7559 431 Production of J3-glucosidase and diauxic usage of sugar mixtures by Candida molischiana Shelby N. Freer and Christopher D. Skory Abstract: The fennentation of cellobiose is a rare trait among yeasts. Of the 308 yeast species that utilize cellobiose aerobically. only 12 species fennent it, and only 2 species, Candida molischiana and Candida wickerhamii, also fennent cellodextrins. Candida molischiana produced l3-glucosidase activity on all carbon sources tested, except glucose, mannose, and fructose. When these sugars were added to cultures growing on cellobiose, the synthesis of l3-glucosidase ceased. However, the total amount of enzyme activity remained constant, indicating that the C. molischiana l3-glucosidase is catabolite repressed and not catabolite inactivated. When grown in medium initially containing glucose plus xylose, cellobiose. maltose, mannitol. or glucitol, C. molischiana preferentially utilized glucose and produced little l3-glucosidase activity until glucose was nearly depleted from the medium. When grown in medium containing cellobiose plus either fructose or mannose. the yeast preferentially utilized the monosaccharides and produced little l3-glucosidase activity. Candida molischiana produced l3-glucosidase and co-utilized cellobiose and xylose. maltose, or trehalose. Glucose and fructose. mannose. or trehalose were co-utilized; however, no l3-glucosidase activity was detected. Thus, the order of substrate preference groups appeared to be (glucose, trehalose. fructose, mannose) > (cellobiose, maltose, xylose) > (mannitol, glucitol). Key words: glucose repression, trehalase. diauxic utilization, yeast, Resume: La fennentation du cellobiose est un phenomene rare chez les levures. Pam1i 308 especes de levures qui utilisaient Ie cellobiose en aerobiose, seulement 12 Ie fennentaient et seulement 2 especes, Candida molischiana et Candida wickerhamii. fennentaient aussi les collodextrines. Candida molischiana avait une activite l3-glucosidase envers toutes les sources de carbone verifiees al'exception du glucose, du mannose et du fructose. Lorsque ces sucres etaient ajoutes ades cultures croissant en presence de cellobiose. la synthese de l3-glucosidase s'arretait, Par contre, l'activite enzymatique globale demeurait constante signifiant que la l3-glucosidase de C. moliochiana etait reprimee mais pas inactivee par un produit du catabolisme. Lorsque C. molischiana etait cultivee dans un milieu contenant au depart du glucose et soit du xylose. du cellobiose, du maltose, du mannitol ou du glucitol. la levure utilisait Ie glucose de fac;on preferentielle et son activite l3-glucosidase demeurait faible jusqu'au moment ou Ie glucose etait apeu pres epuise dan Ie milieu de culture. Lorsque cette levure etait cultivee dans un milieu contenant du cellobiose et soit du fructose ou du mannose. elle utilisait les monosaccharides de fac;on preferentielle et I'activite l3-glucosidase demeurait faible. Candida molischiana produisait une l3-glucosidase et co-utilisait Ie cellobiose et Ie xylose. Ie maltose ou Ie trehalose. Le glucose et Ie fructose, Ie mannose ou Ie trehalose etaient co-utilises, mais aucune activite l3-glucosidase n'etait detectee. L'ordre de preference pour les groupes de substrats apparalt done etre (glucose, trehalose, fructose, mannose) > (cellobiose. maltose. xylose) > (mannitol, glucitol). iV/ots eNs: repression par Ie glucose, trehalase, utilisation diauxique, levure. [Traduit par la redaction] Introduction Received September 26, 1995. Revision received December 16. 1995. Accepted December 19, 1995. Glucose. fructose. and mannose have significant effects upon S.N. Freer and C.D. Skory. Fennentation Biochemistry the expression of numerous genes in yeasts. Many ofthe genes Research Unit, National Center for Agricultural Utilization required for the metabolism of alternate carbon sources Research. Agricultural Research Service. U.S. Department of (sucrose. galactose. maltose. glyceroL and ethanol) are Agriculture (USDA), I 1815 N. University Street, Peoria. repressed during growth on glucose. while several genes IL 61604. U.S.A. involved in glucose utilization are induced during growth on glucose. Both glucose repression and glucose induction ofgene expression in Saccharomyces cerevisiae appears to be primar­ ily regulated at the transcriptional level (Johnston and Carlson 1993; Entian and Barnett 1992; Gancedo 1992; Trumbly 1992). Product names are necessary to report factually on available data. How·ever. the USDA neither guarantees nor warrants Sevetalof the gluconeogenic enzymes (cytosolic malate the standard of the product. and the use of the name by dehydrogenase. phosphopyruvate carboxykinase. isocitrate USDA implies no approval of the product to the exclusion lyase. etc.) are glucose (catabolite) inactivated. Generally. the of others that may also be suitable. inactivation of these enzymes by glucose starts 5-10 min after Can. J. Microbial. 