Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL.

中文翻译：

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使用工程大肠杆菌多酶促级联一锅法生物合成 3'-唾液酸乳糖

在母乳低聚糖 (HMO) 中，含量最丰富且对人体健康有极大益处的低聚糖之一是 3'-唾液酸乳糖 (3'-SL)。鉴于其重要的生理功能和缺乏具有成本效益的生产工艺，我们构建了一个体外多酶辅助因子回收系统，用于从低成本底物生物合成 3'-SL。首先，我们构建了生物合成途径，并通过分子伴侣增加了胞苷单磷酸激酶 (CMK) 的溶解度。我们随后发现 β-半乳糖苷酶 (lacZ) 会影响 3'-SL 的产量，因此在敲除 lacZ 基因后，观察到 3'-SL 的产量增加了 3.3 倍。此外，优化了用于 3'-SL 生产的温度、pH、多磷酸盐浓度和二价金属离子浓度。最后，在优化的条件下建立了高效的生物转化系统。3'-SL 的最大产量达到 38.7 mM，从 N-乙酰神经氨酸（NeuAc，唾液酸，SA）获得了 97.1% 的摩尔产率。结果表明，具有辅因子再生的多酶级联生物合成途径有望作为生产 3'-SL 的工业策略。