Characterization Of β-1,4-Galactosyltransferase From Histophilus Somni And Biosynthesis Of Lacto-N-neotetraose Based On Metabolic Engineering

Lacto-N-neotetraose（LNn T）,constituting the essential components of human milk oligosaccharides（HMOs）,plays important roles such as regulating intestinal microflora and increasing human body immunity.It has been approved as Generally Recognized as Safe（GRAS）by Food and Drug Administration and commercially added to the infant formula.LNn T can be synthesized byβ-1,4-Galactosyltransferase（β-1,4-Gal T）.While there have been only few LNn T-producingβ-1,4-Gal T reported at present.Generally,the expression ofβ-1,4-Gal T easily generated inclusion bodies when using E.coli as a host.In this study,a novelβ-1,4-Gal T from Histophilus somni,referred to as Hs-β-1,4-Gal T,was screened.The enzyme was easily overexpressed in E.coli in soluble form.Accordingly,the purified Hs-β-1,4-Gal T was characterized and its application for LNn T biosynthesis was investigated in vitro and in vivo.Additionally,preliminary crystallographic studies were carried out.Detailed information was shown below:（1）The gene encoding Hs-β-1,4-Gal T was cloned and expressed in E.coli BL21（DE3）,and the recombinant Hs-β-1,4-Gal T was purified by nickel-affinity chromatography.The optimal temperature and p H of Hs-β-1,4-Gal T for transglycosylation activity were measured to be 30°C and 6.0,respectively.It was strictly metal-dependent with Mg²⁺as the optimum metal.The melting temperature（T_m）of recombinant enzyme was determined by Nano DSC as 44.49°C.The in vitro enzymatic production of LNn T was carried out using 1.5 m M UDP-Gal and 1.5m M LNT II as substrates,and the final produced LNn T was 0.43 m M with a conversion ratio of 33%.（2）To in vivo biosynthesize LNn T,the metabolically engineered E.coli BL21（DE3）strain,E42-ΔWNL,constructed previously for the biosynthesis of the LNn T precursor,lacto-N-triose II（LNT II）,was used as initial host.The Hs-β-1,4-Gal T-encoding gene was cloned into vector p ACYCDuet-1,generating p ACYC-Hs-gal T.Combining with recombinant plasmids p RSFDuet-glm MUS and p ETDuet-lgt A,six key enzymes of LNn T synthesis pathway Glm S,Glm M,Glm U,Gal T,Lgt A,and Hs-β-1,4-Gal T were overexpressed.And further introduced to the LNT II-producing E42-ΔWNL strain to realize the LNn T synthesis.When glycerol was used as carbon source,lactose and galactose were used as co-substrates,the titer of LNn T reached 0.66 g/L after 96-h shake-flask fermentation.The host strain E43-Δgal SΔgal R with deleted gal R and gal S encoding transcriptional repressors was incubated in shake flasks for 96h and the yield of LNn T reached 0.72 g/L.（3）Purified Hs-β-1,4-Gal T were obtained by Ni²⁺affinity chromatography and gel filtration chromatography.A single and symmetrical peak was eluted by using gel filtration chromatography,suggesting Hs-β-1,4-Gal T exists in the buffer in a homogeneous aggregated state.SDS-PAGE analysis showed that over 98%purity of the recombinant Hs-β-1,4-Gal T was collected,which could be prepared for further crystallization expriments.Initial screening crystals of Hs-β-1,4-Gal T was carried out by sitting drop vapor diffusion method.The crystals were cultured at 18°C for 2 weeks,and crystallization conditions of Hs-β-1,4-Gal T were optimized by hanging drop method.The X-ray crystallographic structure of Hs-β-1,4-Gal T was collected at around 5（?）resolution.