Efficient Biosynthesis Of Lacto-N-Neotetraose And Lacto-N-Tetraose Based On Metabolic Engineering

Lacto-N-neotetraose（LNnT） and lacto-N-tetraose（LNT）are isomers that consist of D-galactose,D-glucose,and N-acetylglucosamine.They constitute the essential natural human milk oligosaccharides（HMOs）components,each accounting for 2%-7%（w/w）of total HMOs.LNnT and LNT also serve as core structures of HMOs and can be modified to a series of complex fucosylated and/or sialylated HMOs by transferring L-fucose residue atα1,2-,α1,3-,andα1,3/4-linkage and/or sialic acid residue atα2,3-andα2,6-linkage.Their fucosylated and/or sialylated derivatives account for more than 30%（w/w）of total HMOs.They have important physiological functions such as prebiotic effect,immune modulators,antiviral antimicrobials,and intestinal cell responses regulator.LNnT and LNT have been approved as Generally Recognized as Safe（GRAS）by the Food and Drug Administration（FDA）and used as a food ingredient in infant formula.Among the six GRAS notices currently approved by FDA,one is related to the chemically synthesized LNnT,and the other five are approved to synthesize LNnT and LNT by microbial fermentation using Escherichia coli as the host.Because of the advatanges of environmental friendliness,lower cost,and production efficiency,microbial synthesis has gradually become the mainstream method for the production of LNnT and LNT.The use ofβ-1,4-galactosyltransferase（β-1,4-GalT）andβ-1,3-galactosyltransferase（β-1,3-GalT）for LNnT and LNT synthesis from common precursor lacto-N-triose II（LNTⅡ）and the construction of efficient microbial cell factory might be two important factors limiting the efficient synthesis of LNnT and LNT.In this study,E.coli was used as the host strain,and a microbial cell factory was constructed to efficiently produce LNTâ…¡ using genetic engineering tools and metabolic engineering strategies.Then,novelβ-1,4-GalT andβ-1,3-GalT screened by bioinformatics tools were introduced to the engineered E.coli to in vivo synthesize LNnT and LNT,respectively.Detailed information was shown below:（1）The gene lgtA from Neisseria meningitidis encodingβ-1,3-N-acetylglucosaminyltransferase was introduced to E.coli BL21（DE3）to realize the LNTâ…¡ synthesis.After carbon source optimization,the concentration of LNTâ…¡ was 0.53 g/L in shake-flask fermentation.Subsequently,three genes,glm S encoding glucosamine-6-phosphate synthase,glm M encoding glucosamine synthase,and glm U encoding UDP-N-acetylglucosamine pyrophosphorylase,responsible for precursor uridine diphosphate-N-acetylglucosamine（UDP-Glc NAc）supply,were introduced to the engineered strain.It was indicated that these three genes had a combinatorial effect on LNTâ…¡ biosynthesis.The expression strength of glm M,glm S,glm U,and lgt A were optimized by different plasmid copy number combinations and fine-tuned by adjusting the translation rates of these key genes.Based on Western blotting analyses,the appropriate vector and translation regulation could be more conducive to the soluble expression of lgt A and be more effective to synthesize LNTâ…¡.Then,Glm S was mutated to relieve the product feedback inhibition,and the competitive pathway genes,wec B encoding uridine diphosphate-N-acetylglucosamine 2-epimerase,nag B encoding glucosamine-6-phosphate deaminase,and lac Z encodingβ-galactosidase,were deleted to block the futile pathways.The final recombinant strain E42-ΔWNL produced 5.42 g/L LNTâ…¡ by shake-flask fermentation.After the optimization of shake-flask fermentation conditions,LNTâ…¡ titer in 5-L fed-batch cultivation was increased to 46.2 g/L.