The complete sequencing and annotation of the genomes of industrially-important Bacillus species has enhanced our understanding of their properties, and allowed advances in genetic manipulations in other Bacillus species. Post-genomic studies require simple and highly efficient tools to enable genetic manipulation. Here, we summarize the recent progress in genetic engineering strategies for Bacillus species. We review the available genetic tools that have been developed in Bacillus species, as well as methods developed in other species that may also be applicable in Bacillus. Furthermore, we address the limitations and challenges of the existing methods, and discuss the future research prospects in developing novel and useful tools for genetic modification of Bacillus species.

As important genome editing tools, CRISPR/Cas systems, especially those based on type II Cas9 and type V Cas12a, are widely used in genetic and metabolic engineering of bacteria. However, the intrinsic toxicity of Cas9 and Cas12amediated CRISPR/Cas tools can lead to cell death in some strains, which led to the development of endogenous type I and III CRISPR/Cas systems. However, these systems are hindered by complicated development and limited applications. Thus, further development and optimization of CRISPR/Cas systems is needed. Here, we briefly summarize the mechanisms of different types of CRISPR/Cas systems as genetic manipulation tools and compare their features to provide a reference for selecting different CRISPR/Cas tools. Then, we show the use of CRISPR/Cas technology for bacterial strain evolution and metabolic engineering, including genome editing, gene expression regulation and the base editor tool. Finally, we offer a view of future directions for bacterial CRISPR/Cas technology.

The manno endo-1,4-mannosidase (β-mannanase, EC. 3.2.1.78) catalyzes the random hydrolysis of internal (1 → 4)-β-mannosidic linkages in the mannan polymers. A codon optimized β-mannanase gene from Bacillus licheniformis DSM13 was expressed in Bacillus subtilis. When four Sec-dependent and two Tat-dependent signal peptide sequences cloned from B. subtilis were placed upstream of the target gene, the highest activity of β-mannanase was observed using SP as a signal peptide. Then a 1.25-fold activity of β-mannanase was obtained when another copy of groESL operon was inserted into the genome of host strain. Finally, five different promoters were separately used to enhance the synthesis of the target protein. The results showed that promoter P, a modified maltose-inducible promoter, significantly elevated the production of β-mannanase. After 72 h of flask fermentation, the enzyme activity of β-mannanase in the supernatant when using locust bean gum as substrate reached 2207 U/mL. This work provided a promising β-mannanase production strain in industrial application.