• 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.

• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.75-81
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• 1997

Streptomyces chibaensis J-59 did not grow in the culture medium containing only xylose or xylan as a carbon source, because it was defective in xylulokinase production; xylB mutant. S. chibaensis J-59 was able to produce xylanase and $\beta $-xylosidase as well as glucose isomerase. The glucose isomerase in S. chilbaensis J-59 was induced in the medium containing xylan or xylose which could be utilized as an inducer but not sa carbon and energy sources. So we tried to produce glucose isomerase whthout consumption of xylose or xylan as an inducer by using xylB mutant S. chilbaensis J-59. The optimum condition for the production of the glucose isomerase was attained in a culture medium composed of 1% xylan, 0.15% glucose, 1.5% corn steep liquor, 0.1% MaSO$_{4}$ $\CDOT $7H$_{2}$O, and 0.012% CoCL$_{2}$ $\CDOT $ 6H$_{2}$O(pH 7.0). The production of the enzyme reached to a maximum level when the bacteria were cultured for 42 h at 30$\circ $C. The enzyme production in a jar fermentor was increased twice as much as that in a flask culture.

• Microbiology and Biotechnology Letters
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• v.31 no.2
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• pp.111-116
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• 2003

The xylA gene of Bacillus stearothermophilus No. 236 encoding $\beta$-xylosidase was cloned and sequenced previously. The transcriptional start site of the xylA gene cloned in E. coli was identified to be the guanine (G) by primer extension analysis. This supports that the expression of xylA gene is also directed in the E. coli cells by the previously determined transcription initiation signals, -10 sequence (CATAAT) and -35 sequence (TTGTTA) separated by 12 bp. To increase the expression of $\beta$-xylosidase, firstly the spacer region of xylA promoter was extended from 12 to 17 bp, and then the -10 and -35 elements were converted into their respective consensus sequences. The mutant promoters thus obtained were tested for their activities in both the E. coli and B. subtilis host cells. The change of the length of the spacer region from 12 to 17 bp resulted in a 1.6- and 2.5-fold increase in promoter strength in comparison with the wild type promoter in E. coli and B. subtilis cells, respectively. Also, strength of the promoter with the fourth T to A transversion on its -35 element increased in the transcription level by about 35 times compared with that of wild-type promoter. However, surprisingly the 5' end C-to-T transition of the -10 hexamer showed a 5- to 15-fold reduction in $\beta$-xylosidase activity in both E. coli and B. subtilis. Together, the present data demonstrated that the 5' end nucleotide C of the -10 sequence CATAAT and the fourth nucleotide A of the -35 hexamer are two most critical nucleotides for the promoter activity in the context of the xylA promoter.

• Current Research on Agriculture and Life Sciences
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• v.10
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• pp.125-135
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• 1992

Nine strains of xyl mutants that could not utilize xylose as a carbon source were isolated from E. coli JM109 by the treatment of NTG in order to investigate the regulation of xylose operon and to use recipient cells for the cloning of xylR gene. For the characterization of all isolated mutants, colony colors of all mutants on MacConkey-xylose and MacConkey-xylulose agar plate were observed for the utilization of xylose and xylulose, and the growth level and the activity of xylose isomerase and xylulokinase were determined in need. The isolated xylR/T mutants formed the white colony on MacConkey-xy-lose and MacConkey-xylulose agar plate. They did not detect the activity of xylose isomerase, and the activity of xylose isomerase was not restored in transformants of xylR/T mutant with pEX13 which contained xylA gene. xylR and xylT mutants were classified from xylR/T mutants depending upon the growth level in minimal medium. xylT mutants; DH13, DH121 and DH125 could grow a little in that medium, but xylR mutants; DH10, DH53, and DH60 could not grow that medium.