Jun-ichi Sumitani

New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 α-amylase contributes to starch binding and raw starch degrading.

The alpha-amylase from Bacillus sp. no. 195 (BAA) consists of two domains: one is the catalytic domain similar to alpha-amylases from animals and Streptomyces in the N-terminal region; the other is the functionally unknown domain composed of an approx. 90-residue direct repeat in the C-terminal region. The gene coding for BAA was expressed in Streptomyces lividans TK24. Three active forms of the gene products were found. The pH and thermal profiles of BAAs, and their catalytic activities for p-nitrophenyl maltopentaoside and soluble starch, showed almost the same behaviours. The largest, 69 kDa, form (BAA-alpha) was of the same molecular mass as that of the mature protein estimated from the nucleotide sequence, and had raw-starch-binding and -degrading abilities. The second largest, 60 kDa, form (BAA-beta), whose molecular mass was the same as that of the natural enzyme from Bacillus sp. no. 195, was generated by proteolytic processing between the two repeat sequences in the C-terminal region, and had lower activities for raw starch binding and degrading than those of BAA-alpha. The smallest, 50 kDa, form (BAA-gamma) contained only the N-terminal catalytic domain as a result of removal of the C-terminal repeat sequence, which led to loss of binding and degradation of insoluble starches. Thus the starch adsorption capacity and raw-starch-degrading activity of BAAs depends on the existence of the repeat sequence in the C-terminal region. BAA-alpha was specifically adsorbed on starch or dextran (alpha-1,4 or alpha-1,6 glucan), and specifically desorbed with maltose or beta-cyclodextrin. These observations indicated that the repeat sequence of the enzyme was functional in the starch-binding domain (SBD). We propose the designation of the homologues to the SBD of glucoamylase from Aspergillus niger as family I SBDs, the homologues to that of glucoamylase from Rhizopus oryzae as family II, and the homologues of this repeat sequence of BAA as family III.

Cloning and sequencing of the cDNA encoding β-glucosidase 1 from Aspergillus aculeatus ☆

A cDNA was isolated from an Aspergillus aculeatus cDNA library using synthetic oligodeoxyribonucleotide mixtures that corresponded to the internal amino acid (aa) sequence of mature beta-glucosidase 1 (BGL1). Analysis of the nucleotide sequence of the cloned cDNA insert revealed a 2580-bp open reading frame (ORF) that encoded a 860-aa protein. The deduced aa sequence of the ORF shared sequence similarity with several BGL from other microorganisms.

Construction of a recombinant Trichoderma reesei strain expressing Aspergillus aculeatus β-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 β‐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 β‐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 β‐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 β‐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. Biotechnol. Bioeng. 2012;109: 92–99.

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

GH3 (glycoside hydrolase family 3) BGLs (β-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.

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.

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.

Cloning, nucleotide sequence, and transcriptional analysis of Aspergillus aculeatus no. F-50 cellobiohydrolase I (cbhI) gene

Abstract The cbhI gene, coding for a major cellobiohydrolase (CBHI) of Aspergillus aculeatus , was cloned and sequenced. The gene consists of 1620-bp and encodes a protein containing 540 amino acids with a calculated molecular mass of 56,723 Da. CBHI, composed of an N-terminal catalytic domain belonging to family 7 of the glycosyl hydrolases, and a C-terminal cellulose-binding domain (CBD) belonging to family I of the CBDs, showed high similarity with other fungal CBHIs, especially with that of Penicillium janthinellum . The cbhI gene transcription start points in A. aculeatus were defined by primer extension, and the putative promoter sequence was analyzed. This sequence was found to be closely related to the consensus sequences of various fungal genes. Transcription analysis by ribonuclease protection assay revealed that the cbhI gene is induced by low-molecular-weight cellooligosaccharide and repressed by glucose. The results emphasize the possibility that in the A. aculeatus cellulase system, cellobiose is the true inducer and the role of the cbhI gene lies within the cascade regulating cellulase induction.

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

Aspergillus aculeatus β-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 β-linked glucooligosaccharides. Up to a degree of polymerization (DP) 3, rAaBGL1 prefered to hydrolyze β-1,3 linked glucooligosaccharides, but longer than DP 3, preferred β-1,4 glucooligosaccharides (up to DP 5). This result suggested that there were different formation for subsites in the catalytic cleft of AaBGL1 between β-1,3 and β-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.

Overexpression and purification of Aspergillus aculeatus β-mannosidase and analysis of the integrated gene in Aspergillus oryzae

An expression plasmid for the manB gene encoding Aspergillus aculeatus beta-d-mannosidase (MANB) was constructed by using an expression vector carrying an improved promoter. After transformation of A. oryzae by the plasmid, several transformants formed colonies emitting fluorescence on a plate containing 4-methylumbelliferyl beta-d-mannopyranoside (MU-Man) under UV-irradiation. The transformant that displayed the strongest fluorescence, named A. oryzae BMN1, produced about 270 mg MANB/l in liquid culture. Recombinant MANB overproduced in BMN1 was purified by two steps of column chromatography to a single protein band on SDS-polyacrylamide gel electrophoresis and had a molecular weight of 130,000. Analyses by Southern blotting and genomic PCR demonstrated that a single copy of the plasmid was integrated into the chromosome by recombination at the niaD locus.

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 β-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′-CCGN2CCN7G(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.