Biogenic manganese oxides combined with 1-hydroxybenzotriazol and an Mn(II)-oxidizing enzyme from Pleosporales sp. Mn1 oxidize 3,4-dimethoxytoluene to yield 3,4-dimethoxybenzaldehyde.

Using soil samples, we screened for microbes that produce biogenic manganese oxides (BMOs) and isolated Mn(II)-oxidizing fungus, namely Pleosporales sp. Mn1 (Mn1). We purified the Mn(II)-oxidizing enzyme from intracellular extracts of Mn1. The enzyme oxidized Mn(II) most effectively at pH 7.0 and 45 °C. The N-terminal amino acid sequence of the purified enzyme possessed homology with multicopper oxidases in fungi. The properties of the enzyme and the effects of the pH and inhibitors on the Mn(II)-oxidization activity suggested that the enzyme is a member of the multicopper oxidase family. The X-ray diffraction pattern of the BMOs produced by Mn1 showed a strong correlation with that of a typical poorly crystalized vernadite (δ-MnO2). Since BMOs are some of the most reactive materials in the environment, we investigated a potential new application of BMOs as oxidation catalysts. We confirmed that BMOs oxidized aromatic methyl groups when combined with the purified enzyme and a mediator, 1-hydroxybenzotriazole (HBT). BMO oxidation of 3,4-dimethoxytoluene achieved a better yield than that of abiotic MnO2 and white-rot fungus laccase under acidic and neutral pH conditions. Under neutral pH, the BMOs oxidized 3,4-dimethoxytoluene to yield 200-fold more 3,4-dimethoxybenzaldehyde than that of abiotic MnO2. This is the first report to reveal that BMOs combined with a Mn(II)-oxidizing enzyme and mediator can oxidize aromatic hydrocarbons to yield corresponding aldehydes.

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. 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.

Chemical genetic approach using ß-rubromycin reveals that a RIO kinase-like protein is involved in morphological development in Phytophthora infestans.

To characterize the molecular mechanisms underlying life-stage transitions in Phytophthora infestans, we initiated a chemical genetics approach by screening for a stage-specific inhibitor of morphological development from microbial culture extracts prepared mostly from actinomycetes from soil in Japan. Of the more than 700 extracts, one consistently inhibited Ph. infestans cyst germination. Purification and identification of the active compound by ESI-MS, 1H-NMR, and 13C-NMR identified ß-rubromycin as the inhibitor of cyst germination (IC50 = 19.8 µg/L); ß-rubromycin did not inhibit growth on rye media, sporangium formation, zoospore release, cyst formation, or appressorium formation in Ph. infestans. Further analyses revealed that ß-rubromycin inhibited the germination of cysts and oospores in Pythium aphanidermatum. A chemical genetic approach revealed that ß-rubromycin stimulated the expression of RIO kinase-like gene (PITG_04584) by 60-fold in Ph. infestans. Genetic analyses revealed that PITG_04584, which lacks close non-oomycete relatives, was involved in zoosporogenesis, cyst germination, and appressorium formation in Ph. infestans. These data imply that further functional analyses of PITG_04584 may contribute to new methods to suppress diseases caused by oomycetes.

Engineering of the Trichoderma reesei xylanase3 promoter for efficient enzyme expression.

The GH10 xylanase XYNIII is expressed in the hyper-cellulase-producing mutant PC-3-7, but not in the standard strain QM9414 of Trichoderma reesei. The GH11 xylanase gene xyn1 is induced by cellulosic and xylanosic carbon sources while xyn3 is induced only by cellulosic carbon sources in the PC-3-7 strain. In this study, we constructed a modified xyn3 promoter in which we replaced the cis-acting region of the xyn3 promoter by the cis-acting region of the xyn1 promoter. The resulting xyn3 chimeric promoter exhibited improved inductivity against cellulosic carbon over the wild-type promoter and acquired inductivity against xylanosic carbon. Furthermore, PC-3-7 expressing the heterologous ß-glycosidase gene, Aspergillus aculeatus bgl1, under the control of the xyn3 chimeric promoter, showed enhanced saccharification ability through increased cellobiase activity. We also show that the xyn3 chimeric promoter is also functional in the QM9414 strain. Our results indicate that the xyn3 chimeric promoter is very efficient for enzyme expression.

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.

Site-saturation mutagenesis for ß-glucosidase 1 from Aspergillus aculeatus to accelerate the saccharification of alkaline-pretreated bagasse.

