xylB Genes Research Articles

Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.

Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5'-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI857 and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simultaneous and accurate editing of these two genes with an efficiency of 30%. This enabled the edited cells to retain their lysogenic state at 42 °C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNA method holds significant potential for widespread and practical use in synthetic biology.

Development of a novel methanotrophic platform to produce ectoine from methane and lignocellulose-derived sugars

Methane is considered a promising carbon source in the field of biotechnology. Using methane as the carbon source for producing chemicals reduces manufacturing costs and tackles the issue of global warming. Like methane, lignocellulose biomass is also an inexpensive and ample carbon source, which could be used for industrial biomanufacturing. In this study, Methylotuvimicrobium alcaliphilum 20Z was genetically modified to simultaneously consume methane and lignocellulose-derived sugars (glucose and xylose) to produce ectoine, a native osmoprotectant. The xylose utilization pathway, comprising xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli, was first integrated into the chromosome of M. alcaliphilum 20Z. Further expression of the glucose utilization, consisting of glf (glucose facilitator) from Zymmonas mobilis, glk (endogenous glucokinase), and pgi (glucose 6-phosphate isomerase) from E. coli, made the engineered strain 20ZXG consume methane, xylose, and glucose concurrently. Cultivation on three substrates upregulated the Embden–Meyerhof–Parnas and ectoine biosynthesis pathways of 20Z. As a result, the ectoine content produced by 20ZXG grown on the three substrates was 1.7-fold enhanced compared with that obtained on methane solely. Furthermore, the knock-out of ectoine biosynthesis repressor (ectR1) led to an output of 37.93 ± 3.27 mg/g-DCW, which was the highest quantity of ectoine produced after 48 h of cultivation in the presence of three substrates and was 2.3-fold higher than the initial output.

Biochemical characterization of xylanase GH11 isolated from Aspergillus niger BCC14405 (XylB) and its application in xylooligosaccharide production.

To develop an endo-β-1,4-xylanase with high specificity for production of prebiotic xylooligosaccharides that optimally works at moderate temperature desirable to reduce the energy cost in the production process. The xylB gene, encoding for a glycosyl hydrolase family 11 xylanase from a thermoresistant fungus, Aspergillus niger BCC14405 was expressed in a methylotrophic yeast P. pastoris KM71 in a secreted form. The recombinant XylB showed a high specific activity of 3852 and 169 Umg-1 protein on beechwood xylan and arabinoxylan, respectively with no detectable side activities against different forms of cellulose (Avicel Ò PH101 microcrystalline cellulose, phosphoric acid swollen cellulose and carboxymethylcellulose). The enzyme worked optimally at 45°C, pH 6.0. It showed a specific cleavage pattern by releasing xylobiose (X2) as the major product from xylooligosaccharides (X3 to X6) substrates. The highest XOS yield of 708mgg-1 substrate comprising X2, X3 and X6 was obtained from beechwood xylan hydrolysis. The enzyme is potent for XOS production and for saccharification of lignocellulosic biomass.

Herbaspirillum seropedicae expresses non-phosphorylative pathways for D-xylose catabolism.

Herbaspirillum seropedicae is a β-proteobacterium that establishes as an endophyte in various plants. These bacteria can consume diverse carbon sources, including hexoses and pentoses like D-xylose. D-xylose catabolic pathways have been described in some microorganisms, but databases of genes involved in these routes are limited. This is of special interest in biotechnology, considering that D-xylose is the second most abundant sugar in nature and some microorganisms, including H. seropedicae, are able to accumulate poly-3-hydroxybutyrate when consuming this pentose as a carbon source. In this work, we present a study of D-xylose catabolic pathways in H. seropedicae strain Z69 using RNA-seq analysis and subsequent analysis of phenotypes determined in targeted mutants in corresponding identified genes. G5B88_22805 gene, designated xylB, encodes a NAD+-dependent D-xylose dehydrogenase. Mutant Z69∆xylB was still able to grow on D-xylose, although at a reduced rate. This appears to be due to the expression of an L-arabinose dehydrogenase, encoded by the araB gene (G5B88_05250), that can use D-xylose as a substrate. According to our results, H. seropedicae Z69 uses non-phosphorylative pathways to catabolize D-xylose. The lower portion of metabolism involves co-expression of two routes: the Weimberg pathway that produces α-ketoglutarate and a novel pathway recently described that synthesizes pyruvate and glycolate. This novel pathway appears to contribute to D-xylose metabolism, since a mutant in the last step, Z69∆mhpD, was able to grow on this pentose only after an extended lag phase (40-50h). KEY POINTS: • xylB gene (G5B88_22805) encodes a NAD+-dependent D-xylose dehydrogenase. • araB gene (G5B88_05250) encodes a L-arabinose dehydrogenase able to recognize D-xylose. • A novel route involving mhpD gene is preferred for D-xylose catabolism.

Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source.

Xylose is the raw material for the synthesis of many important platform compounds. At present, xylose is commercially produced by chemical extraction. However, there are still some bottlenecks in the extraction of xylose, including complicated operation processes and the chemical substances introduced, leading to the high cost of xylose and of synthesizing the downstream compounds of xylose. The current market price of xylose is 8× that of glucose, so using low-cost glucose as the substrate to produce the downstream compounds of xylose can theoretically reduce the cost by 70%. Here, we designed a pathway for the biosynthesis of xylose from glucose in Escherichia coli. This biosynthetic pathway was achieved by overexpressing five genes, namely, zwf, pgl, gnd, rpe, and xylA, while replacing the native xylulose kinase gene xylB with araL from B. subtilis, which displays phosphatase activity toward d-xylulose 5-phosphate. The yield of xylose was increased to 3.3 g/L by optimizing the metabolic pathway. Furthermore, xylitol was successfully synthesized by introducing the xyl1 gene, which suggested that the biosynthetic pathway of xylose from glucose is universally applicable for the synthesis of xylose downstream compounds. This is the first study to synthesize xylose and its downstream compounds by using glucose as a substrate, which not only reduces the cost of raw materials, but also alleviates carbon catabolite repression (CCR), providing a new idea for the synthesis of downstream compounds of xylose.

Whole-Genome Sequence Data Analysis of Anoxybacillus kamchatkensis NASTPD13 Isolated from Hot Spring of Myagdi, Nepal.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

Improved Plasmid-Based Inducible and Constitutive Gene Expression in Corynebacterium glutamicum.

Corynebacterium glutamicum has been safely used in white biotechnology for the last 60 years and the portfolio of new pathways and products is increasing rapidly. Hence, expression vectors play a central role in discovering endogenous gene functions and in establishing heterologous gene expression. In this work, new expression vectors were designed based on two strategies: (i) a library screening of constitutive native and synthetic promoters and (ii) an increase of the plasmid copy number. Both strategies were combined and resulted in a very strong expression and overproduction of the fluorescence protein GfpUV. As a second test case, the improved vector for constitutive expression was used to overexpress the endogenous xylulokinase gene xylB in a synthetic operon with xylose isomerase gene xylA from Xanthomonas campestris. The xylose isomerase activity in crude extracts was increased by about three-fold as compared to that of the parental vector. In terms of application, the improved vector for constitutive xylA and xylB expression was used for production of the N-methylated amino acid sarcosine from monomethylamine, acetate, and xylose. As a consequence, the volumetric productivity of sarcosine production was 50% higher as compared to that of the strain carrying the parental vector.

Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin

Residual streams from lignocellulosic processes contain sugar mixtures of glucose, xylose, and mannose. Here, the industrial workhorse Corynebacterium glutamicum was explored as a research platform for the rational utilization of a multiple sugar substrate. The endogenous manA gene was overexpressed to enhance mannose utilization. The overexpression of the xylA gene from Xanthomonas campestris in combination with the endogenous xylB gene enabled xylose consumption by C. glutamicum. Furthermore, riboflavin production was triggered by overexpressing the sigH gene from C. glutamicum. The resulting strains were studied during batch fermentations in flasks and 2 L lab-scale bioreactors separately using glucose, mannose, xylose, and a mixture of these three sugars as a carbon source. The production of riboflavin and consumption of sugars were improved during fed-batch fermentation thanks to a dynamic inoculation strategy of manA overexpressing strain and xylAB overexpressing strain. The final riboflavin titer, yield, and volumetric productivity from the sugar mixture were 27 mg L−1, 0.52 mg g−1, and 0.25 mg L−1 h−1, respectively. It reached a 56% higher volumetric productivity with 45% less by-product formation compared with an equivalent process inoculated with a single strain overexpressing the genes xylAB and manA combined. The results indicate the advantages of dynamic multi strains processes for the conversion of sugar mixtures.

