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Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus.

ABSTRACT: The xylose isomerase (xylA) and the xylulose kinase (xylB) genes from Streptomyces rubiginosus were isolated, and their nucleotide sequences were determined. The xylA and xylB genes encode proteins of 388 and 481 amino acids, respectively. These two genes are transcribed divergently from within a 114-nucleotide sequence separating the coding regions. Regulation of the xyl genes in S. rubiginosus was examined by fusing their promoters to the Pseudomonas putida catechol dioxygenase gene and integrating the fusions into the minicircle integration site on the S. rubiginosus chromosome. The expression of catechol dioxygenase was then measured under a variety of conditions. The results indicated that transcription of the xyl genes was induced by D-xylose and repressed by glucose. Data from quantitative S1 mapping were consistent with this conclusion and suggested that xylA had one and xylB had two transcription initiation sites. The transcription initiation site of xylA was 40 bp upstream of the coding region. The two transcription initiation sites of xylB were 20 and 41 bp 5' of its translation initiation codon. Under control of appropriate regulatory elements, the cloned xyl genes are capable of complementing either Escherichia coli xylose isomerase- or xylulose kinase-deficient strains. The deduced amino acid sequence of the S. rubiginosus xylA protein is highly homologous to sequences of other microbial xylose isomerases.

SUBMITTER: Wong HC

PROVIDER: S-EPMC209037 | biostudies-other | 1991 Nov

REPOSITORIES: biostudies-other

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Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus.

Wong H C HC Ting Y Y Lin H C HC Reichert F F Myambo K K Watt K W KW Toy P L PL Drummond R J RJ

Journal of bacteriology 19911101 21

The xylose isomerase (xylA) and the xylulose kinase (xylB) genes from Streptomyces rubiginosus were isolated, and their nucleotide sequences were determined. The xylA and xylB genes encode proteins of 388 and 481 amino acids, respectively. These two genes are transcribed divergently from within a 114-nucleotide sequence separating the coding regions. Regulation of the xyl genes in S. rubiginosus was examined by fusing their promoters to the Pseudomonas putida catechol dioxygenase gene and integr ...[more]

PMID: 1657868

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Project description:BackgroundGenetic tools including constitutive and inducible promoters have been developed over the last few decades for strain engineering in Streptomyces. Inducible promoters are useful for controlling gene expression, however only a limited number are applicable to Streptomyces. The aim of this study is to develop a controllable protein expression system based on an inducible promoter using sugar inducer, which has not yet been widely applied in Streptomyces.ResultsTo determine a candidate promoter, inducible protein expression was first examined in Streptomyces avermitilis MA-4680 using various carbon sources. Xylose isomerase (xylA) promoter derived from xylose (xyl) operon was selected due to strong expression of xylose isomerase (XylA) in the presence of D-xylose. Next, a xylose-inducible protein expression system was constructed by investigating heterologous protein expression (chitobiase as a model protein) driven by the xylA promoter in Streptomyces lividans. Chitobiase activity was detected at high levels in S. lividans strain harboring an expression vector with xylA promoter (pXC), under both xylose-induced and non-induced conditions. Thus, S. avermitilis xylR gene, which encodes a putative repressor of xyl operon, was introduced into constructed vectors in order to control protein expression by D-xylose. Among strains constructed in the study, XCPR strain harboring pXCPR vector exhibited strict regulation of protein expression. Chitobiase activity in the XCPR strain was observed to be 24 times higher under xylose-induced conditions than that under non-induced conditions.ConclusionIn this study, a strictly regulated protein expression system was developed based on a xylose-induced system. As far as we could ascertain, this is the first report of engineered inducible protein expression in Streptomyces by means of a xylose-induced system. This system might be applicable for controllable expression of toxic products or in the field of synthetic biology using Streptomyces strains.

Dataset Information

Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus.

Publications

Genetic organization and regulation of the xylose degradation genes in Streptomyces rubiginosus.

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