Multi-Path Optimization for Efficient Production of 2'-Fucosyllactose in an Engineered Escherichia coli C41 (DE3) Derivative

Abstract

2'-fucosyllactose (2'-FL), one of the simplest but most abundant oligosaccharides in human milk, has been demonstrated to have many positive benefits for the healthy development of newborns. However, the high-cost production and limited availability restrict its widespread use in infant nutrition and further research on its potential functions. In this study, on the basis of previous achievements, we developed a powerful cell factory by using a lacZ-mutant Escherichia coli C41 (DE3)ΔZ to ulteriorly increase 2'-FL production by feeding inexpensive glycerol. Initially, we co-expressed the genes for GDP-L-fucose biosynthesis and heterologous α-1,2-fucosyltransferase in C41(DE3)ΔZ through different plasmid-based expression combinations, functionally constructing a preferred route for 2'-FL biosynthesis. To further boost the carbon flux from GDP-L-fucose toward 2'-FL synthesis, deletion of chromosomal genes (wcaJ, nudD, and nudK) involved in the degradation of the precursors GDP-L-fucose and GDP-mannose were performed. Notably, the co-introduction of two heterologous positive regulators, RcsA and RcsB, was confirmed to be more conducive to GDP-L-fucose formation and thus 2'-FL production. Further a genomic integration of an individual copy of α-1,2-fucosyltransferase gene, as well as the preliminary optimization of fermentation conditions enabled the resulting engineered strain to achieve a high titer and yield. By collectively taking into account the intracellular lactose utilization, GDP-L-fucose availability, and fucosylation activity for 2'-FL production, ultimately a highest titer of 2'-FL in our optimized conditions reached 6.86 g/L with a yield of 0.92 mol/mol from lactose in the batch fermentation. Moreover, the feasibility of mass production was demonstrated in a 50-L fed-batch fermentation system in which a maximum titer of 66.80 g/L 2'-FL was achieved with a yield of 0.89 mol 2'-FL/mol lactose and a productivity of approximately 0.95 g/L/h 2'-FL. As a proof of concept, our preliminary 2'-FL production demonstrated a superior production performance, which will provide a promising candidate process for further industrial production.

Keywords: 2′-fucosyllactose; Escherichia coli; GDP-D-mannose; GDP-L-fucose; fucosylation.

Figure 1

Schematic representation of 2′-FL production by metabolically engineered E. coli in this study. The intracellular enzymes are abbreviated as follows: Zwf, glucose-6-phosphate dehydrogenase; Pgl, 6-phosphogluconolactonase; Gnd, 6-phosphogluconate dehydrogenase; Pgi, phosphoglucose isomerase; PfkA(B), 6-phosphofructokinase-1(2); ManA, mannose 6-phosphate isomerase; ManB, phosphomannomutase; ManC, α-D-mannose 1-phosphate guanylyltransferase; Gmd, GDP-mannose 6-dehydrogenase; WcaG, GDP-L-fucose synthase; WcaJ, UDP-glucose: undecaprenyl-phosphate glucose-1-phosphate transferase; NudK, GDP-mannose hydrolase; NudD, GDP-mannose mannosyl hydrolase; RcsA, positive transcriptional regulator A; RcsB, positive transcriptional regulator B; LacZ, β-galactosidase; LacY, lactose permease; FutC, α-1,2-fucosyltransferase.

Figure 2

Production of 2′-FL in the recombinant E. coli C41 (DE3)ΔZ by fed–batch fermentation. (A) Specific activities of β-galactosidase from the C41 (DE3) and C41 (DE3)ΔZ strains. (B–D) Time profiles of recombinant strains, C41ΔZ/pA, C41ΔZ/pC, and C41ΔZ/pR during the batch fermentations. Symbols are: lactose (), glycerol (), 2′-FL (), and DCW (). Brown vertical arrow indicates the time point for IPTG induction. Purple vertical arrow indicates the time point for lactose addition. (E,F) LC/MS identification of 2′-FL produced by the recombinant E. coli C41 (DE3)ΔZ. The red arrows represent the 2′-FL debris from the mass spectrometer.

Figure 3

Effect of the modulations of intracellular GDP-L-fucose availability and fucosylation activity on 2′-FL production. (A–E) Time profiles of the engineered strains, C41ΔZW/pR, C41ΔZD/pR, C41ΔZK/pR, C41ΔZD/pRA, and C41ΔZD/pRAB during the batch fermentations. (F) Time profile of the strain C41ΔZD-F/pRAB harboring a genomic integration of an individual copy of futC during the batch fermentation. Symbols are: lactose (), glycerol (), 2′-FL (), and DCW (). Brown vertical arrow indicates the time point for IPTG induction. Purple vertical arrow indicates the time point for lactose addition.

Figure 4

Improving 2′-FL production by optimizing the fermentation conditions. Biomass (A) and 2′-FL concentration (B) of the strain C41ΔZWD-F/pRAB at different IPTG concentrations. Biomass (C) and 2′-FL concentration (D) of the strain C41ΔZWD-F/pRAB at different culture temperatures.

Figure 5

Time profiles of the fed-batch fermentation by the strain E.coli C41ΔZWD-F/pRAB in 50-L bioreactor. (A) Cultivation was carried out at 25°C and 0.2 mM IPTG Induction. (B) Cultivation was carried out at 28°C and 0.3 mM IPTG Induction. Symbols are: lactose (), glycerol (), 2′-FL (), and DCW (). Brown vertical arrow indicates the time point for IPTG induction. Purple vertical arrow indicates the time point for lactose addition.