Heterologous expression of genes for bioconversion of xylose to xylonic acid in Corynebacterium glutamicum and optimization of the bioprocess

In bacterial system, direct conversion of xylose to xylonic acid is mediated through NAD-dependent xylose dehydrogenase (xylB) and xylonolactonase (xylC) genes. Heterologous expression of these genes from Caulobacter crescentus into recombinant Corynebacterium glutamicum ATCC 13032 and C. glutamicum ATCC 31831 (with an innate pentose transporter, araE) resulted in an efficient bioconversion process to produce xylonic acid from xylose. Process parameters including the design of production medium was optimized using a statistical tool, Response Surface Methodology (RSM). Maximum xylonic acid of 56.32 g/L from 60 g/L xylose, i.e. about 76.67% of the maximum theoretical yield was obtained after 120 h fermentation from pure xylose with recombinant C. glutamicum ATCC 31831 containing the plasmid pVWEx1 xylB. Under the same condition, the production with recombinant C. glutamicum ATCC 13032 (with pVWEx1 xylB) was 50.66 g/L, i.e. 69% of the theoretical yield. There was no significant improvement in production with the simultaneous expression of xylB and xylC genes together indicating xylose dehydrogenase activity as one of the rate limiting factor in the bioconversion. Finally, proof of concept experiment in utilizing biomass derived pentose sugar, xylose, for xylonic acid production was also carried out and obtained 42.94 g/L xylonic acid from 60 g/L xylose. These results promise a significant value addition for the future bio refinery programs.

Restriction-deficient mutants and marker-less genomic modification for metabolic engineering of the solvent producer Clostridium saccharobutylicum

BackgroundClostridium saccharobutylicum NCP 262 is a solventogenic bacterium that has been used for the industrial production of acetone, butanol, and ethanol. The lack of a genetic manipulation system for C. saccharobutylicum currently limits (i) the use of metabolic pathway engineering to improve the yield, titer, and productivity of n-butanol production by this microorganism, and (ii) functional genomics studies to better understand its physiology.ResultsIn this study, a marker-less deletion system was developed for C. saccharobutylicum using the codBA operon genes from Clostridium ljungdahlii as a counterselection marker. The codB gene encodes a cytosine permease, while codA encodes a cytosine deaminase that converts 5-fluorocytosine to 5-fluorouracil, which is toxic to the cell. To introduce a marker-less genomic modification, we constructed a suicide vector containing: the catP gene for thiamphenicol resistance; the codBA operon genes for counterselection; fused DNA segments both upstream and downstream of the chromosomal deletion target. This vector was introduced into C. saccharobutylicum by tri-parental conjugation. Single crossover integrants are selected on plates supplemented with thiamphenicol and colistin, and, subsequently, double-crossover mutants whose targeted chromosomal sequence has been deleted were identified by counterselection on plates containing 5-fluorocytosine. Using this marker-less deletion system, we constructed the restriction-deficient mutant C. saccharobutylicum ΔhsdR1ΔhsdR2ΔhsdR3, which we named C. saccharobutylicum Ch2. This triple mutant exhibits high transformation efficiency with unmethylated DNA. To demonstrate its applicability to metabolic engineering, the method was first used to delete the xylB gene to study its role in xylose and arabinose metabolism. Furthermore, we also deleted the ptb and buk genes to create a butyrate metabolism-negative mutant of C. saccharobutylicum that produces n-butanol at high yield.ConclusionsThe plasmid vectors and the method introduced here, together with the restriction-deficient strains described in this work, for the first time, allow for efficient marker-less genomic modification of C. saccharobutylicum and, therefore, represent valuable tools for the genetic and metabolic engineering of this industrially important solvent-producing organism.

Engineering cellulolytic bacterium Clostridium thermocellum to co-ferment cellulose- and hemicellulose-derived sugars simultaneously.

