Spatial organization of pathway enzymes via self‐assembly to improve 2′‐fucosyllactose biosynthesis in engineered Escherichia coli

Abstract

As one of the most abundant components in human milk oligosaccharides, 2′‐fucosyllactose (2′‐FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine‐tuning the expression of pathway enzymes and achieved a striking increase of 2′‐FL production, directly facilitating the metabolic flux toward the key intermediate GDP‐l‐fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2′‐FL. Specifically, we first fine‐tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2′‐FL synthesis. Then, two key enzymes (GDP‐mannose 4,6‐dehydratase and GDP‐ l‐fucose synthase) were self‐assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD–RIDD (RI anchoring disruptor–RI dimer D/D domains), resulting in noticeable improvement of 2′‐FL production. Next, to further strengthen the metabolic flux toward 2′‐FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher–SpyTag or PDZ–PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2′‐FL biosynthesis and showed a 2.1‐fold increase of 2′‐FL production compared with a strain expressing free‐floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2′‐FL in shake flask fermentation and was capable of producing 25.1 g/L 2′‐FL by fed‐batch cultivation. This work provides novel approaches for further improvement and large‐scale production of 2′‐FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain.

Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli

“…28 In our previous study, self-assembly of three enzymes (ManC, Gmd, and WcaG) for GDP-L-fucose synthesis into a trienzyme complex by two orthogonal interacting pairs (RIDD−RIAD and PDZ−PDZlig) effectively streamlined the metabolic flux toward GDP-L-fucose and led to a noticeable increase of 2′-FL production. 29 To this end, we speculate that compartmentalizing relevant enzymes into constructed compartments will increase the biosynthesis of 2′-FL. Specifically, peptide RIAD was fused to the N-or C-terminus of pathway enzymes, generating fusion enzymes of ManC-RA, ManB-RA, RA-Gmd, and RA-WcaG, respectively.…”

“…A previous study has proved that the supply of GDP- l -fucose is a rate-limiting step for fucosyllactose synthesis . In our previous study, self-assembly of three enzymes (ManC, Gmd, and WcaG) for GDP- l -fucose synthesis into a trienzyme complex by two orthogonal interacting pairs (RIDD–RIAD and PDZ–PDZlig) effectively streamlined the metabolic flux toward GDP- l -fucose and led to a noticeable increase of 2′-FL production . To this end, we speculate that compartmentalizing relevant enzymes into constructed compartments will increase the biosynthesis of 2′-FL.…”

“…Further, the yield of WPSG15 also witnessed a significant increase and reached 0.653 mol 2′-FL/mol lactose, which was 95.5% higher than that obtained by the control strain WPSG12 (0.334 mol 2′-FL/mol lactose). These results are consistent with previous study that the clustering of pathway enzymes for GDP- l -fucose synthesis can effectively promote 2′-FL production …”

“…It should be noted that modification of the C-terminus of Gmd and WcaG can severely impair their activities. 29 Moreover, gene f utC encoding an α-1,2-fucosyltransferase derived from Helicobacter pylori was subcloned to plasmid pCDFDuet-1, yielding plasmid pCD-f utC. This plasmid was then transduced into engineered strain BWL.…”

“…These results are consistent with previous study that the clustering of pathway enzymes for GDP-L-fucose synthesis can effectively promote 2′-FL production. 29 To evaluate the effects of overexpressing RGG-RD as well as tagging RIAD to pathway enzymes on cell growth and 2′-FL production, WPSG10 and WPSG11 were also constructed (Figure S8). By comparing the OD 600 and 2′-FL titers of WPSG10 and WPSG12, we concluded that the overexpression of scaffold protein RGG-RD resulted in a slight increase in the cell growth and 2′-FL production (Figure 5C).…”