Lacto--fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2′-fucosyllactose (2′-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B and antiviral effects against and . Microbial production of HMOs by engineered is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the -acetylglucosamine moiety of the lacto--tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2′-FL. In this study, we constructed LNFP I-producing strains of with various α1,2-fucTs, and observed undesired 2′-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2′-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2′-FL production. Therefore, to decrease the formation of byproduct 2′-FL, we designed 15 variants of FsFucT from sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2′-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154–171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154–171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2′-FL, while the strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2′-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2′-FL by combining protein engineering of α1,2-FucT designed through structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

中文翻译：

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使用工程α-1,2-岩藻糖基转移酶在大肠杆菌中选择性微生物生产乳-N-岩藻五糖 I

乳岩藻糖五糖 I (LNFP I) 是母乳中第二丰富的岩藻糖基化母乳寡糖 (HMO)，仅次于 2'-岩藻糖基乳糖 (2'-FL)。研究报告称，LNFP I 对 B 组具有抗菌活性，对 和 具有抗病毒作用。通过工程微生物生产 HMO 是一种有吸引力的低成本工艺，但很少有研究调查长链 HMO 的生产，包括五糖 LNFP I。LNFP I 是通过 α1,2-岩藻糖基转移反应合成的 - 乙酰氨基葡萄糖部分。乳糖-四糖骨架，由 α1,2-岩藻糖基转移酶 (α1,2-FucT) 催化。然而，α1,2-FucT 竞争性地将岩藻糖转移为乳糖，导致副产物 2'-FL 的形成。在本研究中，我们构建了具有各种 α1,2-fucT 的 LNFP I 生产菌株，并在补料分批发酵过程中观察到了不需要的 2'-FL 积累，尽管在试管测定中，一些菌株产生了没有 2'-FL 的 LNFP I。佛罗里达州。我们假设 α1,2-FucT 的混杂底物选择性导致了 2'-FL 的产生。因此，为了减少副产物2'-FL的形成，我们从sp设计了15个FsFucT变体。FSC1006 采用理性和半理性设计方法。与野生型 FsFucT 相比，其中 5 个 FsFucT 变体的 2'-FL 产量减少了两倍，同时保持了相当的 LNFP I 产量水平。这些设计包括酶环区域（残基 154-171）或特定残基（Q7、H162 和 L164）中的取代，直接或间接影响底物结合。特别是，表达 FsFucT_S3 变体的菌株，具有形成 α-螺旋结构的取代环区域（残基 154-171），实现了 19.6 g/L LNFP I 和 0.04 g/L 2'-FL 的积累，而表达野生型FsFucT的菌株在Fed-bach发酵过程中积累了12.2 g/L的LNFP I和5.85 g/L的2'-FL。因此，我们通过α1,2-FucT的结构建模和与各种配体的对接模拟设计，结合α1,2-FucT的蛋白质工程，成功地证明了五糖LNFP I的选择性和高效生产，且没有副产物2′-FL ，通过宿主细胞的代谢工程。