Institut für Mikrobiologie, Heinrich-Heine-Universität, Universitätsstr. 1, Geb. 26.12.01, D-40225 Düsseldorf, Germany1
Department of Applied Microbiology, Lund University, 22100 Lund, Sweden2
Author for correspondence: Eckhard Boles. Tel: +49 211 81 12778. Fax: +49 211 81 15370. e-mail: boles{at}uni-duesseldorf.de
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ABSTRACT |
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Keywords: Saccharomyces cerevisiae, xylose uptake, glucose uptake, xylose fermentation, heterologous expression
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INTRODUCTION |
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Efforts to establish a xylose-utilizing pathway in S. cerevisiae by insertion of the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis or other organisms have resulted in only poor ethanol production from xylose (Kötter & Ciriacy, 1993 ; Tantirungkij et al., 1993
; Walfridsson et al., 1995
). Various steps, including the uptake of xylose, have been suggested to limit the metabolism of xylose in metabolically engineered S. cerevisiae (Kötter & Ciriacy, 1993
; Eliasson et al., 2000
). Uptake of xylose by S. cerevisiae has been proposed to be mediated more or less unspecifically by its hexose-transport system. This is composed of a large family of 18 related transporter proteins called Hxts and additional sugar transporters with broader substrate specificity (Boles & Hollenberg, 1997
; Wieczorke et al., 1999
). In xylose-uptake experiments with S. cerevisiae cells, two kinetically distinct uptake components with Km values of about 0·19 M and 1·5 M were determined, demonstrating that the monosaccharide transport system in S. cerevisiae has nearly a 200-fold lower affinity for xylose than for glucose (Kotyk, 1967
; Lagunas et al., 1982
; Kötter & Ciriacy, 1993
).
To improve the xylose-uptake activity of S. cerevisiae it is necessary to identify the specific transporters mediating uptake of xylose. Additionally, heterologous xylose transporters from other organisms may be expressed in S. cerevisiae to improve xylose uptake. Using S. cerevisiae TMB3201, expressing a functional xylose utilization pathway but lacking the complete monosaccharide-transport system, we have characterized the xylose-transporting properties of monosaccharide transporters from yeast, bacteria and plants, by growth and [14C]xylose uptake. Especially the high-affinity glucose transporters of S. cerevisiae can mediate uptake of xylose. Nevertheless, overexpression of these transporters in the xylose-fermenting yeast strain PUA-X1,2 did not increase its growth rate on xylose nor its xylose fermentation rate.
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METHODS |
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Construction of plasmids.
The 0·4 kb SacI/SpeI MET25 promoter fragment of multicopy plasmid p426MET25 (Mumberg et al., 1994 ) was replaced by a 0·4 kb DNA fragment containing an HXT7 promoter fragment from -392 bp to -1 bp that was amplified by PCR with primers P426H7-1 (5'-CTAGAGCTCG TAGGAACAAT TTCGG-3') and P426H7-2 (5'-CGACTAGTGT GATGGTGATG GTGATGCATG TTAACTTTTT GATTAAAATT AAAAAAACTT-3'), and plasmid YEpkHXT7 (Krampe et al., 1998
) as the template, resulting in plasmid p4H7. All monosaccharide-transporter genes were cloned by recombination-cloning into p4H7 using the strategies described by Wieczorke et al. (1999)
. The yeast hexose-transporter genes were amplified by whole-cell PCR from strain FY1679 with GENEPAIRS primers obtained from Research Genetics. The Arabidopsis thaliana STP2 and STP3 ORFs were amplified from plasmids pUC19STP2 and pUC19STP3 using the primers F-ATSTP2 (5'-GGAATTCCAG CTGACCACCA TGGCTGTTGG TTCGATGA-3'), R-ATSTP2 (5'-GATCCCCGGG AATTGCCATG CTAGTCTTTG AAATATTTCT TCC-3'), F-ATSTP3 (5'-GGAATTCCAG CTGACCACCA TGGTAGCAGA AGAAGCAAG-3') and R-ATSTP3 (5'-GATCCCCGGG AATTGCCATG TCAATGGCTA AGAATGGTG-3'), respectively. The xylE ORF was amplified from Escherichia coli strain JM109 with primers F-XYLE (5'-GGAATTCCAG CTGACCACCA TGAATACCCA GTATAATTCC-3') and R-XYLE (5'-GATCCCCGGG AATTGCCATG TTACAGCGTA GCAGTTTGTT G-3'). PCR products were further amplified with primers T3 (5'-GAATAAACAC AAAAACAAAA AGTTTTTTTA ATTTTAAGGA ATTCCAGCTG ACCACC-3') and T2-ORFs (5'-GGGGGAGGGC GTGAATGTAA GCGTGACATA ACTAATTACA TGACTCGAGG ATCCCCGGGA ATTGCCATG-3'). The predicted A. thaliana xylose-proton symporter ORF was amplified from a seedling cDNA bank (Minet et al., 1992
) with primers F7-AT5G (5'-TAATTTTAAT CAAAAAATGT GTTTTAAATC GAGGCCTGAC-3') and R-AT5G (5'-CCCCGGGAAT TGCCATGTCA CTTCAAGATT TTTGATTC-3'), and the PCR product was further amplified with primers T71-ORFs (5'-AACACAAAAA CAAAAAGTTT TTTTAATTTT AATCAAAAA-3') and T2-ORFs. p4H7 was linearized with BamHI and HindIII and transformed into yeast strain EBY.VW4000, together with the PCR-amplified sugar-transporter genes, selecting for uracil prototrophy on a medium containing 2% maltose after homologous recombination in yeast. Plasmids were reisolated, amplified in E. coli SURE (Stratagene), and analysed by restriction enzyme mapping or sequencing (Seqlab). The resulting plasmids were named pTH[name of gene] (e.g. pTHHXT1, pTHHXT3). Construction of plasmids YEpkHXT7, p426MET25-HXT11 and pHL125-GAL2 is described elsewhere (Krampe et al., 1998
; Wieczorke et al., 1999
; Liang & Gaber, 1996
).
