| As one of the most important cell factories, Aspergillus niger was widely used for industrial enzymes and organic acids production. Due to the fact that the previous research of A. niger was mainly focused on the fermentation process and single omic (like transcriptomics or proteomics), the metabolic flux distribution and the interplays among different omics were not fully mining, thus making systematic engineering of A. niger difficult. At present, the quick progress in omics quantification analysis allows the accumulation of varied omics data easy. Obviuosly, the era of big data in omics of industrial bio-process is coming. It becomes more and more important to extract valuable information from the integration analysis of different omics. To make the most of complex omics data, the vertical omics methodology, based on 13C metabolic flux analysis (13C MFA) and genome scale network metabolic model (GSMM), was put forward. To begin with, the GSMM of A. niger was iteratively updated based on the latest genome annotation information and physiological information. The model was then evaluated and verified using 13C flux and transcripomics study. Then, the comprehensive model could be established by integrating varied layer of omics with the optimized GSMM of high-quality, which help to estimate the feasible metabolic targets for the rational design of strains. The metabolic mechanism of glucoamylase production by A. niger was studied using vertical omics integrative analysis.With model strain A. niger CBS 513.88, the effects of shear stress and oxygen supply on A. niger growth and enzyme production were investigated in 5 L fermenter. Belonging to filamentous fungi, A. niger was sensitive to shear stress, as the agitation increased to 600 rpm, the normal growth of A. niger was inhibited. The fed-batch cultivation using the industrial glucoamylase production was adopted. According to the changes in real-time profiles, the fermentation process could be divided into phases of exponential and oxygen limited. During the exponential phase, the strain grew fast and the OUR increased from 0 to 16 mmol O2/L.h quickly. Meanwhile, the broth viscosity increased rapidly. During the oxygen limited phase, the strain growth became slower. Accordingly, the OUR slowly increased to its peak value and then decreased to a stable value. By contrast, the formation rate of glucoamylase was maintained at a relatively higher rate. The linkage experiment using 15 L and 5 L bioreactor revealed that the higher oxygen supply could promote the specific growth rate (μ) as well as the specific glucoamylase production rate (qp). Furthermore, it was found that the citric acid formation obviously increased during oxygen limited condition. To estimate the relations between μ and qp, the chemostat cultivations using high-producing strain A. niger DS 03043 under 8 dilution rate from 0.02 to 0.136 h-1 were conducted. The results showed that there existed two distinct relations between qpand μ. When μ<0.068 h-1. there existed linear positive correlation between qp and μ If μ>0.068 h-1. qp was nearly kept at 0.026 g/gDCW.h, indicating that the supply of precursors and energy was not sufficient to sustain the higher synthesis rate of glucoamylase.Next, the key steps in sample pretreatment for A. niger intracellular metabolomics analysis were optimized. With the aid of the quick-sampling equipment, the influences of four quenching solution and three extracting solution were investigated. It showed that -30℃,40%(v/v) methanol and 75%(v/v) ethanol were the best quenching and extracting solution respectively. For the sample preparation before GC-MS measurement, the influences of three organic solvents and three derivatizing agents on quantitative analysis of 24 standards (mainly amino acids and organic acids). It was found that pyridine and N-Methyl-N-(tert-butyldimethylsilyl)-1,1,1-trifluoroacetamide;N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) +1%t-Butyldimethylchlorosilane were the best solvent and derivatizing agent respectively. Meanwhile, the metabolites leakage during quenching using -30℃ 40% methonal was evaluated and the result showed that except the pro, gly and ala, there were no obvious metabolites leakage for other metabolites. According to isotope dilution mass spectrometry (IDMS) principle, the internal standard lines for A. niger metabolomics analysis were established using uniformly (U)13C-labeled metabolites as internal standards. Compared with external standard lines, the internal standard line improved the reliability in A. niger metabolomics analysis. Subsequently, the cell samples from four chemostat cultivations with different dilution rate were analyzed. The data indicated that the metabolomics analysis could reflect the differences in metabolism for A. niger under varied dilution rates.In this paper, we investigate the metabolic differences and regulation mechanisms between a high glucoamylase-producing strain A. niger DS 03043 and its wild-type parent strain A. niger CBS 513.88 via an integrated isotope-assisted metabolomics and 13C metabolic flux analysis approach. We found that A. niger DS 03043 had higher cell growth, glucose uptake, and glucoamylase production rates but lower oxalic acid and citric acid secretion rates. Consistent with above phenotype changes, A. niger DS 03043 was characterized by an increased carbon