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The cellobiose-responsive regulator ClbR, a Zn(II)2Cys6 binuclear-cluster transcription factor, is a positive regulator of carbohydrate-active enzyme (CAZyme) genes responsive to cellulose in Aspergillus aculeatus. Because Zn(II)2Cys6 transcription factors tend to dimerize with proteins of the same family, we searched for a counterpart of ClbR and identified ClbR2, which is 42% identical to ClbR, as an interacting partner of ClbR by yeast two-hybrid screening. Genetic analyses suggested that ClbR and ClbR2 cooperatively regulate the expression of CAZyme genes in response to cellulose and 1,4-β-mannobiose in A. aculeatus. CAZyme genes under the control of the transcription factor ManR were regulated by ClbR and ClbR2, whereas those controlled by the transcription factor XlnR were regulated by ClbR, but not ClbR2. These findings suggest that ClbR participates in multiple regulatory pathways in A. aculeatus by altering an interacting factor., (© The Author(s) 2023. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Carbon catabolite repression (CCR) enables preferential utilization of easily metabolizable carbon sources, implying the presence of mechanisms to ensure discriminatory gene repression depending on the ambient carbon sources. However, the mechanisms for such hierarchical repression are not precisely understood. In this report, we examined how deletion of pkaA and ganB, which encode cAMP signaling factors, and creA, which encodes a well-characterized repressor of CCR, affects CCR of hemicellulase genes in the filamentous fungus Aspergillus nidulans. β-Xylanase production increased not only in ΔcreA but also in ΔpkaA and ΔganB, with the highest level observed in their double deletants, irrespective of the presence or absence of D-glucose. Expression of the β-xylanase genes in the presence of D-glucose was de-repressed in all the deletion mutants, with significantly higher tolerance against D-glucose repression in ΔpkaA and ΔganB than in ΔcreA. In the presence of galactomannan and D-glucose, partial de-repression of β-mannanase production was detected in ΔcreA, but not in ΔpkaA and ΔganB. The double deletion of creA/pkaA and creA/ganB led to earlier production. Release from D-glucose repression of the β-mannanase genes was partial in the single deletants, while nearly full de-repression was observed in ΔcreAΔpkaA and ΔcreAΔganB. The contribution of PkaA and GanB to CCR by D-xylose of the β-mannanase genes was very minor compared to that of CreA. Consequently, the present study revealed that cAMP signaling plays a major role in CCR of hemicellulase gene expression in a manner that is clearly independent from CreA., (© 2022. The Author(s).)

Various carbohydrate-active enzymes in Aspergillus are produced in response to physiological inducers, which is regulated at the transcriptional level. To elucidate the induction mechanisms in Aspergillus, we screened for new regulators involved in cellulose-responsive induction from approximately 10,000 Aspergillus aculeatus T-DNA-inserted mutants. We constructed the T-DNA-inserted mutant library using the host strain harboring the orotidine 5'-monophosphate decarboxylase gene (pyrG) under the control of the FIII-avicelase gene (cbhI) promoter. Thus, candidate mutants deficient in cellulose-responsive induction were positively screened via counter selection against 5-fluoroorotic acid (5-FOA). Among less than two hundred 5-FOA-resistant mutants, one mutant that the T-DNA inserted into the AasepM locus reduced the cbhI expression in response to cellulose. Since AaSepM is similar to Schizosaccharomyces pombe Cdc14p (E-value, 2e-20; identities, 33%), which is a component of the septation initiation network (SIN)-complex, we constructed an AasepM deletion mutant (ΔAasepM). We analyzed the expression of cellulase and xylanase genes in response to cellulose, septation, and conidiation in ΔAasepM. The AasepM deletion leads to delayed septation and decreased formation of the conidium chain in A. aculeatus but does not affect hyphal growth on minimal media. We also confirmed AaSepM's involvement in multiple cellulose-responsive signaling pathways of cellulase and xylanase genes under the control of the ManR-dependent, XlnR-dependent, and ManR- and XlnR-independent signaling pathways. KEY POINTS : • A new regulator for cellulolytic gene expression has been identified. • AaSepM is involved in septation and conidiation in A. aculeatus. • AasepM is involved in multiple cellulose-responsive signaling pathways.

