School of Biology and Biological Engineering, Guangzhou Higher Education Mega Centre, South China University of Technology, Panyu District, Guangzhou, 510006, Guangdong, People's Republic of China.
Appl Microbiol Biotechnol. 2024 Jan 19;108(1):149. doi: 10.1007/s00253-023-12889-3.
In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. KEY POINTS: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.
在本研究中，我们成功地应用了串联启动子和串联信号肽与过表达信号肽酶相结合的策略，从枯草芽孢杆菌、解淀粉芽孢杆菌和地衣芽孢杆菌中高效表达和生产 γ-谷氨酰肽酶（GGT）酶（BsGGT、BaGGT 和 BlGGT）。为了避免由于重复强启动子引起的不稳定性问题，我们组装了来自不同物种的不同同源基因的串联启动子。为了在食品工业中避免使用抗性标记酶，我们首先从表达载体中去除了大肠杆菌的复制原点和相应的抗性标记。然后将质粒转化到枯草芽孢杆菌宿主中，并使用 CRISPR/Cas9n-AID 碱基编辑系统直接编辑和沉默表达质粒中的卡那霉素抗性基因。因此，构建了一种无抗性标记的重组蛋白表达载体，摇瓶发酵中 BlGGT 菌株的酶活可达 53.65 U/mL。将重组 BlGGT 用环氧树脂固定化，重复使用 10 次后酶活保持 82.8%，在 4°C 下储存 2 个月后酶活保持 87.36%。固定化 BlGGT 酶用于连续合成茶氨酸，转化率为 65.38%。这些结果表明，我们的方法是提高酶生产效率和实现食品工业中酶制剂安全生产的有前途的解决方案。
双启动子和双信号肽组合实现重组蛋白的高效表达。
使用 CRISPR/Cas9n-AID 编辑系统构建枯草芽孢杆菌中无抗性标记的小载体。
优化了 BlGGT 用环氧树脂固定化的过程。
