Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Discovery of the 2,4'-Dihydroxy-3'-methoxypropiophenone Biosynthesis Genes in Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae has 27 putative iterative type I polyketide synthase (PKS) gene clusters, but the secondary metabolites produced by them are mostly unknown. Here, we focused on eight clusters that were reported to be expressed at relatively high levels in a transcriptome analysis. By comparing metabolites between an octuple-deletion mutant of these eight PKS gene clusters and its parent strain, we found that A. oryzae produced 2,4'-dihydroxy-3'-methoxypropiophenone (1) and its precursor, 4'-hydroxy-3'-methoxypropiophenone (3) in a specific liquid medium. Furthermore, an iterative type I PKS (PpsB) encoded by AO090102000166 and an acetyl-CoA ligase (PpsA) encoded downstream from ppsB were shown to be essential for their biosynthesis. PpsC, encoded upstream from ppsB, was shown to have 3-binding activity (Kd =26.0±6.2 µM) and is suggested to be involved in the conversion of 3 to 1. This study deepens our understanding of cryptic secondary metabolism in A. oryzae.

A unique Zn(II)2-Cys6-type protein, KpeA, is involved in secondary metabolism and conidiation in Aspergillus oryzae.

Aspergillus oryzae is an important microorganism in the bio- and food industries; therefore, understanding the mechanism underlying its secondary metabolism regulation is important for ensuring its safe use. Here, we screened a novel Zn(II)2-Cys6-type protein-encoding gene, AO090003001186, designated as kpeA (kojic acid production enhancement A), from an A. oryzae disruption mutant library of transcriptional regulators. kpeA is highly conserved among filamentous fungi and encodes a protein with Zn(II)2-Cys6 motif located in the middle of the sequence. Phylogenetic analysis revealed that KpeA was classified into a distal group compared to other fungal Zn(II)2-Cys6-type transcriptional regulators. A Cys to Ala substitution mutant of KpeA showed identical phenotype to the kpeA disruption strain, confirming that KpeA is novel type Zn(II)2-Cys6 binding protein. Colonies of the kpeA disruption strain (ΔkpeA) had longer aerial hyphae and showed decreased conidia production. Microscopic analysis suggested that the reduced vesicle size and conidial head formation in ΔkpeA strain account for the decreased conidia production. Transcriptional levels of brlA and downstream abaA and wetA were decreased in ΔkpeA strain. Moreover, ΔkpeA strain produced 6-fold more kojic acid than the control strains, and the expression of kojR and kojA was increased in ΔkpeA strain. Therefore, KpeA is a novel Zn(II)2-Cys6-type protein likely involved in conidiation and kojic acid production at the transcriptional level.

The Basic-Region Helix-Loop-Helix Transcription Factor DevR Significantly Affects Polysaccharide Metabolism in Aspergillus oryzae.

Basic-region helix-loop-helix (bHLH) proteins are a superfamily of transcription factors that are often involved in the control of growth and differentiation. Recently, it was reported that the bHLH transcription factor DevR is involved in both asexual and sexual development in Aspergillus nidulans and regulates the conidial melanin production in Aspergillus fumigatus In this study, we identified and characterized an Aspergillus oryzae gene that showed high similarity with devR of A. nidulans and A. fumigatus (AodevR). In the AodevR-disrupted strain, growth was delayed and the number of conidia was decreased on Czapek-Dox (CD) minimal agar plates, but the conidiation was partially recovered by adding 0.6 M KCl. Simultaneously, the overexpression of AodevR was induced and resulted in extremely poor growth when the carbon source changed from glucose to polysaccharide (dextrin) in the CD agar plate. Scanning electron microscopy (SEM) indicated that the overexpression of AodevR resulted in extremely thin aberrant hyphal morphology. Conversely, the deletion of AodevR resulted in thicker hyphae and in more resistance to Congo red relative to the control strain. Quantitative reverse transcriptase PCR (RT-PCR) further indicated that AoDevR significantly affects chitin and starch metabolism, and importantly, the overexpression of AodevR inhibited the expression of genes related to starch degradation. A yeast one-hybrid assay suggested that the DevR protein possibly interacted with the promoter of amyR, which encodes a transcription factor involved in amylase production. Importantly, AoDevR is involved in polysaccharide metabolism and affects the growth of the A. oryzae strain.IMPORTANCEAspergillus oryzae is an industrially important filamentous fungus; therefore, a clear understanding of its polysaccharide metabolism and utilization is very important for its industrial utilization. In this study, we revealed that the basic-region helix-loop-helix (bHLH) transcription factor AoDevR is importantly involved in chitin and starch metabolism in A. oryzae The overexpression of AodevR strongly suppressed the expression of amylase-related genes. The results of a yeast one-hybrid assay suggested that the DevR protein potentially interacts with the promoter of amyR, which encodes a transcription factor involved in amylase production and starch utilization. This study provides new insight for further revealing the regulation mechanism of amylase production in A. oryzae.

