Characterization of the promoter of the nitrate transporter-encoding gene nrtA in Aspergillus nidulans

Liu, Yangyi; Li, Haoxiang; Li, Jingyi; Zhou, Yao; Zhou, Zhemin; Wang, Ping; Zhou, Shengmin

doi:10.1007/s00438-020-01700-x

Cited by 3 publications

(5 citation statements)

References 29 publications

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“…Bradford method was used to determine the protein concentration. The activity of GUS was measured using 4-methylumbelliferyl-b-glucuronide (4-MUG) as substrate, and quantified by the fluorescence intensity of the product 4-methylumbelliferone (4-MU) [ 48 ]. The fluorescence intensity of 4-MU was detected with microplate reader with the excitation wavelength of 365 nm and the emission wavelength of 455 nm.…”

Section: Methodsmentioning

confidence: 99%

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation

et al. 2022

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Background The construction of protein expression systems is mainly focused on carbon catabolite repression and quorum-sensing systems. However, each of these regulatory modes has an inherent flaw, which is difficult to overcome. Organisms also prioritize using different nitrogen sources, which is called nitrogen catabolite repression. To date, few gene regulatory systems based on nitrogen catabolite repression have been reported. Results In this study, we constructed a nitrogen switching auto-inducible expression system (NSAES) based on nitrogen catabolite regulation and nitrogen utilization in Aspergillus nidulans. The PniaD promoter that is highly induced by nitrate and inhibition by ammonia was used as the promoter. Glucuronidase was the reporter protein. Glucuronidase expression occurred after ammonium was consumed in an ammonium and nitrate compounding medium, achieving stage auto-switching for cell growth and gene expression. This system maintained a balance between cell growth and protein production to maximize stress products. Expressions of glycosylated and secretory proteins were successfully achieved using this auto-inducible system. Conclusions We described an efficient auto-inducible protein expression system based on nitrogen catabolite regulation. The system could be useful for protein production in the laboratory and industrial applications. Simultaneously, NSAES provides a new auto-inducible expression regulation mode for other filamentous fungi.

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Section: Methodsmentioning

confidence: 99%

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation

et al. 2022

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“…catB.P and trpC.T were cloned from the genomic DNA of A. nidulans A6 with the primers catB.P-F/catB.P-R and trpC.T-2F/trpC.T-2R, respectively. uidA was cloned from E. coli genomic DNA with the primers uidA-F/uidA-R [19]. The resulting DNA fragments, with the linearized pUC19-pyrG, were used to construct pUC19-pyrG-catB.P-uidA-trpC.T.…”

Section: Construction Of Plasmids For Subsequent Recombinant Strain C...mentioning

confidence: 99%

“…For the GUS assay, cell lysates were resuspended in GUS assay buffer (50 mM sodium phosphate buffer pH 7.0, 10 mM β-mercaptoethanol, 10 mM Na 2 EDTA, and 0.1% Triton X-100). After centrifugation, the supernatant was subjected to fluorometric analysis of GUS activity, as described previously [19]. Fluorometric analysis of GUS activity was performed using 4-methylumbelliferyl-b-glucuronide (4-MUG) as a substrate.…”

Section: Quantification Of Intracellular Gfp Levels and β-Glucuronida...mentioning

confidence: 99%

“…The primer pairs RT-catB-F/RT-catB-R and RT-actA-F/RT-actA-R (Table S8) were used to quantify the catB and actA genes, respectively. The relative mRNA levels were normalized to that of the reference gene actA using the 2 −∆∆Ct method of relative quantification [14,19]. The experiment was repeated thrice.…”

Section: Quantitative Real-time Pcrmentioning

confidence: 99%

“…We predicted that the different intracellular levels of PrxA and CatB may be responsible for their varying contribution to H 2 O 2 resistance at different stages of cell growth. To verify this, we constructed catB.P-uidA and prxA.P-uidA strains in which uidA, the β-glucuronidase (GUS) encoding gene [19], was expressed under the promoters of catB and prxA, respectively (Figure S3). GUS activities of catB.P-uidA and prxA.P-uidA strains were used to investigate the promoter activities of both genes during the growth stages from dormant conidia to mature hyphae.…”

Section: Prxa and Catb Play Nonredundant Roles In Defense Against H 2...mentioning

confidence: 99%

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Cytosol Peroxiredoxin and Cell Surface Catalase Differentially Respond to H2O2 Stress in Aspergillus nidulans

et al. 2023

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Both catalase and peroxiredoxin show high activities of H2O2 decomposition and coexist in the same organism; however, their division of labor in defense against H2O2 is unclear. We focused on the major peroxiredoxin (PrxA) and catalase (CatB) in Aspergillus nidulans at different growth stages to discriminate their antioxidant roles. The dormant conidia lacking PrxA showed sensitivity to high concentrations of H2O2 (>100 mM), revealing that PrxA is one of the important antioxidants in dormant conidia. Once the conidia began to swell and germinate, or further develop to young hyphae (9 h to old age), PrxA-deficient cells (ΔprxA) did not survive on plates containing H2O2 concentrations higher than 1 mM, indicating that PrxA is an indispensable antioxidant in the early growth stage. During these early growth stages, absence of CatB did not affect fungal resistance to either high (>1 mM) or low (<1 mM) concentrations of H2O2. In the mature hyphae stage (24 h to old age), however, CatB fulfills the major antioxidant function, especially against high doses of H2O2. PrxA is constitutively expressed throughout the lifespan, whereas CatB levels are low in the early growth stage of the cells developing from swelling conidia to early growth hyphae, providing a molecular basis for their different contributions to H2O2 resistance in different growth stages. Further enzyme activity and cellular localization analysis indicated that CatB needs to be secreted to be functionalized, and this process is confined to the growth stage of mature hyphae. Our results revealed differences in effectiveness and timelines of two primary anti-H2O2 enzymes in fungus.

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Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae

Miki,

Sakai,

Nakagawa

et al. 2024

Journal of Bioscience and Bioengineering

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Characterization of the promoter of the nitrate transporter-encoding gene nrtA in Aspergillus nidulans

Cited by 3 publications

References 29 publications

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation

Development of an auto-inducible expression system by nitrogen sources switching based on the nitrogen catabolite repression regulation

Cytosol Peroxiredoxin and Cell Surface Catalase Differentially Respond to H2O2 Stress in Aspergillus nidulans

Analysis of nitrogen source assimilation in industrial strains of Aspergillus oryzae

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