flux directed to the oxidative pentose phosphate pathway in contrast to reduced flux through TCA cycle, which were confirmed by consistent changes in pool sizes of metabolites. A higher ratio of ATP over AMP in the high producing strain might contribute to the increase in the PP pathway flux as glucosephosphate isomerase was inhibited at higher ATP concentrations. The increase in PP pathway flux could provide more reducing power (NADPH) and precursors for enzyme synthesis. As for the model strain A. niger CBS 513.88, the relatively higher flux in TCA cycle resulted into the accumulation of intracellular NADH, thus the model strain was in a higher redox state, which led to the secretion of oxalic acid and citric acid, as well as the accumulation of intracellular OAA and PEP, which may in turn result in the decrease in the glucose uptake rate. Accodingly, the specific growth rate and enzyme production rate was decreased in A niger CBS 513.88. This result initially revealed the regulation mechanisms of energy and redox metabolism on flux redistribution in A. niger.Based on A. niger GSMM iMA871, we updated the A. niger GSMM by combining the latest genome annotation and literature mining technology. To improve the metabolism coverage of iHL1210, the biosynthesis pathways of vitamins and cofactors were significantly added (including 52 reactions). Compared with iMA871, the reaction number in the updated model iHL 1210 was increased from 1380 to 1771, and the unique ORF number from 871 to 1210. Data from chemostat cultivations and 13C labelled experiments were used to check the iHL1210’s performance. The average correlation coefficients between the predicted fluxes and measured 13C metabolic fluxes was above 0.89. With the updated model, the cell growth on most of carbon and nitrogen sources could be predicted and the percentages of accuracy were 83% and 73% respectively.224 and 153 genes were predicted to be essential genes on synthetic and yeast extract medium respectively by iHL1210. The effects of NADPH supply on the A. niger potential for enzyme production showed that 22 reactions can support the cell growth and enzyme production, and the in vivo existence of 8 reactions could be verified from reported transcriptomic and proteomics studies. By comparison, the simulated maximum yield of glucoamylase is highest if the NADPH is originated from pentose phosphate (PP) pathway. The MOMA simulation also indicated that if the genes of PP pathway, encoding glucose 6-phosphate 1-dehydrogenase and phosphogluconate dehydrogenase, were inactivated, the maximum yield of glucoamylase would be reduced by 16.9%. In summary, the new A. niger GSMM iHL1210 has obvious improvements with respect to the metabolic coverage and prediction performance, which paves the way for systematic metabolic engineering of A. niger.Lastly, guiding by vertical omics analysis, the metabolism of A. niger under different cultivation process were studied using the integrating analysis of metabolomics, fluxomics and transcriptomics. During the oxygen limited phase, A. niger was in higher redox condition, as well as in lack of energy supply, which was characterized by the accumulation of the intermediates from TC A cycle, the downregulation of genes from fatty acid synthesis and the rapid decrease in specific growth rate. Once the oxygen limitation process began, the metabolites pool sizes of intermediates from PP pathway were quickly reduced, accompanied with the downregulation of most genes from PP pathway. Though the pool sizes of the intermediates of upper EMP pathway were also rapidly decreased, the expression of most genes from lower EMP pathway were maintained at a relatively high level. The A. niger GSMM was adopted to predict the possible flux distribution. The results showed that the relative fluxes of PP pathway were nearly stable while the relative fluxes of EMP pathway were increased obviously, which to some extent relieve the energy demand for cell metabolism. To maintain the redox balance, the genes of y-Aminobutyric Acid (GABA) pathway and glyoxylate bypass were obviously activated. The flux simulation also showed that the relative flux through glyoxylate bypass was obviously improved. The GABA pathway and glyoxylate bypass are branches of TCA cycle and the up-regulation in these two pathways could help to reduce the NADH formation. During the oxygen limited phase, the pool sizes of intracellular amino acids exhibited distinct differences. The pool sizes of serine, aspartate and alanine decreased greatly, which were consistent with the down-regulation of the initial genes in synthesis pathway of these amino acids. On the contrary, histidine, lysine, proline, etc., were accumulated and the genes expression for these amino acids synthesis were maintained at a stable level. The effects of addition of extracelluar amino acids showed that those amino acids with quick decrease in pool sizes were the main limited amino acids for glucoamylase synthesis.The above study reveals the metabolic regulating mechanism of energy and reducing power in glucoamylase production by A.niger. Furthermore, the vertical omics integrative analysis based on 13C metabolic flux analysis and GSMM was explored, which provides valuable clues for systematic optimization and design of A. niger microbial cell factory. |
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