<br>通过 De Novo GDP-l-岩藻糖途径在枯草芽孢杆菌 ATCC 6051a 中生物合成 3-岩藻糖基乳糖的 α-1,3-岩藻糖基转移酶的合理设计,Journal of Agricultural and Food Chemistry

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通过 De Novo GDP-l-岩藻糖途径在枯草芽孢杆菌 ATCC 6051a 中生物合成 3-岩藻糖基乳糖的 α-1,3-岩藻糖基转移酶的合理设计
Journal of Agricultural and Food Chemistry ( IF 5.7 ) Pub Date : 2024-01-06 , DOI: 10.1021/acs.jafc.3c07604
Yukang Xie _{1,

2} , Xinying Wu _{1,

3} , Cong Fu _{1,

2} , Haiyan Duan _{1,

2,

3} , Jiping Shi _{1,

2,

3} , Jenny M Blamey _{4,

5} , Junsong Sun _{1,

2,

3}

Affiliation

3-岩藻糖基乳糖 (3-FL) 是母乳中重要的低聚糖和营养素，可通过 α-1,3-岩藻糖基转移酶 (α-1,3-FucT) 使用鸟苷 5'-二磷酸 (GDP) 在微生物细胞中合成- l-岩藻糖和乳糖作为底物。然而，各种来源的已知 α-1,3-FucT 由于溶解度低，其催化效率受到限制。为了提高微生物生产 3-FL，我们在枯草芽孢杆菌( B. subtilis ) 底盘细胞中评估了 α-1,3-FucT 的效率，该细胞被赋予了 GDP- l-岩藻糖的异源合成途径，揭示了幽门螺杆菌( H. pylori ) 中的 FucTa 活性高于任何其他报道的同系物。为了进一步提高FucTa的催化性能，采用了合理的设计方法，包括对通过定向进化获得的M32突变位点进行胞内评估、配体结合位点多样性分析以及蛋白质结构模拟。在获得的变体中，FucTa-Y218 K变体表现出最高的3-FL产量，在摇瓶生长实验中达到7.55 g/L，比野生型酶高3.48倍。随后在 5 L 生物反应器中的发酵优化导致 3-FL 产量达到 36.98 g/L，凸显了所设计的酶和菌株在工业应用中的巨大前景。

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Rational Design of an α-1,3-Fucosyltransferase for the Biosynthesis of 3-Fucosyllactose in Bacillus subtilis ATCC 6051a via De Novo GDP-l-Fucose Pathway

3-Fucosyllactose (3-FL) is an important oligosaccharide and nutrient in breast milk that can be synthesized in microbial cells by α-1,3-fucosyltransferase (α-1,3-FucT) using guanosine 5’-diphosphate (GDP)-l-fucose and lactose as substrates. However, the catalytic efficiency of known α-1,3-FucTs from various sources was limited due to their low solubility. To enhance the microbial production of 3-FL, the efficiencies of α-1,3-FucTs were evaluated and in Bacillus subtilis (B. subtilis) chassis cells that had been endowed with a heterologous synthetic pathway for GDP-l-fucose, revealing that the activity of FucTa from Helicobacter pylori (H. pylori) was higher than that of any of other reported homologues. To further improve the catalytic performance of FucTa, a rational design approach was employed, involving intracellular evaluation of the mutational sites of M32 obtained through directed evolution, analysis of the ligand binding site diversity, and protein structure simulation. Among the obtained variants, the FucTa-Y218 K variant exhibited the highest 3-FL yield, reaching 7.55 g/L in the shake flask growth experiment, which was 3.48-fold higher than that achieved by the wild-type enzyme. Subsequent fermentation optimization in a 5 L bioreactor resulted in a remarkable 3-FL production of 36.98 g/L, highlighting the great prospects of the designed enzyme and the strains for industrial applications.

更新日期：2024-01-06

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