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Biolechnolog)' Lellers, Vol 19, No II. N'Jt'ember 1997,pp, 1119-1122

D-Xylose metabolism in Rhodospofidium tOfu/aides S.N. Freer*, C.D. Skory and R.J. Bothast Fermentation Biochemistry Research, National Center for Agricultural Research, Agriculture Research Service, 1815 N. University St., Peoria, IL 61604

Hofer et al. (Biochem. Biophys. Acta 1971. 252:1-12) presented circumstantial evidence that suggested that Rhodosporidium toruloides produced a xylose isomerase. We were unable to detect this activity in cell-free extracts of this yeast, however, xylose reductase and xylitol dehydrogenase activities were detected.

Introduction ethanol, substantial amounts of xylitOl were also D-Xylose is one of the most abundant pentose sugats produced. It was concluded that D-xylose fermentation found in nature. It is the predominate hemicellulosic was limited by cofactOr (NAD/NADH) imbalance or by sugar of hardwoods and agricultural residues, an insufficient capacity for xylulose conversion by the accounting for up to 25% of the dry weight biomass pentose phosphate pathway (Kotter and Ciriacy, 1993; of some plant species (ladisch et aI., 1983). In plant Tantirungkij et al., 1993). Recently, Ho and Tsao tissues, it exists primarily in the anhydride form, xylan, (995) constructed a Saccharomyces strain that contained that is easily separated and saccharified into monomeric the xylose reductase, xylitol dehydrogenase and xylulose units by eirher mild chemical or enzymatic treatment. kinase genes. This recombinant organism was able to The abundance and ease of isolation of D-xylose make ferment both glucose and D-xylose, however, xylitol was it a potential feedstOck for the production of other useful also produced (Moniruzzaman et aI., 1997). It is chemicals. unknown whether this is the result of a co-factOr imbal­ ance, limiting xylitOl dehydrogenase or xylulokinase The metabolism of D-xylose by bacteria and yeasts has activities, or some Other factor(s). been studied extensively. Bacteria isomerize xylose directly to xylulose with xylose isomerase (EC 5.3.1.5). The presence of D-xylose isomerase has been reported Yeasts initially reduce D-xylose to xylitOl with in the yeasts Candida uti/is (Tomoyeda and Horitsu, NADPH-linked xylose reductase (aldose reductase; EC 1964) and RhodosporidiuTll tomloides (Hofer et al., 1971), 1.1.1.21) and then convert xylitOl to xylulose with as well as the thermophilic fungus i\Ialbmnchea pu/che//a NAD-linked xylitOl dehydrogenase (D-xylulose reduc­ var. sll!jurea (Banerjee et aI., 1994) and the mesophilic tase; EC 1.1.1.9)(Chakravorty et al., 1962; Smiley and fungus Nettrospora crassa (Rawat et al., 1996). To deter­ Bolen, 1982). Thereafter, xylulose is converted to mine the pathway that Rh. tomloides used for D-xylose xylulose 5-phosphate by xylulokinase (EC 2.7.1.17) and metabolism, Hofer et al. (971) prepared acetOne metabolized by D-xylulose-5-phosphate phosphoketo­ powders of cell-free extracts from D-xylose-grown cells. lase (EC 4.1.2.9) and the pentose phosphate pathway The assay for enzyme activity was based upon the disap­ (Evans and Ratledge, 1984). Saccharomyces cerevisiae is pearance of D-xylose from reaction mixtures in. the unable to ferment D-xylose, however, it can ferment absence of added NADH or NADPH, not the produc­ xylulose. When xylose isomerase was added to D-xylose­ tion of xylulose. To measure D-::-'y'lose, it was first containing media, S. cerevisiae produced ethanol, by converted to xylulose by a bacterial },:ylose isomerase and fermenting the xylulose produced by the exogenous then the xylulose was quantified by the cysteinel enzyme (for review, see du Preez, 1994). Bacterial xylose carbazole/sulfuric acid method. Furthermore, xylitOl was isomerases have been cloned into S. cerevisiae, however, not utilized as a carbon source by induced cells, even the expression of acrive enzymes in yeasts has been though it was taken up by the cells. These results unsuccessful (Amore et al., 1989; Sarthy et al., 1987). led Hofer et al. (1971) to conclude that Rh. tomloides S. cerez'isiae transformed with the yeast genes for xylose metabolized D-xylose via xylose isomerase, not by reductase and xylitol dehydrogense tend to utilize xylose reductase and xylitOl dehydrogenase. Due to D-xylose slowly, incompletely and almost entirely the persistant problems associated with recombinant oxidatively. Although these transformants produced yeast containing modified xylose reductase and xylitOl