Identification of a gene cluster for biosynthesis of the sesquiterpene antibiotic, heptelidic acid, in Aspergillus oryzae.

Heptelidic acid (HA), a sesquiterpene lactone, is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recently, we found that HA was produced by Aspergillus oryzae RIB40 and acted as the growth inhibitor of the salt-tolerant lactic acid bacteria in soy sauce brewing. Although several decades have passed since the discovery of HA, the genes involved in its biosynthesis and biosynthetic pathway have not yet been fully identified. In this study, we identified the HA biosynthetic gene cluster (HA cluster) using gene disruption and expression analysis. We also revealed that two transcription regulatory genes adjacent to the HA cluster were responsible for the expression of HA biosynthetic genes in A. oryzae. Interestingly, the HA cluster contained a gene encoding GAPDH (gpdB), which showed much higher resistance to HA than the GAPDH gene (gpdA) located at the other locus, but which did not seem to act as a self-resistant gene.

Production of the plant polyketide curcumin in Aspergillus oryzae: strengthening malonyl-CoA supply for yield improvement.

The filamentous fungus Aspergillus oryzae was recently used as a heterologous host for fungal secondary metabolite production. Here, we aimed to produce the plant polyketide curcumin in A. oryzae. Curcumin is synthesized from feruloyl-coenzyme A (CoA) and malonyl-CoA by curcuminoid synthase (CUS). A. oryzae expressing CUS produced curcumin (64 µg/plate) on an agar medium containing feruloyl-N-acetylcysteamine (a feruloyl-CoA analog). To increase curcumin yield, we attempted to strengthen the supply of malonyl-CoA using two approaches: enhancement of the reaction catalyzed by acetyl-CoA carboxylase (ACC), which produces malonyl-CoA from acetyl-CoA, and inactivation of the acetyl-CoA-consuming sterol biosynthesis pathway. Finally, we succeeded in increasing curcumin yield sixfold by the double disruption of snfA and SCAP; SnfA is a homolog of SNF1, which inhibits ACC activity by phosphorylation in Saccharomyces cerevisiae and SCAP is positively related to sterol biosynthesis in Aspergillus terreus. This study provided useful information for heterologous polyketide production in A. oryzae.

Comparison of the paralogous transcription factors AraR and XlnR in Aspergillus oryzae.

The paralogous transcription factors AraR and XlnR in Aspergillus regulate genes that are involved in degradation of cellulose and hemicellulose and catabolism of pentose. AraR and XlnR target the same genes for pentose catabolism but target different genes encoding enzymes for polysaccharide degradation. To uncover the relationship between these paralogous transcription factors, we examined their contribution to regulation of the PCP genes and compared their preferred recognition sequences. Both AraR and XlnR are involved in induction of all the pentose catabolic genes in A. oryzae except larA encoding L-arabinose reductase, which was regulated by AraR but not by XlnR. DNA-binding studies revealed that the recognition sequences of AraR and XlnR also differ only slightly; AraR prefers CGGDTAAW, while XlnR prefers CGGNTAAW. All the pentose catabolic genes possess at least one recognition site to which both AraR and XlnR can bind. Cooperative binding by the factors was not observed. Instead, they competed to bind to the shared sites. XlnR bound to the recognition sites mentioned above as a monomer, but bound to the sequence TTAGSCTAA on the xylanase promoters as a dimer. Consequently, AraR and XlnR have significantly similar, but not the same, DNA-binding properties. Such a slight difference in these paralogous transcription factors may lead to complex outputs in enzyme production depending on the concentrations of coexisting inducer molecules in the natural environment.

