| China is the largest producer of citric acid(CA),contributing to more than 70%of worldwide market shares for the competitive cost of CA production and product quality.A.niger YX-1217 is a typical citric acid-producing strain that is used in industry in China,which is different from other CA producing strains because of its high yield,high temperature tolerance and short fermentation time,however no investigation has examined the metabolic mechanism underlying high CA productivity in A.niger YX-1217 and it is not a genetically stable strain and is prone to spontaneous degeneration of CA production.Furthermore,because genetic transformation systems for A.niger YX-1217 are underdeveloped,which greatly limited studies of the molecular mechanisms and metabonomics development of A.niger,it is of great significance to study the mechanism of this strain with high CA production and metabolic engineering for improving its productive characteristics of the strain or further expanding its use in the production of organic acids.In this study,the morphology,transcriptomes,proteomics of A.niger YX-1217 and degenerative YX-1217G were compared and analyzed to explore the metabolic profile associated with high CA yield,and the genetic transformation platform of A.niger YX-1217 was successfully established based on Agrobacterium tumefaciens mediated transformation,which is an opportunity to further improve itaconic acid production by increasing the conversion of citric acid.(1)Morphological phenotypes of A.niger YX-1217 strain with high C A production capacityDifferences of the spore morphology between A.niger YX-1217 and YX-1217G was compared by scanning electron microscopy(SEM)and transmission electron microscopy(TEM).The results showed that the surface of A.niger YX-1217 had many "ridges" with ordered array which made the spores seem rough and its melanin layer was very integrated and compact,while in A.niger YX-1217G the ridges had disappeared,or partly collapsed which made the spore surface of A.niger YX-1217G become smooth and its melanin layer was partly missing and became thinner and relatively loose;the morphological evolution of A.niger YX-1217 in submerged CA fermentation process was investigated in this study using optical microscope,and the results indicated that CA production was along with the formation of pellets.The nucleus of pellets was about 80-90 ?m,and the hyphae were branched,knobby shape with swelled tips,obvious diaphragm,and the extension phase of hyphae could be up to 30 ?m;the smaller and more pellet morphology would favorable to increase CA productivity,which showed that the pellet morphology is directly related to CA production.(2)Transcriptomic analysis of Aspergillus niger strains reveals the mechanism underlying high CA productivityThe transcriptomes of high citric acid-producing A.niger YX-1217 and degenerative YX-1217G were investigated using A.niger ATCC1015 as control at the stage of conidia germination(10 h)and the peak stage of CA production(40 h),and the results revealed that the transcriptional characteristics of A.niger YX-1217 was related to high CA productivity.The transcript of genes involved in carbohydrate hydrolysis and polypeptide degradation pathways,central metabolism,resistance mechanism,transporter system was upregulated compared to the control strains at the stages of 10 h and 40 h.Based on comparative transcriptome data,the center metabolic regulatory model of A.niger YX-1217 related to CA production was constructed,and it showed that abundant hydrolase system,unhindered central metabolic flow,effective resistance mechanism,strong ransport system,a highly efficient electron transport chain,a regeneration system for NAD+/NADP+ apparently was contribute to high CA production in A.niger YX-1217.(3)Proteomic analysis of Aspergillus niger strains reveals the mechanism underlying high CA productivityA.niger YX-1217 is an acid-producing strain with cornmeal as the main medium,and it must have the corresponding metabolic mechanism to efficiently utilize cornmeal.In view of this,the extracellular proteins of A.niger YX-1217 and A.niger YX-1217G were compared and analyzed by two-dimensional electrophoresis(2-DE).The results showed that the amount and concentration of extracellular proteins of A.niger YX-1217G were significantly lower than that of A.niger YX-1217,and six overexpression proteins were identified by mass spectrometry.Then,the differences of intracellular proteins between A.niger YX-1217 and A.niger YX-1217G were analyzed and compared based on ITRAQ technique,and a total of 3,553 proteins were identified.The differential proteins were classified according to the KEGG database and the results showed that the expression level of proteins involved in CA biosynthesis in strain A.niger YX-1217 was significantly higher than that in degenerative strain A.niger YX-1217G,such as pyruvate carboxylase(An04g02090)?citrate synthase(An15g01920)?esterase/lipase(An09g06390)?acyl-CoA dehydrogenase family protein(An17g01150)?v-type proton ATPase subunit B(An02g02020)?ABC metal ion transporter(An03g04060)?glutamine synthetase(An14g01460).The high expression of these proteins was obviously one of the important reasons for the CA production of strain A.niger YX-1217.The transcriptional and proteome data were correlated and the results showed that the transcription and expression levels of most of the genes involved in CA production showed the same trend.(4)Construction of genetic transformation platform for A.niger YX-1217As a filamentous fungus after long-term mutagenesis,genetic transformation systems of A.niger YX-1217 are underdeveloped.To study its metabolic engineering,an optimum genetic transformation system for an industrial A.niger YX-1217 strain mediated by A.tumefaciens was developed in this study.A.tumefaciens AGL-1 was chosen as the mediated strain,the initial A.niger spore concentration was 107 spores/mL suspension,the AS concentration was 300 ?M,and the ratio(v/v)of the initial A.niger spores to A.tumefaciens was 1:9.Using the optimal transformation system,the transformation frequency was 25 transformants per plate using107 conidia.After 10 generations of culture,the transformants could still grow on the resistant plate with hygromycin,which indicated that the transformants were stable.(5)Metabolic engineering of an industrial A.niger YX-1217 strain for itaconic acid productionBased on the genetic transformation technology,the application of CA production by A.niger YX-1217 was developed.The synthesis pathway of itaconic acid was constructed by introducing gene aco encoding aconitase and gene cad encoding aconitate decarboxylase.The results showed that the high efficiency promoter gene glaA encoding glucoamylase of A.niger YX-1217 was screened,and its expression level was far more than the activity of promoter PpkiA and PgpdA using quantitative PCR analysis and high expression of cadA gene;transformant L-1(YX-1217-PglaA-aco-L1-cadA)of the fusion protein,transformant L-2(YX-1217-PglaA-aco-L2-cadA)of the fusion protein and transformant Z-17(YX-1217-PglaA-cadA transformants)was selected to produce itaconic acid by the fed-batch fermentation under three stage control of agitation speed.After the fermentation of 104 h,itaconic acid concentration of L-2 could reach 7.2 g/L,the maximum yield was 0.069 g/L/h,and its itaconic acid concentration was 41.67%higher than Z-17.Under the co-expression of f gene aco and cad,the CA metabolic pathway was extended to the IA metabolic pathway,which made the production ability of this strain more diversified. |
PDF Full Text Request