Bacillus species are widely recognized as good industrial platform strains, which can produce a variety of valuable products including enzymes and functional proteins. Bacillus expression systems gain various competitive edges over other expression systems, with respect to non‐toxicity, convenience for gene modification and high yield of target proteins. Recently, a number of Bacillus expression systems have been developed, and various strategies were conducted to improve the yields of target proteins. In this review, we focused on the strategies of host strain optimization for heterologous protein production, including secretion pathway optimization, RNA and protein stability enhancement, cell growth facilitation, genome streamlining and transcriptional regulator engineering. In addition, Bacillus spore surface display and food‐grade expression systems were developed to expand the application of Bacillus expression system in recent years. Finally, the challenges and prospects of Bacillus expression system were discussed regarding the recent progresses, challenges, and trends in this field.

D-2,3-butanediol has many industrial applications such as chiral reagents, solvents, anti-freeze agents, and low freezing point fuels. Traditional D-2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogenic and not capable of producing D-2,3-butanediol at high optical purity. Bacillus licheniformis is a potential 2,3-butanediol producer but the wild type strain (WX-02) produces a mix of D- and meso-type isomers. BudC in B. licheniformis is annotated as 2,3-butanediol dehydrogenase or acetoin reductase, but no pervious experiment was performed to verify this hypothesis.

Bacillusstrains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed forBacillus licheniformisusing a CRISPR-Cas9 nickase integrated into the genome ofB. licheniformisDW2 with overexpression driven by the P43 promoter. TheyvmCgene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragmentbacABC(42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter geneaprN, which codes for nattokinase inBacillus subtilis, was inserted into the chromosome ofB. licheniformiswith an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacCstrain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC. Finally, the engineered strain DWc9nΔ7 (ΔeprΔwprAΔmprΔaprEΔvprΔbprAΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n inB. licheniformis. This tool could be applied to strain improvement for future research.IMPORTANCEAs important industrial bacteria,Bacillusstrains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration inBacillus licheniformisvia Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.

Zymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production. In this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R2 values more than 0.7 and 0.9, respectively. This study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.

2,3-Butanediol (2,3-BD) can be used as a liquid fuel additive to replace petroleum oil, and as an important platform chemical in the pharmaceutical and plastic industries. Microbial production of 2,3-BD by Bacillus licheniformis presents potential advantages due to its GRAS status, but previous attempts to use this microorganism as a chassis strain resulted in the production of a mix of D-2,3-BD and meso-2,3-BD isomers. The aim of this work was to develop an engineered strain of B. licheniformis suited to produce the high titers of the pure meso-2,3-BD isomer. Glycerol dehydrogenase (Gdh) was identified as the catalyst for D-2,3-BD biosynthesis from its precursor acetoin in B. licheniformis. The gdh gene was, therefore, deleted from the wild-type strain WX-02 to inhibit the flux of acetoin to D-2,3-BD biosynthesis. The acoR gene involved in acetoin degradation through AoDH ES was also deleted to provide adequate flux from acetoin towards meso-2,3-BD. By re-directing the carbon flux distribution, the double-deletion mutant WX-02ΔgdhΔacoR produced 28.2 g/L of meso-2,3-BD isomer with >99 % purity. The titer was 50 % higher than that of the wide type. A bench-scale fermentation by the double-deletion mutant was developed to further improve meso-2,3-BD production. In a fed-batch fermentation, meso-2,3-BD titer reached 98.0 g/L with a purity of >99.0 % and a productivity of 0.94 g/L–h. This work demonstrates the potential of producing meso-2,3-BD with high titer and purity through metabolic engineering of B. licheniformis.