EFFECT OF THE REPLICATION MODE OF A PLASMID ON THE STABILITY OF MULTIMERIC ENDOXYLANASE GENES IN BACILLUS-SUBTILIS

Effect of the replication mode of a plasmid on the stability of tandem ly multimerized endoxylanase genes and a gene dose-dependent expressio n of the endoxylanase were studied in Bacillus subtilis. The structura l genes encoding an endoxylanase, carrying its original promoter and r ibosomal binding sequence, were tandemly multimerized and cloned into the Escherichia coli-B. subtilis shuttle plasmid, pJH27 Delta 88 or pM TL500(e), which has a rolling circle-replicon or a theta (theta)-repli con in B. subtilis, respectively. The cloned dimers in pJH27 Delta 88, which has a rolling circle-replicon, spontaneously rearranged to mono mers in B. subtilis DB104, whereas those in pMTL500e, having a theta ( theta)-replicon, were stably maintained. Expression level of the endox ylanase was proportional to the gene dosage in multimers. The endoxyla nase activity in the supernatant increased from 80 U ml(-1) with pMTL- 1x containing a monomer of the gene to 165 U ml(-1) with pMTL-4x conta ining a tetramer. These results indicate that high level expression of the endoxylanase gene can be obtained by tandemly multimerizing the g enes in a plasmid with a theta (theta)-replicon. (C) 1998 Elsevier Sci ence B.V. All rights reserved.