Comparative genomics of the aconidial Aspergillus niger strain LDM3 predicts genes associated with its high protein secretion capacity

Abstract

Aspergillus niger is widely used as a cell factory for homologous and heterologous protein production. As previous studies reported that reduced sporulation favors protein secretion in A. niger, in this study we conducted a comparative genomic analysis of the non-sporulating industrially exploited A. niger strain LDM3 in China and the model protein secretion strain CBS 513.88 to predict the key genes that might define the genetic basis of LDM3's high protein producing potential in silico. After sequencing using a hybrid approach combining Illumina and PacBio sequencing platforms, a high-quality genome sequence of LDM3 was obtained which harbors 11,209 open reading frames (ORFs) and exhibits large chromosomal rearrangements in comparison to CBS 513.88. An alignment of the two genome sequences revealed that the majority of the 457 ORFs uniquely present in LDM3 possessed predicted functions in redox pathways, protein transport, and protein modification processes. In addition, bioinformatic analyses revealed the presence of 656 ORFs in LDM3 with non-synonymous mutations encoding for proteins related to protein translation, protein modification, protein secretion, metabolism and energy production. We studied the impact of two of these on protein production in the established lab model strain N402. Both tupA and prpA genes were selected because available literature suggested their involvement in asexual sporulation of A. niger. Our co-expression network analysis supportively predicted the role of tupA in protein secretion and the role of prpA in energy generation, respectively. By knockout experiments, we showed that the ΔtupA mutant displayed reduced sporulation (35%) accompanied by higher total protein secretion (65%) compared to its parental strain. Such an effect was, however, not observed in the ΔprpA mutant.

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.…”

“…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 ].…”

Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification

“…starch) to release nutrients [ 7 , 8 ]. The filamentous fungus A. niger is used as microbial cell factory for production of lignocellulolytic enzymes and amylolytic enzymes [ 9 – 12 ]. Amylolytic enzymes that hydrolyze the glycosidic linkages in α-glucans belong to three glycoside hydrolases (GHs) families: GH13 (α-amylases), GH14 (β-amylases) and GH15 (glucoamylases) [ 5 , 13 , 14 ].…”

Aspergillus Metabolome Database for Mass Spectrometry Metabolomics

“…Omic sciences such as genomics [ 19 , 20 , 21 ], transcriptomics [ 22 , 23 , 24 ], proteomics [ 25 , 26 , 27 ], and more recently metabolomics [ 28 , 29 , 30 , 31 ], as well as multi-omics approaches [ 32 , 33 ] have been applied to study the genus Aspergillus . Metabolomics is considered to be the omic that best reflects the phenotype response [ 34 , 35 , 36 ].…”