Optimized Biosynthesis Of N-acetylneuraminic Acid Based On Pathway Engineering And Co-culture Strategy

N-acetylneuraminic acid(NeuAc)is a kind of negatively charged functional monosaccharide located at the end of glycoprotein or glycolipid on the cell surface.It is widely present in various plants,animals and microorganisms.NeuAc plays an important role in many life processes,maintaining the structure of biological macromolecules and transmitting signals between cells and molecules.It is widely used in the medical and food industries and has high commercial value.Currently,enzymatic synthesis and whole-cell biocatalysis are the major methods for NeuAc production.However,the both methods require the addition of expensive precursors,pyruvate and N-acetylglucosamine(GlcNAc),which greatly increases production costs.Therefore,microbial fermentation for producing NeuAc is very promising by using a cheap carbon source such as glucose.Previously,a pathway of NeuAc production with glucose as the sole carbon source was constructed in Escherichia coli.The pathway of NeuAc production was optimized by rational transformation and replacement of key enzymes from different biological sources,but there is still a problem of large accumulation of intermediate GlcNAc during fermentation.Analyzing the N-acetylglucosamine-2-epimerase(Age)and N-acetylneuraminic acid synthase(NeuB)which catalyze the synthesis of NeuAc from GlcNAc in the production pathway,it was found that although the expression intensity of the two enzymes is high,the solubility in the cell is low.Most of the enzyme proteins have formed inclusion bodies with no biological activity.We build NeuB structure by homology modeling using SWISS-MODEL.Then,combined with the known three-dimensional structure of Age protein,we analyzed the aggregation areas of Age and NeuB by SolubiS method and discovered a series of mutation targets that are expected to improve protein solubility.The results showed that some mutants such as Age-L268E,Age-I270K,NeuB-S289P,and NeuB-I290D effectively increased the production of NeuAc,and the engineered strain containing NeuB-I290D had the highest yield of NeuAc.Further modification of other aggregation regions or simultaneous mutation of Age and NeuB has a lower effect on the production of NeuAc than that of NeuB-I290D.The fermentation conditions were optimized in the shake flask,including the nitrogen source of the medium,the induction time,the concentration of manganese ion,and the dissolved oxygen.Under the optimized fermentation conditions,the strain with NeuB-I290D was subjected to shake flask fed-batch fermentation.22.25 g/L NeuAc was accumulated,and 15.08 g/L GlcNAc was also accumulated in the fermentation broth.We designed an E.coli A-E.coli B co-culture system to solve the problem of large accumulation of GlcNAc during the fermentation process.In this system,E.coli A uses glucose as a carbon source to synthesize NeuAc,and another strain of E.coli B uses GlcNAc as a substrate to produce NeuAc.It is expected to reduce the accumulation of intermediate GlcNAc and further increase the production of NeuAc.The ratio of different populations is very important for stable co-culture.Once the growth ratio between the populations is not coordinated,it will seriously affect the production performance of the system.At present,there is no method to accurately control the proportion of co-cultured strains by responding to substrate concentration.We control the growth of strain B through a biosensor capable of sensing the intermediate product GIcNAc-6P,and then precisely regulate the population ratio of the co-culture system.We tried to use riboswitch or transcription factor to build a biosensor that senses GlcNAc-6P.As a result,the transcriptional factor biosensor was more suitable for population proportion regulation.The binding box of the transcription factor NagC was inserted into a specific region near the promoter,obtaining a series of activated biosensors that responded to changes in the concentration of GlcNAc-6P.Next,we tried to use growth inhibitory protein such as RMF,CcdB and MazF to control the growth of the strain,respectively.It was confirmed that MazF could effectively control the growth of strains.The GlcNAc-6P biosensor and MazF constitute a growth control system.In this system,the GlcNAc-6P biosensor was used to control the growth of strain B,and then to control the population ratio of different E.coli in the co-culture system.Finally,we added different concentrations of GlcNAc to characterize the role of the growth control system in co-culture.It was found that with the increase of GlcNAc concentration,the proportion of strain B containing growth control system in the co-culture system increased from 31%to 46%and from 40%to 57%.A strategy to accurately control the population ratio of the co-culture system using biosensors is proposed in this paper.The dynamic regulation of population proportion is preliminarily realized by GlcNAc-6P biosensor.It provides a new idea to solve the problem of population proportion regulation in co-culture.