The Effect Of Adenine On The Synthesis Of Poly Malic Acid From Aureobasidium Pullulans

This thesis studied the high malic acid producing strain A.pullulans CGMCC3337,which was screened by mutagenesis.Firstly,the growth factors affecting polymorphic acid fermentation of A.pullulans were systematically analyzed,then the metabonomics and proteomics methods were used to analyze the mechanism of action,preliminary explored the iconic metabolites and key proteins affecting polymalic acid biosynthesis,which laid a theoretical foundation for further analysis of polymaltoic acid synthesis pathway and fermentation process optimization.The main research contents and experimental results are as follows:(1)The effects of different vitamins and nucleotides on the synthesis of poly-malic acid of A.pullulans were analyzed by single factor method.It was determined that adenine is the key growth factor affecting polymalic acid fermentation.Adding adenine to 0.5 g/L,the yield of pullulan polymalic acid(PMLA)reached 39.25 g/L,which was 33.6%higher than the blank.(2)The mechanism of action of adenine on the synthesis of polymorphic acid of A.pullulans was analyzed by metabolomics and proteomics methods.Compared with the blank group,adenine was added to 0.5 g/L.47 intracellular metabolites detected,By analyzing the metabonomics by principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA),it was found that the differential metabolites include fructose,galactose,pyruvic acid,?-ketoglutaric acid,fumaric acid and the like.Compared with the blank group,398 differential proteins were identified,of which 202 protein expression levels were up-regulated and196 protein expression levels were down-regulated.Up-regulation of protein phosphoglyceratekinase,phosphoglycerate mutase,malatedehydrogenase,malicenzyme,NADHdehydrogenase,ubiquinone-cytochrome c reductase,etc.mainly involved in glycolytic pathway,pyruvate carboxylation pathway And oxidative phosphorylation process.Down-regulation of protein phosphoribosylpyrophosphatekinase,galactose-1-phosphateuridineacyltransferase,pyruvatedehydrogenase,and isocitratedehydrogenaseareprimarilyinvolvedin the tricarboxylic acid cycle and the pullulan synthesis pathway.(3)Combined with metabolomics and proteomics analysis,the key growth factor adenine can increase the expression of key enzymes and key metabolites in the pyruvate carboxylation pathway,which enhances the synthesis of polymalic acid.Biosynthesis;attenuation of the galactose metabolic pathway reduces the synthesis of the byproduct pullulan.