Study On Enzymes Produced By Aspergillus Niger Through Metabonomic And Transcriptomic Analysis By GalA Gene Deletion

Aspergillus niger is one of the important cell factories and is widely used in the production of industrial enzymes and organic acids.In the processing of starch as raw material,the?-glucosidase with transglycosylation ability is being uesd to produce isomaltooligosaccharides.Aspergillus niger producing?-glucosidase is often accompanied by glucoamylase to inhibit its transglycosylation ability,and its metabolic mechanism is not clear.In this study,a mutant strain of Aspergillus niger was obtained by galA deletion using molecular genetic methods,which showed an increase of?-glucosidase ability;Differential transcripts and metabolites caused by gene deletions were analyzed at the metabolome level and transcriptome level.These changes involving metabolic pathway thus confirmed the key factors in the production of?-glucosidase and glucoamylase,then the key metabolites were added to culture to used to verify the effectiveness of the method.The protoplast of A.niger HE01 was transformed by the deletion cassette of galA including galA1-N~R and N~R-galA2,and then A.niger?galA was obtained.The activity changes of?-glucosidase and glucoamylase were analyzed,picking the time for the option of metabolomicsa nd transcriptomics:at the 168 h of fermentation culture,the two strains both showed the highest activity of?-glucosidase and glucoamylase.Aspergillus niger strains were isolates,frozen quickly on the optimal time,one part uesd for the preparation of metabolites,the other for.RNA extraction For one part,freeze-dried samples were extracted by sonication using the methanol aqueous solution for a few times in ice water bath,then ~1HNMR spectra were recorded.The metabilite assignments of ~1H NMR data were identified,there were 26 primary metabolites including carbohydrates,amino acids and cholines.After the normalization of sectional integration of spectral data,The orthogonal signal correction projection to latent structure discriminant analysis(OPLS-DA)was performed using the NMR data,the results is that 7 up-regulated metabolites,including?-glucose andb-glucose,trehalose;alanine,glutamate,betaine;and 4 down-regulated metabolites.including glycine,sarcosine,acetate,ethanol.A relative quantitative analysis of all mRNA in Aspergillus niger strains during the highest activity period of?-glucosidase was performed by high throughput sequencing techniques.In A.niger HE01 and?galA,the transcription level of galA was the highest in all encoded proteins,which were nearly six fold and three fold higher than the internal reference gene GAPDH.The mRNA of galA in?galA was decrease by about half,with 9 genes up-regulated.An enrichment analysis was performed to identify gene ontology terms which were over-represented in the differentially expressed gene set.The mainly tems were related to intracellular amino acid metabolism,transmembrane transport,etc.The expression of HK(hexokinase),IDO(indoleamine 2,3-dioxygenase family protein),YCM2(mitochondrial carrier protein),and AAT(amino acid transporter)were quantified in RT-qPCR.The results were consistent with the transcription data,it is further explained that it can induce gene increase in transmembrane transport-related pathways,as well as the raised the hexokinase activity in?galA.Through exogenous addition of alanine,glycine,glutamic acid,valine as positive control,the?-glucosidase activity of A.niger HE01increased by 42%,50%and 35%,respectively,with of glucoamylase activity increasing 6%,13%and 11%The?-glucosidase activity of A,niger?galA increased by 30%,8%and 23%respectively,with of glucoamylase activity increasing 2%,7%and 4%.The addition of alanine,glycine and glutamic acid to the?--glucosidase activity was more significant than that of glucoamylase,which indicated that the these three amino acids were the key precursors in the synthesis of?-glucosidase.In conclusion,based on NMR metabolomics and high throughput sequencing transcriptomics to investigation on the differences by galA deletion.The use of selected key metabolites to add culture to improve the activity of?-glucoside,Integrated metabolomics and transcriptomics as an efficient method for fingerprinting in genetic disturbances and environmental fluctuations,providing basis research for strains improvement.