Comparative proteomic analysis: SclR is importantly involved in carbohydrate metabolism in Aspergillus oryzae.

The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes in organisms from mammals to microbes. We previously identified the bHLH transcription factor SclR in Aspergillus oryzae and found that the loss of SclR function led to significant phenotypic changes, such as rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. The result implied that SclR is potentially important in both traditional fermentative manufacturing and commercial enzyme production in A. oryzae because of its effect on growth. Therefore, this study presents a comparative assessment at the proteome level of the intracellular differences between an sclR-disrupted strain and a control strain using isobaric tandem mass tag (TMT) labeling for quantification. A total of 5447 proteins were identified, and 568 were differentially expressed proteins (DEPs). Of the DEPs, 251 proteins were increased by 1.5-fold, and 317 proteins were decreased by 1.5-fold in an sclR-disrupted strain compared to the control. The comparison of the quantitative TMT results revealed that SclR was mainly involved in carbon metabolism, especially carbohydrate metabolism. In addition, an enzyme profile by a semi-quantitative method (API-ZYM) indicated that three enzymes (ß-galactosidase, α-glucosidase, and α-mannosidase) were significantly less active in the ∆sclR strain than in the control. Moreover, quantitative RT-PCR showed that the expression of certain genes was changed similarly to their corresponding proteins. These results suggested that a possible function of SclR during growth of A. oryzae is its important involvement in carbohydrate metabolism.

Survival strategy of the salt-tolerant lactic acid bacterium, Tetragenococcus halophilus, to counteract koji mold, Aspergillus oryzae, in soy sauce brewing.

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.

McmA-dependent and -independent regulatory systems governing expression of ClrB-regulated cellulase and hemicellulase genes in Aspergillus nidulans.

Fungal cellulolytic and hemicellulolytic enzymes are promising tools for industrial hydrolysis of cellulosic biomass; however, the regulatory network underlying their production is not well understood. The recent discovery of the transcriptional activators ClrB and McmA in Aspergillus nidulans implied a novel regulatory mechanism driven by their interaction, experimental evidence for which was obtained from transcriptional and DNA-binding analyses in this study. It was found that ClrB was essential for induced expression of all the genes examined in this study, while McmA dependency of their expression was gene-dependent. DNA-binding studies revealed McmA assisted in the recruitment of ClrB to the cellulose-responsive element (CeRE) in the promoters of eglA and eglB, expression of which was significantly reduced in the mcmA mutant. The CCG triplet within the CeRE served as the recognition sequence for the ClrB monomer. In contrast, ClrB did not require McmA for binding as a homodimer to the CGGN8 CCG sequences in the promoter of mndB, expression of which was affected less in the mcmA mutant than in all other examined genes. Thus, there are two types of ClrB-mediated regulation: McmA-assisted and McmA-independent. This novel McmA-ClrB synergistic system provides new insights into the complex regulatory network involved in cellulase and hemicellulase production.

The C2H2-type transcription factor, FlbC, is involved in the transcriptional regulation of Aspergillus oryzae glucoamylase and protease genes specifically expressed in solid-state culture.

Aspergillus oryzae produces a large amount of secreted proteins in solid-state culture, and some proteins such as glucoamylase (GlaB) and acid protease (PepA) are specifically produced in solid-state culture, but rarely in submerged culture. From the disruption mutant library of A. oryzae transcriptional regulators, we successfully identified a disruption mutant showing an extremely low production level of GlaB but a normal level of α-amylase production. This strain was a disruption mutant of the C2H2-type transcription factor, FlbC, which is reported to be involved in the regulation of conidiospore development. Disruption mutants of other upstream regulators comprising a conidiation regulatory network had no apparent effect on GlaB production in solid-state culture. In addition to GlaB, the production of acid protease in solid-state culture was also markedly decreased by flbC disruption. Northern blot analyses revealed that transcripts of glaB and pepA were significantly decreased in the flbC disruption strain. These results suggested that FlbC is involved in the transcriptional regulation of genes specifically expressed under solid-state cultivation conditions, possibly independent of the conidiation regulatory network.

