Construction And Fermentation Optimization Of Cytosine Producing Strain Of Escherichia Coli

Cytosine,scientific name 4-amino-2-hydroxy-pyrimidine,is an important pyrimidine base for the synthesis of DNA and RNA in vivo,which is widely used in the fields of fine chemicals and biomedicine.At present,cytosine is mainly obtained by organic chemical synthesis.This method has some disadvantages such as high cost,low yield and environmental pollution,which is not conducive to the expansion of production.With the development of biology,microbial fermentation has gradually become an ideal method for the study of cytosine production due to its high production efficiency,environmental friendliness,low cost and easy availability of raw materials.In this study,the laboratory uridine engineering bacteria and the cytidine production bacteria provided by the company were used as the starting bacteria to construct the cytosine producing strain by genome editing technology and exogenous plasmid expression.In this study,the uridine engineering strain Escherichia coli U0 was used as the starting strain,and the cytosine synthesis pathway of the strain was reconstructed by CRISPR/CAS9-mediated genome editing technology.First,the cytosine deaminase gene cod A and cytosine osmoase gene cod B were knocked out to block the transformation of cytosine to uracil and reduce cytosine transport into the cell.In the second step,the pyrimidine monophosphate gene ygd H was integrated,and the transcription was controlled by the phage-derived T7promoter to enhance the hydrolysis reaction of CMP to cytosine,and the strain E.coli C3was constructed.Shaking flask fermentation results showed that the yield of cytosine and uridine was 0.86 g/L and 4.82 g/L,which were 3.98%lower than that of starter.The byproduct uracil was 0.97 g/L,37.41%lower than that of the starter,and the biomass(OD₆₀₀)of the strain had no significant change.These results suggest that blocking cytosine degradation and enhancing the metabolic pathway from CMP to cytosine is beneficial to cytosine accumulation.To further improve cytosine production,the key metabolic reactions from uridine acid（UMP）to CMP were enhanced in this study.On the basis of E.coli C3,nucleoside triphosphate pyrophosphate hydrolase gene nud G was integrated to enhance the hydrolysis of cytidyl triphosphate（CTP）to CMP.The mutant uridate kinase gene pyr H^（D93A）was integrated to enhance the transformation of UMP to uridin diphosphate（UDP）.The mutant cytidyl triphosphate synthase（UTP）gene pyr G^（E155K）was integrated to enhance the transformation of UTP to CTP.Fermentation results showed that the cytosine yield of E.coli C6 was 1.34 g/L,which was 55.81%higher than that of E.coli C3.The uridine yield was3.70 g/L,26.29%lower than that of starter.The yield of the byproduct uracil was 0.73 g/L,which was 52.90%lower than that of the starter.These results indicate that enhancing the expression of key enzymes and removing feedback inhibition is beneficial to further accumulation of cytosine.In order to reduce the quantity of uridine and the byproduct uracil,four nucleotide hydrolase genes（ush A,sur E,yjj G and yrf G）were successively knocked out from E.coli C6 strain to construct E.coli C7-C10.The fermentation results showed that cytosine production increased gradually with the deletion of hydrolase gene.The cytosine yield of E.coli C10 was 1.95 g/L,which was 53.54%higher than that of E.coli C6.The uridine yield was 2.89 g/L,which was 25.52%lower than that of E.coli C6 strain.The uracil yield was 0.51 g/L,which was 37.80%lower than that of E.coli C6 strain.These results indicate that hydrolase knockout can weaken the metabolism of UMP to uridine to a certain extent,and thus reduce the production of uracil by product.In this study,Escherichia coli B0 was used as the starting strain,and exogenous recombinant plasmids containing different nucleoside hydrolase genes rih A,rih B and rih C were constructed by homologous recombination method,and the plasmids were introduced into E.coli B0.Compared with E.coli B1 and E.coli B3,E.coli B2 had the highest cytosine yield（1.97 g/L）and the lowest uracil yield（0.48 g/L）.In terms of yield,the specific hydrolase encoded by rih B gene is more inclined to hydrolyze cytidine.Compared with the original strain E.coli B0,the volume of the newly constructed strain decreased significantly,indicating that the presence of plasmids brought a certain burden to the strain growth.In order to reduce the strain growth pressure and improve the bacterial body mass,the middle copy plasmid p S-74 was ligated with hydrolase gene rih B and re-introduced into cytidine producing bacteria to construct strain E.coli B5.Shaking flask fermentation results showed that the cytosine yield of B5 strain was 3.25 g/L,which was 64.14%higher than that of E.coli B2 strain.The byproduct uracil was 0.57 g/L,which was similar to E.coli B2 strain,and the biomass increased by 23.23%.These results indicated that replacing high copy plasmids with low copy number plasmids could effectively reduce the influence of plasmids on bacterial growth,ensure bacterial growth and increase cytosine synthesis efficiency.The cytosine yield of E.coli B5 strain was 45.80g/L,which was 40.84%higher than that of E.coli B2 strain,and the biomass was increased by 20.72%.In conclusion,the selection of nucleoside hydrolase with better cytidine specificity and suitable copy number plasmids can play a positive role in the accumulation of cytosine.The cytosine production strain constructed in this study can achieve the accumulation of cytosine by microbial fermentation,which has a good industrial application prospect compared with the organic synthesis method.