Metabolic Engineering To Synthesize Lacto-N-neotetraose In Bacillus Subtilis

Lacto-N-neotetraose(LNnT)is one of the important ingredients of human milk oligosaccharides,which has the biological functions of enhancing immunity,regulating intestinal flora,and promoting cell maturation.LNnT has been approved by the European Food Safety Authority(EFSA),the European Union(EU)and the Food and Drug Administration of USA as a nutritional fortifier to be added to infant formulas.Currently,the commercial production of LNnT is mainly through chemical synthesis and biosynthesis,the later method only utilizes cheap carbon sources and intracellular renewable donors as raw materials and obtains high economic output with a lower environmental damage cost.Therefore,the biosynthesis has a broader application prospect.LNnT(Galβ1-4GlcNAcβ1-3Galβ1-4Glc)is a linear tetrasaccharide consisting of D-galactose,N-acetylglucosamine,D-galactose and D-glucose,so its synthetic pathway is relatively complicated.At present,there are still some shortcomings in biosynthesis method that limit the efficient synthesis of products,such as insufficient supply of intracellular precursors;imbalanced metabolic flux between synthetic pathways and competitive pathways;excessive metabolic pressure affecting the genetic stability of strains.In this study,Bacillus subtilis was used as the starting strain and the LNnT synthesis pathway was constructed by expressing of heterologous genes;the precursor synthesis pathways were optimized through modular engineering to enhance and balance the supply of intracellular precursors;the expression level of key genes in the competitive pathway were down-regulated by constructing the CRISPRi system,resulting in the balance of metabolic flux between product synthesis and cell growth and the increase of synthesis efficiency of LNnT;finally,through the promoter engineering and the mutation of the Spo0 A promoter,we obtaind a spore-free and plasmid-free LNnT-producing engineering strain.The results of this study are summarized as follows:(1)By co-expressing the β-galactoside permease(LacY)from Escherichia coli K12 and the β-1,3-N-acetylglucosamine aminotransferase(LgtA)and β-1,4-galactosyltransferase(LgtB)from Neisseria meningitidis MC58,the synthesis of LNnT in B.subtilis was achieved with lactose and intracellular uridine diphosphate acetylglucosamine(UDP-GlcNAc)and uridine diphosphate galactose(UDP-Gal)as precursors.The titer of LNnT was 0.61 g/L.Then,by optimizing the expression levels of key enzymes LgtA and LgtB,the LNnT titer was effectively increased to 1.31 g/L.(2)Modular engineering strategy was applied to improve and balance the supply of key precursors of UDP-GlcNAc and UDP-Gal.First,the positive-acting enzymes in the UDP-GlcNAc and UDP-Gal modules were identified by overexpressing related genes or knocking out genes in the branching pathways.Then,the genetic modifications that facilitated the synthesis of LNnT in each module were assembled,resulting in four different levels of each precursor supply.On this basis,the different intensities of the two modules are combined to select the best combination.The optimal combination was that the supply of UDP-GlcNAc and UDP-Gal modules were both at medium-plus level.The titer of LNnT was improved to 1.95 g/L in the shake flask culture.Finally,in continous feeding fermentation,the titer of LNnT was 4.52 g/L in the 3-L fermentor.(3)The expression of key genes in the competitive pathways were down-regulated to an appropriate level by the xylose-induced CRISPRi system,resulting in the weaken of the metabolic flux in the competitive pathways and the balance of metabolic flux distribution between intracellular precursors module and the competitive module(including the glycolysis pathway(EMP),the pentose phosphate pathway(HMP)and the teichoic acid synthesis pathway).The synthesis efficiency of LNnT was effectively improved.The key genes of the competition module are the pfkA,pyk genes in the glycolysis pathway,the zwf gene in the pentose phosphate pathway,and the mnaA gene in the teichoic acid synthesis pathway.Eight sgRNAs with different inhibition efficiencies were designed for each key gene in the competitive pathways.Then,the multiplexed repression of pfkA,pyk,zwf and mnaA genes effectively woke the metabolic flux in the competitive pathways,thereby improving the synthesis of LNnT.Moreover,the rate-limiting step in the conversion of Lacto-N-triose II(LNTII)to LNnT was released by knocking out the tuaD gene encoding UDP-glucose-dehydrogenase and increaseing the expression level of LgtB via adding one copy number into the genome.The influence of addition time and amount of inducer xylose on LNnT production were also investigated to achieve the optimal flux balance among the LNnT synthesis module and the competing module.Finally,the LNnT titer reached 2.30 g/L in the shake flask and 5.41 g/L in the 3-L fermentor.(4)The spore-free and plasmid-free engineering bacteria was constructed through promoter engineering.First,by screening potential endogenous strong promoters from B.subtilis and high-strength promoters of B.subtilis that have been reported,the first promoter library with strong expression potential was constructed.On this basis,the promoters were truncated to 80-100 bp to obtain the second promoter library with shorter sequences.Furthermore,some strong promoters were selected from the second library for key site saturation mutation and screening with flow cytometry to obtain the third promoter library with the highest intensity.By using a series of promoters with different intensities in the constructed strong promoter library to control the expression of key genes lgtA and lgtB,the optimal expression level was achieved and plasmid-free engineering bacteria with high genetic stability was obtained.Then,the spore was eliminated by mutating the Spo0 A promoter.Finally,the LNnT yield reached 2.51 g/L in the shake flask culture.