains derived from cattle, rusa deer and pig. Of the 422 samples analyzed, STEC were detected in 40% (80/200) of cattle, 27.0% (33/122) of deer and 13.0% (13/100) of pigs. STEC isolates belonged to 38 O-serogroups whereby 5.2% (24/462) of the isolates belonged to clinically important EHEC-7 serogroups: O26 (n = 2), O103 (n = 1), O145 (n = 3) and O157 (n = 18). Fourteen serogroups (O26, O51, O84, O91, O100, O104, O110, O117, O145, O146, O156, O157, O177 and ONT) displayed multiple virulence profiles. We also identified two serovars (O117 and O119) in deer which are not well-documented in epidemiological surveys. 73.7% (28/38) of recovered O-serogroups are known to be associated with serious human illnesses including haemolytic uremic syndrome (HUS) and bloody diarrhea. STEC isolates harboring single genotypes stx1, stx2, eae and hlyA accounted for 3.0% (14/462), 9.1% (42/462), 47.6% (220/462) and 1.7% (8/462) of all STEC isolates screened, respectively. Virulence combinations stx1 and stx2 were harboured by 1.3% of isolates while strains with genetic profiles eae/hlyA were the second most prevalent amongst STEC isolates. The full known virulent genotypes (stx2/eae, stx1/stx2/eae, stx1/stx2/hlyA and stx2/eae/hlyA) were present in 22 of the 462 STEC strains. A total of 10 different virulence patterns were recovered amongst animal species. Phylogeny of the gnd gene showed that amongst STEC strains, serovar O100 outlined the main cluster. Fourteen (n = 14) different sequence types (STs) were identified from a panel of twenty (n = 20) STEC isolates. One of the isolate (PG007B) possessed a unique ST (adk 10, fumC 693, gyrB 4, icd 1, mdh 8, purA 8, recA 2) that could not be assigned using MLST databases. None of the ST's recovered in deer were observed in domestic species. Our findings shows that food associated animals found on the tropical island of Mauritius carry a diversity of STEC strains with many serovars known to be associated with human disease. This report indicates that increased awareness, surveillance and hygienic attention at critical stages of the human food chain are warranted.

Reinforcement of Rhodobacter sphaeroides cofactor NADPH to increase the production of farnesol

Farnesol (FOH) is produced by dephosphorylation of farnesyl diphosphate (FPP) derived from two universal building blocks, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). In Rhodobacter sphaeroides these building blocks are generated by MEP pathway, however, many of the biosynthetic reactions and biotransformations in the MEP pathway are limited by low availability of NADPH. Improvement of the amount of intracellular NADPH may enhance the synthesis of FOH. In this study, we utilized the strategies of increasing the production of NADPH and decreasing the consumption of NADPH. The expression of glucose 6-phosphate isomerase (pgi) and glutamate dehydrogenase (gdhA) were inhibited by RNA interference, respectively, and overexpression of 6-glucose phosphate dehydrogenase (zwf) and 6-glucose phosphate dehydrogenase (gnd) in the pentose phosphate pathway were carried out. The results showed that the content of NADPH in the recombinant strains increased significantly, the highest FOH production of RSpgii in the RNA interfered strain was 3.91 mg/g, and the FOH production increased to 3.43 mg/g after zwf gene and gnd gene has been overexpressed. In order to obtain strains with higher FOH production, we used RSpgii as the starting strain, and zwf, gnd and co-overexpressed zwf + gnd gene were overexpressed in RSpgii, respectively. The highest FOH production of the strain RSzgpi reached to 4.48 mg/g which was 2.24 times that of the starting strain RS-GY2.

Genomic and metabolic analysis of Komagataeibacter xylinus DSM 2325 producing bacterial cellulose nanofiber.

