Modification Of L-inalic Acid Biosynthesis Pathway And Fermentation For L-malate Production

L-malic acid is an important natural organic acid and widely used in the pharmaceutical,food,building materials and chemical industry.In this paper,the renewable resources of glucose as raw material,instead of the current industrial production of L-malic acid widely used in the microbial transformation process used fossil raw materials maleic anhydride,the use of specific microbial cell line,biosynthesis of L-malic acid or its polymers,designed to avoid fossil raw materials are not renewable and unsustainable characteristics,cost reduction,energy saving.First,the culture medium components and the culture conditions of shake flask were optimized by fermentation of BK-10.The results showed that glucose 100 g/L,urea 1.5 g/L,KH2PO4 0.2 g/L,ZnSO4 0.2 g/L,MgSO4 0.05 g/L,KCl 0.75 g/L,CaCO330 g/L as the optimum medium,The optimum amount of liquid was 20%and the inoculation amount was 10%.On this basis,adding 0.01%(W/V)Tween 80 could increase the yield of poly malic acid by 17.04%.The yield of malic acid was 79.23 g/L after acid hydrolysis,and the conversion rate was 71.3%,which was 48.7%higher than before.The optimum culture medium and culture conditions were validated on 5L fermentor.The results showed that the optimized conversion rate was 18.33%,the fermentation period was shortened and the production intensity was improved by 52.39%In the previous study,about 10%of the glucose was used in the synthesis of pullulan polysaccharides during the fermentation of polygonatum odoratum.Therefore,by designing homologous fragments of the pullulan synthase gene knocked out in order to achieve the goal of reducing the synthesis of pullulan polysaccharides and increasing the yield of malic acid.However,the experimental results show that the synthesis route of pullulan polysaccharide is located at the initial position of the glycolytic pathway,which is very important for the outbreak of a.aureus.The knockout of the pullulan synthase gene affects the utilization of glucose by bacteria Body can not be normal growth and metabolism,but also no longer produce poly malic acid and other impurities,such as lactic acid,acetic acid,fumaric acid and so on the situation.In addition,the molecular transformation of Aspergillus oryzae,which produces L-malic acid,which can ferment glucose.(C4T318),pyruvate carboxylate(pyc)and malate dehydrogenase(mdh)were extracted by combing and analyzing the L-malic acid biosynthetic pathway in Aspergillus oryzae.The overexpression of these three genes was achieved in Aspergillus oryzae by the expression vector.The results showed that the overexpression of malate transporter could significantly increase the yield of L-malic acid,which was 2 times higher than before.