Kojic acid biosynthesis in Aspergillus oryzae is regulated by a Zn(II)(2)Cys(6) transcriptional activator and induced by kojic acid at the transcriptional level.

A gene encoding the Zn(II)(2)Cys(6) transcriptional factor is clustered with two genes involved in biosynthesis of a secondary metabolite, kojic acid (KA), in Aspergillus oryzae. We determined that the gene was essential for KA production and the transcriptional activation of KA biosynthetic genes, which were triggered by the addition of KA.

Penicillin biosynthesis in Aspergillus oryzae and its overproduction by genetic engineering.

Aspergillus oryzae penicillin biosynthetic genes were clustered. The penicillin production was positively regulated by VeA, a global gene regulator required for transcriptional expression of the penicillin biosynthetic genes. Overexpression of the biosynthetic genes by a strong promoter yielded a greater than 100-fold increase in penicillin production.

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.

A novel vector for positive selection of inserts harboring an open reading frame by translational coupling.

We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can then be selected by exposure to colicin E3. Our method differs from previous methods in that the marker gene is activated without protein fusion, and that selection occurs irrespective of the reading frame of the insert.

Construction of a positive selection marker by a lethal gene with the amber stop codon(s) regulator.

A novel positive selection marker for Escherichia coli transformation was developed. The marker consisted of a DNA fragment encoding the C-terminal ribonuclease domain (CRD) of colicin E3 (colE3) and one or more amber stop codons between the initiation codon and the E3-CRD coding sequence. The toxicity of the marker was controlled by the suppressor activity the host cells possessed. This allowed both effective selection and propagation of the vector possessing the maker by selecting appropriate hosts from among those widely distributed: sup+ strains for selection and sup0 strains for propagation respectively. The insert DNA fragment was introduced onto the vector by replacing the marker DNA. The transformants harboring the vector with an insert grew, but those without an insert were effectively removed by the killing activity of E3-CRD encoded on the marker DNA. The marker was also successfully applied to lambda phage display vector.

Affinity selection of DNA-binding proteins from yeast genomic DNA libraries by improved lambda phage display vector.

Phage display is a useful means of identifying and selecting proteins of interest that bind specific targets. In order to examine the potential of phage display for the genome-wide screening of DNA-binding proteins, we constructed yeast genomic libraries using lambda foo-based vectors devised in this work. After affinity selection using GAL4 UAS(G) as a probe, phages expressing GAL4 were enriched approximately 5 x 10(5)-fold from the library. Approximately 90% of polypeptides encoded in correct translation reading frames by the selected phages were known or putative polynucleotide-binding proteins. This result clearly indicates that the modified lambda phage display vector in combination with our enrichment technique has great potential for the enrichment of DNA-binding proteins in a sequence-specific manner.

A polymerase chain reaction-based method for cloning novel members of a gene family using a combination of degenerate and inhibitory primers.

We have developed a novel method for cloning gene family members by using a polymerase chain reaction technique. The method is based on the amplification of a broad range of homologous genes in combination with the specific inhibition of already cloned genes. To accomplish this, we designed degenerate primers to highly conserved regions among the gene family members, and inhibitory primers to the divergent region at the 3'-margin of each degenerate primer. The 5'-end of the inhibitory primer, the 3'-end of which was aminated, had 3-4 bases overlapping the 3'-end of the degenerate primer. The potential of this method was demonstrated by the successful cloning of a novel member of the yeast MKC7/YAP3 gene family homologue from a filamentous fungus, Aspergillus oryzae, by inhibiting amplification of an already cloned homologue, opsB.