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The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.

ABSTRACT: Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encode D-xylose isomerase and D-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli D-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent D-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. The C-terminal portion of this leader sequence matches the cleavage consensus for lipoproteins and is followed by a 22-residue putative linker sequence rich in serine, threonine, and asparagine. The putative mature 341-amino-acid-residue XylF (calculated molecular mass of 37,069 Da) appears to be a lipoprotein attached to the cell membrane via a lipid anchor covalently linked to the N-terminal cysteine, as demonstrated by metabolic labelling of the recombinant XylF with [14C]palmitate. The induced E. coli avidly bound D-[14C]xylose, yielding additional evidence that T. ethanolicus XylF is the D-xylose-binding protein. On the basis of sequence comparison of XylFs to other monosaccharide-binding proteins, we propose that the sequence signature of binding proteins specific for hexoses and pentoses be refined as (KDQ)(LIVFAG)3IX3(DN)(SGP)X3(GS)X(LIVA) 2X2A. Transcription of the monocistronic 1.3-kb xylF mRNA is inducible by xylose and unaffected by glucose. Primer extension analysis indicated that xylF transcription initiates from two +1 sites, both situated within the xylAB operon. Unlike in similar transport systems in other bacteria, the genes specifying the membrane components (e.g., ATP-binding protein and permease) of the high-affinity D-xylose uptake system are not located in the vicinity of xylF in T. ethanolicus. This is the first report of a gene encoding a xylose-binding protein in a gram-positive or thermophilic bacterium.

SUBMITTER: Erbeznik M

PROVIDER: S-EPMC107324 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.

Erbeznik M M Strobel H J HJ Dawson K A KA Jones C R CR

Journal of bacteriology 19980701 14

Immediately downstream from the Thermoanaerobacter ethanolicus xylAB operon, comprising genes that encode D-xylose isomerase and D-xylulose kinase, lies a 1,101-bp open reading frame that exhibits 61% amino acid sequence identity to the Escherichia coli D-xylose binding periplasmic receptor, XylF, a component of the high-affinity binding-protein-dependent D-xylose transport. The 25-residue N-terminal fragment of the deduced T. ethanolicus XylF has typical features of bacterial leader peptides. T ...[more]

PMID: 9657999

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Project description:Thermoanaerobacter ethanolicus can produce acetate, lactate, hydrogen, and ethanol from sugars resulting from plant carbohydrate polymer degradation at temperatures above 65°C. T. ethanolicus is a promising candidate for thermophilic ethanol fermentations due to the utilization of both pentose and hexose. Although an ethanol balance model in T. ethanolicus has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in ethanol formation have been carried out. To address this issue, we developed a thermostable Cas9-based system for genome editing of T. ethanolicus As a proof of principle, three genes, including the thymidine kinase gene (tdk), acetaldehyde-alcohol dehydrogenase gene (adhE), and redox sensing protein gene (rsp), were chosen as editing targets, and these genes were edited successfully. As a genetic tool, we tested the gene knockout and a small DNA fragment knock-in. After optimization of the transformation strategies, 77% genome-editing efficiency was observed. Furthermore, our in vivo results revealed that redox sensing protein (RSP) plays a more important role in regulation of energy metabolism, including hydrogen production and ethanol formation. The genetic system provides us with an effective strategy to identify genes involved in biosynthesis and energy metabolism.IMPORTANCE Interest in thermophilic microorganisms as emerging metabolic engineering platforms to produce biofuels and chemicals has surged. Thermophilic microbes for biofuels have attracted great attention, due to their tolerance of high temperature and wide range of substrate utilization. On the basis of the biochemical experiments of previous investigation, the formation of ethanol was controlled via transcriptional regulation and influenced by the relevant properties of specific enzymes in T. ethanolicus Thus, there is an urgent need to understand the physiological function of these key enzymes, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Here, we developed a thermostable Cas9-based engineering tool for gene editing in T. ethanolicus The thermostable Cas9-based genome-editing tool may further be applied to metabolically engineer T. ethanolicus to produce biofuels. This genetic system represents an important expansion of the genetic tool box of anaerobic thermophile T. ethanolicus strains.

Dataset Information

The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.

Publications

The D-xylose-binding protein, XylF, from Thermoanaerobacter ethanolicus 39E: cloning, molecular analysis, and expression of the structural gene.

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