CRISPRi-Guided Multiplexed Fine-Tuning of Metabolic Flux for Enhanced Lacto--neotetraose Production in .

Lacto--neotetraose (LNnT), one of the oligosaccharides in human milk, has many beneficial effects on infant health. In a recent work, we have constructed a recombinant strain for the production of LNnT. Here, we further improved LNnT production with a xylose-induced clustered regularly interspaced short palindromic repeats interference system. In particular, the expressions of and genes in the Embden-Meyerhof-Parnas pathway module, gene in the pentose phosphate pathway module, and gene in the teichoic acid synthesis module were downregulated. The LNnT titer was increased from 1.32 to 1.55 g/L. Furthermore, to improve the conversion efficiency of lacto--triose II to LNnT, we knocked out gene in branch pathway and improved the expression of gene, resulting in the further increase of LNnT titer to 2.01 g/L. Finally, the addition time and amount of inducer xylose were optimized, and LNnT titer reached 2.30 g/L in shake flask and 5.41 g/L in 3 L bioreactor.