Metabolic Engineering Of Escherichia Coli For Production Of Mevalonate Acid

The mevalonic acid(MVA)is a kind of organic acid with optical activity,which is an important precursor compound for the synthesis of isoprenoids and has important application value in food,medical,chemical industry and so on.Acetyl coenzyme A is the precursor of mevalonate,and plays an important role in the production of MVA.Based on the extended metabolic network model of Escherichia coli,metabolic pathways with high yield for acetyl coenzyme A synthesis were simulated and the optimal pathway was analyzed and screened.We constructed E.coli engineering strain which synthesized mevalonate based on the optimal synthesis pathway of acetyl coenzyme A,further improved the synthesis ability of mevalonate through metabolic engineering.The simulation design of metabolic pathway for high yield synthesis of acetyl coenzyme A.Using E.coli genome-scale metabolic network model i JO1366 which introduced 498 heterologous reactions from KEGG database,the optimal synthesis pathway of acetyl coenzyme A from glucose was calculated based on the flux balance analysis(FBA),and six new pathways with theoretical carbon molar yield of acetyl coenzyme A from glucose over 90% were obtained.At the same time,combined with the relevant information and literature reports of Enzyme,KEGG and other databases,we found that the NOG pathway is the most feasible pathway among the six pathways.Construction and optimization of mevalonate pathway.A T7 promoter induced plasmid p CDF-mva ES was constructed to express mva E and mva S,which are key genes for mevalonate synthesis.The plasmid p CDF-mva ES was transformed into E.coli BL21 strain and the mevalonate synthesis strain B01 was constructed.By optimizing the concentration of IPTG,the yield of MVA reached 324.30 mg/L.Construction and optimization of NOG pathway.A trc promoter induced plasmid p BHR70 was constructed to express fxpk,which is the key gene of NOG pathway.The plasmid p BHR70 was transformed into strain B01 and the NOG pathway engineering strain B02 was constructed.At the same time,promoters with different intensity were used to regulate the expression intensity of NOG pathway.The plasmid p BHR76 with J23104 as promoter had the highest expression intensity of NOG pathway.The strain was named B06,and the production of MVA reached 447.80mg/L.It had the most obvious effect on the increase of MVA production,which was 38.08% higher than that of B01 strain.Overexpression of key enzymes in non-oxidized pentose phosphate pathway.By inserting strong promoter J23119 into the chromosome of engineering strain B06 to overexpress transketolase and transaldolase genes tal A-tkt B in pentose phosphate pathway,the engineering strain B08 was obtained,and the final production of MVA reached 507.49 mg/L,which was 56.49% higher than that of B01 strain.The system metabolic engineering strategies described in this study also applies to the synthesis of other products using acetyl coenzyme A as precursor in E.coli.