Development of an asporogenic Bacillus licheniformis for the production of keratinase

Aims: Bacillus licheniformis PWD‐1 is a keratin‐degrading, spore‐forming bacterium isolated from a poultry waste digester. A sporulation‐deficient mutant of B. licheniformis PWD‐1, named B. licheniformis WBG, was developed and characterized. Methods and Results: The mutation was generated using the splicing by overlap extension PCR method (Gene SOEing) to create 256 bp deletion in the spoIIAC gene, which encodes an essential sporulation‐specific sigma factor. In vivo gene replacement was accomplished with the use of a temperature‐sensitive plasmid that is able to integrate and excise the nucleotide fragment 256 bp from the B. licheniformis chromosome. PCR analysis and DNA sequencing confirmed the spoIIAC gene deletion. Heat‐treatment assays and electron microscopy verified the absence of spores. Conclusions: This asporogenic strain is able to express normal levels of keratinase when compared with its wild‐type host. Significance and Impact of the Study: In this study, a method of constructing a stable sporulation‐defective strain was developed. It can be potentially useful as a tool to generate asporogenic strains of Bacillus that retain their industrial capabilities for production of exoproteases and other exozymes.