Cellulose and hemicellulose are the most abundant components in plant biomass. A preferred Consolidated Bioprocessing (CBP) system is one which can directly convert both cellulose and hemicellulose into target products without adding the costly hydrolytic enzyme cocktail. In this work, the thermophilic, cellulolytic, and anaerobic bacterium, Clostridium thermocellum DSM 1313, was engineered to grow on xylose in addition to cellulose. Both xylA (encoding for xylose isomerase) and xylB (encoding for xylulokinase) genes from the thermophilic anaerobic bacterium Thermoanaerobacter ethanolicus were introduced to enable xylose utilization while still retaining its inherent ability to grow on 6-carbon substrates. Targeted integration of xylAB into C. thermocellum genome realized simultaneous fermentation of xylose with glucose, with cellobiose (glucose dimer), and with cellulose, respectively, without carbon catabolite repression. We also showed that the respective H2 and ethanol production were twice as much when both xylose and cellulose were consumed simultaneously than when consuming cellulose alone. Moreover, the engineered xylose consumer can also utilize xylo-oligomers (with degree of polymerization of 2-7) in the presence of xylose. Isotopic tracer studies also revealed that the engineered xylose catabolism contributed to the production of ethanol from xylan which is a model hemicellulose in mixed sugar fermentation, demonstrating immense potential of this enhanced CBP strain in co-utilizing both cellulose and hemicellulose for the production of fuels and chemicals.

Metabolic engineering of Corynebacterium glutamicum for efficient production of succinate from lignocellulosic hydrolysate

BackgroundSuccinate has been recognized as one of the most important bio-based building block chemicals due to its numerous potential applications. However, efficient methods for the production of succinate from lignocellulosic feedstock were rarely reported. Nevertheless, Corynebacterium glutamicum was engineered to efficiently produce succinate from glucose in our previous study.ResultsIn this work, C. glutamicum was engineered for efficient succinate production from lignocellulosic hydrolysate. First, xylose utilization of C. glutamicum was optimized by heterologous expression of xylA and xylB genes from different sources. Next, xylA and xylB from Xanthomonas campestris were selected among four candidates to accelerate xylose consumption and cell growth. Subsequently, the optimal xylA and xylB were co-expressed in C. glutamicum strain SAZ3 (ΔldhAΔptaΔpqoΔcatPsod-ppcPsod-pyc) along with genes encoding pyruvate carboxylase, citrate synthase, and a succinate exporter to achieve succinate production from xylose in a two-stage fermentation process. Xylose utilization and succinate production were further improved by overexpressing the endogenous tkt and tal genes and introducing araE from Bacillus subtilis. The final strain C. glutamicum CGS5 showed an excellent ability to produce succinate in two-stage fermentations by co-utilizing a glucose–xylose mixture under anaerobic conditions. A succinate titer of 98.6 g L−1 was produced from corn stalk hydrolysate with a yield of 0.87 g/g total substrates and a productivity of 4.29 g L−1 h−1 during the anaerobic stage.ConclusionThis work introduces an efficient process for the bioconversion of biomass into succinate using a thoroughly engineered strain of C. glutamicum. To the best of our knowledge, this is the highest titer of succinate produced from non-food lignocellulosic feedstock, which highlights that the biosafety level 1 microorganism C. glutamicum is a promising platform for the envisioned lignocellulosic biorefinery.

Simultaneous and Sequential Integration by Cre/loxP Site-Specific Recombination in Saccharomyces cerevisiae.

A Cre/loxP-δ-integration system was developed to allow sequential and simultaneous integration of a multiple gene expression cassette in Saccharomyces cerevisiae. To allow repeated integrations, the reusable Candida glabrata MARKER (CgMARKER) carrying loxP sequences was used, and the integrated CgMARKER was efficiently removed by inducing Cre recombinase. The XYLP and XYLB genes encoding endoxylanase and β-xylosidase, respectively, were used as model genes for xylan metabolism in this system, and the copy number of these genes was increased to 15.8 and 16.9 copies/cell, respectively, by repeated integration. This integration system is a promising approach for the easy construction of yeast strains with enhanced metabolic pathways through multicopy gene expression.

A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes

ABSTRACTThe tolerant yeast Candida glycerinogenes, with high D-xylonate and low-pH tolerances, was used as the host for D-xylonate production at low pH in this study. A low-pH inducible promoter, pGUKd, was engineered using the core promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) combined with the upstream activating sequence of the promoter of the guanylate kinase gene (pGUK1) that had substituted pH-responsive TF binding sites. The recombinant cells that expressed GFP from the hybrid promoter pGUKd displayed dramatically increased fluorescence intensity at pH 2.5, thus verifying that pGUKd is a low-pH inducible promoter. The promoter pGUKd was then used to express the D-xylose dehydrogenase gene xylB, resulting in increased expression levels of xylB at low pH. The recombinant protein exhibited higher specific activities under lower pH conditions and produced 38 g/l D-xylonate at pH 2.5. This rate is much higher than that produced by fermentation at pH 5.5. These results suggest that the novel hybrid promoter pGUKd functions to direct the production of D-xylonate at low pH, and we provide a candidate genetic tool for the stress tolerant yeast C. glycerinogenes.

