| Orotic acid,also known as vitamin B13,has a very key physiological role and can be used in medicine,chemicals,cosmetics and other fields.Chemical synthesis of orotic acid is costly and polluting,and the production of orotic acid by microbial fermentation is a environmentally and friendly alternative.In this study,metabolic engineering strategies and techniques were used to optimize the orotic acid synthesis pathway of E.coli BW25113 wild-type strain.First,the flux of the pathway was enhanced by integrating the T7 promoter in front of key enzyme genes,transforming recombinant plasmids,and increasing precursor supply,the yield of orotic acid reached 82.90 mg/L.In order to avoid the large consumption of orotic acid during the replication of the DNA,the downstream gene pyrE(code phosphoribosyl transferase)of the orotic acid was knocked out to obtain the orotic acid accumulating strain BW-OR13,which increased the yield to 1011.70 mg/L.On this basis,the genes argF and dctA were knocked out in order to knock out the bypass metabolism and modify the orotic acid transport system,but the results were not ideal.Replenishment of uracil by transforming the plasmid expressing phosphoribosyltransferase improved the growth of the strain but reduced the production of orotic acid.The module was further optimized to obtain the highest orotic acid yield of 1126.39 mg/L.To further decrease the accumulation of by-product acetic acid,the medium was optimized to increase the orotic acid yield to 2185.23 mg/L.Activating the glyoxylic acid cycle by knocking out iclR and arcA can reduce the acetic acid by 13.8%,and due to the reduction of the orotic acid synthesis precursor level,the yield is 1467.45 mg/L.The another strategy is to knock out pgi,a key enzyme gene of the glycolytic pathway,which can reduce the carbon metabolic flux of the glycolytic pathway.In the end,the production of acetic acid was reduced by 36.6%compared with the strain BW-OR13,and the production of orotic acid was increased to 2573.75 mg/L. |
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