Effective saccharification of kraft pulp by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae.

Kraft pulp is a promising feedstock for bioproduction. The efficiency of kraft pulp saccharification was improved by using a cellulase cocktail prepared from genetically engineered Aspergillus oryzae. Application of the cellulase cocktail was demonstrated by simultaneous saccharification and fermentation, using kraft pulp and non-cellulolytic yeast.

Aspergillus oryzae-based cell factory for direct kojic acid production from cellulose.

Background: Kojic acid (5-Hydroxy-2-(hydroxymethyl)-4-pyrone) is one of the major secondary metabolites in Aspergillus oryzae. It is widely used in food, pharmaceuticals, and cosmetics. The production cost, however, is too high for its use in many applications.

Identification of regulatory elements in the glucoamylase-encoding gene (glaB) promoter from Aspergillus oryzae.

The Aspergillus oryzae glucoamylase-encoding gene glaB is expressed specifically and strongly only during solid-state cultivation (SSC). To elucidate the basis for the specificity, the glaB promoter was analyzed by electrophoretic gel mobility shift assay (EMSA) which indicated two protein-binding elements from -382 to -353 and from -332 to -313. To confirm that these regions contained cis-elements, deletion analysis of the promoter was undertaken using β-glucuronidase as a reporter.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins.

Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture.

A novel promoter from a hemolysin-like protein encoding the gene, hlyA, was characterized for protein overexpression in Aspergillus oryzae grown in solid-state culture. Using endo-1,4-β-glucanase from A. oryzae (CelA) as the reporter, promoter activity was found to be higher than that of the α-amylase (amyA) and manganese superoxide dismutase (sodM) genes not only in wheat bran solid-state culture but also in liquid culture.

Deletion analysis of the catalase-encoding gene (catB) promoter from Aspergillus oryzae.

The catalase-encoding gene (catB) is expressed strongly in Aspergillus oryzae. To identify the transcription regulatory elements involved in strong expression, we did promoter deletion analysis using beta-glucuronidase (GUS) as a reporter and an electrophoretic gel mobility shift assay (EMSA) systematically. The deletion 200-bp sequence from -1,000 to -800 in the 1,400-bp catB promoter caused a drastic decrease in GUS activity.

The glucoamylase-encoding gene (glaB) is expressed in solid-state culture with a low water content.

Solid-state culture encourages high-level enzyme secretion by Aspergillus oryzae. Using the real-time quantitative reverse transcriptase-polymerase chain reaction, we confirmed that expression of the glucoamylase-encoding gene in A. oryzae cultured in solid-state culture depends on the water content of the culture.

Deletion analysis of the superoxide dismutase (sodM) promoter from Aspergillus oryzae.

The manganese superoxide dismutase gene (sodM) is very highly expressed in Aspergillus oryzae. To elucidate the basis for this high-level expression, deletion analysis of the promoter was undertaken using beta-glucuronidase (GUS) as a reporter. Deletion of a 63-bp sequence from -200 to -138 in the 1,038-bp sodM promoter caused a drastic decrease in GUS activity.

Cloning and expression analysis of two catalase genes from Aspergillus oryzae.

Fungi contain distinct genes encoding the same class of enzyme that are differentially regulated according to conditions. We cloned two catalase genes, catA and catB, from Aspergillus oryzae. The catA gene predicts a 747-amino-acid polypeptide sharing 81% identity with Aspergillus fumigatus catalase (catA) and 77% with Aspergillus nidulans catalase (catA).

Escherichia coli B gamma-glutamylcysteine synthetase: modification, purification, crystallization and preliminary crystallographic analysis.

Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The gammaGCS crystal diffracted to 2.