The Amylolytic Regulator AmyR of Aspergillus niger Is Involved in Sucrose and Inulin Utilization in a Culture-Condition-Dependent Manner

Filamentous fungi degrade complex plant material to its monomeric building blocks, which have many biotechnological applications. Transcription factors play a key role in plant biomass degradation, but little is known about their interactions in the regulation of polysaccharide degradation. Here, we deepened the knowledge about the storage polysaccharide regulators AmyR and InuR in Aspergillus niger. AmyR controls starch degradation, while InuR is involved in sucrose and inulin utilization. In our study, the phenotypes of A. niger parental, ΔamyR, ΔinuR and ΔamyRΔinuR strains were assessed in both solid and liquid media containing sucrose or inulin as carbon source to evaluate the roles of AmyR and InuR and the effect of culture conditions on their functions. In correlation with previous studies, our data showed that AmyR has a minor contribution to sucrose and inulin utilization when InuR is active. In contrast, growth profiles and transcriptomic data showed that the deletion of amyR in the ΔinuR background strain resulted in more pronounced growth reduction on both substrates, mainly evidenced by data originating from solid cultures. Overall, our results show that submerged cultures do not always reflect the role of transcription factors in the natural growth condition, which is better represented on solid substrates. Importance: The type of growth has critical implications in enzyme production by filamentous fungi, a process that is controlled by transcription factors. Submerged cultures are the preferred setups in laboratory and industry and are often used for studying the physiology of fungi. In this study, we showed that the genetic response of A. niger to starch and inulin was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment did not fully match the behavior of the fungus in a solid environment. These results have direct implications in enzyme production and would help industry choose the best approaches to produce specific CAZymes for industrial purposes.

[1]  Diane Bauer,et al.  Unraveling the regulation of sugar beet pulp utilization in the industrially relevant fungus Aspergillus niger , 2022, iScience.

[2]  R. D. de Vries,et al.  The Cultivation Method Affects the Transcriptomic Response of Aspergillus niger to Growth on Sugar Beet Pulp , 2021, Microbiology spectrum.

[3]  R. D. de Vries,et al.  CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass. , 2020, Enzyme and microbial technology.

[4]  Lianggang Huang,et al.  The transcription factor PrtT and its target protease profiles in Aspergillus niger are negatively regulated by carbon sources , 2020, Biotechnology Letters.

[5]  R. D. de Vries,et al.  Genomic and exoproteomic diversity in plant biomass degradation approaches among Aspergilli , 2018, Studies in mycology.

[6]  Adrian Tsang,et al.  Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger , 2018, PloS one.

[7]  R. D. de Vries,et al.  In Silico Analysis of Putative Sugar Transporter Genes in Aspergillus niger Using Phylogeny and Comparative Transcriptomics , 2018, Front. Microbiol..

[8]  R. D. de Vries,et al.  Expression-based clustering of CAZyme-encoding genes of Aspergillus niger , 2017, BMC Genomics.

[9]  J. Visser,et al.  Combinatorial control of gene expression in Aspergillus niger grown on sugar beet pectin , 2017, Scientific Reports.

[10]  R. D. de Vries,et al.  Regulators of plant biomass degradation in ascomycetous fungi , 2017, Biotechnology for Biofuels.

[11]  V. M. D. Martins dos Santos,et al.  Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters , 2015, Biotechnology for Biofuels.

[12]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[13]  R. D. de Vries,et al.  Plant biomass degradation by fungi. , 2014, Fungal genetics and biology : FG & B.

[14]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[15]  Pedro M. Coutinho,et al.  The carbohydrate-active enzymes database (CAZy) in 2013 , 2013, Nucleic Acids Res..

[16]  J. Visser,et al.  A broader role for AmyR in Aspergillus niger: regulation of the utilisation of d-glucose or d-galactose containing oligo- and polysaccharides , 2011, Applied Microbiology and Biotechnology.

[17]  H. Nakano,et al.  High-throughput screening of DNA binding sites for transcription factor AmyR from Aspergillus nidulans using DNA beads display system. , 2010, Journal of bioscience and bioengineering.

[18]  J. Nielsen,et al.  Post-genomic insights into the plant polysaccharide degradation potential of Aspergillus nidulans and comparison to Aspergillus niger and Aspergillus oryzae. , 2009, Fungal genetics and biology : FG & B.

