Metabolic Engineering Of E. Coli DR01 For Riboflavin Production

In this paper,focusing on the purine pathway in riboflavin biosynthesis,Escherichia coli DR01 was used as the original strain to study the related gene modifications from 5-phosphate ribose to riboflavin/FMN,and the effect on the synthesis of riboflavin.Finally,we identified several significant genetic modification targets,and constructed a series of high-yield strains with significantly increased riboflavin production.Firstly,based on the medium copy number plasmid vector(Col E1 replicon),two key genes prs and purF in the purine pathway,and four genes guaA,guaB,ndk and gmk in synthesis pathway from IMP to GTP were overexpressed.The expression plasmids with sRNA of genes purA,guaC and rib F were respectively constructed by sRNA technology to down-regulate the gene expression level.The constructed plasmids were introduced into DR01 strain(the riboflavin titer was 473.5 mg/L)and cultured engineered strains in shake flask.The best results of genetic modification are as follows.The riboflavin production of the strain DR11 S with inhibition of gene rib F reached 1173.4 mg/L,which was 147.8% higher than strain DR01,and the yield was 112.8 mg/g glucose.The riboflavin production of the strain DR03 overexpressing gene prs reached 898.7 mg/L,an increase of 89.8%,and the yield reached 88.7 mg/g glucose.The riboflavin production of strain DR04 overexpressing gene purF reached 762.1 mg/L,an increase of 61%,and the yield was 75 mg/g glucose.The riboflavin production of strain DR05 overexpressing gene guaA reached 748.7 mg/L,an increase of 58.1.%,the yield is 74.9mg / g glucose.After the optimization of initial inoculum of strain DR05,the riboflavin production was further increased to 991.6 mg/L,and the yield was 94.4 mg/g glucose.The riboflavin production of strain DR09 inhibiting gene pur A reached 704 mg/L with an increase of 48.7%,and the yield reached 70.9 mg/g glucose.In addition,the overexpression of genes guaB and ndk increased the production of riboflavin by 24.2% and 30.3%,respectively.Subsequently,the genetic combination modifications of effective genes were carried out,but compared with the single gene modifications,the yield of riboflavin was not further improved,but decreased in varying degrees.We found that the use of medium copy number plasmids for multi-gene expression significantly increased the metabolic burden and plasmid loss rate of the strain.Therefore,a better stable low copy plasmid(pSC101 replicon)was examined to overexpress the genes prs,purF and guaA.The riboflavin production of the corresponding strains was 616.4 mg/L,620.5 mg/L and 725.6 mg/L with increase of 30.2%,31.1% and 53.2%,respectively.The results once again proved that these three genes have significant effect on synthesis of riboflavin.Although the yield is lower than that of the medium copy number plasmid,the metabolic burden of the strain is greatly reduced,and the riboflavin production has increased in unit time,indicating that the assembly of the polygene modification can be performed based on the low copy plasmid.This study not only obtained engineering strains with greatly increased riboflavin production and yield,but also identified several genetic modification targets,which have important effects on riboflavin synthesis.This study laid the foundation for subsequent multi-gene modification combinations and expression module assembly.