高产乳酰-N-三糖Ⅱ大肠杆菌菌株的构建

doi:10.13560/j.cnki.biotech.bull.1985.2025-0690

摘要/Abstract

摘要：

目的利用微生物合成乳酰-N-三糖 Ⅱ（lacto-N-triose, LNT Ⅱ）是实现其工业化生产的可行方法，但目前由于异源酶的表达较差、途径关键酶及限速酶表达不平衡和前体物质合成不足等问题，LNT Ⅱ的产量仍然较低，本研究构建LNT Ⅱ高产菌株，提高LNT Ⅱ的合成能力。方法对比不同促溶蛋白标签对关键酶β-1，3-N-乙酰葡糖胺转移酶LgtA可溶性表达的影响，并精准调控LgtA和限速酶谷氨酰胺-果糖-6-磷酸氨基转移酶GlmS的表达强度，同时对不同来源的谷氨酰胺合成酶GlnA进行筛选，显著提升异源酶的可溶性表达，均衡关键途径酶的表达水平并强化前体供给，最后对LNT Ⅱ发酵培养基成分，包括甘油、IPTG、甜菜碱和乳清酸添加量进行优化。结果将MBP与LgtA融合后，LgtA的溶解度显著提高；通过RBS T7调节关键酶LgtA和限速酶GlmS的翻译强度、表达SGlnA^E304A后有利于LNT Ⅱ的合成和菌株的生长；在甘油添加量为15 mL/L、IPTG添加量为0.1 mmol/L、甜菜碱添加量为3 g/L、乳清酸添加量3 g/L的培养条件下，LNT Ⅱ的产量由4.37 g/L提高至14.12 g/L。结论本研究对LNT Ⅱ生产菌株的代谢工程改造与发酵条件优化，显著提高了LNT Ⅱ的生产水平，并为在大肠杆菌中合成其他种类的HMOs提供了参考。

关键词: 乳酰-N-三糖 Ⅱ, 人乳寡糖, 大肠杆菌, 微生物合成, 促溶蛋白标签

Abstract:

Objective The synthesis of lacto-N-triose Ⅱ (LNT Ⅱ) by microorganisms is a feasible method for its industrial production. However, at present, due to issues such as poor expressions of heterologous enzymes, unbalanced expressions of key and rate-limiting enzymes, and insufficiency of precursors, the production of LNT Ⅱ is still relatively low. This study constructed LNT Ⅱ high-producing strains increasing the synthetic capacity of LNT Ⅱ. Method By comparing the effects of different solubility-promoting protein labels on the soluble expression of the key enzyme β-1, 3-N-acetylglucosaminotransferase (LgtA), regulating the expressions of LgtA and the rate-limiting enzyme glutamine-fructose-6-phosphate aminotransferase (GlmS) precisely, and selecting suitable glutamine synthase (GlnA) from different sources, the soluble expressions of heterologous enzymes were enhanced significantly, the expressions of key pathway enzymes were balanced, the supplies of precursors were strengthened, and the component of LNT Ⅱ fermentation medium such as glycerol, IPTG, betaine and orotic acid concentrations were optimized finally. Result The solubility of LgtA was significantly improved after fused with MBP. Then, by using RBS T7 to regulate the translation of the key enzyme LgtA and the rate-limiting enzyme GlmS and overexpressing SGlnA^E304A, it was beneficial for the synthesis of LNT Ⅱ and the growth of the strain. Finally, the LNT Ⅱ production increased from 4.37 g/L to 14.12 g/L under the culture conditions of 15 mL/L glycerol addition, 0.1 mmol/L IPTG addition, 3 g/L betaine addition, and 3 g/L orotic acid addition. Conclusion This study combined the metabolic engineering and fermentation condition optimization, increasing the LNT Ⅱ production significantly. It provided a reference for the synthesis of other types of HMOs in Escherichia coli.

Key words: lacto-N-triose Ⅱ, human milk oligosaccharides, Escherichia coli, microbial synthesis, solubility-promoting protein tag

何听雨, 逄雨, 张远洋, 孙雪, 李玉, 路福平, 李庆刚. 高产乳酰-N-三糖Ⅱ大肠杆菌菌株的构建[J]. 生物技术通报, 2025, 41(11): 143-152.

HE Ting-yu, PANG Yu, ZHANG Yuan-yang, SUN Xue, LI Yu, LU Fu-ping, LI Qing-gang. Construction of a High-production Lacto -N-triose Ⅱ-producing Escherichia coli Strain[J]. Biotechnology Bulletin, 2025, 41(11): 143-152.

