| Aspergillus niger,one of the efficient cell factories for the production of enzymes,organic acids and food additives,is widely applied in the biochemical industry.In the recent years,the rapid development and application of system biology centered on multi-omics integrative analysis provides researchers a comprehensive approach to better decipher the mechanism of cell metabolism and regulation and predict potential targets for metabolic engineering of industrial strains.Meanwhile,the development of the CRISPR/Cas9 system,an efficient and accurate gene editing method,provides an important guarantee for the genetic engineering of filamentous fungi.Based on the above background,this study investigated the intracellular metabolic changes of Aspergillus niger under oxygen-limited fermentation conditions based on multi-omics integrative analysis and results indicated that the supply of some amino acids may form restrictions for further improvement of glucoamylase production.Therefore,the biosynthesis of these limited amino acids was enhanced by metabolic engineering and the resulting influence on glucoamylase production and intracellular metabolism was investigated.Firstly,the fermentation process of a high-producing A.niger strain for glucoamylase in a 5L fermentor was studied.The transcriptomics and metabolomics data was obtained and multi-omics integrative analysis was carried out combined with the updated genome-scale metabolic model in our lab to explore the metabolic mechanism of the high glucoamylase yield under oxygen-limited phase and predict potential targets for further improvement of glucoamylase production.Results indicated that under oxygen-limited conditions,the expression of the genes involved in glyoxylate cycle and GABA shunt was up-regulated in A.niger to respond to the massive accumulation of NADH resulted from the increased relative fluxes of TCA cycle,thereby maintaining the intracellular redox balance.On the other hand,the cell up-regulated the expression of the genes involved in the energy production,weakened the anabolism and strengthened the catabolism of the fatty acids to relieve the lack of energy supply resulted from oxygen limitation.Furthermore,under oxygen-limited conditions,the relative fluxes of the downstream EMP pathway was increased,which could provide more essential precursors for the biosynthesis of enzyme products.Meanwhile,the protein secretion ability of the cell was also maintained at a high level.In short,multi-omics integrative analysis revealed that the changes in global metabolism under oxygen-limited conditions could provide sufficient energy,precursors and stable redox balance for the high production of glucoamylase.What's more,results of this study showed that the intracellular pools of alanine,glycine,aspartate and glutamate decreased significantly under oxygen-limited conditions.These four amino acids account for high ratios in amino acid composition of glucoamylase and exogenous addition of these amino acids in medium significantly improved glucoamylase production.The above results indicated that these four amino acids probably served as the limited amino acids for glucoamylase production of A.niger.Therefore,in the following work,biosynthesis of these amino acids was elevated by genetic engineering to further improve the glucoamylase production.Secondly,this study optimized the protoplast-mediated transformation(PMT)method in A.niger model strain CBS 513.88(hereinafter referred to as the model strain)to improve the efficiency of genetic engineering.Osmotic stabilizer,enzymatic hydrolysate and enzymatic hydrolysis time are three key factors in PMT method,which were optimized by single factor experiment to efficiently obtain more protoplasts and higher protoplast regenerating rate.After optimization,1.3 M sorbitol was used as osmotic stabilizer in enzymatic hydrolysate and washing buffer,as well as in the re-suspension process of protoplasts.Lysing enzymes were used for cell wall hydrolysis and the whole enzymatic hydrolysis process lasted for 3 hours.Under these optimized conditions,the amount of the protoplasts obtained can reach up to 3×107 protoplasts/ml.Meanwhile,0.95 M sucrose was used as osmotic stabilizer and carbon source in regenerating medium,leading to a protoplast regenerating rate of 11%.The optimized results exceeded the average level of the reported literature.After that,gene kusA was knocked out in the model strain using the optimized PMT method to improve homologous recombination rate,generating the mutant CBS 513.88-?kusA.Then,pigmentation gene olvA was deleted in the mutant CBS 513.88-?kusA and results showed that the homologous recombination rate in the mutant CBS 513.88-?kusA increased from 5.7%to 66%compared with that in the model strain.Although the kusA deficient strain can significantly improve homologous recombination rate,itself has some limitations.Recently,the development of CRISPR/Cas9 system dramatically improved the efficiency and accuracy of gene editing.Therefore,CRISPR/Cas9 mediated homologous recombination method was used and developed in this study to construct mutants.Overexpression cassettes of five genes(An11g02620,An04g00990,An05g00410,An04g06380 and An16g05570,respectively)were inserted in the olvA gene locus in the model strain,generating five mutants namely OE-ala,OE-glu,OE-gly,OE-aspl and OE-asp2,respectively,to elevate the biosynthesis of the four limited amino acids.On the other hand,in the same way,hygromycin resistence gene expression cassette was inserted in the olvA gene locus to additionally construct an olvA deletion strain as the control strain.Finally,the glucoamylase production performance of the five mutants was evaluated at the shake flask level and the 5L fermentor level.In shake flask fermentation,real-time quantitative PCR revealed that all the target genes were successfully overexpressed at the mRNA level while the five mutants exhibited different performance in glucoamylase production.Notably,the results demonstrated that the mutants OE-asp2 and OE-asp1 exhibited significantly increased glucoamylase activity of 114.66 AGI/ml and 103.38 AGI/ml,respectively,which was 23.5%and 11.4%higher than that of the model strain.However,mutant OE-glu exhibited decreased glucoamylase activity of 79.69 AGI/ml and mutants OE-ala and OE-gly exhibited unaltered glucoamylase activity compared to the model strain.On this basis,mutants OE-asp2 and OE-aspl were selected for further fermentation in the 5L fermentor.Results indicated that mutant OE-asp2 exhibited increased glucoamylase activity by 60.3%(234 AGI/ml),with a double glucoamylase yield compared to the model strain while mutant OE-asp1 showed similar performance of glucoamylase production compared with the model strain.Determination of intracellular amino acid pools of the two mutants and the model strain showed that the elevated biosynthesis of cytosolic aspartate in mutant OE-asp2 led to the increase of most amino acid pools including the pool of aspartate,among which the pools of alanine,glycine and leucine increased significantly.It can be speculated that the possible reason for significantly increased glucoamylase production in mutant OE-asp2 may be explained as follows:the altered pools of aspartate and glutamate which resulted from the elevated biosynthesis of cytosolic aspartate,finally led to the increase of most amino acid pools,possibly due to the increased flux of EMP pathway and the regulation of amino acid biosynthesis.Among them,some amino acids serve as important precursors for glucoamylase production.In addition,combined with literature reports,speculations were made to explain the reasons for the decreased and unaltered glucoamylase production performance of other mutants.This study combines the target prediction from multi-omics analysis with the experimental validation,which not only proves the possibility of increasing glucoamylase production by enhancing limited amino acid biosynthesis,but also reveals the complexity of the effects of this strategy on cell metabolism.This systematically conducted study partially reveals the mechanism of resources allocation in cell factory and will provide new clues for the rational design of enzyme production strains. |
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