42: 431-436 (1996). Printed in Canada / Imprime au Canada 432 Can. J. Microbial. Vol. 42, 1996 the addition of glucose to cells growing on nonfermentable The enzyme is produced constitutively. except that glucose. carbon sources and the activity of these enzymes is greatly fructose. and mannose at concentrations > 25 gIL. repress its reduced within I h. Glucose inactivation is irreversible and expression; the aeration state had no effect upon f3-glucosidase appears to depend upon targeting specific enzymes to the expression. Under all conditions tested. diauxic utilization of vacuole where they are inactivated by vacuole protease(s). The glucose-cellobiose mixtures was observed (Freer 1995). mechanism by which glucose triggers this process is unknown Because of the potential utility of these unique enzymes in (Entian and Barnett 1992; Holzer 1976). Although our under­ the biofuels and food and beverage industries. experiments standing of glucose repression in S. cerevisiae has increased were performed to characterize the effect that various sugar greatly in the last 10 years. the exact mechanism ofhow glucose mixtures have upon f3-g1ucosidase production by C. molis­ triggers the cessation of synthesis of specific enzyme is still chiana. The results showed that the f3-glucosidase was catabo­ incomplete and even less is known about the mechanisms that lite (glucose) repressed. while an extracytoplasmic trehalase regulate glucose repression in other yeasts. was constitutively produced. Because of the similarities Many yeasts utilize cellobiose when grown aerobically; between the f3-glucosidase and trehalase. this might be a valu­ however. few yeasts ferment it. Of the approximately able aid in studying the underlying mechanisms of glucose 800 known yeast species. 308 yeasts aerobically utilize cello­ repression in yeast. biose. while only 12 of these species also ferment it (Barnett 1976). The majority of the cellobiose-fermenting yeasts pro­ Materials and methods duce a cytoplasmic f3-IA-glucosidase(s) (EC 3.2.1.21) and ferment only cellobiose. In contrast. two yeast species. Can­ Source of chemicals and yeast dida molischiana and Candida ,vickerhamii (syn. Torulopsis Glucose. peptone. yeast extract. and malt extract were purchased from molischiana and Torulopsis wickerhamii. respectively). pro­ Difco Laboratories. Detroit. Mich. p-Nitrophenyl 13-o-gluco­ duce an extracytoplasmic f3-g1ucosidase(s) that enables them pyranoside (pNPG). 13-0-(+)-cellobiose. xylose. mannose. fructose. to ferment both cellobiose and cellodextrins of degree ofpoly­ maltose. trehalose. glucitol. mannitol. and the glucose detection kit. which was based upon the glucose oxidase - peroxidase reaction. merization 3 to 6 (Gonde et al. 1984; Freer and Detroy 1982; were purchased from Sigma Chemical Co.. St. Louis. Mo. All other LeClere 1984; Freer 1985. 1991; Vasserot et al. 1991). These chemicals were purchased from Fisher Scientific Co.• Fair Lawn. N.J. yeasts have potential for use in the production of fuel alcohol. The yeast NRRL Y-2237 Candida molischiana (Zikes) Meyer et When commercial fungal cellulase enzyme complex is used to Yarrow was obtained from the Agricultural Research Service Culture saccharify cellulose. one ofthe rate-limiting enzymatic steps is Collection. National Center for Agricultural Utilization Research. the conversion of cellobiose to glucose by f3-glucosidase Peoria. Ill. (Desrochers et al. 1981: Sternberg et al. 1977). If yeasts that ferment both glucose and cellobiose are used in the simultane­ Media and culture conditions ous saccharification-fermentation ofcellulose. 10-30% more The basal medium. adjusted to pH 6.0 with HCI. consisted ofpeptone. ethanol is produced (Freer and Detroy 1983; Spindler et al. yeast extract. and malt extract at concentrations of 5. 3. and 3 gIL. 1992) than in fermentations that employ S. cerevisiae (Savarese respectively. The basal medium was prepared at twofold concentration and Young 1978: Blotkamp et al. 1978: for a recent review see and the various carbon sources. dissolved in deionized distilled water. Grohman 1993). which is unable to ferment cellobiose. Addi­ were sterilized separately by autoclaving at 121°C for 20 min and tionally. yeast f3-glucosidases have potential for use in the food combined after cooling. Xylose was filter sterilized. Inocula were and beverage industry for the production offood flavors. Using prepared as described previously (Freer and Detroy 1982) by growing the yeast aerobically for 24 h in 20 mL of basal medium containing Muscat grape marc as a substrate. Vasserot et al. (1991) dem­