Aspergillus aculeatus ß-glucosidase 1 (AaBGL1) is one of the best cellobiose hydrolytic enzymes without transglycosylation products, among ß-glucosidase from various origins, for use in cellulosic biomass conversion with Trichoderma cellulases. However, in our previous report, it was demonstrated that AaBGL1 has lower catalytic efficiency toward cellobiose, which is a major end product from cellulosic biomasses by Trichoderma reesei cellulases, than do gentiobiose and laminaribiose. Thus, we expected that there is room to enhance cellobiose hydrolytic activity of AaBGL1 by increasing catalytic efficiency (k cat/K m) up to that of gentiobiose or laminaribiose for accelerating the saccharification of cellulosic biomasses, and we performed site-saturation mutagenesis targeting nine amino acids supposed to constitute subsite +1 of AaBGL1. We successfully isolated a mutant AaBGL1 (Q201E) having 2.7 times higher k cat/K m toward cellobiose than the WT enzyme. Q201E showed higher activity toward cellotriose and cellotetraose but lower activity toward gentiobiose and laminaribiose than WT. Kinetic analysis of various Q201 mutants toward cellobiose, gentiobiose, and laminaribiose revealed that only the Q201E mutation resulted in improved k cat/K m toward cellobiose. We demonstrated that side chain length and the nondissociated form of the carboxyl group at E201 in Q201E were required for enhancing the activity toward cellooligosaccharides through supporting nucleophile attack by D280 via changing catalytic environment by pH profile of kinetic parameters and mutation analyses. Moreover, we also demonstrated that Q201E produced more effective synergy with cellulases and xylanases than WT in the saccharification of alkaline-pretreated bagasse.

A high performance Trichoderma reesei strain that reveals the importance of xylanase III in cellulosic biomass conversion.

The ability of the Trichoderma reesei X3AB1strain enzyme preparations to convert cellulosic biomass into fermentable sugars is enhanced by the replacement of xyn3 by Aspergillus aculeatus ß-glucosidase 1 gene (aabg1), as shown in our previous study. However, subsequent experiments using T. reesei extracts supplemented with the glycoside hydrolase (GH) family 10 xylanase III (XYN III) and GH Family 11 XYN II showed increased conversion of alkaline treated cellulosic biomass, which is rich in xylan, underscoring the importance of XYN III. To attain optimal saccharifying potential in T. reesei, we constructed two new strains, C1AB1 and E1AB1, in which aabg1 was expressed heterologously by means of the cbh1 or egl1 promoters, respectively, so that the endogenous XYN III synthesis remained intact. Due to the presence of wild-type xyn3 in T. reesei E1AB1, enzymes prepared from this strain were 20-30% more effective in the saccharification of alkaline-pretreated rice straw than enzyme extracts from X3AB1, and also outperformed recent commercial cellulase preparations. Our results demonstrate the importance of XYN III in the conversion of alkaline-pretreated cellulosic biomass by T. reesei.

Characterization of Aspergillus aculeatus ß-glucosidase 1 accelerating cellulose hydrolysis with Trichoderma cellulase system.

Aspergillus aculeatus ß-glucosidase 1 (AaBGL1), which promotes cellulose hydrolysis by Trichoderma cellulase system, was characterized and compared some properties to a commercially supplied orthologue in A. niger (AnBGL) to elucidate advantages of recombinant AaBGL1 (rAaBGL1) for synergistic effect on Trichoderma enzymes. Steady-state kinetic studies revealed that rAaBGL1 showed high catalytic efficiency towards ß-linked glucooligosaccharides. Up to a degree of polymerization (DP) 3, rAaBGL1 prefered to hydrolyze ß-1,3 linked glucooligosaccharides, but longer than DP 3, preferred ß-1,4 glucooligosaccharides (up to DP 5). This result suggested that there were different formation for subsites in the catalytic cleft of AaBGL1 between ß-1,3 and ß-1,4 glucooligosaccharides, therefore rAaBGL1 preferred short chain of laminarioligosaccharides and long chain of cellooligosaccharides on hydrolysis. rAaBGL1 was more insensitive to glucose inhibition and more efficient to hydrolyze the one of major transglycosylation product, gentiobiose than AnBGL, resulting that rAaBGL1 completely hydrolyzed 5% cellobiose to glucose faster than AnBGL. These data indicate that AaBGL1 is valuable for the use of cellulosic biomass conversion.

Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785.

A maltotriose-forming amylase (G3Amy) from Kitasatospora sp. MK-1785 was successfully isolated from a soil sample by inhibiting typical extracellular α-amylases using a proteinaceous α-amylase inhibitor. G3Amy was purified from the MK-1785 culture supernatant and characterized. G3Amy produced maltotriose as the principal product from starch and was categorized as an exo-α-amylase. G3Amy could also transfer maltotriose to phenolic and alcoholic compounds. Therefore, G3Amy can be useful for not only maltotriose manufacture but also maltooligosaccharide-glycoside synthesis. Further, the G3Amy gene was cloned and expressed in Escherichia coli cells. Analysis of its deduced amino acid sequence revealed that G3Amy consisted of an N-terminal GH13 catalytic domain and two C-terminal repeat starch-binding domains belonging to CBM20. It is suggested that natural G3Amy was subjected to proteolysis at N-terminal region of the anterior CBM20 in the C-terminal region. As with natural G3Amy, recombinant G3Amy could produce and transfer maltotriose from starch.

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.

