Designing a Highly Efficient Biosynthetic Route for Lacto- N -Neotetraose Production in Escherichia coli .

Recently, the biosynthesis of human milk oligosaccharides (HMOs) has been attracting increasing attention. Lacto- N -neotetraose (LNnT) is one of the most important neutral-core HMOs with promising health effects for infants. It has received Generally Recognized as Safe (GRAS) status and is the second HMO commercially added in infant formula after 2'-fucosyllactose. In previous studies, a series of engineered Escherichia coli strains have been constructed and optimized to produce high titers of precursor lacto- N -triose II. On the basis of these strains, LNnT-producing strains were constructed by overexpressing the β1,4-galactosyltransferase-encoding gene from Aggregatibacter actinomycetemcomitans NUM4039 (Aa - β1,4 - GalT). Interestingly, an appreciable LNnT titer was obtained by weakening the metabolic flux of the UDP-GlcNAc pathway and simply overexpressing the essential genes lgtA, galE, and Aa - β1 , 4 - GalT in lacZ-, wecB - , and nagB-deleted E. coli . Subsequently, LNnT synthesis was optimized through balancing the expression of these three biosynthetic enzymes. The optimized strain produced LNnT with an extracellular titer of 12.1 g/L in fed-batch cultivation, with the productivity and specific yield of 0.25 g/L·h and 0.27 g/g dry cell weight, respectively.