USE OF HOMOLOGOUS EXPRESSION-SECRETION SIGNALS AND VECTOR-FREE STABLECHROMOSOMAL INTEGRATION IN ENGINEERING OF LACTOBACILLUS-PLANTARUM FORALPHA-AMYLASE AND LEVANASE EXPRESSION

The genuine alpha-amylase gene from Bacillus lichenifonnis (amyL) is n ot expressed in Lactobacillus plantarum, but replacement of the amyL p romoter by a strong L. plantarum promoter leads to efficient expressio n of the gene and secretion of more than 90% of the a-amylase into the culture supernatant. A series of L. plantarum genetic cassettes (tran scription and translation with or without secretion) were cloned by tr anslation fusion of random DNA fragments to the silent amyL coding fra me in the pGIP212 probe vector (P. Hols, A. Baulard, D, Garmyn, B. Del place, S. Hogan, and J. Delcour, Gene 118:21-30, 1992). Five different cassettes were sequenced and found to harbor genetic signals similar to those of other gram-positive bacteria. The functions of the cloned cassettes and the cassettes isolated previously from Enterococcus faec alis were compared in E. faecalis and L. plantarum, respectively. All signals were well recognized in L. plantarum, but cassettes isolated f rom L. plantarum led to a low level of amylase production in E. faecal is, suggesting that the L. plantarum signals are more species specific . Six transcriptional or translational fusions were constructed to exp ress the Bacillus subtilis levanase gene (sacC) in L. plantarum. All o f these constructions were capable of inducing levanase production and secretion in the culture supernatant, and, furthermore, L. plantarum strains harboring the most efficient fusions could grow in MRS medium containing inulin as the major carbon source. Finally, a two-step chro mosomal integration procedure was used to achieve efficient stabilizat ion of an amylase construction without any residual resistance marker or vector sequence.