Yu Xinrui, Zhang Kang, Zhu Xuyang, Lv Huihui, Wu Jing
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China; School of Bioengineering, Jiangnan University, Wuxi 214122, People's Republic of China.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, People's Republic of China; School of Bioengineering, Jiangnan University, Wuxi 214122, People's Republic of China.
Int J Biol Macromol. 2024 Jan;254(Pt 3):127372. doi: 10.1016/j.ijbiomac.2023.127372. Epub 2023 Oct 12.
As a food-safe microorganism, Bacillus subtilis has been widely utilized in the production of food enzyme, where a food-grade expression system without antibiotic is required. However, there is no mature system for such expression, since the recombinant plasmid in existing food-grade expression system is unstable especially in high-density fermentation. In this study, we constructed a food-grade expression system based on the dal gene auxotrophic selection marker. Specifically, maltogenic amylase (AmyM) was expressed in dal deletion strain without antibiotic, yielding an activity of 519 U/mL. To increase the expression of AmyM, the promoter of amyM (gene encoding AmyM) was optimized. Furthermore, we found that excessive expression of dal gene was detrimental to the stability of plasmid, and the ribosome binding site (RBS) of dal was mutated with the reduced synthesis of D-alanine. After that, AmyM activity increased to 1364 U/mL with the 100 % stability of plasmid. The 3-L fermentor cultivation was performed with the highest value ever reported in food-grade microorganisms, an activity of 2388 U/mL, showing the scale-up production capability of this system. Besides, it is also able to apply the system for other food enzymes, which indicating the great generalizability of this system for different application.
作为一种食品安全微生物，枯草芽孢杆菌已被广泛应用于食品酶的生产中，这需要一个无抗生素的食品级表达系统。然而，目前尚无成熟的此类表达系统，因为现有食品级表达系统中的重组质粒不稳定，尤其是在高密度发酵时。在本研究中，我们构建了一种基于dal基因营养缺陷型选择标记的食品级表达系统。具体而言，麦芽糖淀粉酶（AmyM）在无抗生素的dal缺失菌株中表达，活性达到519 U/mL。为了提高AmyM的表达量，对amyM（编码AmyM的基因）的启动子进行了优化。此外，我们发现dal基因的过量表达不利于质粒的稳定性，因此对dal的核糖体结合位点（RBS）进行了突变，从而减少了D-丙氨酸的合成。此后，AmyM活性提高到1364 U/mL，质粒稳定性达到100%。在3-L发酵罐中进行培养，获得了食品级微生物中报道的最高活性值，即2388 U/mL，表明该系统具有放大生产能力。此外，该系统还能够应用于其他食品酶，这表明该系统在不同应用中具有很强的通用性。
