期刊
METABOLIC ENGINEERING
卷 41, 期 -, 页码 23-38出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymben.2017.03.001
关键词
2'(3)-Fucosyllactose; a1,2(1,3)-Fucosyltransferase; GDP-L-fucose; Metabolic engineering; NADPH cofactor; Modular optimization
资金
- National 973 Project of China [2013CB733904, 2012CB721001, 2012CB721101]
- National Natural Science Foundation of China (NSFC) [31530083]
- NSFC [31270133, 31470194, 31371259, 81471904, 31270003, 31400081]
- Tianjin Research Program of Application Foundation and Advanced Technology [15JCQNJC09700]
- Fundamental Research Funds for the Central Universities [65141028]
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 alpha 1,2-fucosyltransferase and alpha 1,3-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 12.43 g/L, respectively. This work provides a platform that enables modular construction, optimization and characterization to facilitate the development of FL-producing cell factories.
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