| Aspergillus niger,a commercial important cell factory,is widely used in the production of organic acids such as citric acid and enzyme preparations such as glucoamylase.Oxygen limitation is the most attractive strategy for industrial glucoamylase fermentation in A.niger.In order to establish a dynamic regulation system for intracellular metabolism in response to oxygen-limited conditions,a critical environmental signal during fermentation,we uncovered the hypoxia-responsive promoters from A.niger and implemented the preliminary research on the structure and function of the promoters.Based on the transcriptome analysis of the glucoamylase fermentation in A.niger,ten upregulated candidate genes under oxygen-limited conditions were selected at first.The transcription levels of these candidate genes under different oxygen availability conditions on flask-level were verified by RT-qRCR,in which two genes showing the highest up-regulation level under low oxygen availability conditions were identified,namely srbB and MIP aquaporin.Their 2000 bp upstream sequences were preliminarily defined as promoter sequences to drive the expression of the enhanced green fluorescent protein(EGFP)used as a reporter gene.Then the cloned plasmids were integrated into the uridine auxotrophic A.niger host and single-copy integrated transformants were selected for subsequent experiments.In this thesis,flask-level cultures with high or low oxygen availability were established,and the performance of the reporter gene under the control of two promoters on diverse oxygen availability conditions was investigated on the protein and the transcription level.However,the fluorescence of PMIPdriven EGFP expression was not detected during submerged fermentation.Thus,its reverse promoter sequence PfhbA was investigated for its hypoxia response ability.Flask-level results indicated that both PsrbB and PfhbA are hypoxic-inducible promoters.Notably,PsrbB is a strong promoter,whose expression level under high oxygen availability is higher than the widely used constitutive promoter PgpdA in A.niger.The highest specific fluorescence intensity of EGFP driven by PsrbB induced by low oxygen availability reached 4.83 times above that of PgpdA and 3.13 folds higher than that under high oxygen availability conditions.Reversely,the specific fluorescence intensity of EGFP controlled by the PfhbA promoter under high oxygen availability conditions was much lower than PgpdA,whereas which notably increased to 3.08 folds compared to that of PgpdA under hypoxia conditions,leading to a ratio of 8.02 between the low and high availability conditions.The above results indicate that PsrbB with high promoter strength enables to play a critical role in regulating the high efficient protein production in A.niger under different oxygen availability conditions.Whereas,PfhbA is a putative ideal hypoxia-inducible promoter,as it is more sensitive to the changed oxygen availability and its basal expression level is lower than PsrbB,revealing its promising role in the dynamic metabolism engineering under hypoxia condition.To investigate the performance of the above two promoters in regulating heterologous protein production,these two promoters were tested to regulate the expression of exogenous βglucuronidase(GUS)in A.niger.The results showed that the expression tendency of GUS regulated by the two promoters was comparable to EGFP.The highest specific enzyme activity of GUS under the control of PfhbA induced by low oxygen availability reached 1.75 times than that of PgpdA and 2.86 fold higher than that under high oxygen availability conditions.Owing to the intense basal expression level of PsrbB,it is less sensitive to hypoxia signal than PfhbA,and the expression of GUS under control of PsrbB is not significant as that of PfhbA.The highest specific enzyme activity of GUS increased to 2.49 folds above its specific fluorescence intensity regulated by PgpdA under hypoxia conditions,leading to a ratio of 1.92 between the low and high availability conditions.In sum,these two promoters exhibit strong and stable hypoxiainducible properties in the regulation of heterologous protein expression.To shed light on the core structural elements of PsrbB,its core region was predicted by bioinformatics analysis in silico.According to the prediction results,PsrbB was truncated into 1690 bp,1024 bp and 588 bp fragments to evaluate their regulation on EGFP.It was observed that when the promoter PsrbB was truncated into 588 bp,it is unable to be induced by hypoxia accompanied by a dramatically reduced gene expression level regulated by this promoter.Therefore,it is hypothesized that the transcription factor binding site associated with hypoxia response and the core promoter region of PsrbB are located in the region of-588 to-1024 bp.Based on the prediction for the binding sites in the-588 to-1024 bp region of PsrbB via several commonly used transcription factor databases,this paper identified binding sites for CAAT-box and stress-responsive elements,and some hypoxia-responsive signaling protein such as sterol regulatory element binding proteins and GABA-dependent transcription factors in this core functional region.In summary,based on oxygen-limited transcriptomic data and flask-level cultivations under low oxygen availability conditions,two hypoxia-inducible promoters PsrbB and PfhbA are identified in A.niger for the first time.Notably,PfhbA is more responsive to hypoxia signals,and exhibits more obvious regulation effect on heterologous protein expression under hypoxia conditions.This dissertation provides a novel and promising tool for the modulation of the target gene under oxygen-limited conditions,which is of great significance for the development of dynamic metabolic regulation strategies for A.niger fermentation. |
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