Identification of integration site library for the development of plasmid-free microbial cell factory in Bacillus...

Bacillus subtilis was widely used for enzyme production and was gradually engineered to biosynthesize value-added chemicals with the development of genetic parts for it. Compared to other genetic parts for expression, the identified integration sites were fewer, which limits the development of B. subtilis. Here, a library of integration sites was developed for B. subtilis. All candidate sites were selected at the 3'-untranslated region of two opposite nonessential genes and separated by essential genes among genome to avoid the destruction for coding sequence of genes and eliminate the homologously recombined strains with the loss of essential genes. The expression of GFP and cell growth were detected for candidate sites to evaluate the gene expression strength and the influence on cell growth. As a result, 12 loci revealed higher gene expression level and cell growth compared with control site amyE, the highest expression site spxA was 1.89 times as high as amyE. Using the developed integration sites library, threefold gene expression range could be achieved without the replacement of promoter and RBS. When the integration site library was used to construct cell factories, the production of lacto-N-triose II and lycopene was increased by 95% and 83%, respectively. In addition, integration site library was also successfully used to increase the enzymatic activity of secretory β-galactosidase by 101% when the strain using spxA locus compared with that using amyE. The developed integration site library could accelerate the construction of stable and plasmid-free cell factories for B. subtilis in the future.