• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.483-489
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• 2004

Pseudomonas sp. S-47 is a bacterium capable of degrading benzoate as well as 4-chlorobenzoate (4CBA). Benzoate and 4CBA are known to be degraded via a meta-cleavage pathway characterized by a series of enzymes encoded by xyl genes. The meta-cleavage pathway operon in Pseudomonas sp. S-47 encodes a set of enzymes which transform benzoate and 4CBA into TCA cycle intermediates via the meta-cleavage of (4-chloro )catechol to produce pyruvate and acetyl-CoA. In the current study, the meta-pathway gene cluster was cloned from the chromosomal DNA of S-47 strain to obtain pCS1, which included the degradation activities for 4CBA and catechol. The genetic organization of the operon was then examined by cloning the meta-pathway genes into a pBluescript SKII(+) vector. As such, the meta-pathway operon from Pseudomonas sp. S-47 was found to contain 13 genes in the order of xylXYZLTEGFlQKIH. The two regulatory genes, xylS and xylR, that control the expression of the meta-pathway operon, were located adjacently downstream of the meta-pathway operon. The xyl genes from strain S-47 exhibited a high nucleoside sequence homology to those from Pseudomonas putida mt-2, except for the xylJQK genes, which were more homologous to the corresponding three genes from P. stutzeri AN10. One open reading frame was found between the xylH and xylS genes, which may playa role of a transposase. Accordingly, the current results suggest that the xyl gene cluster in Pseudomonas sp. S-47 responsible for the complete degradation of benzoate was recombined with the corresponding genes from P. putida mt-2 and P. stutzeri AN10.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.700-705
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• 2003

Pseudomonas sp. S-47 is capable of degrading benzoate and 4-chlorobenzoate as well as catechol and 4-chlorocatechol via the meta-cleavage pathway. The three enzymes of 2-oxopenta-4-enoate hydratase (OEH), acetaldehyde dehydrogenase (acylating) (ADA), and 2-oxo-4-hydroxypentonate aldolase (HOA) encoded by xylJQK genes are responsible for the three steps after the meta-cleavage of catechol. The nucleotide sequence of the xylJQK genes located in the chromosomal DNA was cloned and analyzed. GC content of xylJ, xylQ, and xylK was 65% and consisted of 786, 924, and 1,041 nucleotides, respectively. The deduced amino acid sequences of xylJ, xylQ, and xylK genes from Pseudomonas sp. S-47 showed 93%, 99%, and 99% identity, compared with those of nahT, nahH, and nahI in Pseudomonas stutzeri An10. However, there were only about 53% to 85% identity with xylJQK of Pseudomonas putida mt-2, dmpEFG of P. putida CF600, aphEFG of Comamonas testosteroni TA441, and ipbEGF of P. putida RE204. On the other hand, the xylLTEGF genes located upstream of xylJQK in the strain S-47 showed high homology with those of TOL plasmid from Pseudomonas putida mt-2. These findings suggested that the xylLTEGFIJQK of Pseudomonas sp. S-47 responsible for complete degradation of benzoate and then catechol via the meta-pathway were phylogenetically recombinated from the genes of Pseudomonas putida mt-2 and Pseudomonas stutzeri An10.

• Journal of Microbiology and Biotechnology
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• v.20 no.5
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• pp.946-949
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• 2010

The effects of two different sugars (glucose and xylose) on the expression levels and patterns of the xylose reductase (xyl1), xylitol dehydrogenase (xyl2), and xylulokinase (xyl3) genes were analyzed using Pichia stipitis. A significant increase in mRNA levels of xyl1 was observed after 6 h growth in culture conditions using xylose as a sole carbon source, but expressions of the three genes were not influenced by normal culture media with glucose. In addition, expressions of xyl2 and xyl3 were not observed during the entire culture period during which xylose was added. It also was found that the expression level of xyl1 increased as a function of the xylose concentration (40, 60, and 80 g/l) used in this study, indicating that xyl1 expression sensitively responded to xylose in the culture media. Although the induced level of xyl2 increased slightly after 48 h in the xylose-supplemented culture conditions, the expression of xyl2 was not observed in the xylitol-supplemented culture conditions. Finally, considering the expression of each gene in response to glucose or xylose, the absolute expression levels of the three genes indicate that xyl1 is induced primarily by exposure to xylose.

