Motoo Arai

Direct and Efficient Production of Ethanol from Cellulosic Material with a Yeast Strain Displaying Cellulolytic Enzymes

ABSTRACT For direct and efficient ethanol production from cellulosic materials, we constructed a novel cellulose-degrading yeast strain by genetically codisplaying two cellulolytic enzymes on the cell surface of Saccharomyces cerevisiae. By using a cell surface engineering system based on α-agglutinin, endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414 was displayed on the cell surface as a fusion protein containing an RGSHis6 (Arg-Gly-Ser-His6) peptide tag in the N-terminal region. EGII activity was detected in the cell pellet fraction but not in the culture supernatant. Localization of the RGSHis6-EGII-α-agglutinin fusion protein on the cell surface was confirmed by immunofluorescence microscopy. The yeast strain displaying EGII showed significantly elevated hydrolytic activity toward barley β-glucan, a linear polysaccharide composed of an average of 1,200 glucose residues. In a further step, EGII and β-glucosidase 1 from Aspergillus aculeatus No. F-50 were codisplayed on the cell surface. The resulting yeast cells could grow in synthetic medium containing β-glucan as the sole carbon source and could directly ferment 45 g of β-glucan per liter to produce 16.5 g of ethanol per liter within about 50 h. The yield in terms of grams of ethanol produced per gram of carbohydrate utilized was 0.48 g/g, which corresponds to 93.3% of the theoretical yield. This result indicates that efficient simultaneous saccharification and fermentation of cellulose to ethanol are carried out by a recombinant yeast cells displaying cellulolytic enzymes.

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.

Isolation of azo-dye-degrading microorganisms and their application to white discharge printing of fabric.

Three bacterial strains, which degraded azo dyes, were isolated from soil and sewage samples. The strains were identified as Bacillus sp. OY1-2, Xanthomonas sp. NR25-2 and Pseudomonas sp. PR41-1. The bacteria produced azo-dyes-degrading enzymes. That catalyzed the reduction of methyl red and produced dimethyl p-phenylenediamine and o-aminobenzoic acid. The enzymes could thus be applied to white discharge printing of azo-dyed fabric by owing to there.

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.

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.

Alteration of substrate specificity of fructosyl-amino acid oxidase from Fusarium oxysporum

Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as

Cloning, nucleotide sequence, and expression of the Clostridium thermocellum cellodextrin phosphorylase gene and its application to synthesis of cellulase inhibitors

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