Cyclopiazonic acid biosynthesis gene cluster gene cpaM is required for speradine A biosynthesis.

Speradine A is a derivative of cyclopiazonic acid (CPA) found in culture of an Aspergillus tamarii isolate. Heterologous expression of a predicted methyltransferase gene, cpaM, in the cpa biosynthesis gene cluster of A. tamarii resulted in the speradine A production in a 2-oxoCPA producing A. oryzae strain, indicating cpaM is involved in the speradine A biosynthesis.

Efficient formation of heterokaryotic sclerotia in the filamentous fungus Aspergillus oryzae.

Heterokaryon formation by hyphal fusion occurs during a sexual/parasexual cycle in filamentous fungi, and therefore, it is biotechnologically important for crossbreeding. In the industrial filamentous fungus Aspergillus oryzae, a parasexual cycle has been reported, and it was recently suggested that sexual reproduction should be possible. However, as A. oryzae enters into hyphal fusion with a much lower frequency than Neurospora crassa, the process of heterokaryon formation has not been extensively characterized in A. oryzae. Here, we developed a detection system for heterokaryon formation by expressing red or green fluorescent proteins in nuclei and conferring uridine/uracil or adenine auxotrophy to MAT1-1 and MAT1-2 strains of A. oryzae. The heterokaryon formation of A. oryzae was investigated in paired culture using the genetically modified strains. No sclerotial formation was observed in the hyphal contact regions of the two strains with the same auxotrophy, whereas numerous sclerotia were formed between the strains with different auxotrophies. In most of the formed sclerotia, the uridine/uracil and adenine auxotrophies were complemented, and both red and green fluorescence were detected, indicating that heterokaryotic fusants were formed by hyphal fusion before or during sclerotial formation. Moreover, overexpressing the sclR gene, which encodes a transcription factor promoting sclerotial formation, increased the number of heterokaryotic sclerotia formed between the two auxotrophic strains. Notably, these effects in sclerotial formation of heterokaryotic fusants were observed independently of the mating type pairing combinations. Taken together, these findings demonstrated that paring of different auxotrophs and sclR overexpression promote the formation of heterokaryotic sclerotia in A. oryzae.

Purification and characterization of a glutaminase enzyme accounting for the majority of glutaminase activity in Aspergillus sojae under solid-state culture.

Glutaminase, an enzyme that hydrolyzes L-glutamine to L-glutamate, plays an important role in the production of fermented foods by enhancing the umami taste. In this study, we found ten glutaminase genes in the Aspergillus sojae genome by conducting a BLAST search of the characterized glutaminase sequence. We subsequently constructed glutaminase gene disruptants. The glutaminase activity of the gahB disruptant was decreased by approximately 90 % in A. sojae and Aspergillus oryzae, indicating that this enzyme (GahB) accounted for the majority of the glutaminase activity in Aspergillus species. Subsequently, GahB protein was purified from the AsgahB-overexpressing transformant and characterized. The molecular mass was estimated to be approximately 110 and 259 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahB was a dimer. The optimal pH was 9.0, and the optimal temperature was 50 °C. Analysis of substrate specificity revealed that AsGahB had peptidoglutaminase-asparaginase activity, similar to AsGahA, but preferred free L-glutamine to free L-asparagine, C-terminal glutaminyl, and asparaginyl residues in peptides.

ManR, a transcriptional regulator of the ß-mannan utilization system, controls the cellulose utilization system in Aspergillus oryzae.

ManR, a DNA-binding protein possessing the Zn(II)(2)Cys(6) binuclear cluster domain, acts as a positive regulator of mannanolytic enzyme genes in Aspergillus oryzae. In this study, we found that ManR controlled the expression not only of mannanolytic enzyme genes but also of cellulolytic enzyme genes in A. oryzae, based on DNA microarray analysis of a manR-disruptant and a manR-overexpressing strain. A new model for the regulation of cellulase and hemicellulase genes mediated by ManR and XlnR in A. oryzae is proposed.

