Cloning of, and studies on, genes coding for subtilisins Carlsberg and BPN'
The alkaline serine proteases (subtilisins) secreted by the Bacillus spp. can be categorized into two groups on the basis of immunological cross-reactions. A gene coding for the Carlsberg type was cloned from B. licheniformis (III), and one for the BPN'/NOVO type, from B. subtilis (IV). The subtilisin BPN' gene was partially characterized . The sequence and expression data obtained correspond well with those observed by others. In vitro substitution of part of the coding sequence with the gene encoding chloramphenicol transacetylase was followed by targeted integration into the Bacillus chromosome (IV.6). This resulted in a mutant strain having only c.30% exoproteolytic activity compared with wild-type, due specifically to decreased serine protease secretion. Immunologically, and by gel electrophoresis, no subtilisin could be detected. It was shown that the deletion was readily transferred to closely related strains by selection for chloramphenicol resistance. In combination with other mutations, the subtilisin deletion could form the basis for a better Bacillus host for the synthesis of foreign proteins. The subtilisin Carlsberg gene was cloned and characterized. The sequence data obtained (II.6), allowed, for the first time, a comparison of the entire precursor coding sequence with those available for enzymes of the BPN' type (Appendix 1). Overproduction of the gene product in B. subtilis was achieved, both from the endogenous promoter (III.7.5), and from two heterologous promoters (III.7.2.2). It was demonstrated by various techniques that the extra exoprotease production was due specifically to synthesis of the Carlsberg enzyme. Results with the heterologous promoters pointed to a significant delay before the appearance of subtilisin in the medium, probably due to post-transcriptional factors (III.7.3). Using mRNA extracted from B. 1icheniformis, the transcriptional terminator was located precisely (III.8.3). Transcriptional initiation site determination was attempted by two different procedures. Two sites were approximated by the S1 mapping technique (III.8.2). In contrast, cDNA synthesis and sequencing pointed to a single start site (Appendix 3). The relation of these sites to identifiable elements in the sequences up- and downstream from the gene is discussed. The Carlsberg gene was cloned only with difficulty, unlike the homologous gene for subtilisin BPN'. Possible reasons for this, and alternative cloning strategies, are discussed (III.9; V).