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Applied and Environmental Microbiology, November 2000, p. 4817-4821, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Volker Mai and Juergen Wiegel*

Department of Microbiology and Center for Biological Resource Recovery, University of Georgia, Athens, Georgia 30602

Received 9 December 1999/Accepted 25 July 2000

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.

* Corresponding author. Mailing address: Department of Microbiology, Biol. Sciences Bldg. 215, University of Georgia, Athens, GA 30602-2605. Phone and fax: (706) 542-2651. E-mail: jwiegel{at}arches.uga.edu.

Applied and Environmental Microbiology, November 2000, p. 4817-4821, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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