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Tianjin Development Area Teda Street China Laboratory Microbiology Technology Ministry Education Tianjin China
Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 0457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 71, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 0457, PR China. Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 0457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 71, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 0457, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Fucosyllactoses, including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose nutritional additives in infant formula due to their biological functions, such as the promotion of bifidobacteria growth, inhibition of pathogen infection, and improvement of immune response. In this study, we developed a synthetic biology approach to promote the efficient biosynthesis of 2'-FL and 3-FL in engineered Escherichia coli. To boost the production of 2'-FL and 3-FL, multiple modular optimization strategies were applied in a plug-and-play manner. First, comparisons of various exogenous α1,2-fucosyltransferase and α1,3-fucosyltransferase candidates, as well as a series of E. coli host strains, demonstrated that futC and futA from Helicobacter pylori using BL21(DE3) as the host strain yielded the highest titers of 2'-FL and 3-FL.

Subsequently, both the availability of the lactose acceptor substrate and the intracellular pool of the GDP-L-fucose donor substrate were optimized by inactivating competitive (or repressive) pathways and strengthening acceptor (or donor) availability to achieve overproduction. Moreover, the intracellular redox regeneration pathways were engineered to further enhance the production of 2'-FL and 3-FL. Finally, various culture conditions were optimized to achieve the best performance of 2'-FL and 3-FL biosynthesizing strains. 2'-Fucose lactose of 2'-FL and 3-FL were 92 and 123gL, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.Screening and characterization of an α-L-fucosidase from Bacteroides fragilis NCTC9343 for synthesis of fucosyl-N-acetylglucosamine disaccharides.Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Qingdao, 266237, People's Republic of China.

Hospital Affiliated to Shandong First Medical University, Jinan, 21, Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Qingdao, 266237, People's Republic of China. Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, Shandong University, Qingdao, 266237, People's Republic of China. Fucosyl-N-acetylglucosamine disaccharides are present in many biologically important oligosaccharides, such as human milk oligosaccharides, Lewis carbohydrate antigens, and glycans on cell-surface glycoconjugate receptors, and thus have vast potential for infant formulas, prebiotics, and pharmaceutical applications. In this work, in order to screen biocatalysts for enzymatic synthesis of fucosyl-N-acetylglucosamine disaccharides, we performed sequence analysis of 12 putative and one known α-L-fucosidases of Bacteroides fragilis NCTC9343 and constructed a phylogenetic tree of the nine GH29 α-L-fucosidases. After that, five GH29A α-L-fucosidases were cloned, and four of them were successfully heterogeneous expressed and screened for transglycosylation activity, and a GH29A α-L-fucosidase (BF3242) that synthesized a mix of Fuc-α-1,31,6-GlcNAc disaccharides using pNPαFuc as donor and GlcNAc as acceptor was characterized. The effects of initial substrate concentration, pH, temperature, and reaction time on its transglycosylation activity were studied in detail. Under the optimum conditions of5 UmL enzyme, mM pNPαFuc, and 0 mM GlcNAc in sodium buffer (pH 7) at 37 °C for 45 min, BF3242 efficiently synthesized Fuc-α-1,31,6-GlcNAc at a maximum yield of 79% with the ratio of8 for 1,31,6.
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