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Advances in development of a genetic system for Thermoanaerobacterium spp.: expression of genes encoding hydrolytic enzymes, development of a second shuttle vector, and integration of genes into the chromosome.
- Source :
-
Applied and environmental microbiology [Appl Environ Microbiol] 2000 Nov; Vol. 66 (11), pp. 4817-21. - Publication Year :
- 2000
-
Abstract
- Despite recent success in transforming various thermophilic gram-type-positive anaerobes with plasmid DNA, use of shuttle vectors for the expression of genes other than antibiotic resistance markers has not previously been described. We constructed new vectors in order to express heterologous hydrolytic enzymes in our model system, Thermoanaerobacterium saccharolyticum JW/SL-YS485. Transformed Thermoanaerobacterium expressed active enzyme, indicating that this system may function as an alternate expression host, especially for genes with a thermophilic origin. To develop further the genetic system for T. saccharolyticum JW/SL-YS485, two improved Escherichia coli-Thermoanaerobacterium shuttle vectors, pRKM1 and pRUKM, were constructed. Furthermore, the kanamycin resistance cassette alone and the kanamycin resistance cassette plus the cellobiohydrolase gene (cbhA) from Clostridium thermocellum JW20 were integrated into the xylanase gene (xynA) region of the Thermoanaerobacterium chromosome via homologous recombination using pUC-based suicide vectors pUXK and pUXKC.
- Subjects :
- Bacteria, Anaerobic enzymology
Bacteria, Anaerobic growth & development
Blotting, Southern
Cellulase genetics
Cellulase metabolism
Cellulose 1,4-beta-Cellobiosidase
Glucan 1,3-beta-Glucosidase
Gram-Positive Bacteria enzymology
Gram-Positive Bacteria growth & development
Mannosidases genetics
Mannosidases metabolism
Plasmids
Polymerase Chain Reaction
beta-Glucosidase genetics
beta-Glucosidase metabolism
beta-Mannosidase
Bacteria, Anaerobic genetics
Gene Expression
Genetic Vectors
Gram-Positive Bacteria genetics
Hydrolases genetics
Transformation, Bacterial
Subjects
Details
- Language :
- English
- ISSN :
- 0099-2240
- Volume :
- 66
- Issue :
- 11
- Database :
- MEDLINE
- Journal :
- Applied and environmental microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 11055929
- Full Text :
- https://doi.org/10.1128/AEM.66.11.4817-4821.2000