Zhang Quanwei, Xu Xianhao, Zhang Wei, Huang Ziyang, Wu Yaokang, Liu Yanfeng, Li Jianghua, Du Guocheng, Lv Xueqin, Liu Long
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China; Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China; Institute of Future Food Technology, JITRI, Yixing, 214200, China.
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China; Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.
Metab Eng. 2025 Mar;88:85-93. doi: 10.1016/j.ymben.2024.12.004. Epub 2024 Dec 17.
2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and plays significant roles in gut microbiome balance, neural development, and immunoregulation. However, current fermentation schemes using multiple carbon sources increase production cost and metabolism burden. This study reported the development of an engineered Bacillus subtilis strain that produces 2'-FL using glucose as the sole carbon source. First, a lactose biosynthesis module was constructed by expressing β-1,4-galactosyltransferase gene from Neisseria meningitidis. A 2'-FL titer of 2.53 ± 0.07 g/L was subsequently achieved using glucose as the sole carbon source by the combination of lactose and GDP-L-fucose (GDP-Fuc) biosynthesis modules. Introducing an exogenous nonphosphorylated transport system enhanced the supply of intracellular nonphosphorylated glucose, and the 2'-FL titer increased to 4.94 ± 0.35 g/L. Next, a transcription factor screening platform was designed. Based on this platform, the ligand of the transcription factor LacI was changed from isopropyl β-D-thiogalactoside to lactose. A lactose-responsive genetic circuit was then constructed and used for the dynamic regulation of metabolic fluxes between lactose and GDP-Fuc biosynthesis modules. Ultimately, the 2'-FL titer of the dynamically regulated strain improved by 107% to 9.67 ± 0.65 g/L in shake-flask, and the titer and yield in a 3-L bioreactor reached 30.1 g/L and 0.15 g/g using glucose as the sole carbon source. By using multidimensional engineering strategies, this study constructed a B. subtilis strain capable of efficiently producing 2'-FL with glucose as the sole carbon source, paving the way for the industrial production of 2'-FL with low cost in the future.
2'-岩藻糖基乳糖（2'-FL）是母乳中含量最丰富的低聚糖，在肠道微生物群平衡、神经发育和免疫调节中发挥着重要作用。然而，目前使用多种碳源的发酵方案增加了生产成本和代谢负担。本研究报道了一种工程化枯草芽孢杆菌菌株的开发，该菌株以葡萄糖作为唯一碳源生产2'-FL。首先，通过表达脑膜炎奈瑟菌的β-1,4-半乳糖基转移酶基因构建了乳糖生物合成模块。随后，通过乳糖和GDP-L-岩藻糖（GDP-Fuc）生物合成模块的组合，以葡萄糖作为唯一碳源实现了2'-FL产量为2.53±0.07 g/L。引入外源非磷酸化转运系统增强了细胞内非磷酸化葡萄糖的供应，2'-FL产量提高到4.94±0.35 g/L。接下来，设计了一个转录因子筛选平台。基于该平台，将转录因子LacI的配体从异丙基β-D-硫代半乳糖苷改为乳糖。然后构建了乳糖响应遗传回路，并用于动态调节乳糖和GDP-Fuc生物合成模块之间的代谢通量。最终，在摇瓶中，动态调控菌株的2'-FL产量提高了107%，达到9.67±0.65 g/L，在3-L生物反应器中以葡萄糖作为唯一碳源时，产量和产率分别达到30.1 g/L和0.15 g/g。通过使用多维工程策略，本研究构建了一种能够以葡萄糖作为唯一碳源高效生产2'-FL的枯草芽孢杆菌菌株，为未来低成本工业化生产2'-FL铺平了道路。
