Metabolic engineering strategies of de novo pathway for enhancing 2′‐fucosyllactose synthesis in Escherichia coli

Abstract

Summary 2′‐Fucosyllactose (2′‐FL), one of the most abundant human milk oligosaccharides (HMOs), is used as a promising infant formula ingredient owing to its multiple health benefits for newborns. However, limited availability and high‐cost preparation have restricted its extensive use and intensive research on its potential functions. In this work, a powerful Escherichia coli cell factory was developed to ulteriorly increase 2′‐FL production. Initially, a modular pathway engineering was strengthened to balance the synthesis pathway through different plasmid combinations with a resulting maximum 2′‐FL titre of 1.45 g l −1 . To further facilitate the metabolic flux from GDP‐ l ‐fucose towards 2′‐FL, the CRISPR‐Cas9 system was utilized to inactivate the genes including lacZ and wcaJ , increasing the titre by 6.59‐fold. Notably, the co‐introduction of NADPH and GTP regeneration pathways was confirmed to be more conducive to 2′‐FL formation, achieving a 2′‐FL titre of 2.24 g l −1 . Moreover, comparisons of various exogenous α 1,2‐fucosyltransferase candidates revealed that futC from Helicobacter pylori generated the highest titre of 2′‐FL. Finally, the viability of scaled‐up production of 2′‐FL was evidenced in a 3 l bioreactor with a maximum titre of 22.3 g l −1 2′‐FL and a yield of 0.53 mole 2′‐FL mole −1 lactose.

Strategies for Enhancing Microbial Production of 2′-Fucosyllactose, the Most Abundant Human Milk Oligosaccharide

“…In a study by Li et al, LacY was initially included in part of an engineering module for optimization of 2′-FL-production . In a study by Lin et al, overexpression of LacY only slightly enhanced 2′-FL synthesis in an engineered de novo 2′-FL-producing E. coli .…”

“…Enhancement of 2′-FL production was observed in most of the engineered strains, among which the highest enhancement was generated by introducing zwf and pntAB , encoding for glucose-6-phosphate dehydrogenase and membrane-bound transhydrogenase, respectively . In another study, nine genes related to GTP and NADPH regeneration were selected for expression, and the engineered E. coli with coexpression of zwf and the inosine-guanosine kinase-encoding gene gsk showed the highest enhancement in de novo 2′-FL production . In addition, GTP regeneration by expression of gsk and ndk could enhance the salvage 2′-FL production in engineered E. coli …”

“…Several enzymes are involved in the colanic acid pathway from GDP- l -fucose, in which UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferase (WcaJ, EC 2.7.8.31) is the enzyme responsible for the first step . Deletion of competitive pathway gene wcaJ was widely performed to prevent GDP- l -fucose loss and further improve 2′-FL production in engineered E. coli . ,,,− ,, In addition, Lee et al studied the effect of additional deletion of waaF encoding for ADP-heptose:LPS heptosyltransferase II, which might influence the GDP- l -fucose transport. Deletion of waaF was confirmed to favor colanic acid synthesis .…”

High-Level De Novo Biosynthesis of 2′-Fucosyllactose by Metabolically Engineered Escherichia coli

“…The representative α1,2-fucosyltransferase from Helicobacter pylori FutC or FucT2, E. coli O128 WbsJ, B. fragilis Wcfb, and E. coli O126 WbgL were commonly used to produce 2′-FL. 14,15 Some key genes involved in 2′-FL production were overexpressed by ways of plasmids and/or chromosome integration. 8,16 Other genes involved in competitive pathways were downregulated or deleted from genome.…”

“…8,16 Other genes involved in competitive pathways were downregulated or deleted from genome. 14,15 In addition, Parschat et al constructed an engineered E. coli to not only endogenously synthesize lactose but also support the strain growth and efficiently produced 2′-FL using sucrose as the only carbon source, which could reduce the cost greatly by replacing lactose with sucrose. 17 On the basis of above-mentioned studies, a highly efficient biosynthetic route for 2′-FL production was redesigned via the de novo pathway of GDP-L-fucose using engineered Escherichia coli BL21(DE3) (Figure 1).…”