Analysis of Expressed Sequence Tags from the Fungus Aspergillus oryzae Cultured Under Different Conditions

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories

Translocated duplication of a targeted chromosomal segment enhances gene expression at the duplicated site and results in phenotypic changes in Aspergillus oryzae

Abstract Background Translocated chromosomal duplications occur spontaneously in many organisms; segmental duplications of large chromosomal regions are expected to result in phenotypic changes because of gene dosage effects. Therefore, experimentally generated segmental duplications in targeted chromosomal regions can be used to study phenotypic changes and determine the functions of unknown genes in these regions. Previously, we performed tandem duplication of a targeted chromosomal segment in Aspergillus oryzae. However, in tandem chromosomal duplication, duplication of chromosomal ends and multiple chromosomal duplication are difficult. In this study, we aimed to generate fungal strains with a translocated duplication or triplication of a targeted chromosomal region via break-induced replication. Results Double-strand breaks were introduced into chromosomes of parental strains by treating protoplast cells with I-SceI meganuclease. Subsequently, strains were generated by nonreciprocal translocation of a 1.4-Mb duplicated region of chromosome 2 to the end of chromosome 4. Another strain, containing a triplicated region of chromosome 2, was generated by translocating a 1.4-Mb region of chromosome 2 onto the ends of chromosomes 4 and 7. Phenotypic analyses of the strains containing segmental duplication or triplication of chromosome 2 showed remarkable increases in protease and amylase activities in solid-state cultures. Protease activity was further increased in strains containing the duplication and triplication after overexpression of the transcriptional activator of proteases prtT. This indicates that the gene-dosage effect and resulting phenotypes of the duplicated chromosomal region were enhanced by multiple duplications, and by the combination of the structural gene and its regulatory genes. Gene expression analysis, conducted using oligonucleotide microarrays, showed increased transcription of a large population of genes located in duplicated or triplicated chromosomal regions. Conclusion In this study, we performed translocated chromosomal duplications and triplications of a 1.4-Mb targeted region of chromosome 2. Strains containing a duplication of chromosome 2 showed significant increases in protease and amylase activities; these enzymatic activities were further increased in the strain containing a triplication of chromosome 2. This indicates that segmental duplications of chromosomes enhance gene-dosage effects, and that the resulting phenotypes play important phenotypic roles in A. oryzae

Identification of a Basic Helix-Loop-Helix-Type Transcription Regulator Gene in Aspergillus oryzae by Systematically Deleting Large Chromosomal Segments▿ †

We previously developed two methods (loop-out and replacement-type recombination) for generating large-scale chromosomal deletions that can be applied to more effective chromosomal engineering in Aspergillus oryzae. In this study, the replacement-type method is used to systematically delete large chromosomal DNA segments to identify essential and nonessential regions in chromosome 7 (2.93 Mb), which is the smallest A. oryzae chromosome and contains a large number of nonsyntenic blocks. We constructed 12 mutants harboring deletions that spanned 16- to 150-kb segments of chromosome 7 and scored phenotypic changes in the resulting mutants. Among the deletion mutants, strains designated Δ5 and Δ7 displayed clear phenotypic changes involving growth and conidiation. In particular, the Δ5 mutant exhibited vigorous growth and conidiation, potentially beneficial characteristics for certain industrial applications. Further deletion analysis allowed identification of the AO090011000215 gene as the gene responsible for the Δ5 mutant phenotype. The AO090011000215 gene was predicted to encode a helix-loop-helix binding protein belonging to the bHLH family of transcription factors. These results illustrate the potential of the approach for identifying novel functional genes

Generation of Large Chromosomal Deletions in Koji Molds Aspergillus oryzae and Aspergillus sojae via a Loop-Out Recombination▿

We established a technique for efficiently generating large chromosomal deletions in the koji molds Aspergillus oryzae and A. sojae by using a ku70-deficient strain and a bidirectional marker. The approach allowed deletion of 200-kb and 100-kb sections of A. oryzae and A. sojae, respectively. The deleted regions contained putative aflatoxin biosynthetic gene clusters. The large genomic deletions generated by a loop-out deletion method (resolution-type recombination) enabled us to construct multiple deletions in the koji molds by marker recycling. No additional sequence remained in the resultant deletion strains, a feature of considerable value for breeding of food-grade microorganisms. Frequencies of chromosomal deletions tended to decrease in proportion to the length of the deletion range. Deletion efficiency was also affected by the location of the deleted region. Further, comparative genome hybridization analysis showed that no unintended deletion or chromosomal rearrangement occurred in the deletion strain. Strains with large deletions that were previously extremely laborious to construct in the wild-type ku70+ strain due to the low frequency of homologous recombination were efficiently obtained from Δku70 strains in this study. The technique described here may be broadly applicable for the genomic engineering and molecular breeding of filamentous fungi