Ruminiclostridium josui Fae1A is a modular enzyme consisting of an N-terminal signal peptide, family-1 carbohydrate esterase module (CE1), family-6 carbohydrate-binding module (CBM6), and dockerin module in that order. Recombinant CE1 and CBM6 polypeptides were collectively and separately produced as RjFae1A, RjCE1, and RjCBM6. RjFae1A showed higher feruloyl esterase activity than RjCE1 towards insoluble wheat arabinoxylan, but the latter was more active towards small synthetic substrates than the former. This suggests that CBM6 in RjFae1A plays an important role in releasing ferulic acid from the native substrate. RjCBM6 showed a higher affinity for soluble wheat arabinoxylan than for rye arabinoxylan and beechwood xylan in native affinity polyacrylamide gel electrophoresis. Isothermal titration calorimetry analysis demonstrated that RjCBM6 recognized a xylopyranosyl residue at the nonreducing ends of xylooligosaccharides. Moreover, it showed exceptional affinity for 2 3 -α-l-arabinofuranosyl-xylotriose (A 2 XX) among the tested branched arabinoxylooligosaccharides. Fluorometric titration analysis demonstrated that xylobiose and A 2 XX competitively bound to RjCBM6, and both bound to the same site in RjCBM6. RjCBM6's preference for the xylopyranosyl residue at the nonreducing end of xylan chains explains why the positive effect of CBM6 on RjFae1A activity was observed only during short incubation but not after extended incubation., Competing Interests: Conflict of Competing Interest We have no conflict of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To better understand the light regulation of ligninolytic systems in Trametes polyzona KU-RNW027, ligninolytic enzymes-encoding genes were identified and analyzed to determine their transcriptional regulatory elements. Elements of light regulation were investigated in submerged culture. Three ligninolytic enzyme-encoding genes, mnp1 , mnp2 , and lac1 , were found. Cloning of the genes encoding MnP1 and MnP2 revealed distinct deduced amino acid sequences with 90% and 86% similarity to MnPs in Lenzites gibbosa , respectively. These were classified as new members of short-type hybrid MnPs in subfamily A.2 class II fungal secretion heme peroxidase. A light responsive element (LRE), composed of a 5'-CCRCCC-3' motif in both mnp promoters, is reported. Light enhanced MnP activity 1.5 times but not laccase activity. The mnp gene expressions under light condition increased 6.5- and 3.8-fold, respectively. Regulation of laccase gene expression by light was inconsistent with the absence of LREs in their promoter. Blue light did not affect gene expressions but impacted their stability. Reductions of MnP and laccase production under blue light were observed. The details of the molecular mechanisms underlying enzyme production in this white-rot fungus provide useful knowledge for wood degradation relative to illumination condition. These novel observations demonstrate the potential of enhancing ligninolytic enzyme production by this fungus for applications with an eco-friendly approach to bioremediation., Competing Interests: The authors declare no conflict of interest.

The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cellulase production in filamentous fungi is repressed by various carbon sources. In our preliminary survey in Aspergillus nidulans, degree of de-repression differed depending on carbon sources in a mutant of creA, encoding the transcriptional repressor for carbon catabolite repression (CCR). To further understand mechanisms of CCR of cellulase production, we compared the effects of creA deletion with deletion of protein kinase A (pkaA) and G (ganB) genes, which constitute a nutrient sensing and signaling pathway. In plate culture with carboxymethyl cellulose and D-glucose, deletion of pkaA and ganB, but not creA, led to significant de-repression of cellulase production. In submerged culture with cellobiose and D-glucose or 2-deoxyglucose, both creA or pkaA single deletion led to partial de-repression of cellulase genes with the highest level by their double deletion, while ganB deletion caused de-repression comparable to that of the creA/pkaA double deletion. With ball-milled cellulose and D-glucose, partial de-repression was detected by deletion of creA but not of pkaA or ganB. The creA/pkaA or creA/ganB double deletion led to earlier expression than the creA deletion. Furthermore, the effect of each deletion with D-xylose or L-arabinose as the repressing carbon source was significantly different from that with D-glucose, D-fructose, and D-mannose. Consequently, this study revealed that PkaA and GanB participate in CreA-independent CCR and that contribution of CreA, PkaA, and GanB in CCR differs depending on the inducers, repressing carbon sources, and culture conditions (plate or submerged). Further study of CreA-independent mechanisms is needed to fully understand CCR in filamentous fungi.