图/表 7

表1 融合蛋白的理化性质

Table 1 Physicochemical properties of fusion proteins

融合蛋白名称 Name of fusion protein	理论等电点 Theoretical isoelectric point	负电荷氨基酸残基总数 Total number of negatively charged amino acid residues	正电荷氨基酸残基总数 Total number of positively charged amino acid residues	总平均亲水性 Overall average hydrophilicity
TrxA-Nm58LgtA^{R13H，L24M，R205C}	6.48	64	61	-0.412
SUMO-Nm58LgtA^{R13H，L24M，R205C}	6.38	69	65	-0.621
GST-Nm58LgtA^{R13H，L24M，R205C}	6.99	81	80	-0.478
MBP-Nm58LgtA^{R13H，L24M，R205C}	6.24	99	93	-0.441
AHP-Nm58LgtA^{R13H，L24M，R205C}	7.75	50	51	-0.552

图1 融合蛋白的亲疏水性能分析A-E：分别表示添加促溶标签的融合蛋白TrxA-Nm58LgtAR13H，L24M，R205C、SUMO-Nm58LgtAR13H，L24M，R205C、GST-Nm58LgtAR13H，L24M，R205C、MBP-Nm58LgtAR13H，L24M，R205C和AHP-Nm58LgtAR13H，L24M，R205C的亲疏水性分析，图中纵坐标Score代表疏水性值。正值表示疏水性区域，负值表示亲水性区域。横坐标代表氨基酸在该蛋白序列中的位置

Fig. 1 Analysis of hydrophilicity and hydrophobicity of fusion proteinsA-E: Indicate the hydrophilicity analysis of the fusion proteins TrxA-Nm58LgtAR13H,L24M,R205C, SUMO-Nm58LgtAR13H,L24M,R205C, GST-Nm58LgtAR13H,L24M,R205C, MBP-Nm58LgtAR13H,L24M,R205C and AHP-Nm58LgtAR13H,L24M,R205C with solubility-promoting tags, respectively. In the figures, the vertical axis score indicates the hydrophobicity value. Positive values indicate hydrophobic regions, and negative values indicate hydrophilic regions. The horizontal axis indicates the position of the amino acid in the protein sequence

图2 不同促溶蛋白标签对LNT Ⅱ产量的影响A：融合蛋白示意图；B：Q1-Q5的菌株生长情况和LNT Ⅱ产量；C：融合蛋白胶图

Fig. 2 Influence of different solubility-promoting protein tags on the production of LNT ⅡA: Schematic diagram of fusion protein; B: growth and LNT Ⅱ production of strains Q1 to Q5; C: fusion protein gel diagram. M: Marker; 1: Nm58LgtAR13H,L24M,R205C; 2: TrxA-Nm58LgtAR13H,L24M,R205C; 3: SUMO-Nm58LgtAR13H,L24M,R205C; 4: GST-Nm58LgtAR13H,L24M,R205C; 5: MBP-Nm58LgtAR13H,L24M,R205C; 6: AHP-Nm58LgtAR13H,L24M,R205C

图3 LNT II代谢途径图

Fig. 3 Metabolic pathway of LNT II

图4 LgtA和GlmS*的表达强度对LNT Ⅱ合成的影响A：质粒过表达MBP-Nm58LgtAR13H，L24M，R205C和GlmS*与不同RBS组合示意图；B：Z1-Z36的菌株生长情况和LNT Ⅱ产量

Fig. 4 Influences of the expression intensities of LgtA and GlmS* on the synthesis of LNT ⅡA: Schematic diagrams of plasmids overexpressing MBP-Nm58LgtAR13H,L24M,R205C and GlmS* with various RBSs. B: Growth and LNT Ⅱ production of strains Z1 to Z36

图5 不同来源的GlnA对LNT Ⅱ合成的影响A：过表达不同来源GlnA示意图；B：G0-G2的菌株生长情况和LNT Ⅱ产量

Fig. 5 Influences of GlnA from different sources on the synthesis of LNT ⅡA: Schematic diagram of overexpressing GlnA from different sources. B: Growth and LNT Ⅱ production of strains G0 to G2

图6 甘油、IPTG、甜菜碱和乳清酸添加量对LNT Ⅱ合成的影响

Fig. 6 Effects of glycerol, IPTG, betaine and orotic acid addition on the synthesis of LNT Ⅱ

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