© 1997 Chapman & H'lll BiolfC!JIIO/Ogy Lmm· Vo/19· ;'\0 11 . 1997 1119 S.N. Freer et al. dehydrogenase genes and the lack of success in getting cysteine-HCl, 2 mM D-xylose and 0.1 mL of crude baererial xylose isomerase expressed in an acrive form enzyme preparation. After incubation of the reaction in yeasr, we decided to reexamine the presence of xylose mixture at 30°C for 60 min, xylulose was assayed by isomerase activity in Rh. tomloides. A xylose isomerase the cysteine/carbazole/sulfuric acid method (Dische and gene from a yeast species might be a good candidate Borenfteund, 1951),' Idemical results were obtained for cloning and expressing in Saccharomyces. We were with both buffer systems. Xylose reductase activity was unable to deteer xylose isomerase activity in cell-free assayed in reaction mixtures (1.0 mL) containing extracrs from D-xylose-grown Rh. tomloides cells, 50 mM Tris-HCl (pH 7.5), 50 mM D-xylose, 0.34 mM however xylose reductase and xylitol dehydrogenase NADPH and 0.025 mL of enzyme preparation. The activities were readily detectable. oxidation of NADPH was followed specrrophotometri­ cally as a decrease in absorbance at 340 nm. Xylitol Materials and methods dehydrogenase was assayed in a similar manner, except Organism and media 50 mM xylitol and 2 mM NAD- was used in the reac­ The yeasts used in this study were obtained from the tion mixture and the formation of NADH was followed Agriculture Research Service Culture CoUection, by rhe inctease in absorbance at 340 nm. Background National Cemer for Agricultural Urilization Research, reductase and dehydrogenase activities were also Peoria, IL. Rhodosporidil/m tomloides (syn. Rhodotomla measured and included imo rhe calculations. One unit gll/tinis) NRRL Y-17,902 was originally deposired by of enzyme activity represems 1 f.l.mole of cofactor M. HOfer wirh The American Type Culture Collection converted pet minute. (ATCC 26,194). All of the experimems reponed herein were also performed using the rype strain, Rhodotomla Analytical methods gll/tinis NRRL Y-2,502. However, since the data were Sugar and sugar alcohol concemrations wete analyzed similar, only the data obtained using NRRL Y-17,902 by HPLC using a HPX-87H column (Bio-Rad are reponed. Cells were cultivated as described previ­ Laboratories) and a diffetential refractOmeter. Protein ously (Kotyk and Hofer, 1965), using eirher D-glucose concenttations were determined with the Bio-Rad or D-xylose as the sole carbon source. Cultures were Protein Assay kit, rhat is based on the Bradford method, grown aerobically in 250 ml baffled flasks containing using bovine serum albumin as the Standard. 50 ml medium in a 250 rpm rotary shaker adjusted to 29°C. Results and discussion Growth of Rh. toru/oides on D-xylose Cell extracts Many yeast produce xylirol when grown on D-xylose as An acerone powder was prepared from the cell-free a sole carbon source. If Rh. tom/aide5 does not contain supernatam of D-xylose-grown ceUs as described by xylose reductase, but ,rather COntains xylose isomerase Hofer et al. (1971). Also, D-xylose- and D-glucose­ (Hofer et aI., 1971), it is unlikely that it would produce grown cells were harvesred by cemrifugarion at xylitol. For xylirol ro be produced, it would first have 8,000 X g for 10 min, washed mice with srerile distilled to be isomerized ro Aylulose and then reduced by a xylu­ warer and suspended in 100 mM Tris-HCl buffer (pH lose reductase (xylirol dehydrogenase). The results

