J. FERMENT. BIOENG.,

Abstracts of the Articles Printed in Seibutsu-kogaku Kaishi

Vol. 76. No. 5 (1998)

Development of High-Frequency Transformation Methods in Asper- gillus oryzae. KENJI OZEKI,* YOKO ISHIMARU (NAGAMAXJ), AKIO KODA, TOSHITAKA MINETOKI, KAZUHIKO OHBUCHI, MASAAKI HAMACHI, and CHIEKO KUMAGAI (General Research Laboratory, Ozeki Corp., 4-9 Imazu Dezaike-cho, Nishinomiya, Hyogo 663-8227) Seibutsu-kogaku 16: 187-193. 1998.

High transformation efficiency was obtained by an improvement of transformation procedures in Aspergillus oryzae. When Yatalase was used as the protoplasting enzyme and the resultant protoplasts were stored at 5°C for 1 d, the efficiency of transformation using in- tegration vectors showed increases of 4.8-fold in A. oryzae argB_ and 11 -fold in A. oryzae niaD-, compared to the usual method (iovozym 234 and CELLULASE R-10 were used as the nrotonlasting enzvmes. and the resultant competent cells were transformed ;mmedi%ely). The efficiency of transformation using plasmid (AMAI) vectors showed increases of 5.6-fold in A. oryzae argB- and 4.2-fold in A. oryzae niaD-, compared to the usual method.

* Corresponding author.

Improvement of Fermentative Activity of Sake Yeast by Giving Resistance to Clotrimazole. HIROKO MIZOGUCHI,* MUTSUMI WATANABE, AKIRA NISHIMURA, and KYOICHI KONDO (Research and Development Dept., Hakutsuru Sake Brewing Co. Ltd., 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe, Hyogo 658-0041) Seibutsu-kogaku 76: 194-199. 1998.

Mutants resistant to clotrimazole (CTZ) were isolated from a non-urea forming sake yeast strain, HL-163, and an isoamyl acetate highly producing strain, Ffe-13. Most of the CTZ-resistant mutants fermented sake mash faster, and produced higher amounts of alcohol than their parent strains, while keeping their parental advantages for sake making. CTZ-resistant mutants were also resistant to cyclo- heximide, sulfometuron methyl, cerulenin, chloramphenicol, and oligomycin. Since the chemical structures of these drugs and their actions toward yeast cell are widely different from each other, CTZ- resistant mutants showed pleiotropic drug resistance (PDR) pheno- types. In a Northern analysis, the transcript of PDRS, a gene encod- ing an ATP-binding cassette (ABC) transporter, which extrudes the drugs out of yeast cell, was overexpressed in CTZ-resistant mutants. These observations indicated that CTZ-resistant mutants with im- proved fermentative activity must have a certain mutation related to PDR.

* Corresponding author.

Purification and Characterization of ,&V-Acetylhexosaminidase from Streptomyces sp. No. 499. -Note- MITSUHIRO FUJIMORI* and AKIHIRO OKITANI (Department of Food Science and Technology, Nippon Veterinary and Animal Science University, 1-7-I Kyonan-cho, Musashino-shi, Tokyo 180-8602) Seibutsu-kogaku 76: 200-204. 1998.

In the course of our screening program for chitinase and ,B-N- acetylhexosaminidase (HexoNAcase) from the fermented broth of about 800 stock cultures of Streptomyces sp. isolated from soil sam- ples, the highest amounts of both enzymes were found to be produced in the broth by Streptomyces sp. no. 499 isolated from a soil sample collected at Toyonaka-shi, Osaka, Japan. The enzymes was produced

by Streptomyces sp. no. 499 cultivated with chitin as the growth sub- strate. HexoNAcase from the filtrate of the broth was purified by chro- matography using, successively, Amberlite CC-50, P-cellulose, and Sephadex G-100 columns. The enzyme was completely purified, its purity being comfirmed by SDS-polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was demonstrated to be 52,000 by a gel chromatography and 57,ooO by SDS-PAGE. Its optimum pH and temperature for hydrolytic action were 6.0 and 55°C towards p-nitrophenyl-B-N-acetylglucosaminide, and 5.0 and 55°C towards p-nitrophenyl+N-acetylgalactosaminide. The enzyme was confirmed to readily hydrolyze chitobiose, chitotriose, and chitotetraose in that order, releasing N-acetylglucosamine in the mode of the exo-type hydrolase.

* Corresponding author.

Molecular Characterization of the Structural Elements of Chloreila Chromosomes: Modeling of Plant Artificial Chromosomes. -Monograph- Ta~snr Y-A (Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, 1-4-I Kagamiyama, Higashi- Hiroshima 739-8527) Seibutsu-kogaku 76: 205-216. 1998.

The future growth of plant biotechnology will be built upon a better understanding of complex, multigene traits, including the pathways of natural-product biosynthesis and the mechanisms of resistance against environmental stresses, along with a better integra- tion of biochemistry, physiology, genetics, and developmental and cel- lular biology. Development of new tools and methods for the transfer of multiple genes (sometimes in the order of a hundred) to plants will be required for various purposes. The plant artificial chromosome (PAC) promises to be a powerful candidate as a next-generation vector in plant transformation. Learning from the success of YACs (yeast artificial chromosomes) in the cloning of huge human DNAs, it is clear that a minimum of three types of chromosome elements are needed to construct a linear, mitotically stable artificial chromosome: a centromere, telomeres, and origins of replication. The natures of the corresponding elements in plant chromosomes are, however, less well understood. To obtain an experimental model for molecular and structural analyses, we screened a variety of organisms by pulsed-field gel electrophoresis for the smallest plant-type chromosome. Species of the unicellular green alga Chlorella were found to contain very small genomes of approximately 39 Mbp, almost a quarter the size of the Arabidopsis genome. In the case of Chlorella vulgaris C-169, 16 chromosomes ranging from 980Kbp to 4.0Mbp in size could be resolved by CHEF gel electrophoresis under ordinary conditions. The smallest chromosome of this strain (chromosome I, 980 Kbp) would serve as an excellent experimental subject for structural analysis because it can be routinely isolated easily and in quantity. With this chromosomal DNA, a set of overlapping cosmid clones (a contig) has been established. Telomeres obtained from this chromosome con- sist of 5’-TTTAGGG repeats running from the centromere toward the termini, a sequence identical to that reported for higher plants. Putative replication origins and centromeric components isolated as curved DNA from C-169 chromosome I were characterized by sequencing and localizing on the chromosome. By combining the Chlorella chromosomal structural elements thus obtained, various forms of artificial chromosomes can be now designed to be introduced and stably maintained in plants.

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