Development of a new method for d-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD+ and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.

Production of Acetoin through Simultaneous Utilization of Glucose, Xylose, and Arabinose by Engineered Bacillus subtilis.

Glucose, xylose and arabinose are the three most abundant monosaccharide found in lignocellulosic biomass. Effectively and simultaneously utilization of these sugars by microorganisms for production of the biofuels and bio-chemicals is essential toward directly fermentation of the lignocellulosic biomass. In our previous study, the recombinant Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain was already shown to efficiently utilize xylose for production of acetoin, with a yield of 0.36 g/g xylose. In the current study, the Bacillus subtilis168ARSRCPΔacoAΔbdhA strain was further engineered to produce acetoin from a glucose, xylose, and arabinose mixtures. To accomplish this, the endogenous xylose transport protein AraE, the exogenous xylose isomerase gene xylA and the xylulokinase gene xylB from E. coli were co-overexpressed in the Bacillus subtilis 168ARSRCPΔacoAΔbdhA strain, which enabled the resulting strain, denoted ZB02, to simultaneously utilize glucose and xylose. Unexpectedly, the ZB02 strain could simultaneously utilize glucose and arabinose also. Further results indicated that the transcriptional inhibition of the arabinose transport protein gene araE was the main limiting factor for arabinose utilization in the presence of glucose. Additionally, the arabinose operon in B. subtilis could be activated by the addition of arabinose, even in the presence of glucose. Through fed-batch fermentation, strain ZB02 could simultaneously utilize glucose, xylose, and arabinose, with an average sugar consumption rate of 3.00 g/l/h and an average production of 62.2 g/l acetoin at a rate of 0.864 g/l/h. Finally, the strain produced 11.2 g/l acetoin from lignocellulosic hydrolysate (containing 20.6g/l glucose, 12.1 g/l xylose and 0.45 g/l arabinose) in flask cultivation, with an acetoin yield of 0.34 g/g total sugar. The result demonstrates that this strain has good potential for the utilization of lignocellulosic hydrolysate for production of acetoin.

Overexpressions of xylA and xylB in Klebsiella pneumoniae Lead to Enhanced 1,3-Propanediol Production by Cofermentation of Glycerol and Xylose.

1,3-Propanediol (1,3-PD) is a valuable platform compound. Many studies have shown that the supplement of NADH plays a key role in the bioproduction of 1,3-PD from Klebsiella pneumoniae. In this study, the xylA and xylB genes from Escherichia coli were overexpressed individually or simultaneously in K. pneumoniae to improve the production of 1,3-PD by cofermentation of glycerol and xylose. Compared with the parent strain, the xylose consumption was significantly increased by the introduction of these two genes. The 1,3-PD titers were raised from 17.9 g/l to 23.5, 23.9, and 24.4 g/l, respectively, by the overexpression of xylA and xylB as well as their coexpression. The glycerol conversion rate (mol/mol) was enhanced from 54.1% to 73.8%. The concentration of 2,3-butanediol was increased by 50% at the middle stage but drastically decreased after that. The NADH and NADH/NAD(+) ratio were improved. This report suggests that overexpression of xylA or xylB is an effective strategy to improve the xylose assimilation rate to provide abundant reducing power for the biosynthesis of 1,3-PD in K. pneumoniae.

Engineered xylose utilization enhances bio-products productivity in the cyanobacterium Synechocystis sp. PCC 6803

Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from Escherichia coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation.

Pooled genetic analysis reveals an association of SNPs of only a few genes with risk predisposition to ischemic stroke in a Chinese population.

Up to date, series of studies on ischemic stroke had resulted in inconsistent conclusion. In this study, we aimed to evaluate which specific genes were associated with increased predisposition to stroke in a Chinese population. To identify the specific genes and polymorphisms associated with predisposition to ischemic stroke, we performed high throughput sequenom based next-generation sequencing from 743 patient with history of ischemic stroke. Variants SNPs in nNOS, renalase, MTHFR, CELSR1 and XYLB genes were found significantly associated with IS, thus suggesting involvement of these loci to IS in Chinese patients. Because of different locations on chromosomes, haplotype analyses were not feasible. Our results demonstrate the top level genes that are related to normal vascular physiology, viz. genes of nitric oxide synthesis, endothelial health and smooth muscle biology. Further validation studies are awaited.