Engineering Bacillus subtilis for efficient production of 3′-sialyllactose

• Published in: Systems Microbiology and Biomanufacturing 2024, Vol.4 (1), p.294-306
• Main Authors: Zhu, Yonglian, Wu, Yaokang, Yu, Wenwen, Zhang, Quanwei, Liu, Yanfeng, Li, Jianghua, Du, Guocheng, Lv, Xueqin, Liu, Long
• Format: Article
• Language: English
• Subjects: Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Food Microbiology; Life Sciences; Metabolomics; Microbiology; Original Article
• ISSN: 2662-7655; 2662-7663
• DOI: 10.1007/s43393-023-00174-3

3′-Sialyllactose (3′-SL) promotes the proliferation of the bifidobacterial population and shapes gut microbiota. Thus, it can be used in the infant formula industry. In this study, an engineered Bacillus subtilis 168 modified by previous research can synthesize Neu5Ac, an important precursor for 3′-SL biosynthesis, named 3D6.2. Firstly, neuA and nst from Neisseria meningitidis were introduced into 3D6.2 to realize the de novo biosynthesis of 3′-SL from glucose and lactose. Subsequently, the 3′-SL synthetic pathway was optimized with a push–pull-restrain strategy. The promoters and ribosome binding site sequences of neuA and nst were optimized to increase the expression of the two enzymes. Moreover, the β-galactosidase gene was deleted to reduce the consumption of competitive lactose. As a result, the 3′-SL titer was found to improve from 3.8 to 344.7 mg/L. Furthermore, several protein scaffolds were chosen to improve the catalytic efficiency of NeuA and Nst, and the ratio of these two enzymes in the synthetic scaffolds was optimized. Thus, a final 3′-SL titer of 1252.1 mg/L was obtained. These results demonstrate the potential for improving 3′-SL production in B. subtilis in the future.