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Additional file 1: Table S1. Factors and levels of Plackett-Burman experiment. Table S2. Factors and levels of Central Composite design. Table S3. Plackett-Burma test design and results. Table S4. The variance analysis of Plackett-Burman test. Table S5. The design and results of steepest ascent experiment. Table S6. CCD design and results. Table S7. The variance analysis of experiment. Figure S1. The alkaline protease and process pH during the fermentation of different initial medium pH.

Brown spot disease, caused by Alternaria alternate, is one of the most destructive leaf spot diseases that made a major impact on tobacco growing. Biocontrol has attracted increasing attentions as its features of environmental friendship, promoting plant growth, etc. Iturin A, with broad spectrum antifungal activity, is a kind of biosurfactant that mainly produced by Bacillus. In this study, the promoter of iturin A synthetase cluster of Bacillus amyloliquefaciens HZ-12 was respectively replaced by promoters P43, PbacA, PsrfA and Pylb, our results implied that transcriptional level of gene ituD and iturin A titer showed consistent change trends, and PbacA was proven as the most efficient promoter, iturin A titer of which reached 950.08 ± 19.43 mg/L. Furthermore, regulator gene abrB was deleted to release the repression effect of AbrB on PbacA, ituD transcriptional level and iturin A titer were increased by 133.25 % and 20.88 %, respectively. Then, the maximum iturin A titer reached 2013.43 ± 32.86 mg/L by optimizing fermentation medium, increased by 392.15 % compared to the original (408.97 ± 21.35 mg/L). Finally, our results demonstrated that enhancing iturin A synthetic capability benefited the suppression of A. alternate. Collectively, this study provided a promising strain with an efficient fermentation medium for large-scale industrial production of iturin A.

Additional file1: Table S1. Strains and plasmids used in this study. Table S2. Primers used in this study. Table S3. The effects of GC content at the bottom of stem-loop structure, Gibbs free energy ΔG, U-tract length on terminator performances. Table S4. The sequences of different terminators plasmids used in this study. Table S5. Comparison of termination efficiency of different terminators in Bacillus. Figure S1. The secondary structure map of TamyL with a size of 501 bp. Figure S2. The construction map of plasmids with different terminators and dual reporter genes.

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

Tobacco specific nitrosamines (TSNA) mainly consisting of N-nitrosonornicotine (NNN), N-nitrosoanatabine (NAT), N-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are a group of toxic components threatening human health. To inhibit TSNA formation in tobacco leaves, a high nitrite reductive strain with low nitrate reduction ability was isolated and applied to tobacco leaves in an attempt to lower the nitrite precursor of TSNA. By morphology, physiology, biochemistry, and 16S rDNA sequence analysis, the strain DA9 was identified as Bacillus amyloliquefaciens. Under the optimized fermentation parameters (glucose 40 g/L, NH4Cl 4 g/L, corn steep liquor 8 g/L, MnSO4 0.01 g/L, KH2PO4 1.0 g/L, MgSO4 0.3 g/L, initial pH 7.0, inoculum age 6 h, inoculum size 3%, temperature 37 °C), the maximum cell dentisity of 1.2 × 10(9) CFU/mL was obtained at 36 h. The DA9 cell suspensions were applied in the air-curing process of the Burley tobacco (Eyan 6) leaves. The treatment by DA9 cells lowered 32% of the nitrite content and 47% of total TSNA content in the tobacco leaves, and the concentrations of the NNN, NNK, and NAT were decreased by 48%, 12%, and 35%, respectively. Collectively, this study provides a promising strain and a novel strategy for decreasing TSNA during the air-curing process.