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 20 g glucose/L. The cells were harvested aseptically by centrifugation. onstrated that the C. molischiana f3-g1ucosidase produced washed once with water. and resuspended in 10 mL of sterile water. monoterpenols from monoterpene heterosides. Thirty millilitres of experimental medium in 50-mL flasks were A major obstacle in the production of f3-g1ucosidase is that inoculated with 0.225 mL ofcell suspension. Flasks were capped with glucose represses the expression of f3-glucosidase(s) in most either cotton (aerobic growth) or serum stoppers and vented with yeasts. For example. glucose at concentrations > 2 gIL 26-gauge sterile needles (anaerobic growth). The cultures were incu repressed the expression of f3-g1ucosidase in Kluyveromyces bated at 28°C on a rotary shaker at 250 rpm. All results presented lactis (Herman and Halvorson 1963a. 1963b) and Kluyveromy herein represent aerobic growth conditions. since it was detennined ces fragi/is x Kluyveromyces dobzharskii (MacQuillan et al. that anaerobic growth of C. molischiana with fennentable sugars 1960: MacQuillan and Halvorson 1962). These cytoplasmic (glucose. fructose. mannose. cellobiose. trehalose. and cellodextrins) enzymes are produced constitutively when the yeasts are grown yielded identical results (data not shown). on carbon sources other than glucose. fructose. and mannose (MacQuillan et al. 1960: MacQuillan and Halvorson 1962). Growth, carbohydrate, J3-glucosidase, and trehalase The regulation of f3-g1ucosidase expression by C. wickerhamii analyses is similar. but not identical. to that described in the above Growth was measured by the increase in optical density at 600 nm. To organisms. in that both glucose and the aeration state of the assay for 13-glucosidase activity. appropriately diluted cells plus medium were incubated with 0.5 mL of2.5 mM pNPG in 0.1 M acetic culture appear to regulate f3-glucosidase expression. When acid (adjusted to pH 3.5 with HCI) for 15 min at 28°C. The reactions grown aerobically. f3-glucosidase is produced constitutively. were tenninated by the addition of 0.5 mL of 0.4 M glycine-NaOH except that glucose at concentrations greater than 50 gIL (pH 10.8) and the amount ofp-nitrophenol produced was detennined represses its expression. When grown anaerobically. f3-glucosi spectrophotometrically at 400 nm (Freer and Detroy 1983). One unit dase is produced even in the presence of 100 g glucoselL (Freer of enzyme activity represented 1 !Lmol of product (p-nitrophenol) and Detroy 1985). The C. molischiana f3-g1ucosidase appears fonned per minute. To assay for trehalase activity. appropriately to be regulated in a similar manner to the Kluyveromyces spp. diluted samples were incubated with 0.2 mL of 50 mM trehalose in Freer and Skory 433
Table 1. Effect of glucose, fructose and mannose upon Fig. 1. Test for catabolite inactivation. After 24 h of growth in C. molischiana l3-glucosidase activity.a medium containing 50 g cellobiose/L, C. molischiana cultures were diluted with either basal medium or medium containing Additions I3-Glucosidase activity (%)" glucose. fructose, or mannose. Samples were taken various times and growth (e), l3-glucosidase activity (A). cellobiose (0). None 100.0' and monosaccharide concentrations (_) were measured. 10 mM glucose 91.3 100 mM glucose 56.6 250 mM glucose 38.2 60 6 50 10 mM fructose 102.0 9 Cellob;",," 100 mM fructose 98.0 50 1 5 250 mM fructose 98.0 10 mM mannose s::: 40 4 100.9 0 100 mM mannose 98.1 :.;:: ctl 30 3:::J 250 mM mannose 90.3 .. E ...... s::: o Ql 20 22- a - Candida lI10lischiana was grown for 5 days in YM medium -u that initially contained 50 g cellobioselL. .Js::: E 0 10 1f "The substrate (pNPG) concentration was 2.5 mM. 'QU :.;:: 0- 'The control contained 3.57 U/mL of l3-glucosidase activity.