（2）We successfully screened a novelβ-1,4-GalT originated from Aggregatibacter actinomycetemcomitans NUM4039（Aa-β-1,4-GalT）using N.meningitidisβ-1,4-galactosyltransferase（Lgt B）and Histophilus somniβ-1,4-galactosyltransferase（Hs-β-1,4-GalT）as amino acid sequence templates.The gene encoding Aa-β-1,4-GalT was cloned and expressed in E.coli BL21（DE3）,and the recombinant Aa-β-1,4-GalT was purified by nickel-affinity chromatography.The optimal temperature and p H of Aa-β-1,4-GalT for transglycosylation activity were measured to be 30°C and 8.0.The recombinant enzyme had a relative weak thermostability,and the half-life（t1/2）at 30,35,and 40°C were determined as 8.77,5.10,and0.87 h,respectively.It was strictly metal-dependent with Mg²⁺as the optimum metal.The Km,Vmax,kcat,and kcat/Km toward LNTâ…¡ were determined to be 6.0 m M,10.87μM min^-1,0.16min^-1,and 0.03 m M^-1min^-1,respectively,and those for uridine diphosphate galactose（UDP-Gal）were 0.38 m M,2.67μM min^-1,0.04 min^-1,and 0.10 m M^-1min^-1,respectively.Under the optimum conditions,the total,transgalactosylation,and hydrolysis activity were 10.3,3.7,and6.6 U/mg,respectively.Subsequently,the concentrations of substrates LNTâ…¡ and UDP-Gal and the amount of the enzyme were optimized for LNnT synthesis.The in vitro enzymatic reaction reached equilibrium after 5 h when LNTâ…¡,UDP-Gal,and the enzyme content were20 m M,60 m M,and 9 U/m L,respectively.The final produced LNnT was 13 m M with a 65%conversion ratio.（3）The Aa-β-1,4-GalT-encoding gene and gal E encoding uridine diphosphate-glucose 4-epimerase were co-introduced to the LNTâ…¡-producing E42-ΔWNL strain to realize the LNnT synthesis.Using shake-flask fermentation,the titers of LNTâ…¡ and LNnT reached 4.59 and 0.18g/L,respectively.When the strain EWNL with weakened supply of precursor UDP-Glc NAc was used as the host by introducing key genes lgt A,Aa-β-1,4-GalT,and gal E,generating the recombinant strain EA02,the LNnT titer was improved to 0.42 g/L and LNTâ…¡ titer was reduced to 3.11 g/L.The expression strength of lgt A,Aa-β-1,4-GalT,and gal E were optimized by different plasmid copy number combinations.The final strain EA19 produced 1.91 g/L LNTâ…¡ and 0.91 g/L LNnT.Fed-batch cultivation was carried out in a 3-L bioreactor.The LNnT and LNTâ…¡ titers reached to 12.1 and 9.36 g/L,respectively.（4）In order to verify the ability of recombinant strain E42-ΔWNL to biosynthesize LNT,genes encoding theβ-1,3-GalT previously reported from E.coli O55:H7（Wbg O）or Chromobacterium violaceum（Cvβ3GalT）along with gal E were constructed on vector p CDFDuet-1 and introduced into E42-ΔWNL,yielding the recombinant strains EL01 and EL03,respectively.After shaking-flask fermentation for 96 h,the LNT titers were 2.81 and 0.77 g/L,respectively.We successfully screened a novel LNT-producingβ-1,3-galactosyltransferase originated from Pseudogulbenkiania ferrooxidans（Pf-β-1,4-GalT）using Wbg O and Cvβ3GalT as amino acid sequence templates.The genes Pf-β-1,3-GalT and gal E were co-constructed in p CDFDuet-1,and introduced to E42-ΔWNL,generating EL02.Using shaking-flask fermentation,the titers of LNTâ…¡ and LNT were 0.37 and 3.04 g/L,respectively.LNT titer in3-L fed-batch cultivation reached to 25.49 g/L,and there was a small amount of LNTⅡ（3.44g/L）remained in the broth.