Crystal structures of glycoside hydrolase family 3 ß-glucosidase 1 from Aspergillus aculeatus.

GH3 (glycoside hydrolase family 3) BGLs (ß-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion.

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.

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). 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.

Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterparts.

Although the capabilities of Trichoderma reesei cellulases have been greatly improved, these enzymes are still too costly for commercial use. The aim of this research was to assess the biomass saccharification capability of JN11, a recombinant cellulase, compared with that of the commercially available cellulases Accellerase 1500 and Cellic CTec. The activities of JN11, Accellerase 1500, and Cellic CTec were compared by using various types of cellulosic biomass, including rice straw, Erianthus, eucalyptus, and Japanese cedar. JN11 had higher saccharification capability for rice straw, Erianthus, eucalyptus, and Japanese cedar compared with the commercial cellulases. The JN11 saccharification of cellulosic biomasses, including hemicellulose (NaOH-pretreated biomasses), resulted in high glucose and xylose yields because of the high xylanase/xylosidase activity of JN11. Moreover, even JN11 saccharification of hemicellulose-free biomasses (sulfuric acid-, hydrothermally, and steam exploded-pretreated biomasses) resulted in high glucose yields. The cellulase activity of JN11, however, was comparable to that of its commercial counterparts. These findings indicate that the saccharification ability of cellulase is unrelated to its cellulase activity when measured against Avicel, CMC, pNP-lactoside, and other substrates. JN11 showed high activity for all types of pretreated cellulosic biomasses, indicating its usefulness for saccharification of various cellulosic biomasses.

XlnR-independent signaling pathway regulates both cellulase and xylanase genes in response to cellobiose in Aspergillus aculeatus.

The expression levels of the cellulase and xylanase genes between the host strain and an xlnR disruptant were compared by quantitative RT-PCR (qPCR) to identify the genes controlled by XlnR-independent signaling pathway. The cellulose induction of the FI-carboxymethyl cellulase (cmc1) and FIb-xylanase (xynIb) genes was controlled by XlnR; in contrast, the cellulose induction of the FIII-avicelase (cbhI), FII-carboxymethyl cellulase (cmc2), and FIa-xylanase (xynIa) genes was controlled by an XlnR-independent signaling pathway. To gain deeper insight into the XlnR-independent signaling pathway, the expression profile of cbhI was analyzed as a representative target gene. Cellobiose together with 1-deoxynojirimycin (DNJ), a glucosidase inhibitor, induced cbhI the most efficiently among disaccharides composed of ß-glucosidic bonds. Furthermore, cellobiose with DNJ induced the transcription of cmc2 and xynIa, whereas cmc1 and xynIb were not induced. GUS reporter fusion analyses of truncated and mutated cbhI promoters revealed that three regions were necessary for effective cellulose-induced transcription, all of which contained the conserved sequence 5'-CCGN(2)CCN(7)G(C/A)-3' within the CeRE, which has been identified as the upstream activating element essential for expression of eglA in A. nidulans (Endo et al. 2008). The data therefore delineate a pathway in which A. aculeatus perceives the presence of cellobiose, thereby activating a signaling pathway that drives cellulase and hemicellulase gene expression under the control of the XlnR-independent regulation through CeRE.

Halophilic characterization of starch-binding domain from Kocuria varians α-amylase.

The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85°C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70°C in the presence of 0.2-2.0M NaCl. The refolding rate was enhanced with higher salt concentration.

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus ß-glucosidase 1 for efficient biomass conversion.

To develop a Trichoderma reesei strain appropriate for the saccharification of pretreated cellulosic biomass, a recombinant T. reesei strain, X3AB1, was constructed that expressed an Aspergillus aculeatus ß-glucosidase 1 with high specific activity under the control of the xyn3 promoter. The culture supernatant from T. reesei X3AB1 grown on 1% Avicel as a carbon source had 63- and 25-fold higher ß-glucosidase activity against cellobiose compared to that of the parent strain PC-3-7 and that of the T. reesei recombinant strain expressing an endogenous ß-glucosidase I, respectively. Further, the xylanase activity was 30% lower than that of PC-3-7 due to the absence of xyn3. X3AB1 grown on 1% Avicel-0.5% xylan medium produced 2.3- and 3.3-fold more xylanase and ß-xylosidase, respectively, than X3AB1 grown on 1% Avicel. The supernatant from X3AB1 grown on Avicel and xylan saccharified NaOH-pretreated rice straw efficiently at a low enzyme dose, indicating that the strain has good potential for use in cellulosic biomass conversion processes.

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.

Development of a homologous transformation system for Aspergillus aculeatus based on the sC gene encoding ATP-sulfurylase.

A homologous transformation system was developed using the endogenous ATP-sulfurylase gene, AasC, as a selectable marker in Aspergillus aculeatus. Spontaneous mutation was proved to be beneficial in isolating AasC-deficient mutants. Molecular analysis of sC(+) transformants revealed that the frequency of single copy integration at ATP-sulfurylase locus was more than 40%.