Lacto-N-新四糖(LNnT),作为人乳寡糖(HMO)中的中性核心结构,由于其特殊的生理功能而引起了广泛的关注。在使用细胞工厂方法合成LNnT的过程中,糖基转移酶的底物混杂导致产生更长的寡糖衍生物。这里,来自脑膜炎奈瑟球菌(LgtA)的β1,3-N-乙酰氨基葡萄糖转移酶的合理修饰有效地降低了长链LNnT衍生物的浓度。具体来说,最佳β1,4-半乳糖基转移酶(β1,4-GalT)是从七个已知的候选物中选择的,能够在大肠杆菌BL21(DE3)中高效合成LNnT。此外,研究了乳糖浓度对LNnT及其较长衍生物分布模式的影响。LgtA的修改是在计算辅助下进行的,涉及基于分子对接的丙氨酸扫描以识别底物结合袋,并对关键氨基酸实施大的空间位阻以阻碍LNnT进入。在LgtA的位置223和228处实施饱和诱变产生有利的突变变体,其不影响LNnT合成,同时显著减少较长寡糖衍生物的产生。最有效的突变体,N223I,将长链衍生物的摩尔比降低了近70%,展示了LNnT生产的前景看好,副产品减少。
Lacto-N-neotetraose (LNnT), as a neutral core structure within human milk oligosaccharides (HMOs), has garnered widespread attention due to its exceptional physiological functions. In the process of LNnT synthesis using cellular factory approaches, substrate promiscuity of glycosyltransferases leads to the production of longer oligosaccharide derivatives. Here, rational modification of β1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis (LgtA) effectively decreased the concentration of long-chain LNnT derivatives. Specifically, the optimal β1,4-galactosyltransferase (β1,4-GalT) was selected from seven known candidates, enabling the efficient synthesis of LNnT in Escherichia coli BL21(DE3). Furthermore, the influence of lactose concentration on the distribution patterns of LNnT and its longer derivatives was investigated. The modification of LgtA was conducted with computational assistance, involving alanine scanning based on molecular docking to identify the substrate binding pocket and implementing large steric hindrance on crucial amino acids to obstruct LNnT entry. The implementation of saturation mutagenesis at positions 223 and 228 of LgtA yielded advantageous mutant variants that did not affect LNnT synthesis while significantly reducing the production of longer oligosaccharide derivatives. The most effective mutant, N223I, reduced the molar ratio of long derivatives by nearly 70 %, showcasing promising prospects for LNnT production with diminished byproducts.