Synthesis Of L-malic Acid By Gene Edition Of E.coli MG1655

L-malic acid is an intermediate product in the TCA cycle of organisms and has high application value in food,medicine and chemical industries.At present,there are three main methods for producing L-malic acid in the industry: chemical synthesis,immobilization(enzyme conversion)and microbial fermentation.As people’s requirements for the industrial production environment continue to increase,the cost of raw materials for chemical synthesis and immobilization methods continues to rise,and L-malic acid is used more and more widely in the food industry,microbial fermentation has become the most potential way.The production method of malic acid is also a hot spot of research.In order to obtain a high-yielding strain of L-malic acid,the wild-type E.coli MG1655 was modified at the genome level through metabolic engineering,and the malic acid yield reached 66% of the theoretical yield through aerobic fermentation.The transformation is mainly divided into three parts:(1)First use CRISPR/Cas9 gene editing technology to knock out the poxB,pta-ackA,ldhA,and pflB genes of E.coli MG1655,block the by-product metabolic pathway of pyruvate,increase the flux of the TCA cycle,and get the project strain M04.After fermentation,the output of pyruvate reached 5.68g/L,and the output of L-malic acid reached 4.09g/L.(2)Then,on the basis of strain M04,the malic enzyme genes mae A and mae B were knocked out to prevent L-malic acid from flowing to pyruvate,and strain M06 was obtained.Compared with strain M04,the production of pyruvate decreased by 1.98g/L,and the production of L-malic acid increased by 4.21g/L.It proves that the knock-out of the pyruvate replenishment pathway allows more metabolic flux to flow to L-malic acid.(3)Finally,continue to knock out the pfkA gene on the basis of the strain M06.By blocking the glycolytic pathway,glucose is metabolized through the pentose phosphate pathway to produce more NADPH and increase the intracellular reducing power.Obtain mutant strain M07.After shaking flask fermentation for 48 hours,the L-malic acid production of strain M07 reached 9.893 g/L,and the yield was 0.66 mol/mol malic acid/glucose.Compared with the wild type,the growth rate and glucose consumption of strain M07 decreased,but the accumulation of L-malic acid increased by 9.081 g/L,while the by-products of lactic acid and acetic acid decreased by 94% and 77%,respectively.High purity malic acid is advantageous.