• Microbiology and Biotechnology Letters
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• v.47 no.4
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• pp.667-672
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• 2019

We compared two integration systems for stable expression of heterologous genes in Saccharomyces cerevisiae. A Candida glabrata-derived gene was used as the selective marker for the Cre/loxP system, and XYLP, XYLB, GRE3, and XYL2 genes were used as model heterologous genes and ligated into the universal pRS-CMT vector. The resulting pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids were sequentially integrated into yeast chromosome VII by four integration processes (marker rescue and gene integration). The four introduced genes were successfully expressed. Further, the pRS-PBG2 plasmid harboring expression cassettes for the four genes was constructed for one-step integration. The four genes that were introduced were stably maintained as a gene cluster and were simultaneously expressed. The one-step integration was more effective for the simultaneous integration and expression of the four genes related to xylan/xylose metabolism. This method will enable the generation of a useful biosystem through appropriate use of gene integration methods.

• Journal of Microbiology and Biotechnology
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• v.18 no.5
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• pp.837-844
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• 2008

Glucose (xylose) isomerases from Streptomyces sp. have been used for the production of high fructose corn syrup for industrial purposes. An 11-kb DNA fragment containing the xyl gene cluster was isolated from Streptomyces lividans TK24 and its nucleotide sequences were analyzed. It was found that the xyl gene cluster contained a putative transcriptional repressor (xylR), xylulokinase (xylB), and xylose isomerase (xylA) genes. The transcriptional directions of the xylB and xylA genes were divergent, which is consistent to those found in other streptomycetes. A gene encoding XylR was located downstream of the xylB gene in the same direction, and its mutant strain produced xylose isomerase regardless of xylose in the media. The enzyme expression level in the mutant was 4.6 times higher than that in the parent strain under xylose-induced condition. Even in the absence of xylose, the mutant strain produce over 60% of enzyme compared with the xylose-induced condition. Gel mobility shift assay showed that XylR was able to bind to the putative xyl promoter, and its binding was inhibited by the addition of xylose in vitro. This result suggested that XylR acts as a repressor in the S. lividans xylose operon.

• Journal of Microbiology and Biotechnology
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• v.25 no.9
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• pp.1476-1484
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• 2015

Three endoxylanase-encoding genes from the moderately themophilic chemoorganotrophic bacterium Melioribacter roseus were cloned and expressed in Escherichia coli. Genes xyl2091 (Mros_2091) and xyl2495 (Mros_2495) encode GH10 family hydrolases, whereas xyl2090 (Mros_2090) represents the GH30 family. In addition to catalytic domains, Xyl2090 and Xyl2091 contain carbohydrate-binding modules that could facilitate their binding to xylans and Por sorting domains associated with the sorting of proteins from the periplasm to the outer membrane, where they are covalently attached. Recombinant endoxylanase Xyl2495 exhibited a high specific activity of 1,920 U/mg on birchwood xylan at 40℃. It is active at low temperatures, exhibiting more than 30% of the maximal activity even at 0℃. Endoxylanases Xyl2090 and Xyl2091 have lower specific activities but higher temperature optima at 80℃ and 65℃, respectively. Analysis of xylan hydrolysis products revealed that Xyl2090 generates xylo-oligosaccharides longer than xylopentaose. Xylose and xylobiose are the major products of xylan hydrolysis by the recombinant Xyl2091 and Xyl2495. No activity against cellulose was observed for all enzymes. The presence of three xylanases ensures efficient xylan hydrolysis by M. roseus. The highly processive "free" endoxylanase Xyl2495 could hydrolyze xylan under moderate temperatures. Xylan hydrolysis at elevated temperatures could be accomplished by concerted action of two cell-bound xylanases; Xyl2090 that probably degrades xylans to long xylo-oligosaccharides, and Xyl2091 hydrolyzing them to xylose and xylobiose. The new endoxylanases could be useful for saccharification of lignocellulosic biomass in biofuels production, bleaching of paper pulp, and obtaining low molecular weight xylooligosaccharides.