Fig. 2. Utilization of glucose, fructose and mannose. Candida Fig. 4. Utilization of cellobiose. maltose. xylose. glucitol. llIo1ischiana was grown in medium containing fructose, and mannitol. Candida llIo1ischiana was grown in medium mannose, glucose + fructose, or glucose + mannose. containing cellobiose (D) and maltose. xylose. glucitol. or Growth (e).I3-glucosidase activity (A), glucose (_). and mannitol (_). Residual medium carbohydrates (including fructose or mannose (D) were measured. xylitol (+)). growth (e). and l3-glucosidase activity (A) were measured. 60 6 c: 50 25 9 Fructose/L 25 9 Mannose/L 5 ~ 6 o c: 50 ,50 9 Celiobiose/L 40 9 Celiobiose/L 5 +:: 40 4 :::J o ~5 9 Maltose/L 25 9 Xylose/L ~ E +:: (5 ~ 30 3 :5 E 40 l\ 4 ? ... ..c: 30 ',.., 3 :5 :::Jgw 2i o Ql -t.l ~8 10 1 '> ...I c: 20 2 -~ E 0 ~:::J ~ oU ~~~~~~~1~ $!~ ~~ 0 10 CD...I O__+-I---1F=:l1bo.111__-"~I-f--+-+-+-I--T-iO« ~ «- ~ 6 Ql l.; 50 5 -s rJl .c: Ql e -c 40 4 'iii J1;j 00 5~ C) >. 0 o ...... "0 ~ 4~ "§ 30 ---~.. 3 15 C)>. o .c: ~ 20 2 c:;; o W 3~ cc. .c ... W 2~ U 10 1 Cll cc. U o~~~:±:::t::±Wd:::cl::!s;t::tl 0 10 1 o 2 4 6 0 2 4 6 o 0 Time (days) o 2 4 6 a 2 4 6 Time (days)
Fig. 3. Utilization of cellobiose, fructose, and mannose. Candida llIolischiana was grown in medium containing cellobiose (D) and either fructose or mannose (_). Residual Fig. 5. Utilization of glucose. cellobiose. and trehalose. Candida medium carbohydrates. growth (e) and l3-glucosidase molischiana was grown in medium containing trehalose (D) activity (A) were measured. and either glucose (_) or cellobiose (0). Residual medium carbohydrates. growth (e). and l3-glucosidase activity (A) were measured. 60 50 9 Celiobiose/L _ 50 9 Celiobiose/L 6 ::J -::J.c::_- 25 9 Fructose/L --"\25 9 Mannose/L E 50 5 2- ~- ~ 60 6 ::J '. ':; 25 9 Trehalose/L E 40 .~ ~ 4 ;; Co) 50 5 2 ~...... "'- Fig. 6. Utilization of glucose and trehalose. Candida lIlo1ischiana was grown in medium containing either trehalose (0) and (or) glucose (_). Residual medium carbohydrates. growth (e), and l3-glucosidase activity (A) were measured. c: 80 75 9 Glucose/L 65 9 Glucose/L 50 9 Glucose/L 4 0 10 9 Trehalose/L 25 9 Trehalose/L :;:::; ctS 60 3 ..J l- -E I- c: o -Cll 40 2 ::J -(,,) - >. .J c: ~ E 0 20 1 oU :;:::;> 0- (,,) co..J 0 _0)<- o < Cll .c: Cll f1l - 80 25 9 Glucose/L 10 9 Glucose/L 4 ctS ~~ 50 9 Trehalose/L 65 9 Trehalose/L 32 o I- 1-"'0 60, 3 f1l C)>. ~ 0 .c: (,,) 0 40 2 :::::l .Q G l- I ctS co. (.) 20 1 0 0 0 2 4 6 0 2 4 6 0 2 4 6 Time (days) glucose, fructose, and mannose repressed the expression of the were utilized in the presence of an excess of glucose (65 gIL). C. lIIo1ischiana l3-glucosidase and none of the carbon sources As the amount of trehalose initially present in the media tested overcame this repression. increased, glucose tended to increase, indicating that some of When grown in medium initially containing cellobiose and the trehalose was being converted to glucose. Thus, the treha maltose, C. lIIo1ischiana produced l3-glucosidase activity and lase produced by C. molischiana does not appear to be glucose coutilized the substrates (Fig. 4). No free glucose was detected (catabolite) repressed and the enzyme activity appears to be in the culture broths. Co-utilization also occurred in the culture resistant to end-product inhibition (i.e., the enzyme functioned grown in cellobiose and xylose: however, a large amount of in the presence of 65 gIL glucose). xylitol was detected in this culture (Fig. 4), as well as when the In summary, like the enzymes responsible for the metabolism organism was grown on xylose alone (data not shown). When of sucrose, galactose, maltose, glycerol, and ethanol in S. cere C. lIIo1ischiana was grown on mixtures of xylose and glucose, visiae (Johnston and Carlson 1993: Entian and Barnett 1992; fructose, or mannose, xylose utilization and xylitol formation Gancedo 1992; Trumbly 1992) and cellobiose in several was not observed (data not shown). Candida lIIolischiana Kluyveromyces spp. (Herman and Halvorson 1963a, 1963b: utilizes glucitol or mannitol as a sole carbon source (Freer MacQuillan et al. 