Exploration and characterization of hypoxia-inducible endogenous promoters in Aspergillus niger

Xiao, Xianzun; Ouyang, Liming; Qi, Jie; Chu, Ju

doi:10.1007/s00253-021-11417-5

Cited by 6 publications

(8 citation statements)

References 51 publications

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“…In contrast to bacteria and single-celled eukaryotes [ 21 , 22 ], the heterogeneous cultures with complex pelleted and dispersed morphologies made it impossible to measure biomass by cell turbidity. However, determining cell dry weight is usually tedious and time consuming [ 15 , 16 , 25 ]. To eliminate the influence of cell biomass variance and simplify this promoter strength detection procedure, the fluorescence value distribution of conidia populations determined by flow cytometry was applied in this study to reflect the promoter activity ( Figure 1 ).…”

Section: Discussionmentioning

confidence: 99%

“…Compared to well-established methods in bacteria and single-celled eukaryotes, such as Saccharomyces cerevisiae [ 21 , 22 ], accurate and efficient promoter evaluation approaches are rarely available for filamentous fungi due to the filamentous physiological features of irregular morphologies, which limits the discovery and evaluation of promoters. Traditionally, some enzymes are applied as reporters for promoter evaluation in filamentous fungi, including luciferase [ 12 , 13 ], β-galactosidase of E. coli [ 16 , 23 , 24 ], and β-glucuronidase of E. coli [ 15 , 25 ]. However, such enzyme-based approaches involve many labor-intensive and time-consuming steps for activity detection, for example, releasing target proteins from a mycelial culture and determining dry cell weight for accurate enzymatic activity data normalization.…”

Section: Introductionmentioning

confidence: 99%

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Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production

Zheng

Wang

et al. 2022

JoF

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Aspergillus niger is an important industrial workhorse for the biomanufacturing of organic acids, proteins, etc. Well-controlled genetic regulatory elements, including promoters, are vital for strain engineering, but available strong promoters for A. niger are limited. Herein, to efficiently assess promoters, we developed an accurate and intuitive fluorescent-auxotrophic selection workflow based on mCherry, pyrG, CRISPR/Cas9 system, and flow cytometry. With this workflow, we characterized six endogenous constitutive promoters in A. niger. The endogenous glyceraldehyde-3-phosphate dehydrogenase promoter PgpdAg showed a 2.28-fold increase in promoter activity compared with the most frequently used strong promoter PgpdAd from A. nidulans. Six predicted conserved motifs, including the gpdA-box, were verified to be essential for the PgpdAg activity. To demonstrate its application, the promoter PgpdAg was used for enhancing the expression of citrate exporter cexA in a citric acid-producing isolate D353.8. Compared with the cexA controlled by PgpdAd, the transcription level of the cexA gene driven by PgpdAg increased by 2.19-fold, which is consistent with the promoter activity assessment. Moreover, following cexA overexpression, several genes involved in carbohydrate transport and metabolism were synergically upregulated, resulting in up to a 2.48-fold increase in citric acid titer compared with that of the parent strain. This study provides an intuitive workflow to speed up the quantitative evaluation of A. niger promoters and strong constitutive promoters for fungal cell factory construction and strain engineering.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production

Zheng

Wang

et al. 2022

JoF

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“…The sampling and quantification of intracellular metabolites was slightly modified based on Wang et al [ 19 ]. 1–2 mL culture broth (precisely weighed) was transferred by a rapid sampling protocol from bioreactor into 10 mL precooled quench solution (60% v/v methanol at − 30 °C).…”

Section: Methodsmentioning

confidence: 99%

“…The quantification methods of gene copy number and RNA level were adapted from Xiao et al [ 19 ]. To determine copy number of target genes in positive transformants, the genome DNA was extracted as described in Arentshorst et al [ 17 ].…”

Section: Methodsmentioning

confidence: 99%

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

Xiao

Ouyang

et al. 2022

Microb Cell Fact

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Background Our recent multi-omics analyses of glucoamylase biosynthesis in Aspergillus niger (A. niger) suggested that lipid catabolism was significantly up-regulated during high-yield period under oxygen limitation. Since the catabolism of fatty acids can provide energy compounds such as ATP and important precursors such as acetyl-CoA, we speculated that enhancement of this pathway might be beneficial to glucoamylase overproduction. Results Based on previous transcriptome data, we selected and individually overexpressed five candidate genes involved in fatty acid degradation under the control of the Tet-on gene switch in A. niger. Overexpression of the fadE, fadA and cyp genes increased the final specific enzyme activity and total secreted protein on shake flask by 21.3 ~ 31.3% and 16.0 ~ 24.2%, respectively. And a better inducible effect by doxycycline was obtained from early logarithmic growth phase (18 h) than stationary phase (42 h). Similar with flask-level results, the glucoamylase content and total extracellular protein in engineered strains OE-fadE (overexpressing fadE) and OE-fadA (overexpressing fadA) on maltose-limited chemostat cultivation were improved by 31.2 ~ 34.1% and 35.1 ~ 38.8% compared to parental strain B36. Meanwhile, intracellular free fatty acids were correspondingly decreased by 41.6 ~ 44.6%. The metabolomic analysis demonstrated intracellular amino acids pools increased 24.86% and 18.49% in two engineered strains OE-fadE and OE-fadA compared to B36. Flux simulation revealed that increased ATP, acetyl-CoA and NADH was supplied into TCA cycle to improve amino acids synthesis for glucoamylase overproduction. Conclusion This study suggested for the first time that glucoamylase production was significantly improved in A. niger by overexpression of genes fadE and fadA involved in fatty acids degradation pathway. Harnessing the intracellular fatty acids could be a strategy to improve enzyme production in Aspergillus niger cell factory.

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“…In general, the construction and screening of promoter libraries require stable and widely expressed reporter genes as quantitative standards in order to realize the quick and accurate determination of promoter activities. The commonly used reporter genes for filamentous fungi include genes encoding fluorescent protein, [21,22] β-glucuronidase (GUS), [23,24] and β-galactosidase (β-gal). [25] Bidirectional promoters are located between two adjacent genes in opposite transcription directions.…”

Section: Key Pointsmentioning

confidence: 99%

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum

Liu,

Li,

Zhang

et al. 2024

Biotechnology Journal

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Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter PAngpdA. Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high‐yielding strains AG‐6, AG‐18, and AG‐41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac‐∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression.

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Exploration and characterization of hypoxia-inducible endogenous promoters in Aspergillus niger

Cited by 6 publications

References 51 publications

Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production

Evaluation of Aspergillus niger Six Constitutive Strong Promoters by Fluorescent-Auxotrophic Selection Coupled with Flow Cytometry: A Case for Citric Acid Production

Enhancement of fatty acid degradation pathway promoted glucoamylase synthesis in Aspergillus niger

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum

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