Genetic Characterization of Mutations Related to Conidiophore Stalk Length Development in Aspergillus niger Laboratory Strain N402

Abstract

Aspergillus niger is an important filamentous fungus in industrial biotechnology for the production of citric acid and enzymes. In the late 1980s, the A. niger N400/NRRL3 strain was selected for both fundamental and applied studies in relation to several processes including gluconic acid and protein production. To facilitate handling of A. niger, the N400 wild-type strain was UV mutagenized in two consecutive rounds to generate N401 and N402. N402 was used as a reference laboratory strain and exhibits the phenotypes with reduced conidiophore stalk length and reduced radial growth. The conidiophore stalk length and radial growth of A. niger strain N400 were determined and compared to N401 and N402. The length of N400 conidiophore stalks (2.52 ± 0.40 mm) was reduced in N401 and N402 to 0.66 ± 0.14 mm and 0.34 ± 0.06 mm, respectively. Whereas N400 reached a colony diameter of 6.7 ± 0.2 cm after 7 days, N401 and N402 displayed reduced radial growth phenotype (4.3 ± 0.1 and 4.1 ± 0.1, respectively). To identify the mutations (dubbed cspA and cspB) responsible for the phenotypes of N401 and N402, the genomes were sequenced and compared to the N400 genome sequence. A parasexual cross was performed between N400 and N402 derivatives to isolate segregants which allowed cosegregation analysis of single nucleotide polymorphisms and insertions and deletions among the segregants. The shorter conidiophore stalk and reduced radial growth in N401 (cspA) was found to be caused by a 9-kb deletion on chromosome III and was further narrowed down to a truncation of NRRL3_03857 which encodes a kinesin-like protein homologous to the A. nidulans UncA protein. The mutation responsible for the further shortening of conidiophore stalks in N402 (cspB) was found to be caused by a missense mutation on chromosome V in a hitherto unstudied C2H2 transcription factor encoded by the gene NRRL3_06646. The importance of these two genes in relation to conidiophore stalk length and radial growth was confirmed by single and double gene deletion studies. The mutations in the laboratory strain N402 should be taken into consideration when studying phenotypes in the N402 background.

Genome sequences of 24 Aspergillus niger sensu stricto strains to study strain diversity, heterokaryon compatibility, and sexual reproduction

“…Strains in this clade are best known for their organic acid production, such as the citric acid producer A. niger ATCC1015 ( Andersen et al 2011 ) and strain CBS131.51 (synonym CBS769.97, ATCC6275), which was part of a patent from 1,977 for citric acid production. Additionally, this clade contains A. niger NRRL3 (synonym N400) and A. niger ATCC64974 (synonym N402) which have been initially selected for gluconic acid production, and thereafter have extensively been used in laboratory studies ( Bos et al 1988 ; Demirci et al 2021 ).…”

A Special Phenotype of Aconidial Aspergillus niger SH2 and Its Mechanism of Formation via CRISPRi

“…The strains used in this study are listed in Supplemental information-S1 Table S1. The following media were used in this study: Luria-Bertani medium (LB) containing (g L −1 ) tryptone (10), yeast extract (5), and NaCl (10); Czapek-Dox medium (CD) containing (g L −1 ) glucose (20), KCl (2), MgSO 4 •7H 2 O (0.5), KH 2 PO 4 (1), NaNO 3 (3), FeSO 4 •7H 2 O (0.01), pH 5.5; Hyper osmosis CD medium (HCD) containing (g L −1 ) sucrose (342.3), KCl (2), MgSO 4 •7H 2 O (0.5), KH 2 PO 4 (1), NaNO 3 (3), FeSO 4 •7H 2 O (0.01), pH 5.5; N-acetyl-D-glucosamine Czapek-Dox medium (NCD) containing (g L −1 ) N-acetyl-D-glucosamine (20), KCl (2), MgSO 4 •7H 2 O (0.5), KH 2 PO 4 (1), NaNO 3 (3), FeSO 4 •7H 2 O (0.01), pH 5.5; N-acetyl-D-glucosamine and glucose Czapek-Dox medium (NGCD) containing (g L −1 ) N-acetyl-D-glucosamine (20), glucose (20), KCl (2), MgSO 4 •7H 2 O (0.5), KH 2 PO 4 (1), NaNO 3 (3), FeSO 4 •7H 2 O (0.01), pH 5.5; Dextrose peptone yeast extract medium (DPY) containing (g L −1 ) glucose (20), tryptone (10), yeast extract (5), KH 2 PO 4 (5), MgSO 4 •7H 2 O (0.5); Potato dextrose agar medium (PDA) containing (g L −1 ) potato extract powder (300), glucose (20), agar (2), chloramphenicol (0.1). The solid medium and semi-solid medium contained 2% (w:v) and 0.5% (w:v) agar, respectively.…”

“…The aims of the present study were: (1) to explore chemical agents such as N-acetyl-Dglucosamine (GlcNAc) with the potential for inducing the production of pure homokaryotic strains (i.e., the spore-like propagule) from aconidial Aspergillus niger SH2; (2) to determine the life activities of homokaryotic strains (spore-like propagule), such as drug resistance, genetic transformation, and germination; (3) to form a CRISPRi system in aconidial Aspergillus niger SH2; (4) to analyze the effect of genes or pathways on mediating the spore-like propagule response to N-acetyl-D-glucosamine using transcriptomic analysis and the CRISPRi system; and (5) to determine the essential genes for controlling phenotypic change in Aspergillus niger SH2. This study was expected to improve our understanding of the critical factors involved in the mechanism for phenotypic change in Aspergillus niger SH2 and provide a fine model for researching phenotypic change in filamentous fungi.…”

“…Fungi are one of the most used species in the industry for enzyme production [ 1 , 2 ]. The filamentous fungus Aspergillus niger is widely used as a cellular factory to produce native or heterologous proteins, organic acids, drugs, etc.…”

“…The filamentous fungus Aspergillus niger is widely used as a cellular factory to produce native or heterologous proteins, organic acids, drugs, etc. [ 1 , 3 ], due to its secretion efficiency, high-production capacity, capability of carrying out posttranslational modifications, and metabolic diversity [ 4 , 5 ]. However, the high productivity of the Aspergillus niger is strongly related to its specific morphological form [ 6 , 7 ].…”