Identification of the glutaminase genes of Aspergillus sojae involved in glutamate production during soy sauce fermentation.

Glutaminase, an enzyme that catalyzes the conversion of L-glutamine to L-glutamate, enhances the umami taste in soy sauce. The Aspergillus sojae genome contains 10 glutaminase genes. In this study, we estimated that approximately 60% of the glutamate in soy sauce is produced through the glutaminase reaction. To determine which glutaminase is involved in soy sauce glutamate production, we prepared soy sauces using single and multiple glutaminase gene disruptants of A. sojae. The glutamate concentration in soy sauce prepared using the ΔgahA-ΔgahB-ΔggtA-Δgls disruptant was approximately 60% lower than that in the control strain, whereas it was decreased by approximately 20-30% in the ΔgahA-ΔgahB disruptant. However, the glutamate concentration was unchanged in the soy sauces prepared using the ΔgahA-ΔggtA-Δgls and ΔgahB-ΔggtA-Δgls disruptants. These results indicate that four glutaminases are involved in glutamate production in soy sauce, and that the peptidoglutaminase activities of GahA and GahB increase the glutamate concentration in soy sauce.

Analysis of the functions of recombination-related genes in the generation of large chromosomal deletions by loop-out recombination in Aspergillus oryzae.

Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure.

srdA mutations suppress the rseA/cpsA deletion mutant conidiation defect in Aspergillus nidulans.

Conidiation is an important reproductive process in Aspergillus. We previously reported, in A. nidulans, that the deletion of a putative glycosyltransferase gene, rseA/cpsA, causes an increase in the production of extracellular hydrolases and a severe reduction in conidiation. The aim of this study was to obtain novel genetic factors involved in the repression of conidiation in the rseA deletion mutant. We isolated mutants in which the rseA deletion mutant conidiation defect is suppressed and performed a comparative genomic analysis of these mutants. A gene encoding a putative transcription factor was identified as the associated candidate causative gene. The candidate gene was designated as srdA (suppressor gene for the conidiation defect of the rseA deletion mutant). The conidiation efficiency of the rseAsrdA double-deletion mutant was increased. Introduction of wild-type srdA into the suppressor mutants caused a conidiation defect similar to that of the rseA deletion mutant. Notably, the conidiation efficiencies of the rseAsrdA double-deletion and srdA single-deletion mutants were higher than that of the wild-type strain. These results indicate that srdA is a novel genetic factor that strongly represses conidiation of the rseA deletion mutant, and a putative transcriptional regulator, SrdA is a negative regulator of conidiation in A. nidulans.

A further study on chromosome minimization by protoplast fusion in Aspergillus oryzae.

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1-12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 µg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.

ManR, a novel Zn(II)2Cys6 transcriptional activator, controls the ß-mannan utilization system in Aspergillus oryzae.

Fungal endo-ß-mannanases (ß-mannanases) are widely used as industrial enzymes; however, no transcriptional regulator of ß-mannanases has been identified in fungi or other eukaryotic cells to date. To identify a transcriptional regulator of ß-mannanases in Aspergillus oryzae, a gene-disruptant library of transcriptional regulators was screened for mutants exhibiting reduced ß-mannanase activity by using konjac glucomannan as the substrate, and ManR, a Zn(II)(2)Cys(6) type DNA binding protein was identified. Moreover, a manR-overexpressing strain showed significantly increased ß-mannanase activity. DNA microarray analysis of the manR-disruptant strain further indicated that when konjac glucomannan is used as the carbon source, ManR positively regulates the gene expression of not only ß-mannanase, but also the enzymes involved in the degradation of galactomannans and glucomannans such as α-galactosidase, ß-mannosidase, acetylmannan esterase, and ß-glucosidase. Furthermore, we demonstrated that the presence of 1,4-ß-d-mannobiose increased the expression of the endo-ß-mannanase gene (manG, AO090010000122), and that ManR plays a key role in the inducible expression of manG in A. oryzae. Therefore, we conclude that ManR is a positive regulator of the ß-mannan utilization system in A. oryzae. This is the first study to identify a transcriptional regulator of this system in eukaryotic cells.