Lacto--neotetraose (LNnT) is a neutral human milk oligosaccharide with important biological functions. However, the low LNnT productivity and the incomplete conversion of the intermediate lacto--tetraose II (LNT II) currently limited the sustainable biosynthesis of LNnT. First, the LNnT biosynthetic module was integrated in . Next, the LNnT export system was optimized to alleviate the inhibition of intracellular LNnT synthesis. Furthermore, by utilizing rate-limiting enzyme diagnosis, the expressions of LNnT synthesis pathway genes were finely regulated to further enhance the production yield of LNnT. Subsequently, a strategy of cofermentation using a glucose/glycerol (4:6, g/g) mixed feed was employed to regulate carbon flux distribution. Finally, by overexpressing key transferases, LNnT and LNT II titers reached 112.47 and 7.42 g/L, respectively, in a 5 L fermenter, and 107.4 and 2.08 g/L, respectively, in a 1000 L fermenter. These are the highest reported titers of LNnT to date, indicating its significant potential for industrial production.
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