Metabolic Engineering of Escherichia coli for Lacto-N-triose II Production with High Productivity.
Metabolic Engineering of Escherichia coli for Lacto-N-triose II Production with High Productivity.
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用于高产乳-N-丙糖 II 的大肠杆菌代谢工程。
DOI：
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发表时间：
2021
影响因子：
6.1
通讯作者：
W. Mu
中科院分区：
文献类型：
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作者：
Yingying Zhu;L. Wan;Jiawei Meng;Guocong Luo;Geng Chen;Hao Wu;Wenli Zhang;W. Mu
乳糖-N-丙糖II（LNT II）是人乳低聚糖（HMOs）的核心结构单元，因其在生产复合HMOs中的营养潜力和应用而引起了广泛关注。在这项研究中，大肠杆菌代谢工程，以有效地生产LNT II使用甘油作为碳源和乳糖作为底物。强化UDP-N-乙酰葡萄糖胺（UDP-GlcNAc）生物合成途径，引入β-1，3-N-乙酰葡萄糖胺转移酶（LgtA），构建LNT II高产基础菌株。为了增加LNT II的滴度和产量，进行拷贝数和核糖体结合位点序列的组合优化以调节途径酶的基因表达强度和翻译速率。接着，将多点突变引入葡萄糖胺-6-磷酸合酶（GlmS）以减轻反馈抑制。然后，通过删除相关基因来阻断无效途径，构建了一系列工程菌株。最后对培养条件进行了优化。摇瓶培养中，LNT Ⅱ的效价从0.53 g/L逐步提高到5.52 g/L。最终工程菌流加培养LNT II产量为46.2 g/L，LNT II产量和含量分别为0.77 g/（L.h）和0.95 g/g细胞干重。
Lacto-N-triose II (LNT II), a core structural unit of human milk oligosaccharides (HMOs), has attracted substantial attention for its nutraceutical potentials and applications in the production of complex HMOs. In this study, Escherichia coli was metabolically engineered to efficiently produce LNT II using glycerol as a carbon source and lactose as a substrate. The UDP-N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway was strengthened, and β-1,3-N-acetylglucosaminyltransferase (LgtA) was introduced to construct an LNT II-producing base strain. To increase the titer and yield of LNT II, combinatorial optimization of the copy number and the ribosomal binding site sequence was performed to tune the gene expression strength and translation rates of the pathway enzymes. Next, multipoint mutations were introduced to glucosamine-6-phosphatesynthase (GlmS) to relieve the feedback inhibition. Then, a series of engineered strains were constructed by blocking the futile pathways by the deletion of the relevant genes. Finally, the culture conditions were optimized. LNT II titer was improved step-by-step from 0.53 to 5.52 g/L in shake-flask cultivations. Fed-batch culture of the final engineered strain produced 46.2 g/L of LNT II, with an LNT II productivity and content of 0.77 g/(L·h) and 0.95 g/g dry cell weight, respectively.