cAMP signaling factors regulate carbon catabolite repression of hemicellulase genes in Aspergillus nidulans.

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.

A component of the septation initiation network complex, AaSepM, is involved in multiple cellulose-responsive signaling pathways in Aspergillus aculeatus.

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.

Significance of a family-6 carbohydrate-binding module in a modular feruloyl esterase for removing ferulic acid from insoluble wheat arabinoxylan.

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.

Light Regulation of Two New Manganese Peroxidase-Encoding Genes in KU-RNW027.

To better understand the light regulation of ligninolytic systems in 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, , , and , were found.

Two Manganese Peroxidases and a Laccase of KU-RNW027 with Novel Properties for Dye and Pharmaceutical Product Degradation in Redox Mediator-Free System.

Two manganese peroxidases (MnPs), MnP1 and MnP2, and a laccase, Lac1, were purified from KU-RNW027. Both MnPs showed high stability in organic solvents which triggered their activities. Metal ions activated both MnPs at certain concentrations.

CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica.

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.

The modular arabinanolytic enzyme Abf43A-Abf43B-Abf43C from Ruminiclostridium josui consists of three GH43 modules classified in different subfamilies.

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.

CreA-independent carbon catabolite repression of cellulase genes by trimeric G-protein and protein kinase A in Aspergillus nidulans.

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.

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.

Function of a laminin_G_3 module as a carbohydrate-binding module in an arabinofuranosidase from Ruminiclostridium josui.

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 -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.

Ruminiclostridium josui Abf62A-Axe6A: A tri-functional xylanolytic enzyme exhibiting α-l-arabinofuranosidase, endoxylanase, and acetylxylan esterase activities.

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.

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.

Development of an efficient host-vector system of Ruminiclostridium josui.

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.

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.

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.

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.

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.

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.

Regulation of genes encoding cellulolytic enzymes by Pal-PacC signaling in Aspergillus nidulans.

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.

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.

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).

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 (P ( CBHI )) and the orotidine 5'-phosphate decarboxylase gene (pyrG).

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.