Bacterial cellulose nanofiber (CNF) is a polymer with a wide range of potential industrial applications. Several Komagataeibacter species, including Komagataeibacter xylinus as a model organism, produce CNF. However, the industrial application of CNF has been hampered by inefficient CNF production, necessitating metabolic engineering for the enhanced CNF production. Here, we present complete genome sequence and a genome-scale metabolic model KxyMBEL1810 of K. xylinus DSM 2325 for metabolic engineering applications. Genome analysis of this bacterium revealed that a set of genes associated with CNF biosynthesis and regulation were present in this bacterium, which were also conserved in another six representative Komagataeibacter species having complete genome information. To better understand the metabolic characteristics of K. xylinus DSM 2325, KxyMBEL1810 was reconstructed using genome annotation data, relevant computational resources and experimental growth data generated in this study. Random sampling and correlation analysis of the KxyMBEL1810 predicted pgi and gnd genes as novel overexpression targets for the enhanced CNF production. Among engineered K. xylinus strains individually overexpressing heterologous pgi and gnd genes, either from Escherichia coli or Corynebacterium glutamicum, batch fermentation of a strain overexpressing the E. coli pgi gene produced 3.15 g/L of CNF in a complex medium containing glucose, which was the best CNF concentration achieved in this study, and 115.8% higher than that (1.46 g/L) obtained from the control strain. Genome sequence data and KxyMBEL1810 generated in this study should be useful resources for metabolic engineering of K. xylinus for the enhanced CNF production.

Colitose-containing O-polysaccharide structure and O-antigen gene cluster of Escherichia albertii HK18069 related to those of Escherichia coli O55 and E. coli O128

A 3,6-dideoxy-l-xylo-hexose (colitose)-containing partially O-acetylated branched polysaccharide was obtained by mild acid hydrolysis (2% HOAc, 100 °C, 2 h) of the lipopolysaccharide of Escherichia albertii HK18069 followed by gel-permeation chromatography on Sephadex G-50 Superfine. Part of colitose residues (~40%) was cleaved upon hydrolysis, and the full cleavage was achieved by prolonged hydrolysis (8 h) under the same conditions and resulted in a modified linear polysaccharide. Structure of the O-polysaccharide of E. albertii HK18069 was established by 1D and 2D 1H and 13C NMR spectroscopy applied to both initial and modified O-deacetylated and colitose-free polysaccharides:▪ where β-d-Galp is mono-O-acetylated at position either 3 (~50%) or 4 (~30%). The O-antigen gene cluster of E. albertii HK18069 between conserved galF and gnd genes together with flanking regions was sequenced, and predicted functions of the genes were found to be consistent with the O-polysaccharide structure established. The O-polysaccharide structure and the O-antigen gene cluster of E. albertii HK18069 are related to those of Esherichia coli O55 and E. coli O128 reported earlier. It is proposed to create for strain HK18069 a new E. albertii O-serogroup, O8.

Deletion of gene gnd encoding 6-phosphogluconate dehydrogenase promotes L-serine biosynthesis in a genetically engineered strain of Methylobacterium sp. MB200.

To identify potential target genes involved in L-serine biosynthesis in Methylobacterium sp. MB200 and to evaluate the gnd genetically-engineered strains for L-serine production. Five genes that are not associated with the central metabolic pathway but with L-serine biosynthesis were identified from Methylobacterium sp. MB200 mutants. Gene gnd, encoding 6-phosphogluconate dehydrogenase (PGDH), was selected for further evaluation. The gnd deletion mutant showed a 600% increase in D-serine tolerance and an 80% decrease in PGDH activity compared to Methylobacterium sp. MB200. gnd over-expression did not affect D-serine tolerance, whereas it did increase enzyme-activity up to 136%. Additionally, analysis revealed that in Methylobacterium sp. MB200, L-serine inhibited PGDH activity. The deletion of gnd did not affect growth, whereas it did enhance the biosynthesis of L-serine, resulting in a 225% increase in production of L-serine compared to the wild-type. gnd, one of the five genes identified here that is associated with L-serine synthesis, can be developed as a potential candidate for metabolic engineering to promote L-serine synthesis in Methylobacterium sp. MB200.

