The Construction And Fermentation Of Recombinant Escherichia Coli For L-Malic Acid Production

L-malic acid is an important natural organic acid, and as a part of TCA cycle, it is widelyused in food, medicine and chemical industry, and has a huge market prospect. Microbialproduction of L-malic acid by fermentation has a lot of advantages and becomes one of hotesttopics in recent years. There are a large number of by-products besides L-malic acid in thecomponent of wide type strains fermentation production, but L-malic acid producing strainconstructed by genetic engineering method can eliminate the metabolic side products anddistributing carbon flow to the target product as much as possible. Because of well-knowngenetic background and being improved to produce many products like succinic acid andlactic acid, Escherichia coli has caused concern of many researchers all over the world as apotential L-malic acid producing strain.In this research, based on the succinic acid synthesis pathway of recombinant E. colistrain B0013-1050, the synthesize pathway of L-malic acid was constructed by using Redhomologus recombination technology and Xer/dif recombinase system. In order to eliminatedownstream products, the gene fumB, fumC, maeB was deleted from chromosome, and arecombinant E. coli strain2030was obtained finally. This strain can produce L-malic acid inanaerobic fermentation, the major side products were succinic acid and pyruvic acid. In a15Lfermentor, at the beginning of anaerobic fermentation, sodium hydrogen carbonate was addedto supply CO₂, this distributed more carbon flow to L-malic acid, and then reduced the yieldof pyruvic acid. After30h anaerobic fermentation, a production of12.5g/L L-malic acid wasachieved, the productivity and yield was0.42g/（L·h） and52.1%, respectively.In order to strengthen the L-malic acid synthesis pathway, malate dehydrogenaseencoded gene mdh from Aspergillus flavus was cloned and expressed in recombinant E. colistrain to distribute more carbon flow to L-malic acid. Cloned gene mdh to plasmids withdifferent copy numbers and then transform them into the recombinant strain E. coli2030. Theresult showed that a low expression level of malate dehydrogenase promoted theaccumulation of L-malic acid more effectively.In order to achieve steady inheritance of gene mdh and control a low level of malatedehydrogenase, the gene was integrant expressed in the recombinant E. coli strain2030toconstructed a recombinant strain E. coli2040. This strain obtained a higher yield of L-malicacid in the shake-flask fermentation, and in15L fermentor, after30h anaerobic fermentation,a production of14g/L L-malic acid was achieved, the yield and productivity was enhanced to60.3%and0.47g/（L·h）. Compared with the reports of other countries, there was a certain gapin the production and yield, so subsequent research was needed to improved the producingability of the recombinant E. coli strain.