Journal
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
Volume 68, Issue 8, Pages 2477-2484Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.jafc.9b07642
Keywords
CRISPR interference; gene knockdown; metabolic flux redirection; lacto-N-neotetraose; Bacillus subtilis
Funding
- National Natural Science Foundation of China [31622001, 31871784, 31870069, 21676119, 31671845]
- Key Research and Development Program of China [2018YFA0900300]
- Postgraduate Research & Practice Innovation Program of Jiangsu Province [ICYCX19_1825]
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Lacto-N-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 Bacillus subtilis 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 pfkA and pyk genes in the Embden-Meyerhof-Pamas pathway module, zwf gene in the pentose phosphate pathway module, and mnaA 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-N-triose II to LNnT, we knocked out tuaD gene in branch pathway and improved the expression of lgtB 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.
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