多细胞耦合转化N-乙酰氨基葡萄糖和乳糖生产唾液酸乳糖

Multicellular coupling fermentation for 3ʹ-sialyllactose conversion using N-acetyl-glucosamine and lactose

摘要:

唾液酸乳糖是母乳寡糖(human milk oligosaccharides, HMOs)中含量最丰富的唾液酸化低聚糖之一，对婴幼儿的健康发育有重要作用，但是其目前缺乏高效、廉价的生产工艺。针对该问题，本研究建立了多菌株两步法耦合生物合成唾液酸乳糖方法。第一步构建2株工程菌，大肠杆菌JM109(DE3)/pET28a-BT0453和JM109(DE3)/pET28a-nanA，用于耦合合成中间产物N-乙酰神经氨酸，在2株工程菌株细胞生物量比为1:1，反应时间为32 h的条件下，得到的N-乙酰神经氨酸最高产量为20.4 g/L。第二步向上述发酵液中添加大肠杆菌JM109(DE3)/pET28a-neuA、JM109(DE3)/ pET28a-nst和面包酵母，通过三菌株耦合发酵合成3ʹ-唾液酸乳糖(3ʹ-sialyllactose, 3ʹ-SL)。在底物N-乙酰氨基葡萄糖、乳糖浓度均为200 mmol/L，面包酵母细胞生物量为150 g/L，辅因子Mg²⁺浓度为20 mmol/L的优化条件下，发酵24 h，发酵液中3ʹ-唾液酸乳糖的最高产量达到55.04 g/L，底物N-乙酰氨基葡萄糖的转化率为43.47%。研究结果为低成本生产3ʹ-唾液酸乳糖提供了一条新的技术路线。

关键词:3ʹ;-唾液酸乳糖;N-乙酰氨基葡萄糖;耦合发酵;两步法合成;母乳寡糖

Abstract:

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker’s yeast were added to the above fermentation broth to synthesize 3ʹ-sialyllactose (3ʹ-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker’s yeast, 20 mmol/L Mg²⁺, the maximum yield of 3ʹ-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3ʹ-SL.

Key words:3ʹ;-sialyllactose;N-acetyl-glucosamine;coupled fermentation;two-step synthesis;human milk oligosaccharides