The abnA gene from Ruminiclostridium josui encodes the large modular arabinanolytic enzyme, Abf43A-Abf43B-Abf43C, consisting of an N-terminal signal peptide, a Laminin_G_3 module, a GH43_22 module, a Laminin_G_3 module, a Big_4 module, a GH43_26 module, a GH43_34 module and a dockerin module in order with a calculated molecular weight of 204,108. Three truncated enzymes were recombinantly produced in Escherichia coli and biochemically characterized, RjAbf43A consisting of the first Laminin_G_3 module and GH43_22 module, RjAbf43B consisting of the second Laminin_G_3 module, Big_4 module and GH43_26 module, and RjAbf43C consisting of the GH43_34 module. RjAbf43A showed a strong α-l-arabinofuranosidase activity toward sugar beet arabinan, highly branched arabinan but not linear arabinan, thus it acted in the removal of arabinose side chains from sugar beet arabinan. By contrast, RjAbf43B showed a strong exo-α-1,5-l-arabinofuranosidase activity toward linear arabinan and arabinooligosaccharides whereas RjAbf43C showed low activity toward these substrates. Although RjAbf43B was activated by the presence of some metal ions such as Zn 2+ , Mg 2+ and Ni 2+ , RjAbf43A was inhibited by these ions. RjAbf43A and RjAbf43B attacked sugar beet arabinan in a synergistic manner. By comparison, RjAbf43A-Abf43B containing both GH43_22 and GH43_26 modules showed lower hydrolytic activity toward sugar beet arabinan but higher activity toward sugar beet fiber than the sum of the individual activities of RjAbf43A and RjAbf43B, suggesting that the coexistence of two distinct GH43 modules in a single polypeptide is important for the efficient hydrolysis of an insoluble and natural polysaccharide but not a soluble substrate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from Trametes polyzona KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations. The two MnPs and Lac1, played important roles in dye degradation and pharmaceutical products deactivation in a redox mediator-free system. They completely degraded Remazol brilliant blue (25 mg/L) in 10-30 min and showed high degradation activities to Remazol navy blue and Remazol brilliant yellow, while Lac1 could remove 75% of Remazol red. These three purified enzymes effectively deactivated tetracycline, doxycycline, amoxicillin, and ciprofloxacin. Optimal reaction conditions were 50 °C and pH 4.5. The two MnPs were activated by organic solvents and metal ions, indicating the efficacy of using T. polyzona KU-RNW027 for bioremediation of aromatic compounds in environments polluted with organic solvents and metal ions with no need for redox mediator supplements.

Pectinolytic enzymes are used in diverse industrial applications. We sought to isolate a pectate lyase from Aspergillus luchuensis var. saitoi, a filamentous fungus used in traditional food and beverage preparation in Japan. The identified enzyme, named AsPelA, is orthologous to PelA from A. luchuensis mut. kawachii (AkPelA); the enzymes exhibit 99% amino acid sequence identity, with Ile140 and Val197 of AsPelA being replaced by Val and Asp in AkPelA, respectively. AsPelA activity decreased to 71%, 61%, and 46% of maximal activity after 60-min incubation at 60 °C, 70 °C, and 80 °C, whereas AkPelA activity dropped to 16%, 10%, and 8.5%, respectively, indicating that AsPelA is more thermostable than AkPelA. Furthermore, AsPelA was stable within a neutral-to-alkaline pH range, as well as in the presence of organic solvents, detergents, and metal ions. Our findings suggest that AsPelA represents a candidate pectate lyase for applications in food, paper, and textile industries., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Laminin_G_3 modules can exist together with family-43 catalytic modules of glycoside hydrolase (GH43), but their functions are unknown. Here, a laminin_G_3 module and a GH43 module derived from a Ruminiclostridium josui modular arabinofuranosidase Abf43A-Abf43B-Abf43C were produced individually as RjLG3 and RjGH43_22, respectively, or combined as RjGH43-1 to gain insights into their activities. Isothermal calorimetry analysis showed that RjLG3 has high affinity toward 3 2 -α-l-arabinofuranosyl-(1,5)-α-l-arabinotriose but not for α-1,5-linked arabinooligosaccharides, which suggests that RjLG3 interacts specifically with a branched arabinofuranosyl residue of an arabinooligosaccharide but not an arabinofuranosyl residue at the end of α-1,5-linked arabinooligosaccharides. RjGH43-1 (with CBM) shows higher activity toward sugar beet arabinan than RjGH43_22 (without CBM), which suggests that the LG3 module in RjGH43-1 plays an important role in substrate hydrolysis as a carbohydrate-binding module., (© 2018 Federation of European Biochemical Societies.)

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.