7.5) containing 1 roM MnS04 and 0.5 mM DTT. The (Fig. 1) indicated that deteCtable levels of xylirol were cellular paste was homogenized with 30 g of OA5 mm produced by Rh. tortt/oides when grown in synthetic glass beads for 3 min at 4000 rpm in a Braun homog­ medium containing 3% (w/v) D-xylose as the sole enizer. After centrifugation at 8,000 X g for 20 min, carbon source. In 7 days, the organism utiljzed about NH4S04 was added to vatious degrees of saturation. 15 g D-xylose I-I and produced about 2.5 g xylitol I-I. After 1 hr at 4°C, the precipitares were collected by Thus, this yeast must possess either xylose isomerase cemrifugation at 8,000 X g for 20 min. The pellets were and xylulose redUCtase, or xylose reductase and xylitol suspended in 50 mM Tris/HCl buffer (pH 7.5) and dehydrogenase. assayed for activity. D-xylose metabolism enzymes Enzyme assays Rh. tortlloides was grown in synthetic medium containing Xylose isomerase aCtIVIry was assayed in reaction 4% (w/v) D-xylose for 3 days. The ce11s were harvested, mixtures (1.0 m]) comaining either 50 mM Tris-HCl disrupted and an acetone powder of the ce11-free extracr (pH 7.5), 1 mM MnS04, 0.5 mM DTT , 2 mM D­ was prepared identically ro that previously described by xylose and 0.1 mL of crude enzyme preparation or Hofer et al. (971). The results (Table 1) showed that , 80 mM borate buffer (pH 8.2), 1 mM ,MnS04 0.15% xylose isomerase acrivity was nor detectable in eithet

1120 BioredJ!lolog)' Letters· Vol 19 . No 11 . 1997 D-Xylose metabolism in Rhodosporidium roruloides

6 30 xylose isomerase actlvlty, we did detect xylose reduc­ :3 tase and xylitol dehydrogenase activities, at rates of 25 c 5 .2 36 and 52 Tlmole substrate per min per mg protein, ~ respectively, in our acetone powder. This is about 7- to 4 20 C III U lO-fold faster than the rate obtained by HOfer et at. :3 c .r:: 0 (1971). In a positive control experiment, xylose ~ 3 15 U e isomerase was readily detected in cell-free extracts of c.:> ;g 2 10 >. the xylose-utilizing, ruminal bacteria Selenomonas rtlmi­ ~ III nantium D (data not shown). til 5 0 >. x 0 In another attempt to detect xylose isomerase activity 2 3 4 5 6 7 in Rh. torttloides, culrures grown for three days with Time (Days) either D-xylose or D-glucose as the sole carbon source were harvested and homogenized as described in the Figure 1 Growth (n), D-xylose utilization (.) and xylitol Materials and Methods. Ammonium sulfate was added production (e) by Rh. toru/oides grown in synthetic medium ro the cell-free supernatants ro various degrees of satu­ initially containing 30 g D-xylose per liter. ration. Again, we were unable ro deteCt any xylose isomerase activity, while xylose reductase and xylitol dehydrogenase activities were readily detected (Table 2). Table 1 Xylose isomerase, xylose reductase and xylitol dehydrogenase activities of crude homogenates From D-xylose-grown cells, little xylose reductase and an acetone powder of D-xylose-grown activity precipitated at or below 60% ammonium sulfate Rh. toru/oides cells saturation, while the xylirol dehydrogenase activity precipitated at 60% of saturation. Although none of the Activity' activities were detectable in the crude cell-free prepa­ Sample XI" XF/O X/Y' rations from D-glucose-grown cells (data not shown), a Crude extract NDc 0.034 0.108 small amount of both xylose reductase and :l..)'lirol dehy­ Acetone powder

Table 2 Xylose isomerase, xylose reductase and xylitol dehydrogenase activities of ammonium sulfate fractionated cell-free homogenates of D-xylose- and D-glucose-grown Rh. toru/oides cells.

