Combinatorial Regulation Of L-serine Synthesis In Corynebacterium Glutamicum

L-serine is an extremely important amino acid API,which can be used to improve neurological function and treat brain injury.The microbial fermentation of L-serine has become a research hotspot due to its environmental.However,the microbial fermentation of L-serine has not yet been industrialized.Corynebacterium glutamicum is approved as a safe strain for producing food and drug by FDA.The recombinant strain A36 with higher L-serine titer was constructed in our laboratory previously.However,slow cell growth and L-serine yield of the strain A36 remained a problem.This study used A36 as the parental strain,through enhanced transcription level of three key enzymes in L-serine synthesis pathway and coupled heterologous L-serine synthesis enzyme of Escherichia coli.Firstly,the promoters of three key enzymes,including PGDH（encoded by ser A）,PSAT（encoded by ser C）and PSP（encoded by ser B）were replaced by a strong promoter dap-e11 to enhance the L-serine synthesis.Subsequently,by coupling the heterologous key enzymes of serine synthesis pathway to make the reaction easier to proceed.Finally,the high-yielding L-serine strains were obtained,and the fermentation medium was optimized to further improve L-serine production.The main findings of this study were as follows:（1）Combinatorial regulation of L-serine synthesis pathway key enzymes:the original promoters’strength of serine synthesis pathway key enzymes in strain A36（PGDH encoded by ser A,PSAT encoded by ser C and PSP encoded by ser B）was evaluated by probe plasmid.The results suggested that the original promoter’s strength of three key enzymes were relatively lower and had a large regulatory interval.The original promoters of ser A,ser B and ser C were replaced by strong promoter dap-e11 respectively,and resulted in strains A36-A,A36-C and A36-B with L-serine titer as 35.83 g/L,33.29 g/L and 34.09 g/L,increased by17.21%,8.90%and 11.51%compared with the parent strain A36 respectively.The recombinant strains A36-AC,A36-AB,A36-BC and A36-ACB were constructed by combinatorial replacing the promoters of the three key enzymes as the strong promoter dap-e11.The L-serine titer of A36-AC,A36-AB,A36-BC and A36-ACB was 45.15 g/L,43.89 g/L,38.22 g/L and 45.83 g/L,increased by 47.69%,43.57%,25.02%and 49.92%compared with the strain A36 respectively.While the maximum biomass OD₅₆₂ of A36-AC,A36-AB,A36-BC and A36-ACB was 44.76,48.05,48.34 and 46.56,decreased by 18.77%,12.79%,12.27%and 15.50%compared with the strain A36 respectively.The results indicated that the promoter of PGDH replaced as dap-e11 had a significant effect to L-serine titer.（2）Over-pressing heterologous PGDH derived from E.coli:according the previous studies,to reduce the thermodynamic free energy of L-serine synthesis and make the reaction easier to perform,heterologous PGDH was overexpressed in strain A36.The recombinant strain A36-PGDH was constructed by overexpressing the heterologous PGDH in E.coli by using the C.glutamicum-E.coli shuttle plasmid p DXW-10（p D）.The results showed that the L-serine titer of A36-PGDH was 29.65 g/L at 120 h,there was no significant change compared with the strain A36.The growth rate of strain A36-PGDH was significantly higher than the strain A36 before 60 h in fermentation.The maximum biomass OD₅₆₂ of the strain A36-PGDH reached 63.08,increased by 14.48%compared with the strain A36.The results of PGDH enzyme activity assay showed that the specific enzyme activity of the strain A36-PGDH（3.42 U/g）was 41.32%higher than the strain A36（2.42 U/g）,indicated that the heterologous expression of PGDH increased its enzyme activity effectively.（3）Combinatorial enhancement of L-serine synthesis pathway:the recombinant strains A36-ACP,A36-ABP,A36-BCP and A36-ACBP were successfully constructed by further combining the above strategies,including replacing the promoters of key enzymes and heterologous expressing PGDH in C.glutamicum.The maximum biomass OD₅₆₂ of the strain A36-ACP,A36-ABP,A36-BCP and A36-ACBP was 62.73,51.71,57.74 and 57.37respectively.The maximum biomass OD₅₆₂ of the strain A36-ACP increased by 13.85%compared with strain the strain A36.The L-serine titer of the strain A36-ACP,A36-ABP,A36-BCP and A36-ACBP was 38.24 g/L,37.15 g/L,39.23 g/L and 43.64 g/L.The L-serine titer of A36-ACBP increased by 42.76%compared with the strain A36.The maximum biomass OD₅₆₂ of the strain A36-ACBP increased by 23.23%compared with the strain A36-ACB without overexpressing the heterologous PGDH.（4）Fermentation optimisation of recombinant strains:the recombinant strain A36-ACBP was selected for fermentation optimization.The optimized medium was obtained by orthogonal optimization of sucrose,（NH₄）₂SO₄,biotin and 2-KG concentration.The maximum biomass OD₅₆₂ and the L-serine titer of A36-ACBP in the optimized medium were64.20 and 53.13 g/L,increased by 16.52%and 73.80%compared with the parent strain A36respectively.A fed-batch fermentation of A36-ACBP was conducted in a 5 L fermenter and the L-serine titer reached 62.23 g/L,which was the highest L-serine titer by fermentation in C.glutamicum reported so far.This study lay the foundation for the industrial microbial fermentation production of L-serine.