• Journal of Industrial Technology
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• v.29 no.A
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• pp.161-167
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• 2009

The bifunctional Xylanase-Cellulase hybrid protein was constructed by gene fusion. Two genes corresponding to endoxylanase gene (xylS) and endocellulase gene (celA) were amplified by PCR from Bacillus licleniformis NBL420. It was then linked through splicing by overlap extension (SOE) by PCR method. The two resulting fused hybrids, xyl/cel and cel/xyl, which differ by its orientation, were confirmed by its nucleotide sequencings. One of two fusion genes, xyl/cel was successfully expressed into pET22b(+) vector (pxyl/cel) with bifunctional xylanase-cellulase activity. On the contrary, the other cel/xyl fusion protein showed only cellulase activity with much decreased xylanase activity. Enzymatic properties of Xyl/Cel fusion protein were investigated regarding optimum pH, optimum temp, thermostability, and pH stability. It was revealed that Xyl/Cel fusion protein retained the bifunctional xylanase-cellulase activities eventhough two enzymes were connected with each other directly. These informations could be useful for construction of other hybrid proteins as well as increased range of substrate utilization.

• Journal of Microbiology and Biotechnology
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• v.26 no.7
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• pp.1252-1258
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• 2016

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.

• Journal of Life Science
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• v.28 no.11
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• pp.1277-1284
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• 2018

In this study, a repeated yeast integrative plasmid (R-YIp) harboring Cre/loxP system was constructed to integrate various gene expression cassettes into the yeast chromosome. The R-YIp system contains a reusable selective marker (CgTRP1), loxP sequence, and target sequence for integration. Therefore, many gene expression cassettes can be integrated into the same position of the same yeast chromosome. In the present study, several model enzymes involving xylan/xylose metabolism were examined, including endoxylanase (XYLP), ${\beta}$-xylosidase (XYLB), xylose reductase (GRE3) and xylitol dehydrogenase (XYL2). Efficient expression of these genes was obtained using two promoters (GAL10p and ADH1p) and various plasmids (pGMF-GENE and pAMF-GENE plasmids) were constructed. The XYLP, XYLB, GRE3, and XYL2 genes were efficiently expressed under the control of the GAL10 promoter. Subsequently, R-YIps containing the GAL10p-GENE-GAL7t cassette were constructed, resulting in pRS-XylP, pRS-XylB, pRS-Gre3, and pRS-Xyl2 plasmids. These plasmids were sequentially integrated into chromosome VII of a Saccharomyces cerevisiae strain by repeated gene integration and selective marker rescue. These genes were integrated by the R-YIp system and were stably expressed in the yeast transformants to produce active recombinant enzymes. Therefore, we expect that the R-YIp system will be able to overcome current limitations of the host cells and allow selective marker selection for the integration of various genes into the yeast chromosome.

• Journal of Microbiology and Biotechnology
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• v.34 no.3
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• pp.700-709
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• 2024

Polyhydroxybutyrate (PHB) production from lignocellulosic biomass is economically beneficial. Because lignocellulosic biomass is a mixture rich in glucose and xylose, Escherichia coli, which prefers glucose, needs to overcome glucose repression for efficient biosugar use. To avoid glucose repression, here, we overexpressed a xylose regulator (xylR) in an E. coli strain expressing bktB, phaB, and phaC from Cupriavidus necator and evaluated the effect of xylR on PHB production. XylR overexpression increased xylose consumption from 0% to 46.53% and produced 4.45-fold more PHB than the control strain without xylR in a 1% sugar mixture of glucose and xylose (1:1). When the xylR-overexpressed strain was applied to sugars from lignocellulosic biomass, cell growth and PHB production of the strain showed a 4.7-fold increase from the control strain, yielding 2.58 ± 0.02 g/l PHB and 4.43 ± 0.28 g/l dry cell weight in a 1% hydrolysate mixture. XylR overexpression increased the expression of xylose operon genes by up to 1.7-fold. Moreover, the effect of xylR was substantially different in various E. coli strains. Overall, the results showed the effect of xylR overexpression on PHB production in a non-native PHB producer and the possible application of xylR for xylose utilization in E. coli.