Ruminiclostridium josui Abf62A-Axe6A is a modular enzyme comprising (in order from the N-terminus): an N-terminal signal peptide, a glycoside hydrolase family 62 (GH62) catalytic module, a family 6 carbohydrate binding module (CBM6), a dockerin module and an additional carbohydrate esterase family 6 catalytic module (CE6). In this study, three Abf62A-Axe6A derivatives were constructed, overexpressed in Escherichia coli, purified, and biochemically characterized: RjAbf62A-Axe6A, containing all four modules but lacking the signal peptide; RjAbf62A-CBM6, containing the GH62 and CBM6 modules; and RjAxe6A, containing only CE6. RjAbf62A-Axe6A was highly active toward arabinoxylan and moderately active toward sugar beet arabinan, and released mainly arabinose. Analysis of the arabinoxylooligosaccharide hydrolysis products revealed that RjAbf62A-Axe6A released α-1,2- and α-1,3-linked arabinofuranose from both singly and doubly substituted xylosyl residues. Furthermore, RjAbf62A-Axe6A exhibited a weak activity toward linear 1,5-α-l arabinan and arabinooligosaccharides, indicating that it is capable of cleaving α-1,5-linkage. Surprisingly, RjAbf62A-Axe6A also demonstrated an endoxylanase activity toward birchwood and beechwood xylans and xylooligosaccharides. Although RjAbf62A-CBM6 exhibited a similar substrate specificity to RjAbf62A-Axe6A, RjAbf62A-CBM6 showed lower activities toward soluble arabinoxylans, birchwood and beechwood xylans and arabinoxylooligosaccharides but not toward insoluble arabinoxylan. RjAbf62A-Axe6A is the first reported GH62 enzyme with α-l-arabinofuranosidase and endoxylanase activities. Although both RjAbf62A-Axe6A and RjAxe6A had acetylxylan esterase activities, RjAbf62A-Axe6 exhibited a higher activity toward insoluble wheat arabinoxylan compared with RjAxe6., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Although Ruminiclostridium josui (formerly Clostridium josui), a strictly anaerobic mesophilic cellulolytic bacterium, is a promising candidate for biomass utilization via consolidated bioprocessing, its host-vector system has not yet been established. The existence of a restriction and modification system is a significant barrier to the transformation of R. josui. Here, we partially purified restriction endonuclease RjoI from R. josui cell extract using column chromatography. Further characterization showed that RjoI is an isoschizomer of DpnI, recognizing the sequence 5'-G met ATC-3', where the A nucleotide is Dam-methylated. RjoI cleaved the recognition sequence between the A and T nucleotides, producing blunt ends. We then successfully introduced plasmids prepared from Escherichia coli C2925 (dam - /dcm - ) into R. josui by electroporation. The highest transformation efficiency of 6.6 × 10 3 transformants/μg of DNA was obtained using a square-wave pulse (750 V, 1 ms). When the R. josui cel48A gene, devoid of the dockerin-encoding region, cloned into newly developed plasmid pKKM801 was introduced into R. josui, a truncated form of RjCel48A, RjCel48AΔdoc, was detected in the culture supernatant but not in the intracellular fraction. This is the first report on the establishment of fundamental technology for molecular breeding of R. josui., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the past decade, various transcriptional activators of cellulolytic enzyme genes have been identified in Ascomycete fungi. The regulatory system of cellulolytic enzymes is not only partially conserved, but also significantly diverse. For example, Trichoderma reesei has a system distinct from those of Aspergillus and Neurospora crassa-the former utilizes Xyr1 (the Aspergillus XlnR ortholog) as the major regulator of cellulolytic enzyme genes, while the latter uses CLR-2/ClrB/ManR orthologs. XlnR/Xyr1 and CLR-2/ClrB/ManR are evolutionarily distant from each other. Regulatory mechanisms that are controlled by CLR-2, ClrB, and ManR are also significantly different, although they are orthologous factors. Expression of clr-2 requires the activation of another transcription factor, CLR-1, by cellobiose, while CLR-2 is constitutively active for transactivation. By contrast, ClrB activation requires cellobiose. While ClrB mainly regulates cellulolytic genes, ManR is essential for the activation of not only cellulolytic but also mannanolytic enzyme genes. In this review, we summarize XlnR/Xyr1- and CLR-2/ClrB/ManR-dependent regulation in N. crassa, A. nidulans, A. oryzae, and T. reesei and emphasize the conservation and diversity of the regulatory systems for cellulolytic enzyme genes in these Ascomycete fungi. In addition, we discuss the role of McmA, another transcription factor that plays an important role in recruiting ClrB to the promoters in A. nidulans.

