화학공학소재연구정보센터
Biotechnology and Bioengineering, Vol.116, No.12, 3324-3332, 2019
Biosynthesis of the human milk oligosaccharide 3-fucosyllactose in metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis and beta-galactosidase modification
3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2 '-fucosyllactose (2 '-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function. Whereas the biological production of 2 '-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of alpha-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2 '-FL, and lower digestibility of 3-FL than 2 '-FL by bifidobacteria. In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in k(cat) and the decrease in K-m. We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP-L-fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZ Delta m15) for better utilization of D-lactose. Production using the lacZ Delta m15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g center dot L-1 center dot hr(-1) and the specific 3-FL yield of 0.5 g/g dry cell weight.