Rational Construction of a Robust Bacillus amyloliquefaciens Cell Factory for Acid-Stable α Amylase Production

With the development of synthetic biology and biotechnology, chassis engineering has become the main means of industrial protein production, but it has been limited by the lack of efficient gene editing methods and effective engineering strategies. Bacillus amyloliquefaciens shows potential for expressing heterologous proteins, but its cells undergo early autolysis, hindering further application. In this study, an autolysis-related prophage gene cluster was rationally deleted by establishing an efficient CRISPR-nCas9 editing process, and the prophage mutant strain was constructed, which prevented cell lysis. Based on the prophage mutant strain, we screened secondary metabolite biosynthetic gene clusters that hindered the expression of heterologous proteins, and we made reasonable deletions to further improve their efficient expression. Finally, an optimized yield of acid-stable α amylase (2,46,089.21 U/mL) was obtained in a 5-L fed-batch fermentation. Therefore, we successfully constructed an ideal candidate strain for the expression of heterologous proteins, which provides an important research basis for the development of more chassis strains.