Systematic Metabolic Engineering Of Kosakonia Sp. For Production Of 2’-fucosyllactose

2’-fucosyllatose(2’-FL)is a rare fuco-oligosaccharide and the highest content of human milk oligosaccharide in breast milk.It has been approved by the European Union and FDA as a new type of infant milk powder additive.2’-FL has a variety of important physiological activities such as inhibiting inflammation,promoting the development of the immune system,and regulating intestinal microbes,and has broad application prospects in the fields of food and medicine.Microbial method is one of the main methods for producing 2’-FL.At present,2’-FL biosynthesis has been achieved in a variety of model chassis(including Escherichia coli,Saccharomyces cerevisiae,etc.).However,the current construction of synthetic pathways mostly adopts the strategy of inducing expression of multiple foreign genes,which usually leads to problems such as poor genetic stability and heavy metabolic burden of recombinant bacteria.This article takes the Fuco Pol high-producing strain Kosakonia sp.CCTCC M2018092 isolated in the early stage of the laboratory as the research object.The biosynthesis of fucopolysaccharide(Fuco Pol)is strengthened through the engineering transformation of by-product metabolism and the design of reactor stirring system.At the same time,it takes advantage of its natural fucosyl donor to design and construct a non-model chassis for 2’-FL biosynthesis.The main research results of this paper are as follows:1.Gene knockout of the by-product pathway of fucopolysaccharide(Fuco Pol)synthesis and analysis of production increase mechanism:Genomic analysis was performed on the metabolic pathways of Kosakonia sp.strain synthesizing Fuco Pol,the by-product glucan synthesis key gene glucosyltransferase gene(mdo H)was mined,and the mdo H gene was knocked out using the optimized CRISPR-Cas9 genome editing method.The production of Fuco Pol in shake flask fermentation reached 5.95±0.31 g/L,an increase of 31.5% compared to the original strain.Transcriptome analysis found that mdo H gene knockout mainly affects the differential gene expression of membrane transport,energy metabolism,amino acid metabolism and carbohydrate metabolism pathways,and the key genes of glycolysis and TCA cycle(such as pdh,aco,ogdh,etc.)are down-regulated to 0.2-0.5 times,the key genes for GDP-fucose and Fuco Pol synthesis(such as man B,man C,wca J,etc.)were up-regulated to 1.2-1.8 times.RT-q PCR further verified the expression of key genes(man B,man C,wca J,wca E)Up-regulation trend(p<0.001).The results of the study showed that knock-out of the byproduct pathway gene mdo H effectively regulates carbon flux and promotes Fuco Pol biosynthesis.2.Fuco Pol rheological properties and optimized design of reactor stirring system:Hydrodynamic measurement found that Fuco Pol fermentation broth has shear thinning characteristics and belongs to pseudoplastic fluid.The results of viscoelasticity test indicate that the rheological behavior of Fuco Pol fermentation broth belongs to polymer entanglement solution behavior.Aiming at the high-viscosity fermentation characteristics of Fuco Pol,a frame-type combined stirring blade system(SBF)is designed.The computational fluid dynamics(CFD)simulation evaluation SBF system can effectively improve the mixing efficiency of the fluid in the tank and promote the gas-liquid mixing.The 50 L fermenter fermentation verification found that the use of the SBF system significantly increased the oxygen supply capacity in the fermenter,delayed the time when the dissolved oxygen(DO)fell to zero,and the physiological parameter respiratory quotient(RQ)indicated that the aerobic metabolism was maintained in the high viscosity stage.The final Fuco Pol fermentation production reached 22.79±0.18 g/L,an increase of 34.1% compared to the control stirring system.The results show that the design of the SBF stirring system can effectively improve the flow field environment at the reactor level and strengthen the biosynthesis of Fuco Pol.3.Metabolic engineering transformation of non-model chassis synthesis2’-FL:Taking advantage of the natural fucosyl donor of Kosakonia sp.,knocking out the key gene wca J of the Fuco Pol synthesis pathway,fermentation verification results show that Fuco Pol synthesis is blocked,and the α-1,2-fucosyltransferase Gene(fut C)from Helicobacter pylori is expressed heterologously,optimized the soluble expression of heterologous protein,added lactose substrate for fermentation,identification and detection revealed the target product 2’-FL,the initial production was about240.31±11.92 mg/L.Overexpression of the lactose permease gene(lac Y)enhances the lactose transport of the strain,and knocks out the galactosidase gene(lac Z)to reduce the consumption of lactose by the strain itself.The results of shake flask fermentation showed that the production of 2’-FL reached 445.43±52.10 mg/L,an increase of about85.36% compared with the initial strain.Further optimize the fermentation medium formula,using 40 g/L glycerol,8 g/L tryptone,16 g/L yeast extract,2.31 g/L potassium dihydrogen phosphate,and 12.54 g/L dipotassium hydrogen phosphate as the fermentation medium.The 2’-FL shake flask fermentation production reached 1.14±0.13g/L,which was 375.64% higher than the initial strain and reached the product gram level.Conclusion: In this project,by knocking out the mdo H gene,the Kosakonia sp.chassis system was optimized for multi-level processes,and the host was further modified by metabolic engineering,and finally the 2’-FL biosynthesis with Kosakonia sp.non-model chassis was realized.