Efficient Production of 2′-Fucosyllactose from l-Fucose via Self-Assembling Multienzyme Complexes in Engineered Escherichia coli,ACS Synthetic Biology

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Efficient Production of 2′-Fucosyllactose from l-Fucose via Self-Assembling Multienzyme Complexes in Engineered Escherichia coli
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2021-08-20 , DOI: 10.1021/acssynbio.1c00102
Li Wan ₁ , Yingying Zhu ₁ , Geng Chen ₁ , Guocong Luo ₁ , Wenli Zhang ₁ , Wanmeng Mu _{1,

2}

Affiliation

2′-Fucosyllactose (2′-FL) has been widely used as a nutritional additive in infant formula due to its multifarious nutraceutical and pharmaceutical functions in neonate health. As such, it is essential to develop an efficient and extensive microbial fermentation platform to cater to the needs of the 2′-FL market. In this study, a spatial synthetic biology strategy was employed to promote 2′-FL biosynthesis in recombinant Escherichia coli. First, the salvage pathway for 2′-FL production from l-fucose and lactose was constructed by introducing a bifunctional enzyme l-fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) derived from Bacteroides fragilis and an α-1,2-fucosyltransferase (FutC) derived from Helicobacter pylori into engineered E. coli BL21(DE3). Next, the endogenous genes involved in the degradation and shunting of the substrate and key intermediate were inactivated to improve the availability of precursors for 2′-FL biosynthesis. Moreover, to further improve the yield and titer of 2′-FL, a short peptide pair (RIAD-RIDD) was used to form self-assembling multienzyme complexes in vivo. The spatial localization of peptides and stoichiometry of enzyme assemblies were subsequently optimized to further improve 2′-FL production. Finally, cofactor regeneration was also considered to alleviate the potential cofactor deficiency and redox flux imbalance in the biocatalysis process. Fed-batch fermentation of the final WLS20 strain accumulated 30.5 g/L extracellular 2′-FL with the yield and productivity of 0.661 mol/mol fucose and 0.48 g/L/h, respectively. This research has demonstrated that the application of spatial synthetic biology and metabolic engineering strategies can dramatically enlarge the titer and yield of 2′-FL biosynthesis in engineered E. coli.

中文翻译：

在工程大肠杆菌中通过自组装多酶复合物从 l-岩藻糖高效生产 2'-岩藻糖基乳糖

2'-岩藻糖基乳糖 (2'-FL) 因其在新生儿健康中的多种营养和药物功能而被广泛用作婴儿配方奶粉中的营养添加剂。因此，必须开发一个高效且广泛的微生物发酵平台来满足 2'-FL 市场的需求。在这项研究中，采用空间合成生物学策略来促进重组大肠杆菌中的 2'-FL 生物合成。首先，通过引入源自脆弱拟杆菌的双功能酶l-岩藻糖激酶/ GDP- l-岩藻糖焦磷酸化酶 (Fkp)构建了从l-岩藻糖和乳糖生产 2'-FL的补救途径以及源自幽门螺杆菌的α-1,2-岩藻糖基转移酶（FutC）进入工程大肠杆菌BL21（DE3）。接下来，将参与底物和关键中间体降解和分流的内源基因失活，以提高 2'-FL 生物合成前体的可用性。此外，为了进一步提高2'-FL的产量和效价，使用短肽对（RIAD-RIDD）在体内形成自组装多酶复合物. 随后优化了肽的空间定位和酶组装的化学计量，以进一步改善 2'-FL 的产生。最后，辅因子再生也被认为可以缓解生物催化过程中潜在的辅因子缺乏和氧化还原通量不平衡。最终 WLS20 菌株的补料分批发酵积累了 30.5 g/L 的胞外 2'-FL，产量和产率分别为 0.661 mol/mol 岩藻糖和 0.48 g/L/h。该研究表明，空间合成生物学和代谢工程策略的应用可以显着提高工程大肠杆菌中2'-FL 生物合成的效价和产量。

更新日期：2021-10-15

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