Fine and dynamic tuning the glycolytic flux ratio of an artificial carbon saving pathway for high yield of mevalonate in Escherichia coli

Abstract Background In natural Escherichia coli , glucose is mainly metabolized via the Embden-Meyerhoff-Parnas (EMP) pathway. However, in the metabolic process of conversion of pyruvate to acetyl-CoA, one-third of the carbon is lost at CO 2 . To decrease the loss of glucose in the metabolic process and enhance the carbon conversion efficiency production of desired products by E. coli , we constructed a carbon saving pathway, EP-bifido pathway. As the balance of energy and reducing power was not optimal, we use synthetic biology methods to precisely and dynamically adjust the EMP pathway and pentose phosphate pathway (PPP) flux to improve the production of mevalonate (MVA) via the EP-bifido pathway. Result Here, we enhanced the MVA titer and yield in E. coli in two ways. First, the promoter of the first gene of the PPP, zwf , was replaced with a set of promoters of different strength to enhance PPP flux for NADPH supply. Compared with the previous EP-bifido strains, the zwf -modified strains showed obvious differences in NADPH, NADH, and ATP synthesis levels and production routes. Among them, strain BP10BF accumulated 11.2 g/L of MVA after 72 h of fermentation and the molar conversion rate from glucose reached 62.2%. Second, the expression of pfkA was suppressed at a certain time by the clustered regularly interspaced short palindromic repeats interference (CRISPRi) system to avoid the growth defect caused by pfkA direct knock-out. The resulting MVA yield of strain BiB1F was 8.53 g/L, and the conversion rate from glucose reached 68.7%. Conclusion This is the highest MVA conversion rate reported in shaken flask fermentation. The CRISPRi and promoter fine-tuning provided an effective strategy for metabolic flux redistribution in many metabolic pathways and promotes the chemicals production.