State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu 214122, China.
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China.
J Agric Food Chem. 2024 Mar 13;72(10):5379-5390. doi: 10.1021/acs.jafc.3c09335. Epub 2024 Feb 29.
3'-Sialyllactose (3'-SL) is among the foremost and simplest sialylated breast milk oligosaccharides. In this study, an engineered for high-titer 3'-SL biosynthesis was developed by introducing a multilevel metabolic engineering strategy, including (1) the introduction of precursor CMP-Neu5Ac synthesis pathway and high-performance α2,3-sialyltransferase (α2,3-SiaT) genes into strain BZ to achieve synthesis of 3'-SL; (2) optimizing the expression of -- involved in the UDP-GlcNAc and CMP-Neu5Ac synthesis pathways, and constructing a glutamine cycle system, balancing the precursor pools; (3) analysis of critical intermediates and inactivation of competitive pathway genes to redirect carbon flux to 3'-SL biosynthesis; and (4) enhanced catalytic performance of rate-limiting enzyme α2,3-SiaT by RBS screening, protein tag cloning. The final strain BZAPKA14 yielded 9.04 g/L 3'-SL in a shake flask. In a 3 L bioreactor, fed-batch fermentation generated 44.2 g/L 3'-SL, with an overall yield and lactose conversion of 0.53 g/(L h) and 0.55 mol 3'-SL/mol, respectively.
3'-唾液酸乳糖（3'-SL）是最重要和最简单的母乳低聚糖之一。在本研究中，通过引入多级代谢工程策略，开发了一种用于高产量 3'-SL 生物合成的工程菌，该策略包括：（1）引入前体 CMP-Neu5Ac 合成途径和高性能的α2,3-唾液酸转移酶（α2,3-SiaT）基因到菌株 BZ 中，以实现 3'-SL 的合成；（2）优化涉及 UDP-GlcNAc 和 CMP-Neu5Ac 合成途径的基因的表达，并构建谷氨酰胺循环系统，平衡前体池；（3）分析关键中间产物并失活竞争途径基因，将碳通量重新定向到 3'-SL 生物合成；（4）通过 RBS 筛选和蛋白标签克隆，增强限速酶α2,3-SiaT 的催化性能。最终的菌株 BZAPKA14 在摇瓶中产生了 9.04 g/L 的 3'-SL。在 3 L 生物反应器中，分批补料发酵产生了 44.2 g/L 的 3'-SL，总产率和乳糖转化率分别为 0.53 g/（L h）和 0.55 mol 3'-SL/mol。
