Multi‐level metabolic engineering of Escherichia coli for high‐titer biosynthesis of 2′‐fucosyllactose and 3‐fucosyllactose |
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Authors: | Mengli Li Chenchen Li Yejiao Luo Miaomiao Hu Zhu Liu Tao Zhang |
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Affiliation: | 1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi Jiangsu, China ; 2. Zhejiang Institute for Food and Drug Control, Hangzhou China ; 3. International Joint Laboratory on Food Science and Safety, Jiangnan University, Wuxi Jiangsu, China |
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Abstract: | Fucosyllactoses (FL), including 2′‐fucosyllactose (2′‐FL) and 3‐fucosyllactose (3‐FL), have garnered considerable interest for their value in newborn formula and pharmaceuticals. In this study, an engineered Escherichia coli was developed for high‐titer FL biosynthesis by introducing multi‐level metabolic engineering strategies, including (1) individual construction of the 2′/3‐FL‐producing strains through gene combination optimization of the GDP‐L‐fucose module; (2) screening of rate‐limiting enzymes (α‐1,2‐fucosyltransferase and α‐1,3‐fucosyltransferase); (3) analysis of critical intermediates and inactivation of competing pathways to redirect carbon fluxes to FL biosynthesis; (4) enhancement of the catalytic performance of rate‐limiting enzymes by the RBS screening, fusion peptides and multi‐copy gene cloning. The final strains EC49 and EM47 produced 9.36 g/L for 2′‐FL and 6.28 g/L for 3‐FL in shake flasks with a modified‐M9CA medium. Fed‐batch cultivations of the two strains generated 64.62 g/L of 2′‐FL and 40.68 g/L of 3‐FL in the 3‐L bioreactors, with yields of 0.65 mol 2′‐FL/mol lactose and 0.67 mol 3‐FL/mol lactose, respectively. This research provides a viable platform for other high‐value‐added compounds production in microbial cell factories.An engineered Escherichia coli was developed for high‐titer FL biosynthesis by introducing multi‐level metabolic engineering strategies. Combined with the optimization of metabolic pathways and the performance improvement of rate‐limiting enzymes, 64.62 g/L of 2 ''‐FL and 40.68 g/L of 3‐FL were finally obtained in the 3‐L bioreactors. |
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