Metabolic Engineering Of Aspergillus Niger For Production Of D-Glucaric Acid

D-glucaric acid,a dicarboxylic acid derivative of glucose,is an important platform compound with high added value.It is widely used in disease treatment,chemical synthesis and food processing,and has broad market prospects.In recent years,the microbial method has attracted the attention of experts and scholars because of its advantages of low cost,low pollution and green environmental protection.Aspergillus niger is a food safety strain,because of its clear background,strong acid resistance,can use cheap carbon source and high conversion rate is used in the industrial production of a variety of organic acids,is the ideal glucaric acid production strain.This research in a laboratory building contains Cre/loxP system of Aspergillus niger S834 for starting strain,using metabolic engineering transformation strategy,the building up of glucaric acid synthesis pathway in Aspergillus niger,strengthen the synthesis pathway and secrete pathway,establish cofactors metabolism of the circulation system and reduce the carbon flux to further improve the level of glucaric acid synthesis,The glucaric acid producing strain was constructed to lay a foundation for efficient production of glucaric acid and downstream related products by microorganisms.The specific research results of this paper are as follows:(1)S834 was used as the starting strain to construct the glucaric acid synthesis pathway.Through literature review and BLAST comparison,it was found that Aspergillus niger had a completed glucuronate synthesis pathway,and lacked the glucuronate dehydrogenase that oxidized glucuronate to glucaric acid.Therefore,ppudh derived from Pseudomonas putida KT2440 was successfully expressed in Aspergillus niger.The first generation of glucaric acid producing strain S985 was obtained,and its shaking flask fermentation yield was 18.74 mg/L.(2)To enhance the synthesis and exosecretion of Aspergillus niger,promote the accumulation of glucaric acid.The self-derived anmioxA,Saccharomyces Cerevisiae S288C ScJEN1 and self-derived aninoA were successfully expressed in Aspergillus niger successively,making the yield of the fourth-generation transformed strain S2086 reach 102.10 mg/L.Compared with S985,the yield was 4.5 times,indicating that the synthesis pathway of glucaric acid in Aspergillus niger was strengthened.Enhancing the extracellular transport of glucaric acid could reduce the intracellular synthesis pressure and increase the yield.(3)Construct cofactor recycling system to maintain intracellular redox balance and promote the synthesis of glucaric acid.The llnox derived from Lactococcus lactis subsp.cremoris MG1363 was successfully expressed in Aspergillus niger,which provided NAD~+ for the synthesis of glucaric acid.The yield of the fifth generation strain S2390 was 115.65mg/L,which was 13.3%higher than that of the previous generation strain,indicating that the establishment of intracellular redox balance system could further improve the yield of glucaric acid.(4)Weaken branch metabolism and improve the synthesis level of glucaric acid.On the basis of S2390,the expression of pfkA and zwf was successfully weakened,and the carbon flux of the glucaric acid synthesis pathway was improved.The yield of the seventh generation of modified strain S2635 reached 313.65 mg/L,which was 15.7 times higher than S985.It indicates that the branch metabolism of weakened carbon source is of great significance to the increase of yield.