Metabolic engineering of mevalonate-producing Escherichia coli strains based on thermodynamic analysis

Thermodynamic states of the central metabolism in a metabolically engineered Escherichia coli strain producing mevalonate (MVA) were studied to identify metabolic reactions with regulatory function for improvement of the specific rate of MVA production. Intracellular concentrations of metabolites were determined for E. coli strains expressing Enterococcus faecalis genes mvaE and mvaS (strain MV) by gas chromatography (GC)- and liquid chromatography (LC)-mass spectrometry (MS). Mixtures of 13C-labeled metabolites served as internal standards were prepared from E. coli cultured in a completely 13C-labeled medium. Based on the concentration data, the change in Gibbs energy (ΔG) and substrate saturation ([S]/KM) were calculated for each metabolic reaction and then compared between the control and MVA-producing strains. The thermodynamic and kinetic analyses showed that further activation of thermodynamically feasible reactions in the upper part of glycolysis and the pentose phosphate pathway seems difficult and that metabolic bypassing to the Entner–Doudoroff pathway was a promising strategy to improve the acetyl coenzyme A (AcCoA) and NADPH supply required for MVA biosynthesis. Strain MV-ΔGndΔGntR was constructed by deletion of the gnd and gntR genes, which respectively encode 6-phosphogluconate dehydrogenase and a negative regulator of the expression of two enzyme genes responsible for the Entner–Doudoroff pathway. Cultivation in the nongrowth phase revealed that the yield and specific production rate of MVA increased to 0.49 ± 0.01 Cmol (Cmol glucose)−1 and 2.61 ± 0.10 mmol (g dry cell weight)−1 h−1, which were 113% and 158% that of the MV strain, respectively.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization.

Structure elucidation and gene cluster characterization of the O-antigen of Escherichia coli O80

Mild alkaline degradation of the lipopolysaccharide of Escherichia coli O80 afforded a polysaccharide, which was studied by sugar analysis, selective cleavage of glycosidic linkages, and 1H and 13C NMR spectroscopy. Solvolysis of the polysaccharide with CF3CO2H cleaved the linkages of α-Fuc and β-linked GlcNAc and GalNAc residues to give two disaccharides. The following structure of the hexasaccharide repeating unit of the O-polysaccharide was established: [Display omitted] The polysaccharide repeat also contains a minor O-acetyl group but its position was not determined. The O-antigen gene cluster of E. coli O80 between the conserved galF and gnd genes was analyzed and found to be consistent with the O-polysaccharide structure established.

O antigen of FranconibacterpulverisG3872 (O1) is a 4-deoxy-d-arabino-hexose-containing polysaccharide synthesized by the ABC-transporter-dependent pathway.

Franconibacter (Enterobacter, Cronobacter) pulveris bacteria share several typical characteristics with, and hence pose a challenge for the detection of, Cronobacter sakazakii, an emerging opportunistic pathogen, which can cause severe infections in neonates. A structurally variable O-specific polysaccharide (OPS) called O antigen provides the major basis for the typing of Gram-negative bacteria. We investigated the structure and genetics of the O antigen of F. pulveris G3872 (designated O1). An OPS was isolated by mild alkaline degradation of the LPS, whereas the same polysaccharide and its oligosaccharide fragments were obtained by mild acid degradation. Studies by sugar analysis and NMR spectroscopy showed that the OPS contained d-ribose, l-rhamnose (l-Rha) and a rarely occurring monosaccharide 4-deoxy-d-arabino-hexose, and the OPS structure was established. The O-antigen gene cluster of F. pulveris G3872 between JUMPStart and gnd genes includes putative genes for glycosyltransferases, ATP-binding cassette (ABC)-transporter genes wzm and wzt, and genes for the synthesis of l-Rha, but no genes for the synthesis of 4-deoxy-d-arabino-hexose. A mutation test with the wzm gene confirmed that the OPS is synthesized and exported by the ABC-transporter-dependent pathway. A trifunctional transferase was suggested to catalyse formation of two glycosidic linkages and add a methyl group to the non-reducing end of the OPS to terminate the chain elongation. A carbohydrate-binding module that presumably recognizes the terminal methyl-modified monosaccharide was found at the C-terminus of Wzt. Primers specific for F. pulveris G3872 were designed based on the wzm gene, which has potential to be used for identification and detection of the O1 serogroup.

Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35gl−1 and 1.40gl−1 from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.