We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 Aspergillus aculeatus T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (cbhI) promoter and the orotidine 5'-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and cbhI expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (dppIV) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and cbhI expression. The deletion of dppIV resulted in a significant reduction in the cellulose-responsive expression of both cbhI as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in A. aculeatus. In contrast, the dppIV deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in A. aculeatus.

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 CGGN 8 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., (© 2016 John Wiley & Sons Ltd.)

SRF-MADS proteins are transcription factors conserved among eukaryotes that regulate a variety of cellular functions; however, their physiological roles are still not well understood in filamentous fungi. Effects of a mutation in mcmA gene that encodes the sole SRF-MADS protein in the fungus Aspergillus nidulans were examined by RNA sequencing. Sequencing data revealed that expression levels of cellulase genes were significantly decreased by the mutation as reported previously. However, expression levels of various hemicellulolytic enzyme genes, several extracellular protease genes, the nosA and rosA genes involved in sexual development, and AN4394 encoding an ortholog of EcdR involved in Aspergillus oryzae conidiation, were also significantly decreased by the mutation. As expected from the RNA sequencing data, the mcmA mutant had reduced protease production, cleistothecial development, and conidiation. This is the first report describing the involvement of SRF-MADS proteins in protease production in fungi, and asexual and sexual development in Aspergillus.

For identifying the genes that are regulated by a transcription factor (TF), we have established an analytical pipeline that combines genomic systematic evolution of ligands by exponential enrichment (gSELEX)-Seq and RNA-Seq. Here, SELEX was used to select DNA fragments from an Aspergillus nidulans genomic library that bound specifically to AmyR, a TF from A. nidulans. High-throughput sequencing data were obtained for the DNAs enriched through the selection, following which various in silico analyses were performed. Mapping reads to the genome revealed the binding motifs including the canonical AmyR-binding motif, CGGN8CGG, as well as the candidate promoters controlled by AmyR. In parallel, differentially expressed genes related to AmyR were identified by using RNA-Seq analysis with samples from A. nidulans WT and amyR deletant. By obtaining the intersecting set of genes detected using both gSELEX-Seq and RNA-Seq, the genes directly regulated by AmyR in A. nidulans can be identified with high reliability. This analytical pipeline is a robust platform for comprehensive genome-wide identification of the genes that are regulated by a target TF.

Cellulosic biomass represents a valuable potential substitute for fossil-based fuels. As such, there is a strong need to develop efficient biotechnological processes for the enzymatic hydrolysis of cellulosic biomass via the optimization of cellulase production by fungi. Ambient pH is an important factor affecting the industrial production of cellulase. In the present study, we demonstrate that several Aspergillus nidulans genes encoding cellulolytic enzymes are regulated by Pal-PacC-mediated pH signaling, as evidenced by the decreased cellulase productivity of the palC mutant and pacC deletants of A. nidulans. The deletion of pacC was observed to result in delayed induction and decreased expression of the cellulase genes based on time course expression analysis. The genome-wide identification of PacC-regulated genes under cellobiose-induced conditions demonstrated that genes expressed in a PacC-dependent manner included 82 % of ClrB (a transcriptional activator of the cellulase genes)-regulated genes, including orthologs of various transporter and β-glucosidase genes considered to be involved in cellobiose uptake or production of stronger inducer molecules. Together with the significant overlap between ClrB- and PacC-regulated genes, the results suggest that PacC-mediated regulation of the cellulase genes involves not only direct regulation by binding to their promoter regions but also indirect regulation via modulation of the expression of genes involved in ClrB-dependent transcriptional activation. Our findings are expected to contribute to the development of more efficient industrial cellulase production methods.

ClbR is a Zn(II)2Cys6 transcriptional activator that controls the expression of cellulase-related genes in response to Avicel and cellobiose in Aspergillus aculeatus. A clbR-overexpressing strain (clbR-OE) that expresses the clbR gene at levels sevenfold higher than the control strain sustainably produced xylanolytic and cellulolytic activities during 10-day cultivation of A. aculeatus, enabling synchronization of xylanolytic and cellulolytic activities at a maximum level. However, clbR overexpression did not simultaneously increase levels of all xylanolytic and cellulolytic enzymes. Peptide mass fingerprint analysis revealed markedly increased production of FIa-xylanase in clbR-OE, whereas expression of FIII-avicelase and FII-carboxymethyl cellulase was unaffected and expression of hydrocellulase was lower in clbR-OE than in the control. Northern blot analysis confirmed that these effects of clbR overexpression on enzyme production were mediated at the transcriptional level. These data suggest that ClbR participates in diverse signaling pathways to control the expression of cellulosic biomass-degrading enzymes in A. aculeatus.

