Dynamic Flux Regulation Of Artificial Carbon-saving EP-bifido Pathway Improves Mevalonate Production In E.coli

Glucose is mainly metabolized through the Embden-meyerhof-parnas pathway in E.coli.However,during the metabolism of pyruvate to acetyl-CoA,1/3 of the carbon is lost.In order to reduce the loss of glucose in the metabolic process and enhance the product conversion efficiency of bacteria,we constructed the EP-bifido pathway.In the EP-bifido pathway,we reduced carbon loss by knocking out pfkA(encoding the phosphofructokinase A subunit)to reduce the carbon flux from pyruvate to acetyl-CoA;at the same time we introduced fxpk(encoding bifunctional phosphate transketone Enzyme),overexpression of fbp(encoding fructose-1,6-diphosphatase)constructing a bifido carbon rearrangement pathway.EP-bifido has obtained good results in the fermentation of PHA,PHB,MVA products,but because the unreached energy and reducing power balance,the EP-bifido pathway still has good potential for optimization.In this paper,we improved the yield and yield of MVA from two aspects.On the one hand,we replaced the promoter of the first gene zwf of the Pentose phosphate pathway(PPP)to enhance the metabolic flow of PPP and increase the supply of NADPH.In addition to selecting five promoters with different strengths from IGEM,we took advantage of the sugar phosphate stress phenomenon of the host strain Bw25113 △pfkA,cloned and used the promoter in the SgrR-SgrS operon to regulate the sugar phosphate stress of the bacteria Mechanism to dynamically adjust the flux of PPP.Compared with the wild-type strains,the synthesis and contribution ratios of NADPH,NADH and ATP were significantly changed.Strain BP10BF strain accumulated 11.2 g/L of MVA after 72 hours of fermentation,and the molar conversion rate reached 62.2%.On this basis,we further improved the conversion rate by changing the expression intensity of the MVA production pathway.After overexpressing the mevalonate production pathway,the MVA yield of the BPB02F strain reached 11.14g/L,and the conversion rate reached 68.4%.On the other hand,we have introduced a CRISPRi(Clustered regularly interspaced short palindromic repeats interference)system to suppress the expression intensity of pfkA in order to avoid metabolic disturbance and growth inhibition caused by pfkA gene knockout.We designed three effective CRISPRi inhibitory target sites and conducted combinatorial fermentation experiments.We found that the introduction of the CRISPRi system had a certain degree of inhibition on bacterial growth but CRIPSRi system did improve the conversion rate of product.The MVA yield of strain BiB1F reached 8.53 g/L,and the conversion rate reached 68.7%.This also proves that the yield and yield of fermentation products are not completely positively correlated.Considering the microtoxicity and complexity of the operation of adding an inducer,we used the metabolic regulation function of catabolic repressor/activator protein to change the promoter sequence of pfkA,so that the expression of pfkA was affected by Cra Regulates down-regulation of promoters to achieve self-induced suppression.Fermentation experiments of Cra-regulated pfkA showed that after the pfkA promoter was replaced with Cra-regulated promoter,the physicochemical properties of the cells veried a lot.The upper limit of growth of some modified strains was significantly increased,and all strains showed the ability to consume sugar quickly.The sugar consumption of BMB02F,which consumed the most sugar,reached 102.5 g at 72 hours of fermentation.The capacity of MVA production and accumulation of bacteria has also been unprecedentedly improved.Among them,the yield of MVA produced by strain BMBF 72h fermentation reached 52.66g/L,and the conversion rate reached 64.96%,and the yield of MVA produced by strain BRBF 72h fermentation reached 55.31g/L,conversion rate Up to 63.00%.Through optimized EP-bifido system,the yield of shake flask fermentation MVA reached 55.31g/L.By using Cra regulatory promoter to regulate the expression of pfkA,the bacteria not only accumulated enough biomass,but also timely introducted enough flux into the PPP,allowed product to take into account both in titre and yield.This yield is the highest reported for MVA shake flask fermentation currently.