Directed Evolution Of ?-1,3-fucosyltransferase By A Single-cell Ultra-high-throughput Screening Method

Fucosylation is involved in a variety of biological and pathological processes in eukaryotic organisms including tissue development,angiogenesis,fertilization,cell adhesion,inflammation and tumor metastasis,which are catalyzed by fucosyltransferases(FucTs).Research has shown that FucTs have significant applied interest for the enzymatic and microbial synthesis of fucosyl therapeutic cancer vaccine and functional ingredients.Therefore,the development of high throughput screening method of FucTs and the directed evolution of enzyme activity not only provide excellent properties of mutant enzymes,but also have important significances for understanding the structure-function relationship of FucTs molecules.In order to improve its catalytic activity and provide insights into structure-function relationship of FucTs,a new fluorescence-based cell sorting(FACS)ultra-high-throughput screening methodology for?-1,3-fucosyltransferase(?-1,3-FucT)with fast screening speed(>10~7 times/h)is first developed.Based on the system,a series of key parameters including fluorescence diffusion efficiency,the ratio of donor and receptor,inclubation time and enzyme reaction time are optimized,which improves the operation efficiency of the system.The mode sreeening,which the Escherichia coli expressing?-1,3-fucosyltransferase(?-1,3-FucT)are separated from the cells without the enzyme activity in flow cytometry,indicates that the systems has high enrichment rate(>10~4).We further develop a method of Cast-PCR to get quantitative assessment of high/low activity proved that the screening method for 2 folds higher activity cells can enrich nearly 20 times from the lower activity by just one round FACS sorting,which has a good sorting resolution.Then the mutant S7(S45F/D127N/R128E H131I/Y199N/E340D/V368A)with enzyme activity of the catalytic activity of natural substrate-LacNAc and non natural substrate Lactose increased by 5 times and 8.5 times respectively is obtained by 3 rounds of evolution for random mutagenesis and semi-rational library.The mutation site were put into the crystal structure of?-1,3-FucT and found that most of the mutations are near by the pocket of receptor substrate,which provides insight to reveal the substrate combination and the catalytic mechanism.At last,we designed a fluorescent receptor for?-1,2-fucosyltransferase(?-1,2-FucT),which not only expand the application of the technology but also lay the foundation for the molecular directed evolution of the other glycosyltransferase.