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. 2022 Aug 30:10:977215.
doi: 10.3389/fbioe.2022.977215. eCollection 2022.

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens

Cong Jiang  1 , Changwen Ye  2 , Yongfeng Liu  3 , Kuo Huang  2 , Xuedeng Jiang  1 , Dian Zou  1 , Lu Li  4 , Wenyuan Han  1 , Xuetuan Wei  1
Affiliations

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens

Cong Jiang et al. Front Bioeng Biotechnol. .

Abstract

Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.

Keywords: alkaline protease; bacillus amyloliquefaciens; fermentation optimization; promoter screening; recombinant expression.

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Conflict of interest statement

YL was employed by GeneMind Biosciences Company Limited. CY and KH were employed by China National Tobacco Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Sequence analysis of bsp-1 gene and deduced amino acids. (A) Nucleotide (upper line) and deduced amino acid (lower line) sequences of the protease BSP-1. The predicted signal peptide, propeptide and mature peptide are marked with arrows. (B) Amino acid sequence alignment of the protease BSP-1, with subtilisin (DJ-4, DFE) from B. amyloliquefaciens and subtilisin (NAT, BSF1) from B. subtilis. The signal peptide, pro-peptide, and mature peptide (mature) were indicated with arrows. “.” indicated the same residue as the first sequence. The catalytic center residues (Asp-32, His-64, and Ser-221) were boxed. The initial amino acid of the mature peptide was numbered as + 1.
FIGURE 2
FIGURE 2
Heterologous expression of protease gene bsp-1 in B amyloliquefaciens BAX-9. (A) PCR analysis of recombinant strain colonies. M: Marker; 1-2: P BAX-9/pHY-300 and BAX-9/pHY-Ppqq/BSP-1. (B) SDS-PAGE of analysis of BAX-9/pHY-Ppqq/BSP-1. M: Marker; 1-2: BAX-9/pHY-300 and BAX-9/pHY-Ppqq/BSP-1.
FIGURE 3
FIGURE 3
Illustration of pHY300-PX/BSP-1, where the promoter sequence can be easily replaced.
FIGURE 4
FIGURE 4
Skim milk agar plates of the fermentation supernatant of different strains. CK: BAX-9/pHY-300, Psra: BAX-9/pHY-Psra/BSP-1, Psrs: BAX-9/pHY-Psrs/BSP-1, Prnp: BAX-9/pHY-Prnp/BSP-1, Pffs: BAX-9/pHY-Pffs/BSP-1, Pcsp: BAX-9/pHY-Pcsp/BSP-1, Ppqq: BAX-9/pHY-Pscp/BSP-1, Pscp: BAX-9/pHY-Pscp/BSP-1, Phyp: BAX-9/pHY-Phyp/BSP-1, Psrf: BAX-9/pHY-Psrf/BSP-1, Pitu: BAX-9/pHY-Pitu/BSP-1, Pfen: BAX-9/pHY-Pfen/BSP-1, Pbac: BAX-9/pHY-Pbac/BSP-1, P43: BAX-9/pHY-P43/BSP-1, PtrnQ: BAX-9/pHY-PtrnQ/BSP-1.
FIGURE 5
FIGURE 5
SDS-PAGE analysis of the fermentation supernatant of different strains. M: Marker; 1-8: BAX-9/pHY-300, BAX-9/pHY-P43/BSP-1, BAX-9/pHY-Pscp/BSP-1, BAX-9/pHY-Pcsp/BSP-1, BAX-9/pHY-Ppqq/BSP-1, BAX-9/pHY-Psrf/BSP-1, BAX-9/pHY-Pitu/BSP-1, BAX-9/pHY-Pfen/BSP-1.
FIGURE 6
FIGURE 6
Determination of enzyme activity and biomass of recombinant strains. Asterisks show the significant difference (p < 0.05) compared with the control.
FIGURE 7
FIGURE 7
RT-PCR results of recombinant strains. Asterisks show the significant difference (p < 0.05) compared with the control.
FIGURE 8
FIGURE 8
Enzyme activity and culture growth of BAX-9/pHY-P43/BSP-1 under fermentation using the optimized fermentation medium.
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