2'-Fucosyllactose (2'-FL) is a fucosylated human milk oligosaccharides, which has many im-portant physiological functions such as preventing intestinal diseases, improving immunity and promoting brain development. Based on the genome level, this study constructed 2'-FL synthetic chassis cells by knocking out of 2'-FL precursor synthesis related metabolic genes in Escherichia coli BL21 (DE3) , such as β-galactosidase (lacZ), undecaprenyl-phosphate glucose-1-phosphate transferase(wcaJ) and GDP-mannose mannosyl hydrolase(nudD); on this basis, the exogenous gene α-1,2-Fucosyltransferase (wbgL) and phosphomannomutase(manB), man-nose-1-phosphate guanylyltransferas(manC), GDP-mannose 4,6-dehydratase(gmd) and GDP-L-fucose syn-thase(wcaG) were introduced into the genome of E. coli to explore the effect of overexpression of 2'-FL synthesis gene on 2'-FL yield at the genomic level. The results showed that the accumulation of 2'-FL reached 1.99 g·L-1 in shake-flask fermentation with B-05 strain. The optimal fermentation conditions were determined by shake-flask fermentation optimization: IPTG induction concentration was 0.4 mmol·L-1, OD600 was 2.4, and induction tem-perature was 28 ℃. Under these conditions, the synthesis amount of 2'-FL in shake-flask fermentation reached 3.92 g·L-1. In 5 L fed-batch fermentation, the content of 2'-FL reached 43.48 g·L-1. The results showed that the efficient synthesis of 2'-FL was achieved by introducing exogenous 2'-FL-related synthetic genes into the ge-nome for overexpression, which provides data support for the construction of integrated 2'-FL strains.
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