Emi Kunitake

A novel transcriptional regulator, ClbR, controls the cellobiose- and cellulose-responsive induction of cellulase and xylanase genes regulated by two distinct signaling pathways in Aspergillus 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 (PCBHI) 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)2Cys6-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.

Agrobacterium tumefaciens-mediated transformation of Aspergillus aculeatus for insertional mutagenesis

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.

Reversible impairment of the ku80 gene by a recyclable marker in Aspergillus 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.

Conservation and diversity of the regulators of cellulolytic enzyme genes in Ascomycete fungi

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.

Effects of clbR overexpression on enzyme production in Aspergillus aculeatus vary depending on the cellulosic biomass-degrading enzyme species

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. Graphical Abstract Effects of clbR overexpression on enzyme production in Aspergillus aculeatus vary depending on the cellulosic biomass degrading enzyme species.

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 CGGN8CCG 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.

Involvement of an SRF-MADS protein McmA in regulation of extracellular enzyme production and asexual/sexual development in Aspergillus nidulans

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. Graphical abstract McmA regulated cellulase/protease production and asexual/sexual development. The figure shows impaired development of fruiting bodies (cleistothecia) in the mcmA mutant.

A Robust Analytical Pipeline for Genome-Wide Identification of the Genes Regulated by a Transcription Factor: Combinatorial Analysis Performed Using gSELEX-Seq and RNA-Seq.

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.

Dipeptidyl peptidase IV is involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes in Aspergillus aculeatus

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. Graphical abstract Dashed lines with arrows indicate putative signaling pathways.

Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi

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.