Partial characterization of the Streptomyces lividans xlnB promoter and its use for expression of a thermostable xylanase from Thermotoga maritima.
Xylanase activity assays were used to screen a Streptomyces coelicolor genomic library in Escherichia coli, and a xylanase gene that is 99% identical to the xylanase B gene (xlnB) of S. lividans (GenBank accession no. M64552) was identified. The promoter region of this gene was identified by using a transcriptional fusion between the upstream region of the S. coelicolor xlnB gene and the xylE reporter gene. Transcription from the xlnB promoter was found to be induced by xylan and repressed by glucose. A single apparent transcription start site was identified by both primer extension analysis and in vitro run off transcription assays. Analysis of deletions of the promoter identified a region required for glucose repression. By using the transcriptional and protein localization signals of the Streptomyces xlnB gene, an in-frame translational fusion between the end of the xlnB signal sequence and the ATG of the Thermotoga maritima xynA gene was constructed. The xynA gene encodes a thermostable xylanase that has been demonstrated to be useful in the bleaching of Kraft pulp. The xlnB-xynA gene fusion was expressed in Streptomyces, and the activity of the protein produced was thermostable and was localized to the supernatant fraction of harvested cells.
Chen
C C
CC
Department of Genetics, University of Georgia, Athens, Georgia 30602-7223, USA.
Westpheling
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D043364:Endo-1,4-beta Xylanases; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*; D013379:Substrate Specificity; D013816:Thermodynamics; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics*; D014158:Transcription, Genetic; D014995:Xylosidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases