Synthesis Of GDP-Fucose With Combinatorial Enzymes

GDP-fucose,namely guanosine 5?-diphosphate-L-fucose,as a glycosyl donor and metabolic intermediate was widely found in various types of organisms.It is mainly involved in the biosynthesis of oligosaccharides in prokaryotes and used as an important glycosyl donor for protein glycosylation and as a precursor for the synthesis of other sugar nucleotidesin eukaryotes.GDP-fucose is an important intermediate for the synthesis of human milk oligosaccharides.It plays an important role in the synthesis of functional oligosaccharides and has broad application prospects in industrial production.Therefore,the synthesis of GDP-fucose has important theoretical significance and practical applicationvalue.In this study,the enzymes related to synthesis of GDP-fucose were cloned from Escherichia coli BL21(DE3)and Escherichia coli K12 and expressed in Escherichia coli BL21(DE3).The main research results are as follows:1.The genes related to synthesis of GDP-fucose named manB,manC,gmd and fcl were cloned from Escherichia coli BL21(DE3)and Escherichia K12.The genes were ligated with pGEX-4T-1,pGEX-4T-2,pGEX-6P-1 and pGEX-6P-1 to form the recombinant plasmids,and transformed into Escherichia coli BL21(DE3)component cells to get recombinant strains.The recombinant strains were induced at 25~oC with 0.1 mM IPTG for24 h.After that,the cells were harvest and the recombinant enzymes were purified from the supernatant of lysis solution by GST-Sefinose Resin column.manB-pGEX-4T-1 had no enzyme activity,and the remaining three proteins had lower crude protein content and purification efficiency after analysis.The protein content of the crude enzyme solution was 42.13 mg,47.73mg,and 48.95 mg,respectively,and the purification efficiency was 1.0%,1.6%,and 5.17%,respectively.2.TherecombinantengineeredstrainsofmanB-pET-28a-BL21(DE3),manC-pET-28a-BL21(DE3),gmd-pET-28a-BL21(DE3)and fcl-pET-28a-BL21(DE3)were constructed.mpglk-pET-28a was completely synthesized by Suzhou Hongxun Biotechnology Co.,Ltd.The enzymes were induced by IPTG to achieve soluble expression of the protein.The selection from the vector,medium,induction time,and IPTG induction concentration were optimized for The enzymesrelated to synthesis of GDP-fucose.By comparing the TB medium with the LB medium,the TB medium was selected to provide nutrients for the growth of the recombinant strain.By optimizing the inducing concentration and induction time of IPTG,the optimal protein expression conditions were obtained.mpglk was induced 0.02 mM for 48 h,manB was 0.08 mM for 48 h,manC was 0.04 mM for 36 h,gmd was 0 mM for 36 h,and fcl was 0.08 mM for 36 h.The final optimized yields of the five GDP-fucose synthesis related enzymes were 1.58,2.27,3.31,3.80,and 3.79 times than were not optimized.3.The synthesis of mannose-6-phosphate,an intermediate in GDP-fucose synthesis,was optimized to determine the optimal reaction conditions:5 mM mannose,10 mM sodium hexametaphosphate,5 mM MgCl2,0.5 mg/mL mpglk,100?L PBS buffer system,reaction at37 ~oC for 8 h.The yield of mannose-6-phosphate reached 80%.Simplify the third step of the intermediate product GDP-mannose synthesis step,by adding the substrate mannose and mpglk,manB,and manC in the reaction system at the same time,GDP-mannose can be generated;and the final product GDP-fucoseis synthesized by a combined enzymatic method.The substrate mannose and the first three steps of GDP-fucose synthesisrelated enzymes(mpglk,manB,manC)were added to the reaction system for 12 hours.After the reaction was completed,add gmd at 37°C for 2 hours.Fcl were added at 37 ~oC for 2 hours.GDP-fucose can achieve the highest conversion rate of 1.50%.