Lacto-N-neotetraose (LNnT) is one of the important components of human milk oligosaccharides (HMOs), which has been approved by the Food and Drug Administration of the United States and the European Union to be added to food as a new resource food ingredient. To achieve the lacto-N-neotetraose producing strains and efficient synthesis of the products, recombinant strains containing overexpression of the key enzyme genes lgtA, NmlgtB, glmS and galE were constructed in this study. The recombinant bacteria were cultured in IPTG-induced shake flasks for fermentation and combinatorial screening of the modular LNnT synthesis pathway using plasmids of different copy numbers. The results showed that the optimal plasmid combination of pET-glmS-galE and pRSF-lgtA-NmlgtB resulted in a yield of 1.168 g/L of LNnT production by strain SA1. Further, the fermentation conditions were optimized by changing the type of medium, the induction temperature, the IPTG concentration and the moment of induction. The results showed that the optimal fermentation medium of the recombinant strain was DM medium, with an induction temperature of 25 ℃, IPTG concentration of 0.2 mmol/L, and induction time of 8 h. The yield of LNnT fermentation in shake flasks of the engineered bacterium SA1 was 2.50 g/L, an increase of 114% compared to the pre-optimization period. The yield of LNnT reached 14.25 g/L by fermentation in 3 L fermenters, which is the highest yield reported so far. This study provides a methodological idea for the industrial production of LNnT and provides an important reference value for the preparation of HMOs by microbial fermentation.
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