食品科学2025，Vol.46Issue(11)：87-93,7.DOI:10.7506/spkx1002-6630-20241222-185
代谢工程改造大肠杆菌通过补救途径合成3-岩藻糖基乳糖
Metabolic Engineering of Escherichia coli to Synthesize 3-Fucosyllactose via the Salvage Pathway
摘要
Abstract
3-Fucosyllactose(3-FL)is a typical human milk oligosaccharides(HMOs)and plays a significant physiological function in the growth and brain development of infants.In this study,efficient biosynthesis of 3-FL was achieved by metabolic engineering of Escherichia coli BL21star(DE3).The α-1,3-fucosyltransferase gene futA from Helicobacter pylori and the L-fucokinase/GDP-L-fucose pyrophosphorylase gene fkp from Bacteroides fragilis were co-introduced into the initial strain,E.coli BL21star(DE3)ΔlacZΔwcaJ,resulting in the production of 1.01 g/L of 3-FL in a shake flask.By using the CRISPR/Cas9 system to delete the genes related to the fucose metabolism pathway,including the L-fucose isomerase gene fucI,the L-fucose kinase gene fucK and the L-rhamnose isomerase gene rhaA,the yield of 3-FL increased to 1.36 g/L.The α-1,3-fucosyltransferase FT1 from H.pylori NCTC11637 was the most effective in 3-FL biosynthesis,and the recombinant strain BLWFA-T expressing FT1 produced 1.56 g/L of 3-FL.Subsequently,the expression level of the fucose metabolism pathway-related genes was optimized,and the recombinant strain BLWFR-T2 expressing fkp through plasmid pETDuet-1 and expressing FT1 through plasmid pRSFDuet-1 produced 2.58 g/L of 3-FL.Finally,the regeneration of the cofactor guanosine triphosphate(GTP)was enhanced,and the recombinant strain BLWFR-T7 overexpressing the guanylate kinase gene gmk and the inosine-guanosine kinase gene gsk produced the highest level of 3-FL(3.01 g/L).After fed-batch fermentation for 78 h,BLWFA-T7 produced the highest yield of 3-FL(27.82 g/L)in a 5 L fermentor.This study provides a research basis for efficient microbial synthesis of 3-FL and other HMOs.关键词
3-岩藻糖基乳糖/大肠杆菌/补救途径/生物合成/代谢工程Key words
3-fucosyllactose/Escherichia coli/salvage pathway/biosynthesis/metabolic engineering分类
生物工程引用本文复制引用
梁山泉,何滋,江正强,杨绍青..代谢工程改造大肠杆菌通过补救途径合成3-岩藻糖基乳糖[J].食品科学,2025,46(11):87-93,7.基金项目
国家自然科学基金重点项目(22338013) （22338013）
"十四五"国家重点研发计划重点专项(2022YFC2104902) （2022YFC2104902）