1960: MacQuillan and Halvorson 1962), the 1995), but when grown in combination with cellobiose, the C. molischiana l3-glucosidase is glucose (catabolite) repressed. cultures preferentially utilized cellobiose and produced 13 Glucose, fructose, and mannose repressed the expression of glucosidase activity (Fig. 4). this enzyme. None of the carbon sources tested relieved the Previous results showed that C. lIIo1ischiana produced 13 repression, and unlike the C. ,vickerhamii l3-glucosidase (Freer glucosidase activity when trehalose was used as the sole carbon and Detroy 1985), the aeration state had no effect upon the source (Freer 1995). With trehalose-cellobiose mixtures, expression of the C. molischiana l3-glucosidase (data not C. molischiana utilized trehalose preferentially over cellobiose shown). (Fig. 5). Surprisingly, C. molischiana also appeared to prefer The order of substrate preference groups for C. molischiana entially utilize trehalose over glucose (Fig. 5). When grown in appeared to be (glucose, trehalose, fructose, mannose) > medium initially containing 50 g glucoselL and 25 g treha (cellobiose, maltose, xylose) > (glucitol, mannitol). The result 10seIL, the glucose concentration remained essentially constant that C. molischiana co-utilized glucose and trehalose was un for the first 3 days of growth, while the concentration of expected (Fig. 6). Other than the anaerobic use of glucose and trehalose was reduced to about 4 gIL. To determine if this was cellobiose by C. ,vickerhamii (Freer and Detroy 1985), this is truly diauxic utilization of the glucose-trehalose mixtures, the only other example, of which we are aware, of a yeast C. molischiana was grown in medium initially containing vari co-utilizing glucose and a disaccharide. ous concentrations of glucose and trehalose. The results The majority ofthe C. molischiana trehalase activity appears (Fig. 6) indicate that C. molischiana co-utilizes these carbon to be associated with the cell surface. Cells plus media con sources. Relatively small concentrations of trehalose (10 gIL) tained 2.6 U/mL of trehalase activity, while the isolated cells 436 Can. J. Microbial. Vol. 42,1996 and clarified media contained 2.0 and 0.4 U/mL of activity, Freer. S.N., and Detroy. R.W. 1985. Regulation of 13-1 ,4-g1ucosidase respectively. The location of the enzyme(s), as well as the fact expression by Candida wickerhalllii. Appl. Environ. Microbiol. 50: that glucose accumulated in the medium when C. molischiana 152-159. Freer. S.N., and Greene, R.V. 1990. Transport of glucose and cello was grown in medium containing high concentrations oftreha biose by Candida wickerhamii and C1avispora lusitaniae. J. BioI. lose, suggests that C. molischiana might not transport trehalose Chern. 265: 12864 - 12868. across its cytoplasmic membrane but, rather, first hydrolyzes Gancedo, J.M. 1992. Carbon catabolite repression in yeasts. Eur. trehalose and then transports the resultant glucose. This is J. Biochem. 206: 297-313. believed to be the mechanism by which C. molischiana and Gonde. P.. Blondin, B.. LeClere, M., Ratomahenina, R., Arnaud, A., C. lvickerhamii metabolize cellobiose (Freer and Detroy 1983; and Galzy, P. 1984. Fermentation ofcellodextrins by different yeast LeClere et ai. 1984: Freer and Greene 1990). strains. Appl. Environ. Microbiol. 48: 265-269. The majority ofthe yeast trehalases studied to date have been Gonde, P., Ratomahenina, R., Arnaud, A., and Galzy, P. 1985. Puri fication and properties of a exocellular l3-glucosidase of Candida intracellular enzymes whose primary function appears to be to lIlolischiana (Zikes) Meyer and Yarrow capable of hydrolyzing control the intracellular trehalose concentration, which is soluble cellodextrins. Can. J. Biochem. Cell BioI. 63: 1160-1166. thought to play a role as a reserve carbohydrate, in thermo Grohmann. K. 1993. Simultaneous saccharification and fern1entation tolerance and in the germination of spores (Elbein 1974; of cellulosic substrates to ethanol. In Bioconversion of forest and Thevelein 1984: Wiemken 1990). 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