The transformation of Aspergillus oryzae has been achieved with a plasmid carrying the Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OCTase). The frequency of transformation was relatively low (0.7 transformants/^g DNA)but the transformed phenotype was extremely stable for manygenerations without selective pressure. Southern blot analysis revealed that transformation had occurred by integration of multiple tandem copies of plasmid DNAinto the host genome through non-homologous recombination. There was no evidence of the existence of free plasmid in the transformants. The number of integrated copies of the plasmid ranged from 15 to 60. The specific activity of OCTase in the cellfree extract was proportional to the copy number of the plasmid, indicating that most of the integrated argB gene was expressed. Aspergillus oryzae has been important in fermentation industries in Japan, i.e., sake, shoyu and miso manufacture as well as the production of industrial enzymes. Strain improvement in A. oryzae has been carried out by mutation and protoplast fusion,1} but not yet by genetic transformation, since vectors carrying selectable markers for the development of a transformation system are not readily available.

Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae RIB40 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding beta-glucuronidase (GUS) down-stream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.

No abstract

Useful cryophilic killer strains of Saccharomyces cerevisiae were bred by back-crossing KL-88, which is a wild killer sake yeast, with a repetitive parent WL-7and is one of the best cryophilic wine yeasts. The hybrids maybe aid in producing good wines in pure cultures at relatively low temperature (13~15°C).A haploid of KL-88 was crossed with haploids of WL-7to yield first-generation hybrids. Haploids possessing both killing activity and SO2 tolerance were selected from the hybrids and back-crossed with a haploid of WL-7 to yield second-generation hybrids. Several strains of killer hybrids were comparable to WL-7 in fermentation ability at 15°C, SO2 tolerance, TTCstain, and growth on jS-alanine mediumat 35°C. One strain (2HYL-2) aided in producing wine as good as with WL-7, in fermentation at 15°C on pilot-plant scale.The hybrid killed only Saccharomyces yeasts in grape must. In the last-stage of fermenting Koshu grape must with the killer hybrid (2HYL-2), no sensitive yeasts were detected, because they were killed by the killer yeast. Moreover, most of the neutral yeasts, such as Kloeckera and Torulopsis, that were abundant in grape juice before the addition of the starter yeast, disappeared in the last-stage.On the other hand, many sensitive pseudo-film-forming yeasts of the genus Saccharomyces were detected in the last-stage of fermenting must with WL-7strain. These results show the possibility of fermenting musts with only a single strain"of cryophilic killer yeasts at a relatively low temperature without contaminating other yeast strains.Because grape musts are usually not pas-tolerance by back-crossing KL-88 strain, a teurized before fermentation in wine making, wild killer sake yeast, with OC-2 strain, one of many wild yeasts from natural grapes are the excellent wine yeasts, for repetitive parent brought to fermenting musts. Ohara and age. One ofthehybrids, 2HY-1strain, successNonomura1} reported that many yeast strains fully killed spontaneous wild yeasts of other than the yeast strain inoculated were Saccharomyces in grape musts containing SO2; participating in the fermentation ofmusts in a consequently, no film formation occurred in Japanese winery. Undesirable yeasts in the the resultant wine. must can harm the wine (off odor, excessive However, the hybrid was slow in fermenvolatile acid, incomplete fermentation, tur-tation at low temperature, around 15°C. It is bidity, etc.).2>3) Furthermore, bottled wine can highly desirable that fermentation proceeds begin fermentation again or film can form on smoothly at relatively low temperature to prothe surface of table wine during storage if duce wine of good quality, especially in white processing has been faulty, e.g., lack of SO2 or wine. Because Koshu grapes, a typical variety improper topping. Such phenomenawere for white wine in Japan, are usually harvested mainly caused by the growth of pseudo-film-late in cooler weather, the fermentation is apt forming yeasts, belonging to Saccharomyces to be delayed. Accordingly, cryophilic killer oviformis, S. ...

A 5.3-kbp fragment of the KHS gene was cloned from a genomic bank of Saccharomyces cerevisiae No. 115 constructed with an E. coli as the host and YEp13 as the vector. A non-killer yeast strain was transformed to a killer strain with the multi-copy vector containing the KHS gene, and the transformant could secrete 3-4 times more killer toxin into culture media than the donor, strain No. 115. The KHS toxin was purified 80-fold from the culture filtrate by gel filtration and column chromatography. The nucleotide sequence of a 2.8-kbp fragment of the KHS DNA that was enough for the expression of the killer activity was identified, and we found an open reading frame consisted of 2124 bp. Comparison of the open reading frame and N-terminal amino acid sequence of purified KHS toxin, suggested that the presumed peptide from the KHS gene might be processed between 36Gln and 37Ala before secretion.