Construction of a High-production Lacto -N-triose Ⅱ-producing Escherichia coli Strain

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

Abstract

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

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.

Figures/Tables 7

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

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

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

Fig. 3 Metabolic pathway of LNT II

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

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

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

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