Efficient production of 2'-fucosyllactose in unconventional yeast Yarrowia lipolytica

Abstract

2'-Fucosyllactose (2'-FL) has great application value as a nutritional component and the whole cell biosynthesis of 2'-FL has become the focus of current research. Yarrowia lipolytica has great potential in oligosaccharide synthesis and large-scale fermentation. In this study, systematic engineering of Y. lipolytica for efficient 2'-FL production was performed. By fusing different protein tags, the synthesis of 2'-FL was optimized and the ubiquitin tag was demonstrated to be the best choice to increase the 2'-FL production. By iterative integration of the related genes, increasing the precursor supply, and promoting NADPH regeneration, the 2'-FL synthesis was further improved. The final 2'-FL titer, 41.10 g/L, was obtained in the strain F5-1. Our work reports the highest 2'-FL production in Y. lipolytica, and demonstrates that Y. lipolytica is an efficient microbial chassis for the synthesis of oligosaccharides.

Keywords: 2′-Fucosyllactose; Iterative integration; NADPH regeneration; Pathway enhancement; Yarrowia lipolytica.

Graphical abstract

Fig. 1

Effect of expression of FutC fused with different protein tags on 2′-FL titer. (A) Schematic digram of the 2′-FL biosynthetic pathway. (B) Effect of different protein tags on 2′-FL production. The data was derived from the results of three repeated experiments.

Fig. 2

Enhanced 2′-FL production by iterative integration. (A) Plasmid construction for co-overexpression of Ub-hpFutC, klLAC12, maGMD and hpGMER. (B) 2′-FL production in strains obtained after the first, second, and third rounds of plasmid integration, respectively. The dashed line indicates the average level of 2′-FL production for each group of strains. The data was derived from the results of three repeated experiments.

Fig. 3

Enhanced 2′-FL production by enhancing GDP-d-mannose synthesis pathway. (A) Schematic view of the strategies to strengthen GDP-d-mannose synthesis by co-overexpressing of PMI40, SEC53, and PSA1. (B) 2′-FL production of the strains F3-5 and the strain F4-2. (C) 2′-FL yield on glucose and 2′-FL titer (g/g DCW) of the strain F3-5 and the strain F4-2. The data was derived from the results of three repeated experiments. T-test was performed to determine p values. ***p < 0.001; ****p < 0.0001.

Fig. 4

The enhancement of NADPH regeneration pathway promotes 2′-FL accumulation. (A) Schematic view of the enhanced NADPH regeneration pathway. (B) 2′-FL production and 2′-FL yield on glucose of the strains overexpressing ZWF1, PGL, and GND1, respectively. (C) 2′-FL titer of the strain F4-2 and the strain F5-1. (D) 2′-FL yield on glucose and OD₆₀₀ of the strain F4-2 and the strain F5-1. The data was derived from the results of three repeated experiments. T-test was performed to determine p values. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5

Biosynthesis of 2’ -FL by fed-batch fermentation. (A) Cell density (OD₆₀₀), glucose concentration and lactose concentration in the culture medium during fermentation. (B) 2’ -FL production during fermentation.