Metabolic Engineering Of Bacillus Subtilis For Efficient N-acetylneuraminic Acid Biosynthesis

N-Acetylneuraminic acid(NeuAc)is an important functional sugar.It is widely used as a nutritional health care product and medicine to promote infant brain development and maintain elderly brain health.NeuAc is approved as a new food additive ingredient by the US,EU and China.Currently,the methods of NeuAc production are mainly extraction and whole cell catalysis.However,the extraction method has the problems of low content of NeuAc in the natural raw materials.The whole cell catalytic method has the dilemma that the substrate is expensive and not easy to be industrialized,which limits the market supply of NeuAc.Therefore,it is an efficient and sustainable NeuAc synthesis strategy to synthesize NeuAc de novo from cheap carbon sources such as glucose through microbial fermentation of food safety hosts.Currently,there are several key difficulties in the de novo synthesis of NeuAc.First,the synthesis of NeuAc by non-safe host has potential safety hazards.There are thermodynamic bottlenecks and key speed-limiting steps limit the synthesis efficiency.Secondly,the NeuAc synthetisis enhanced by a single pathway is easy to cause the metabolic pressure of cells to limit the efficiency of NeuAc synthesis.The metabolic pressure and synthetic efficiency of the three synthetic pathways of NeuAc have not been systematically resolved.Thirdly,insufficient supply of the key precursor of phosphoenolpyruvate(PEP)for NeuAc synthesis limits the efficiency of NeuAc synthesis.In order to solve the above three problems,first,we successfully constructed the NeuAc synthesis pathway in a generally recognized as safe(GRAS)host.The thermodynamic bottleneck is further resolved by measuring the concentration of key substances,combined with the strategy of self-assembly of enzyme proteins to construct substrate channels and the replacement of the thermodynamic bottleneck-free approach,to remove the thermodynamic bottleneck.At the same time,through the optimization of different promoters to verify the relationship between the expression level of key enzymes in the NeuAc synthesis pathway and the catalytic efficiency,identify the rate-limiting steps of the synthesis pathway,and further releve the rate-limiting steps of NeuAc synthesis through enzyme-directed evolution and strategies for screening highly active enzymes from different sources.Secondly,by analyzing the theoretical yield,synthesis efficiency and metabolic pressure of the three NeuAc synthesis pathways,a multi-pathway synthesis strategy was designed to relieve the metabolic pressure of cells and improve the efficiency of NeuAc synthesis.Finally,by analyzing,constructing and optimizing the dual-carbon source co-utilization system of glucose and glycerole,the insufficient supply of PEP was relieved,and the efficiency of NeuAc synthesis was improved.The main research contents of this paper are as follows:(1)On the basis of N-acetylglucosamine(GlcNAc)high-yield engineering bacteria BSGN6,further introduce glucosamine 6-phosphate acetyltransferase(GNA1),N-acetylglucosamine 2-isomerase(AGE)and N-Acetylneuraminic synthase(NeuB),which uses glucose as a substrate,GlcNAc and PEP as precursors to synthesize NeuAc,successfully obtained a NeuAc yield of 0.33 g/L.Secondly,the two supply modules of GlcNAc and PEP were strengthened and balanced by modular engineering,NeuAc titer increased to 1.65 g/L.Thirdly,a system for the common utilization of glucose and malate double carbon source was designed to block the glycolysis pathway while introducing the Entner-Doudoroff pathway,and optimizing the expression of malic enzyme(YtsJ)to balance NeuAc biosynthesis and cell growth,NeuAc titer increased to 2.18 g/L.(2)The intracellular key metabolite concentration was measured to calculate the thermodynamic properties of key enzymes in the NeuAc synthesis pathway,and it was found that there was a thermodynamic bottleneck in the reversible reaction catalyzed by AGE.Through the optimization of the expression levels of key enzymes in the NeuAc synthesis pathway and the analysis of intermediate product yield models,it was found that the NeuB enzyme derived from E.coli has a kinetic bottleneck.To address the thermodynamic bottleneck problem,the thermodynamic limitations were alleviated by replacing the original AGE pathway with the NeuC pathway without thermodynamic bottlenecks,and the NeuAc titer was increased from 2.18 g/L to 3.36 g/L.For the kinetic bottleneck of the NeuB enzyme,the high-throughput screening strategy based on the NeuAc biosensor and flow cytometry was used to realize the directed evolution of the NeuB enzyme,and screened different sources of NeuB enzyme with better kinetic performance.As a result of that the kinetic bottleneck of the NeuAc synthesis pathway was alleviated by replacing the high catalytic activity NeuB enzyme from Neisseria meningitidis,which increased the NeuAc production from 2.18 g/L to 4.04 g/L.Furthermore,when the NeuC pathway with thermodynamic advantages and the Nem NeuB enzyme with kinetic advantages were integrated,the NeuAc titer reached 5.67 g/L.(3)When the expression of NeuC pathway is further enhanced,the cell growth and sugar consumption rate are significantly reduced,thus limiting the possibility of continuing to enhance the NeuC pathway to obtain high-yield NeuAc strains.Therefore,a strategy based on the three NEu Ac synthesis pathways(the AGE pathway,NeuC pathway and NanE pathway)with different strengths was designed to further improve the efficiency of NeuAc synthesis.First,the stoichiometric data of the three NeuAc synthesis pathways were analyzed.The highest theoretical yield of the NeuAc synthesis pathway reached 0.75 g NeuAc/g glucose under the consideration of glucose consumption for growth,and there was no cofactor imbalance problem.Further different expression levels of the key enzymes of the NeuAc synthesis pathway GNA1,AGE,NeuC,NanE and Nem NeuB were obtained by controlled under different strength promoters.It was found that the overexpression of the three enzyme proteins GNA1,AGE and NanE had little effect on cell growth,but the overexpression of NeuC and Nem NeuB enzyme proteins seriously limited cell growth.On this basis,two synthetic levels of high and low in each synthesis pathway were designed for modular optimization,and finally the low-level AGE and NeuC pathways,and the high-level NanE pathway were assembled,and the highest NeuAc output reached 7.87 g/L,38.8%higher than the single NeuC pathway.(4)The insufficient supply of PEP severely limits the efficiency of NeuAc synthesis.In order to solve this problem,we tried to build an efficient supply system of PEP in Bacillus subtilis through a multi-carbon source co-utilization strategy based on glucose,malate,gluconate and glycerol.First,by optimizing the expression level of phosphoenolpyruvate kinase Pck A and the amount of malic acid added to build a dual-carbon source co-utilization system of glucose and malate,the NeuAc titer was significantly increased to 12.05 g/L.However,there are problems that the high cost of malate and the extremely rapid consumption of malate was not conducive to industrialization.Second,the glucose and gluconate co-utilization system was constructed by replacing the original promoter with the P₄₃ promoter,and the NeuAc titer reached 11.46 g/L.However,there is a problem that the low carbon source yield leads to high industrialization costs.Therefore,a NeuAc efficient synthesis system based on a double carbon source of glucose and glycerol was designed and constructed.First,the Glp K from different sources was optimized through different strength promoters.The Glp K-eco enzyme from E.coli was more conducive to NeuAc synthesis.The titer of NeuAc reached 11.93 g/L,increased by 51.6%.On this basis,by constructing and optimizing the down regulation system of key metabolic genes aroA,zwf,galE,and murB based on gluconic acid bisoensor and CRISPRi,NeuAc production reached 14.99 g/L,which was 25.6%higher than that of the control strain.And the highest yield of NeuAc reached 22.14 g/L in the 3L fermentor.