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.
ASJC Scopus subject areas
- Food Science
- Applied Microbiology and Biotechnology