De novo biosynthesis of 6\'-sialyllactose from lactose and sialic acid using co-culture of engineered E. coli and S. cerevisiae

• 1、Jiangnan University,Jiangsu WuXi,214122

Abstract：6\'-Sialyllactose is the most abundant sialyllactose in human milk oligosaccharides (HMOs), which plays an important role in the growth and development of infants. Achieving efficient production of 6\'-sialyllactose has always been a difficult and challenging task in the field of oligosaccharide synthesis. To address this problem, in this paper, we first constructed the engineered strain E. coli JM109(DE3)/pET28a-plst6 for the expression of α-2,6-sialic acid transferase, and explored the optimal conditions for the synthesis of 6\'-sialyllactose by coupled fermentation of E. coli JM109(DE3)/pET28a-plst6, baker\'s yeast, and E. coli JM109(DE3)/pET28a-neuA at the level of shake flasks and 7L fermenters. Then, an engineering strain E. coli JM109(DE3)/pETDeut-1-plst6-neuA was constructed to co-express α-2,6-sialyltransferase and CMP-Neu5Ac synthetase. The effect of coupling this strain with baker\'s yeast to synthesize 6?-sialyllactose was evaluated and optimized. The results showed that the highest yield of 6?-sialyllactose in the fermentation broth reached 35.52 g/L with 97.97% conversion of sialic acid under the shaking flask level three-strain coupled fermentation mode with a substrate sialic acid concentration of 60 mM, a lactose concentration of 160 mM, a baker\'s yeast biomass of 150 g/L, a temperature of 30 C, and an Mg2+ concentration of 20 mM. The synthesis of 6?-sialyllactose was achieved by a three-bacteria coupled fermentation conversion at the level of a 7 L fermenter. The maximum yield of 6?-sialyllactose in the fermentation broth reached 43.79 g/L after 24 hours of fermentation, with a 72.66% conversion of sialic acid. Under the two-strain coupled fermentation mode, shaking flask level fermentation was carried out for 30 hours with a 6?-sialyllactose concentration of 35.58 g/L, and the conversion of sialic acid was 93.69%; at the level of 7L fermenter, the highest yield of 52.29 g/L was achieved after 16 hours of fermentation, with a conversion rate of 97.99%. The results of this study provide a technological basis for the large-scale mass production of 6?-sialyllactose.

Keywords： genetic engineering；6?-sialyllactose； coupled fermentation； high-density culture； human milk oligosaccharides