Production Of Lacto-N-triose Ⅱ By Metabolically Engineered Escherichia Coli

Human milk oligosaccharides（HMOs）are important nutrients with a variety of physiological activities in breast milk,which can promote the growth and development of infants and improve their immunity.Among them,Lacto-N-triose（LNTⅡ）is the skeleton precursor of two types of tetrasaccharides in HMOs,type 1 structure Lacto-N-tetraose（LNT）and type 2 structure Lacto-N-neotetraose（LNn T）.Therefore,how to economically and efficiently produce LNT on an industrial scale is becoming a current research hotspot.Microbial cell factories,which combine various synthetic biology and metabolic engineering methods to eco-friendly and sustainably produce high-value chemicals with cheap and renewable biomass materials,is currently an effective strategy for large-scale production of LNTⅡ.In this study,N-acetylglucosamine（Glc NAc）from chitin was used as carbon source and donor substrate,while lactose was used as receptor substrate.A new method for the production of LNT II by microbial metabolism and fermentation from renewable resources was developed,which provided theoretical support for further research.The main contents of this paper are as follows:（1）Firstly,the LNT II synthesis pathway with Glc NAc as carbon source and substrate was constructed in Escherichia coli BL21（DE3）.The effect of different overexpression strategies of target pathway enzymes on LNT II product was explored.The experimental results showed that EHD01 without enzyme Lgt A displayed no accumulation of LNT II,while EHD02（only overexpressing Lgt A）reached 0.12 g/L extracellular LNT II.Based on EHD02,strain EHD03（overexpressing Glm U and Lgt A）,EHD04（overexpressing Glm M,Glm U,and Lgt A）,and EHD05（overexpressing Glm M,Glm U,Nag A,and Lgt A）were constructed,with titers of 0.24,0.60 and 2.44 g/L,respectively.The results of shake flask fermentation showed that the production of LNT II was significantly increased by strengthening the expression of these enzymes.（2）The genome of host strain was modified by CRISPR-Cas9 gene-editing technology.Gene lac Z（responsible for the degradation of lactose）and nan E（metabolic bypass gene）were knocked out successively,giving strain E.coli BL21（DE3）Δlac Z and E.coli BL21（DE3）Δlac ZΔnan E.EHD07,EHD08,and EHD09 were obtained by strengthening the expression of four pathway enzymes in aforementioned engineered strains.Shake flask fermentation was conducted to verify the effect of genome modification.The results showed that compared with EHD07 and EHD08,the LNT II titer of EHD09was increased by 1.05-fold and 0.58-fold,respectively,which indicated that the deletion of both lac Z and nan E was more favorable for the production of LNT II.（3）Using Gibson Assembly DNA molecular cloning method,genes encoding pathway enzymes for LNT II biosynthesis were constructed on plasmids with different copy number and were further introduced into E.coli BL21（DE3）Δlac ZΔnan E,resulting in 24 strains EHD09-EHD32.Finally,EHD29（harboring plasmid p RSFDuet-nag A-glm M,p ETDuet-glm U,and p ETDuet-lgt A（Cm^R））achieved the optimal LNT II titer,reaching 3.70 g/L,with a yield of0.618 mol LNT II/mol lactose.（4）The fermentation conditions of strain EHD29 were optimized.Different concentrations of inducer（isopropylβ-D-1-Thiogalactopyranoside,IPTG）（0.5 m M,1.0 m M,2 m M,and 3m M）and various induction temperatures（20°C,25°C,and 28°C）were carried out orthogonally.Finally,the highest titer of LNT II reached 6.94 g/L,which was 89%higher than that obtained under initial fermentation condition.Fed-batch cultivation was also conducted in a 3 L fermentor.After 76 h of fermentation,LNT II titer reached 15.8 g/L,and the maximum OD₆₀₀was 67.8.