Microbial Synthesis of l-Fucose with High Productivity by a Metabolically Engineered Escherichia coli

Abstract

l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2′-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI, fucK, and rhaA. The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2′-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

Efficient Biosynthesis of Lacto-N-Biose I, a Building Block of Type I Human Milk Oligosaccharides, by a Metabolically Engineered Escherichia coli

“…Interestingly, L-fucose biosynthesis was slightly impacted when the lactose concentration varied from 3 to 15 g/L, and the intermediate 2′-FL was not detectable and lactose was consumed very little under all conditions, indicating that only a small amount of added lactose was enough to drive L-fucose synthesis and lactose recycling. 27 Herein, we studied the effect of the concentration of added lactose on LNB biosynthesis by the strain ENP05L. Similar to lactose recycling in L-fucose biosynthesis reported previously, the added lactose concentration almost did not affect the LNB titer, and the concentrations of the residual LNTri II and LNT.…”

“…In a previous work, Meng et al. developed the l -fucose biosynthesis via the route of “lactose →2′-FL → l -fucose” and enhanced the gene expression of the 2′-FL degradation enzyme via additional genomic integration, by which l -fucose was highly efficiently produced without detectable 2′-FL residue . Similar attempts could be made in the future to further enhance LNB production.…”

“…The effect of the lactose concentration was investigated. Interestingly, l -fucose biosynthesis was slightly impacted when the lactose concentration varied from 3 to 15 g/L, and the intermediate 2′-FL was not detectable and lactose was consumed very little under all conditions, indicating that only a small amount of added lactose was enough to drive l -fucose synthesis and lactose recycling …”

“…This phenomenon of lactose recycling was also observed in the microbial synthesis of L-fucose reported previously. 26,27 Meng et al constructed a two-step route of "lactose →2′fucosyllactose (2′-FL) → L-fucose", including one fucosylation step for 2′-FL synthesis and one degradation step for L-fucose synthesis, releasing lactose. Lactose concentration almost remained stable during L-fucose biosynthesis, and the intermediate 2′-FL was almost undetectable, indicating that the 2′-FL degradation activity was very high.…”

High-Level Production of l-Fucose by Plasmid-Free or Antibiotic-Independent Metabolically Engineered Escherichia coli Strains

“…The plasmid pCD-AfcA was constructed previously, carrying a codon-optimized α-L-fucosidase gene afcA from Bifidobacterium bifidum. 19 Then, these three fragments were assembled via overlap extension PCR using the primer pairs FucIK-OL-F/R to generate the integration cassette, which contains constitutive promoter P J23119 , afcA, T7 terminator, and two homologous arms corresponding to the upstream and downstream regions of the f ucIf ucK gene cluster. To delete the f ucI-fucK gene cluster and integrate the afcA, plasmid pcrEG-FucIK and the integration cassette were electroporated into the MGC07 strain harboring pEcCpf1, which had been induced by 10 mM L-arabinose.…”