Isolation of the btsT gene encoding thioesterase and construction of vector for its insertion mutagenesis in Bacillus licheniformis

Abstract

Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to thioesterases. The mechanism and function of thioesterases in peptide synthesis is not known. Antibiotic bacitracin, produced by Bacillus licheniformis, is synthesised by a large multienzyme complex composed of the three bacitracin synthetases BA1, BA2 and BA3. Genes encoding bacitracin synthetases are organised in the bac operon. Upstream of the bac operon is btsT gene encoding thioesterase. In order to investigate the influence of thioesterase on bacitracin biosynthesis 1.2 kb fragment was isolated from chromosomal DNA B. licheniformis by using PCR . This fragment consists of almost entire btsT gene and complete promotor region of bac operon. Fragment has been hybridised with B. licheniformis BA1 chromosomal DNA. In addition the Tc resistance gene (tetM) was inserted into the fragment to construt vector for insertion mutagenesis of the btsT gene.