The cellobiose- and cellulose-responsive induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes is not regulated by XlnR in Aspergillus aculeatus, which suggests that this fungus possesses an unknown cellulase gene-activating pathway. To identify the regulatory factors involved in this pathway, we constructed a random insertional mutagenesis library using Agrobacterium tumefaciens-mediated transformation of A. aculeatus NCP2, which harbors a transcriptional fusion between the cbhI promoter (P ( CBHI )) and the orotidine 5'-phosphate decarboxylase gene (pyrG). Of the ~6,000 transformants screened, one 5-FOA-resistant transformant, S4-22, grew poorly on cellulose-containing media and exhibited reduced cellobiose-induced expression of cbhI. Southern blot analysis and nucleotide sequencing of the flanking regions of the T-DNA inserted in S4-22 indicated that the T-DNA was inserted within the coding region of a previously unreported Zn(II)(2)Cys(6)-transcription factor, which we designated the cellobiose response regulator (ClbR). The disruption of the clbR gene resulted in a significant reduction in the expression of cbhI and cmc2 in response to cellobiose and cellulose. Interestingly, the cellulose-responsive induction of FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes that are under the control of XlnR, was also reduced in the clbR-deficient mutant, but there was no effect on the induction of these genes in response to D-xylose or L-arabinose. These data demonstrate that ClbR participates in both XlnR-dependent and XlnR-independent cellobiose- and cellulose-responsive induction signaling pathways in A. aculeatus.

Auxotrophic mutants of Aspergillus can be isolated in the presence of counter-selective compounds, but the process is laborious. We developed a method to enable reversible impairment of the ku80 gene (Aaku80) in the imperfect fungus Aspergillus aculeatus. Aaku80 was replaced with a selection marker, orotidine 5'-phosphate decarboxylase (pyrG), followed by excision of pyrG between direct repeats (DR) to yield the Aaku80 deletion mutant (MR12). The gene-targeting efficiency at the ornithine carbamoyltransferase (argB) locus was drastically elevated from 3% to 96% in MR12. The frequency of marker recycling depended on DR length. One uridine auxotroph was obtained from 3.3 × 105, 1.4 × 105, and 9.2 × 103 conidia from strains harboring 20-, 98-, and 495-bp DRs, respectively. Because these strains maintained the short DRs after 5 d of cultivation, we investigated whether Aaku80 function was disrupted by pyrG insertion with the 20-bp DR and restored after excision of pyrG. The Aaku80 disruption mutant (coku80) was bred by inserting pyrG sandwiched between 20-bp DRs into the second intron of Aaku80, followed by excision of pyrG between the DRs to yield the coku80rec strain. Analyses of homologous recombination frequency and methyl methanesulfonate sensitivity demonstrated that Aaku80 function was disrupted in coku80 but restored in coku80rec. Furthermore, pyrG was maintained in coku80 at least for ten generations. These data indicated that reversible impairment of ku80 in A. aculeatus is useful for functional genomics in cases where genetic segregation is not feasible.

Agrobacterium tumefaciens-mediated transformation (AMT) was applied to Aspergillus aculeatus. Transformants carrying the T-DNA from a binary vector pBIG2RHPH2 were sufficiently mitotically stable to allow functional genomic analyses. The AMT technique was optimized by altering the concentration of acetosyringone, the ratio and concentration of A. tumefaciens and A. aculeatus cells, the duration of co-cultivation, and the status of A. aculeatus cells when using conidia, protoplasts, or germlings. On average, 30 transformants per 104 conidia or 217 transformants per 107 conidia were obtained under the optimized conditions when A. tumefaciens co-cultured with fungi using solid or liquid induction media (IM). Although the transformation frequency in liquid IM was 100-fold lower than that on solid IM, the AMT method using liquid IM is better suited for high-throughput insertional mutagenesis because the transformants can be isolated on fewer selection media plates by concentrating the transformed germlings. The production of two albino A. aculeatus mutants by AMT confirmed that the inserted T-DNA disrupted the polyketide synthase gene AapksP, which is involved in pigment production. Considering the efficiency of AMT and the correlation between the phenotypes and genotypes of the transformants, the established AMT technique offers a highly efficient means for characterizing the gene function in A. aculeatus.