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D-Xylose Metabolism in Rhodospofidium Tofu/Aides S.N 8164 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.
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