Activity" Xylose Isomerase Xylose Reductase Xylitol Dehydrogenase Sample Glucose D-Xylose Glucose D-Xylose Glucose D-Xylose

20% NH.SO. 40% NH.SO. 60% NH.SO. 80% NH.SO. NADH NADP+

"Activity is expressed as p.mole of NAD+ or NADPH converted per min per mg protein. "NO is not detected.

Bioteebl/%g)' Letters· Vol 19 . ,\"0 11 . 1997 1121 S.N. Freer er al.

Likewise, in rhe NAD-linked xylirol dehydrogenase du Preez, Jc. (994). Enzyme Microb. Technol. 16:944-956 reaction, a product accumulated thar had the same Evans, CT, and Ratledge, C (1984). Arch. Microbiol. 139:4H-52 Ho, NWY, and Tsao, GT (995). The Parent Cooperarion Treary retention time as xylulose. If co-facror was omitted from (PCT) Parent No. W095/13362 the reaction, it did not appear that any D-xylose was Hofer, M, Beu, A, and Koryk, A (971). Biochim. Biophys. consumed, nor were any new products detected (data Acta. 2552:1-12 not shown). Kotter, P, and Ciriacy, M (993). Appl. Microbiol. Biorechnol. 38:776- 783 Koryk, A, and HOfer, M (1965). Biochim. Biophys. Acta. Thus, we were unable to confirm the report of Hofer 102:410-422. et al. (1971). We detected no evidence of a xylose iso­ Ladisch, MR, Lin, KW, Voloch, M and Tsao, GT (983). Enzyme merase in Rh. torlt/oides, however, we did detect xylose Microb. Technol. 5:82-102 reductase and xylirol dehydrogenase activities. If Rh. Moniruzzaman, M, Dien, BS, Skory, CD, Chen, ZD, Hespell, tom/oides possesses a xylose isomerase, it cannot playa RB, Ho, NWY, Dale, BE, and Borhasr, R] (997). World). Microbiol. Biorhehnol. 13:341-346 majot role in the metabolism of D-xylose by this yeast. Rawar, U, Phadrara, S, Deshpande, V, and Rao, M (996). Biorechno!. Lett. 18:1267-1270 References Smiley, KL, and Bolen, PL (982). Biorechno!. Len. 4:607-610 Amore, R, Wilhelm, M, and Hollenberg, CP (1989). App!. Sarrhy, AV, McConaughy, BL, Lobo, Z, Sundstrom, JA, Furlong, Micrbiol. Biorechnol. 30:351-357 CE, and Hall, BD (1987). App!. Environ. Microbiol. Banerjee, S, Archana, A, and Sryanarayana, T (994). Curro 53:1996-2000 Microbiol. 29:349-352 Tanrirungkij, M, Nakashima, N, Seki, T, and Yoshida, T (1993). Chakravorry, M, Veiga, LA, Bacila, M and Horecker, BL (1962). ). Ferment. Bioeng. 75:83-88 J. BioI. Chem. 237:1014-1020 Tomoyeda, M, and Horirsu, H (1964). Agr. BioI. Chern. Dische, Z and Borenfreund, E (951).] Bioi Chern 192:119-131 28:139-143

Received 3 July 1997; Revisions requested 31 July 1997; Revisions received 18 September 1997; Accepted 22 September 1997

Supplied by the U.S. Department of Agriculture, National Center for Agricultural Utilization Research, Peoria, Illinois.

1122 Biotechnology Letten· Vol 19 . )\"0 11 . 1997