Metabolic Engineering Of Aspergillus Niger For The Production Of L-malic Acid At Low PH

Malic acid is widely used in food,health care,medicine,daily chemical and other fields.In recent years,the production of malic acid by microbial fermentation has attracted much attention because of its advantages such as wide source of substrates,low production cost,high yield and environmental protection.At present,the product of malic acid by microbial fermentation requires the addition of CaCO3 to maintain the pH of medium at about 6,which not only increases the cost of raw materials,but also increases the technological difficulty and purification cost of downstream separation and extraction of malic acid.Therefore,it is necessary to explore a method to produce malic acid by fermentation at low pH.In this paper,We used the Aspergillus niger ATCC 1015 containing the Cre/loxP system as the starting strain to constructed strain of malic acid producting under low pH through the elimination of oxalic acid and citric acid by-products,the reconstruction of intracellular glyoxylic acid metabolic pathway and strengthen the malic acid transport system.The main research results are as follows:(1)The knockout vector of peroxins corresponding to A.niger isocitrate lyase on peroxosomes was constructed with plasmid pLH594 constructed in the laboratory as the starting vector.The expression vectors of malate synthase gene(ms*),isoccitrate lyase gene(icl),citrate lyase gene(acl)and cisaconitase gene(aco*)were constructed with plasmid pLH331 as the starting vector.The genes were knocked out and overexpressed successively by Agrobacterium-mediated transformation.(2)The by-products oxalic acid and citric acid were successfully eliminated by knocking out the oxaloacetate hydrolasee gene(oahA)and citric acid transporter encoding gene(cexA).(3)The glyoxylate was reconstructed in the cytoplasm by successively expressing malate synthase(MS),isoccitrate lyase(ICL),citrate lyase(ACL)and cisaconitase(ACO)located in the cytoplasm.Through the overexpression of the binary carboxylic acid transporter on the cell membrane(DCT1)to strengthen the malic acid transport system.The yield of malic acid increased from 2.49 g/L to 5.94 g/L,which was 2.4 times higher than that of original strain S469.