Isolation and characterization of mutants of Trichosporon adeninovorans resistant to 2‐deoxy‐D‐glucose

Abstract

We isolated stable mutants from Trichosporon adeninovorans resistant to 2-deoxy-~-glucose. All mutants were altered in the C-catabolite repression and produced different amounts of glucoamylase and 8-glucosidase when grown on several carbon sources. In addition to the C-catabolite derepression of these extracellular enzymes, a repression defect .of isocitrate lyase was found.Several yeast species have been investigated as potentially valuable candidates for the production of single cell protein or ethanol from starch. Therefore, in various laboratories extracellular amylolytic enzymes of yeasts have become subject of study (CLEMENTI et al. 1980, SA-CORREIA and VAN UDEN 1981, SPENCER-MARTINS 1982, TUCKER et al. 1984, SILLS et al. 1984a, MODENA et al. 1986, CLEMENTI and ROSS 1986.Mutants resistant to carbon catabolite repression by glucose have been investigated for the genetical characterization and regulation of production of extracellular enzymes. These mutants were isolated by selecting for 2-deoxy-~-glucose or glucoseamine resistance (ZIMMERMANN and SCHEEL 1977, VAN UDEN et al. 1980, CABECA-SILVA 1982, MCCANN and BARNETT 1984, SILLS et al. 1984b, MANCZINGER 1985, BROWN et al. 1987, DE MOT and VERACHTERT 1987.Recently we characterized an extracellular glucoamylase of a new yeast species, Trichosporon adeninovorans, described by MIDDELHOVEN et al. (1984) (BUTTNER et al. 1987). In this paper we report on the isolation and characterization of carbon catabolite derepression mutants from T. adeninovorans using 2-deoxy-~-glucose. Materials and methodsStrain and growth conditions: Trichosporon adeninovorans LS3 is a strain from our laboratory. Yeast cells were grown at 30 "C on a rotary shaker in 500-ml-flasks containing 200 ml of a minimal salt medium (TANAKA et al. 1967) supplemented with 1 mg biotin and 1 mg thiamine per litre. Ethanol, hexadecane, glucose, galactose, maltose or soluble starch were used as carbon source (1 %). The growth was followed by measuring the absorbance at 650 nm.Induction and isolation ofmutants: Cells ofa 12-h-culture werecentrifhgated and resuspended in 0.9 % NaCl solution. Mutants resistant to repression of starch hydrolysis were isolated after N-methyl-N -nitro-"-nitrosoguanidine mutagenesis. The concentration of the mutagen was 2.5 mM and a cell concentration of 10*/ml was used. After 2 h at 25 "C the cells were centrifugated and washed with 0.9 % NaCl solution. After resuspension the cells were plated on mineral salt medium containing 1 %corn starch and 0.1 % 2-deoxy-~-glucose, solidified with 2 % agar. Mutant colonies were selected after 72 h.Enzyme preparation: To determine some enzyme activities either culture supernatant, cell-free extract or permiabilized cells were used. To prepare cell-free extract yeast was resuspended in 50 mM Tris/HCl buffer (pH 8.0), disrupted by passing them twice through a X-pressure cell and centrifugated at 20,000 x g for 20 min. Permiabilized cells were produced by cooling of yeast cells overnight at -20 "C in 100 mM sodium acetate buffer (pH 5.0) ...

Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms

“…A better understanding of how glucose regulates gene expression in yeast was driven both by a need to understand the mechanism of gene regulation at a fundamental level [19], but also because of the need to ameliorate carbon source usage of various yeasts (eg. starch, xylose) [20][21][22], for industrial needs such as brewing [23,24] or dough leavening [25], or to ameliorate the expression of enzymes of industrial interest that are sometimes glucose-repressed [26,27].…”

A history of research on yeasts 10: foundations of yeast genetics1

“…Sphaeroplast fusion has been used for genetic mapping41. A number of auxotrophic mutants have been obtained and the yeast appears to have four chromosomes42, 185, 323, 324 (for reviews, see37, 380).…”