Evolution Of Riboswitch Threshold And Its Application In N-acetylneuraminic Acid Production

N-acetylneuraminic acid(NeuAc)is a common sialic acid that is widely existed in nature and can be used to synthesize other sialic acids.It is mainly present at the end of glycosylated sugar chains and plays an important role in cell biological recognition,which like other sialic acids.In addition,NeuAc can also be used as health food and medicine,which has high commercial value.At present,there are many problems in the production method of NeuAc.Therefore,the production of NeuAc has many advantages by using a cheap carbon source such as glucose,without adding any precursor.Based on the NeuAc production pathway constructed with glucose as the sole carbon source in the laboratory,the rate-limiting enzymes of NeuAc production were found that were N-acetylglucosamine-2-isomerase and neuraminic acid synthase.Two N-acetylglucosamine-2-isomerases and three neuraminic acid synthases with the reported highest enzymatic activity were selected to construct six NeuAc production pathways.Because the optimal temperatures of these enzymes are different,six pathways were constructed to perform fermentation in shake flask at 25℃,30℃,and 37℃,respectively.The plasmid PBac after replacing isoenzyme on PB with Agea and NeuBc was fermented at 30℃ to produce 1.9 g/L NeuAc,which is 1.5 times the yield of NeuAc produced using PB at 37℃.It proves that Age and NeuB were indeed the key enzymes in the NeuAc production pathway.The expression intensity of key enzymes was optimized based on the replacement of key enzymes,and 3.7 g/L of NeuAc was produced after fermentation,which was 1.2 times that before the replacement.The continued increase in expression intensity did not further increase NeuAc production,demonstrating that the restriction of NeuAc production may have shifted to supply of another precursor PEP Then the PEP metabolic pathway of the strain was modified and six key genes were selected for knockout,which further increased the production of NeuAc.Metabolic flux of PEP to pyruvate was reduced in pykA-knockout strain.Because pykA is not the main pyruvate kinase,it will not excessively affect the metabolism of the strain,making NeuAc yield the highest.Other knockout have a strong impact on metabolism.On the basis of deleting pykA,continue to carry out other deletions.Among them,knocking out pykA and ptsG at the same time makes NeuAc reach the highest yield.In order to further improve glucose transport,the glucose-promoting protein glfzm of Z.mobilis was introduced,which caused NeuAc to accumulate rapidly in the early stage,and 3.5 g/L of NeuAc was produced in 24 hours,which was 1.4 times that before the glfzm gene was introduced.However,the introduction of glfzm led to the accumulation of a large amount of pyruvate,which further inhibited the normal metabolism of the strain.We have made various attempts to reduce the accumulation of pyruvate,but there was still a large amount of pyruvate accumulation.The possible cause was severe imbalance in metabolism caused by rigid transformation of strain metabolism.Therefore,directed evolution and high-throughput screening were considerd to obtain strains with increased NeuAc production.Therefore,directed evolution may be a better method.As a flexible regulating element,riboswitch has been widely used in metabolic engineering,but its performance often needs to be improved and optimized.The NeuAc riboswitch biosensor has been constructed earlier in our laboratory and applied to increase NeuAc production in metabolic engineering,but its lower threshold limits theincrease in NeuAc production.There is currently no strategy to increase the riboswitch threshold.In this study,a method for the evolution of riboswitches was established.TetA is a bifunctional protein that can transport Ni2+ into cells and tetracycline out of cells.The NeuAc riboswitch was linked to the 5’ end of gene of TetA so that the expression level of the TetA protein responded to the concentration of NeuAc.The optimal screening concentration was determined by adding different concentrations of Ni2+ and tetracycline.The screening conditions were further validated in shake flasks.To verify the screening effect,a model library was established.By mixing riboswitches with two negative.controls which had no riboswitches,and using determined screening conditions,the strains containing riboswitches were enriched to more than 90%.In this way,we established for the first time a method that can effectively evolve the riboswitch threshold.Next,the established riboswitch evolution method in vivo was used to evolve the NeuAc riboswitch,and an improved riboswitch with high threshold,high sensitivity,and large dynamic range obtaind.The structural simulations of the original riboswitches and mutants revealed that only minor change was produced in the structure between the original riboswitches and mutants.Molecular dynamics simulation was performed to calculate the bind free energy of the riboswitch mutant for NeuAc.The results prove that the mutation of the riboswitch will not reduce the ligand binding capacity of the riboswitch,and the increase in the riboswitch threshold is caused by a decrease in the proportion of riboswitches that can form an effective cleavage conformation.Flow cytometry analysis proves that the riboswitch after mutation still responds in the same mode without affecting the application of riboswitch in metabolic engineering.The production of NeuAc can be increase by optimizing the expression of the enzyme in NeuAc pathway,using the obtained riboswitch,which proved that the enhanced expression of the pathway was beneficial to the increase of NeuAc production.Further evolution of the key enzyme Agea mutations further increased NeuAc production.After analysis,we propose that Agea mutations reduce the bind ability between protein and ManNAc and make the reaction proceed towards ManNAc synthesisIn summary,NeuAc production was improved through rational improvement in this paper.After designing and verifying,the method of riboswitch in vivo evolution was established for the first time.This method has the advantages of simple operation,short test period and high screening efficiency.In the field of sensor threshold optimization,this method is the first of its kind,and also provides ideas for the evolution of other biosensors.It is of great significance in increasing the range of biosensor applications.