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Applied and Environmental Microbiology, December 2001, p. 5425-5430, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5425-5430.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Construction of a Xylanase-Producing Strain of Brevibacterium lactofermentum by Stable Integration of an Engineered xysA Gene from Streptomyces halstedii JM8

Sirin A. I. Adham, Ana B. Campelo, Angelina Ramos, and José A. Gil*

Departamento de Ecología, Genética y Microbiología, Área de Microbiología, Facultad de Biología, Universidad de León, León, Spain

Received 26 July 2001/Accepted 14 September 2001

A xylanolytic strain of Brevibacterium lactofermentum containing the Streptomyces halstedii His-tagged xysA gene was generated. The new strain contains DNA derived from S. halstedii, expresses xylanolytic activity, and was obtained by an integrative process mediated by a conjugative plasmid targeted to a dispensable chromosomal region located downstream from the essential cell division gene ftsZ. The His-tagged Xys1 enzyme was constitutively expressed under the control of the kan promoter from Tn5 and was easily purified by use of Ni-nitrilotriacetic acid-agarose. The new strain is stable for more than 200 generations, lacks any known antibiotic resistance gene, and does not need any selective pressure to maintain the integrated gene. This strategy can be used to integrate any gene into the B. lactofermentum chromosome and to maintain it stably without the use of antibiotics for selection.

* Corresponding author. Mailing address: Departamento de Ecología, Genética y Microbiología, Área de Microbiología, Facultad de Biología, Universidad de León, 24071 León, Spain. Phone: 34-987-291503. Fax: 34-987-291479. E-mail: degjgs{at}unileon.es.

Applied and Environmental Microbiology, December 2001, p. 5425-5430, Vol. 67, No. 12
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.12.5425-5430.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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