[19]  Masashi Kato,et al.  Inducer-Dependent Nuclear Localization of a Zn(II)2Cys6 Transcriptional Activator, AmyR, in Aspergillus nidulans , 2009, Bioscience, biotechnology, and biochemistry.

[20]  Guanglei Liu,et al.  Inulinase-expressing microorganisms and applications of inulinases , 2009, Applied Microbiology and Biotechnology.

[21]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[22]  Peter J. Punt,et al.  Aspergillus niger genome-wide analysis reveals a large number of novel alpha-glucan acting enzymes with unexpected expression profiles , 2008, Molecular Genetics and Genomics.

[23]  J. A. Roubos,et al.  Identification of InuR, a new Zn(II)2Cys6 transcriptional activator involved in the regulation of inulinolytic genes in Aspergillus niger , 2007, Molecular Genetics and Genomics.

[24]  Vera Meyer,et al.  Highly efficient gene targeting in the Aspergillus niger kusA mutant. , 2007, Journal of biotechnology.

[25]  T. Rabilloud,et al.  Silver staining of proteins in polyacrylamide gels , 2006, Nature Protocols.

[26]  L. Dijkhuizen,et al.  Database mining and transcriptional analysis of genes encoding inulin-modifying enzymes of Aspergillus niger. , 2006, Microbiology.

[27]  Ronald P. de Vries,et al.  A New Black Aspergillus Species, A. vadensis, Is a Promising Host for Homologous and Heterologous Protein Production , 2004, Applied and Environmental Microbiology.

[28]  B. Henrissat,et al.  The Three-dimensional Structure of Invertase (β-Fructosidase) from Thermotoga maritima Reveals a Bimodular Arrangement and an Evolutionary Relationship between Retaining and Inverting Glycosidases* , 2004, Journal of Biological Chemistry.

[29]  Tetsuo Kobayashi,et al.  Mode of AmyR Binding to the CGGN8AGG Sequence in the Aspergillus oryzaetaaG2 Promoter , 2004, Bioscience, biotechnology, and biochemistry.

[30]  J. Visser,et al.  Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides , 2001, Microbiology and Molecular Biology Reviews.

[31]  J. Nielsen,et al.  Influence of carbon source on α-amylase production by Aspergillus oryzae , 2001, Applied Microbiology and Biotechnology.

[32]  J. Visser,et al.  Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation. , 2001, European journal of biochemistry.

[33]  Masashi Kato,et al.  Characterization of the amyR gene encoding a transcriptional activator for the amylase genes in Aspergillus nidulans , 2001, Current Genetics.

[34]  K. Gomi,et al.  Molecular Cloning and Characterization of a Transcriptional Activator Gene, amyR, Involved in the Amylolytic Gene Expression in Aspergillus oryzae , 2000, Bioscience, biotechnology, and biochemistry.

[35]  K. L. Petersen,et al.  A new transcriptional activator for amylase genes in Aspergillus , 1999, Molecular and General Genetics MGG.

[36]  C. R. Soccol,et al.  Recent developments in microbial inulinases , 1999, Applied biochemistry and biotechnology.

[37]  L. Boddy,et al.  Purification and characterisation of an Aspergillus niger invertase and its DNA sequence , 1993, Current Genetics.

[38]  P. Williamson,et al.  Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization , 1992, Journal of bacteriology.

[39]  K. Kwon-Chung,et al.  A zinc finger protein from Candida albicans is involved in sucrose utilization , 1992, Journal of bacteriology.

[40]  S. Armitt,et al.  Mutants ofAspergillus nidulans lacking pyruvate carboxylase , 1972, FEBS letters.

[41]  K. Hildén,et al.  Grand Challenges in Fungal Biotechnology , 2020, Grand Challenges in Biology and Biotechnology.

[42]  R. P. Vries,et al.  The Current Biotechnological Status and Potential of Plant and Algal Biomass Degrading/Modifying Enzymes from Ascomycete Fungi , 2020 .

[43]  R. D. de Vries,et al.  Regulation of plant biomass utilization in Aspergillus. , 2014, Advances in applied microbiology.

[44]  J. Herdu Amylolytic families of glycoside hydrolases: focus on the family GH-57 , 2005 .

[45]  K. Ohta,et al.  Molecular cloning and characterization of an exoinulinase gene from Aspergillus niger strain 12 and its expression in Pichia pastoris. , 2003, Journal of bioscience and bioengineering.

[46]  H. Ronne Glucose repression in fungi. , 1995, Trends in genetics : TIG.