Production Of 3-fucosyllactose By Metabolic Engineering Transformation Of Escherichia Coli

3-Fucosyllactose(3-FL)is one of the important components in breast milk oligosaccharides.Fucosylated HMOs accounts for the highest proportion of HMOs species,while 3-FL is the most important component of fucosylated HMOs.The content accounts for5%.As one of fucosylated HMOs,3-fl is composed of β-Lactose and L-fucose composition,L-fucose linked to position α1,3 of the glucose unit in lactose.3-FL plays an important role in preventing infectious diseases in infants and young children,and as a prebiotic to inhibit the growth of harmful microorganisms,it has received extensive attention due to its application value in infant formula milk powder.At present,the synthesis of 3-FL can be carried out by chemical synthesis,enzymatic synthesis and cell factory synthesis.Chemical synthesis is easy to scale up and industrialize due to its controllability,but the chemical synthesis steps are relatively cumbersome and the yield is low.Toxic reagents will be involved.Enzymatic synthesis is more environmentally friendly,but the precursor substance GDP-L-fucose is difficult to obtain and expensive.The production of 3-fl by cell fermentation has the advantages of green environmental protection,renewable raw materials and low production cost.Wholecell fermentation synthesis of 3-FL requires engineering strains with high fucosylation ability to synthesize 3-FL with high efficiency.The reported α1,3 fucosyltransferases come from a single source,and the initial enzyme activity is relatively low.Therefore,the discovery of novel high-efficiency fucosyltransferases is the key to the production of 3-fucosyllactose by wholecell fermentation.In this study,using BL21(DE3)as the starting strain,the synthetic pathways of GDP-fucose and 3-FL were constructed,and the synthetic pathway of 3-FL was enhanced by knockout of branch genes and overexpression of key enzymes of the metabolic pathway to optimize the 3-FL synthetic pathway.3-FL synthesizes recombinant strains,and uses this strain as a host for screening to discover novel high-efficiency fucosyltransferases,and conduct modular optimization of the new enzymes.After exploring the optimal fermentation conditions of the recombinant strains,batch the fed-feed fermentation experiment further improved the yield of 3-FL.The main research contents are as follows:(1)De novo synthesis pathway construction: the constructed recombinant plasmids pcdfmanc-manb-gmd-wcag and petduet futa(M32)were transferred into BL21(DE3)to overexpress the key enzyme and fucosyltransferase of GDP-L-fucose synthesis pathway.The protein expression was verified by SDS-PAGE and shake flask fermentation.After 72 h of IPTG induction,the yield of 3-FL was 0.24 g/L.This study proved that the de novo anabolic pathway of 3-FL was successfully constructed.(2)Host modification: the competitive pathway related enzymes lac Z and wcaj of 3-FL metabolic pathway were knocked out by crispr-cas9 gene editing technology.The obtained strains were transferred to pcdfmanc man B GMD WCAG and pet futa(M32)for shake flask fermentation experiment.After 72 h of IPTG induction,the yield of 3-fl was 0.43 g/L.It was verified that the knockout of lac Z and wcaj promoted the synthesis of 3-FL.(3)Screening of new enzymes: nine possible enzymes were inferred by blast.The enzyme with α1,3-fucosyltransferase activity was screened for intracellular fermentation by using the3-FL production strain successfully constructed in(2).The results showed that four of them had fucosyltransferase activity,and the 3-FL yield of fut M2 enzyme with the highest yield was 1.98g/L.This study took it as the starting enzyme for subsequent optimization.(4)Optimization of modularization strategy: the recombinant strain was optimized by combined modularization strategy.The key enzymes of GDP-L-fucose synthesis pathway,man B,Manc,GMD,WCAG and fut M2,were divided into two metabolic modules.They were overexpressed with three plasmids with different copy numbers.After verification by shake flask horizontal fermentation,the best module combinations were determined as p CDF-man Cman B-gmd-wca G and p ET-fut M2.(5)Optimization of fermentation conditions: the fermentation conditions of the obtained recombinant strain were optimized.By controlling different induction temperature and final concentration of inducer IPTG,the fermentation of the recombinant strain was verified under the optimal fermentation conditions.Finally,the 3-FL production strain obtained a 3-FL production of 2.54 g/L at the shake flask level,an increase of 28.4%.(6)High density fed batch fermentation: in order to further improve the yield of 3-FL,the high-density fermentation of 3-FL production strain was carried out in the form of fed batch.1l medium was added to the 3 L fermentation tank.After 52 h of fermentation,the final yield of3-FL was 20.3 g/L.