Enhanced production of 2'-fucosyllactose by introducing fucosyllactose exporter and optimizing biosynthetic pathway of GDP-L-fucose in engineered Corynebacterium glutamicum

Abstract

ABSTRACT

Human milk contains a considerable amount of oligosaccharides (about 15 g/L), unlike other mammalian milks. Human milk oligosaccharides (HMOs) consist of 200-300 kinds of oligosaccharides. In particular, 80% of HMOs are fucosylated oligosaccharides, and 2-fucosyllactose (2-FL) is the most abundant oligosaccharide among HMOs. 2-FL is a trisaccharide composed of lactose and fucose. Fucose binds to the galactose of lactose through α1-2 linkage. 2-FL has many physiological functions such as prebiotic effects, inhibition of intestinal adhesion of pathogens and enterotoxin secreted from pathogens and alleviation of the inflammatory responses. Corynebacterium glutamicum, which is approved as GRAS (Generally Recognized As Safe), was used to produce 2'-FL in previous researches. In order to biosynthesize 2'-FL, GDP-L-fucose and lactose are required. The wild-type C. glutamicum lacks the GDP-L-fucose biosynthetic pathway, so the genes for biosynthesis of GDP-L-fucose were introduced. In addition, the lactose permease gene from Escherichia coli was introduced since the wild type C. glutamicum cannot import lactose and the codon-optimized α-1,2 fucosyltransferase gene from Helicobacter pylori was introduced for fucosylation. Using the above-mentioned metabolically engineered strain, 11.5 g/L of 2-FL was produced in fed-batch fermentation. In this study, several attempts have been made to enhance 2'-FL production. First, the ABC transporter permease gene from Bifidobacterium infantis was introduced to export intracellular 2'-FL. As a result, it showed a 38% improvement in 2-FL secretion per gram of cells than the strain constructed previously. The concentration of 2'-FL in the medium was 830 mg/L, which was 52% higher than that of the strain constructed in previous research. Next, the GDP-L-fucose biosynthetic pathway was optimized to enhance the metabolic flux to GDP-L-fucose. In the system for synthesizing GDP-L-fucose using glucose only, the metabolic flux mostly directed to glycolysis for cell growth. In order to solve this problem, the phosphofructokinase A (pfkA) gene was disrupted by the double crossover method using a pK19mobsacB vector, and the ΔP strain that blocked the first major pathway of the glycolysis (Fructose 6-phosphate → Fructose 1,6-bisphosphate) was constructed. By using a mixture of glucose and fructose as a carbon source, a two track system was constructed in which glucose was used for synthesis of GDP-L-fucose and fructose for cell growth. As a result, 2-FL was produced at a concentration of 1.22 g/L in flask fermentation, and 12.6 g/L in fed-batch fermentation. However, cell growth was delayed in the middle of fermentation, and the consumption rate of glucose and fructose continuously changed, making it difficult to maintain a constant concentration during fermentation. To solve this problem, glucose was supplied intermittently while supplying fructose to maintain a constant concentration. As a result, 2.05 g/L of 2-FL was produced in flask fermentation. But, in fed-batch fermentation, cell growth was delayed for a long time when glucose was supplied. The cell adaptation process was added at the pre-culture step in which the cells to be inoculated into the main culture were prepared. After establishing an environment similar to that of the main culture, the cells were cultured up to the mid-log phase and inoculated into the main culture. As a result, the delay of cell growth was solved, and 21.5 g/L of 2-FL was produced in fed-batch fermentation. This result is 87% higher than the amount of 2-FL produced in the previous research. Finally, by changing the culture temperature from 30℃ to 37℃ in the latter stage of the fermentation, the fluidity of the cell membrane was increased to allow the intracellular 2-FL to go out of the cells. As a result, 2.61 g/L of 2-FL was produced in flask fermentation, which is 379% higher than that the amount of 2-FL produced in the previous research. In this study, a microbial bioprocess was developed to produce 2-FL with high titer and productivity. It is believed that the bioprocess developed in this study would provide a technical framework for industrial production of 2-FL.