title: Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement.
abstract: Fucosyllactoses, including 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL), are important oligosaccharides in human milk that are commonly used as 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. The final concentrations of 2'-FL and 3-FL were 9.12 and 12.43g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300457, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, PR China.
authors.forename: Di
authors.initials: D
authors.lastname: Huang
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China.
authors.forename: Kexin
authors.initials: K
authors.lastname: Yang
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China.
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Xu
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Wang
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China.
authors.forename: Ru
authors.initials: R
authors.lastname: Wang
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300457, PR China. Electronic address: liubin1981@nankai.edu.cn.
authors.forename: Bin
authors.initials: B
authors.lastname: Liu
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, Tianjin Economic-Technological Development Area (TEDA), 23 Hongda Street, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300457, PR China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, PR China. Electronic address: fenglu63@nankai.edu.cn.
authors.forename: Lu
authors.initials: L
authors.lastname: Feng
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000201:enzymology / Q000235:genetics*; D060847:Metabolic Engineering; D014312:Trisaccharides / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D014312:Trisaccharides; D005647:Fucosyltransferases; C031420:2'-fucosyllactose
keywords: 2′(3)-Fucosyllactose; GDP-L-fucose; Metabolic engineering; Modular optimization; NADPH cofactor; α1,2(1,3)-Fucosyltransferase