The Design And Regulation Of Lacto-N-neotetraose Metabolism Pathway

Lacto-N-neotetraose(LNnT)is one of the high content human milk oligosaccharides(HMOs).It can be added to infant formula as a food additive,which can enhance human immunity,reduce the incidence of disease infection and improve intestinal function,has a good application prospect.Microbial fermentation is one of the commonly used methods for the synthesis of LNnT,but it has some problems such as low substrate conversion and complex metabolic pathway,which is not conducive to the synthesis of LNnT.Therefore,in this study,recombinant Escherichia coli was used to regulate the metabolic pathway and expression of key enzymes in biological synthesis of LNnT,so as to improve the synthesis of LNnT.In this study,E.coli K12 MG1655 was selected as the starting strain.First,the lgtA(encoding ?-1,3-N-acetylglucosamine aminotransferase)and lgtB(encoding ?-1,4-galactosyltransferase)from Nesseria meningitides were expressed by a plasmid result pCDFDuet-lgtAB.The recombinant plasmid was transformed into the original strain,and the initial titer of LNnT was 0.04 g/L by shaking-flask fermentation,indicating that the de novo synthesis pathway of LNnT was constructed successfully.Lactose is the important substrates for the synthesis of LNnT,in order to improve the conversion rate of the substrate lactose,lac Z was knocked out,and lac Y was over-expressed.As a result,the yield of LNnT on lactose increased from 0.01 mol/mol to 0.09 mol/mol,and the titer of LNnT elevated to 0.41 g/L.In addition,Lacto-N-triose II(LNTII)is a key intermediate product for the synthesis of LNnT,and changes in the expression levels of key rate-limiting enzymes in the metabolic pathway will affect the accumulation of LNTII and the titer of LNnT.The key rate-limiting enzymes in the metabolic pathway were identified by CRISPR-Cas9 and CRISPRi,and the expression of genes pgi,glmS,glmM,glmU,man A,mur A,wec B,nag B encoded the key ratelimiting enzymes was combined to obtain a strain with a high accumulation of LNTII.Then LNnT was synthesized by the recombination strain,and the highest titer reached 1.04 g/L.Finally,the strain M29 AB with the highest titer was obtained by regulating the expression of of genes pgm,gal E,ugd encoded key enzymes in the precursor UDP-galactose(UDP-Gal)metabolic pathway.The titer of LNnT reached 1.2 g/L,which was 93% higher than that of the control strain M0-PAB,and the yield of LNnT on lactose reached 0.28 mol/mol.The optimal fermentation conditions were obtained as follows: substrate lactose concentration 5 g/L,fermentation temperature 30?,fermentation time 60 h.