Study On Breeding Of High Cytidine-producing Strains With Atmospheric Room Temperature Plasma Mutagenesis Technology Combined With CRISPR/Cas9 System

Cytosine nucleoside,also known as cytidine,It is an important raw material for a variety of anti-cancer drugs and health foods.Cytidine is mainly obtained by microbial fermentation,but the current fermentation level of cytidine is not high,which is affected by many factors.Therefore,in order to obtain high-yield strains of cytidine,the first thing to solve is to design and optimize the cytidine metabolism network.Based on this premise,three breeding strategies for cytidine producing bacteria were proposed.In addition,the high-yield cytidine producing strain was constructed by using the high-efficiency mutagenic method,such as ARTP mutagenic technology and CRISPR/cas9.The specific research contents are as follows:1.The mutant of histidine deficient Escherichia coli was selected by atmospheric pressure room temperature plasma.ARTP mutagenesis screening technology has the advantages of better equipment,easier operation method,higher safety factor,and higher mutation frequency.In order to screen high-yield cytidine producing strains,by blocking the diversion of PRPP to histidine metabolism,the amount of synthesis of PRPP,a metabolite at the front of cytidine synthesis,was increased,and then the content of cytidine was increased.E.coli K-12(AthrA)was used as the developing strain,ARTP mutagenesis technology was used to select histidine auxotrophic mutation under the conditions of discharge power 110 W,gas flow rate 10 SLPM and mutagenesis time 70 s.Fungus E.coli K-12(?thrA His-).2.CRISPR/Cas9 system was used to construct a mutant strain of E.coli with cdd gene deletion.In recent years,CRISPR/Cas9 gene editing technology has been used in microbial genome modification to optimize the expression and regulation of microbial genes to increase the yield of chemical and biological products.In the de novo cytidine synthesis pathway of E.coli,blocking the cytidine degradation pathway is a necessary strategy for cytosine synthesis.By knocking out the cdd gene encoding cytidine deaminase and cutting off the degradation pathway of cytidine,the accumulation of cytidine can be increased.Taking E.coli K-12(?thrA His-)as the starting strain,pTargetF and pCas plasmids were selected,in which There are genes encoding?,-red exo,bet and gam.According to the primer designed by the target gene cdd,the pTargetF-cdd plasmid was successfully constructed.After the homology arm was constructed,the pTargetF-cdd and homology arm DNA fragments were electrically transferred into E.coli competent cells containing the plasmid pCas to make the plasmid and homology The arm binds to the host genome and guides the Cas9 bound to it to recognize the target sequence for cleavage,thereby deleting the target gene cdd.Finally,by eliminating pTargetF and pCas,a non-resistant mutant strain E.coli K-12(?thrA His-?cdd).3.CRISPR/Cas9 system was used to construct a mutant strain of E.coli with deletion of the poxB gene.Knocking out the poxB gene in E.coli,pyruvate produced by sugar metabolism is beneficial to cytidine synthesis.By reducing the metabolic shunt of pyruvate to acetic acid,it provides more energy for the growth of bacteria,reduces the harm of acetic acid to the growth of E.coli,and provides a de novo synthesis of cytidine.Taking E.coli K-12(?thrA His-?cdd)as the starting strain,the poxB gene was knocked out using CRISPR/Cas9 and ?-red system.According to the primer designed by the target gene poxB,the pTargetF-poxB plasmid was successfully constructed.After constructing the homology arm,the pTargetF-poxB and homology arm DNA fragments are electrically transferred into E.coli competent cells containing the plasmid pCas,so that the plasmid and homology arm are bound to the host genome,and the Cas9 bound to it is directed to recognize the target The sequence is cut so that the target gene is deleted.Finally,by eliminating pTargetF and pCas,a mutant strain E.coli K-12(?thrA His-?cddApoxB).4.Study on the ability of recombinant strains to ferment cytidine.In this article,the starting bacteria E.coli K-12(?thrA),mutant E.coli K-12(?thrA His-),E.coli K-12(?thrA His-?cdd)and E.coli K-12(?thrA His-?cdd?poxB)Fermentation at 37?,180 rpm for 48 h,analysis of bacterial concentration,glucose consumption rate,pH and cytidine production during fermentation.Compared with the control group E.coli K-12(?thrA),the histidine auxotrophic strain E.coli K-12(?thrA His-)showed no significant difference in growth,and the glucose consumption rate was always lower than that of the control group E.coli K-12(?thrA His').E.coli K-12(?thrA)is 82.78%of the control group.The cytidine yield of E.coli K-12(?thrA)was 0.549±0.015 g/L,and the cytidine concentration in the fermentation broth of the mutant strain was 0.60±0.011 g/L,which was 1.10 times that of the control group.The glycoside conversion rate of E.coli K-12(?thrA His-)was increased by 30.95%.After knocking out the cdd gene,the mutant strain E.coli K-12(?thrA His-?cdd)has a growth curve that is basically the same as that of the starting strain E.coli K-12(?thrA His-),and the glucose consumption is slightly higher than that of the starting strain.It is 1.23 times that of the control group E.coli K-12(?thrA His-).The control strain E.coli K-12(?thrA His-)had a cytidine yield of 0.604±0.011 g/L,and the mutant strain E.coli K-12(?thrA His-?cdd)had a cytidine yield of 0.789±0.016 g/L.E.coli K-12(?thrA His-?cdd is 1.31 times that of the control group E.coli K-12(AthrA His-),and the glycoside conversion rate of E.coli K-12(?thrA His-?cdd)is increased by 8.18%.Using E.coli K-12(?thrA His'?cdd)as the starting strain,the poxB gene was knocked out to obtain the burst strain E.coli K-12(?thrA His-?cdd?poxB).It was found that the growth rate and cell concentration of mutant strain E.coli K-12(AthrA His-?cdd?poxB)were higher than those of control bacteria,and the glucose consumption of E.coli K-12(AthrA His'AcddApoxB)was lower than that of the starting strain,is 88.3%of the control group K-12(?thrA His-?cdd).The control strain E.coli K-12(?thrA His-?cdd)had a cytidine yield of 0.789±0.016 g/L,and the mutant strain E.coli K-12(?thrA His-?cdd?poxB)had a cytidine yield of 0.989±0.018 g/L is 1.25 times that of the control group E.coli K-12(?thrA His-?cdd).The glycoside conversion rate of E.coli K-12(?thrA His-?cdd?poxB)was increased by 41.18%.It shows that the content of cytidine can be increased by blocking the shunt of PRPP to histidine metabolism,increasing the synthesis amount of the front metabolite PRPP,cutting off the degradation pathway of cytidine,and weakening the synthesis pathway of acetate.