Other techniques.
Molecular biology techniques were performed using published procedures (Sambrook et al., 1989 ). Xylose uptake was assayed as described previously (Weierstall et al., 1999
). For the determination of the ethanol production rates, ethanol concentrations in yeast culture supernatants were measured using an enzymic test kit (Roche).
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RESULTS |
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To identify which hexose transporters are able to take up xylose, the growth rates of the transformants in liquid synthetic medium without uracil and with 2% xylose as the sole carbon source were determined. Only transformants expressing the Hxt4, Hxt5, Hxt7 or Gal2 transporters were able to grow in the xylose medium (Fig. 1). In contrast, transformants expressing the transporters Hxt1, 3, 8, 9, 10, 11, 13, 14 and 15 did not grow with 2% xylose. Our results indicated that Hxt4, 5 and 7, and Gal2, are able to mediate the uptake of xylose into S. cerevisiae cells. We did not analyse Hxt2 as it was not possible to clone it behind the HXT7 promoter fragment on vector p4H7. Hxt6, Hxt16 and Hxt17 were not included because they are almost identical to Hxt7, Hxt15 and Hxt13, respectively. HXT12 is a pseudogene (Wieczorke et al., 1999
).
Determination of xylose-uptake activities
To confirm the results obtained in the growth tests, short-term (60 s) xylose uptake assays were performed with radiolabelled xylose. Xylose uptake was linear for at least 60 s under the conditions used (data not shown). Transformants of strain TMB3201 expressing Hxt4, Hxt5, Hxt7 or Gal2, or containing the empty vector p4H7, were grown into the exponential growth phase in synthetic medium without uracil and with 2% maltose. As expected, the hexose-transport-deficient strain TMB3201 with the empty vector was not able to take up xylose (Fig. 2). In contrast, expression of Hxt4, 5 and 7, and Gal2, conferred xylose uptake activities upon the strain. Xylose uptake activity with 50 mM radiolabelled xylose was highest for the strain expressing Hxt7 [48 nmol min-1 (mg dry weight)-1] and lowest for the strain expressing Hxt5 (11 nmol min-1 (mg dry weight)-1] (Fig. 2
). The parental strain CEN.PK2-1C transformed with the empty vector p4H7 exhibited an uptake activity of 15 nmol min-1 (mg dry weight)-1.
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The E. coli xylE gene encodes a xyloseproton symporter (Davis & Henderson, 1987 ). The hexoseproton symporters Hup1 from Chlorella kessleri, and Stp2 and Stp3 from A. thaliana, have been shown to mediate uptake not only of various hexoses but also of xylose (reviewed by Büttner & Sauer, 2000
). Moreover, after completion of the A. thaliana genome sequencing project, a sequence has been found on chromosome 5 encoding a predicted xyloseproton-symporter-like protein targeted to chloroplasts (accession number At5g59250). The xylE, STP2 and STP3 ORFs, as well as the putative xyloseproton symporter sequence lacking the 31 amino acids encoding the chloroplast-targeting sequence, were cloned into the multicopy expression vector p4H7. After transformation of the resulting plasmids and plasmid pNEV-E expressing HUP1 behind the yeast PMA1 promoter (Robl et al., 2000
) into strain TMB3201, Stp2, Stp3 and Hup1, but not as expected XylE and the putative A. thaliana xyloseproton symporter, supported growth of the yeast transformants on synthetic medium with 2% glucose (data not shown). However, none of the transporters restored growth in synthetic medium with 2% xylose, indicating that they are not able to take up xylose into the yeast cells. Moreover, we transformed strain TMB3201 with the P. stipitis genomic DNA library YEpTW (Weierstall et al., 1999
) and an A. thaliana seedling cDNA bank (Minet et al., 1992
), and selected the transformants for growth in synthetic medium with 2% xylose. From a total of more than 18000 and 10000 transformants, respectively, we could not find any transformant able to utilize and to grow on xylose.
Xylose fermentations and growth tests with HXT-overexpressing strains
The S. cerevisiae PUA strains were selected for faster growth on xylulose media (Porep, 1987 ; Kötter & Ciriacy, 1993
). Subsequently, the P. stipitis XYL1 and XYL2 ORFs under control of the S. cerevisiae PDC1 and ADH1 promoters, respectively, were chromosomally integrated in these strains, resulting in PUA-X1,2. In order to see whether overexpression of xylose-transporting proteins could increase the ethanol production rate from xylose by recombinant S. cerevisiae strains, pTHHXT5, YEpkHXT7, pHL125-GAL2 and the empty vector were transformed into the PUA-X1,2 strain. The transformants were pre-grown on synthetic medium with 2% xylose to an OD600 of 1·9. Cells were harvested by centrifugation and washed twice with 2% xylose medium. They were then resuspended in 0·1 vol. of the volume of the same medium, incubated at 30 °C and the ethanol concentration in the medium determined at different time points. As can be seen in Fig. 3
, overexpression of the monosaccharide transporters did not increase the ethanol production rate. Instead, it seemed that ethanol production was even slightly delayed, especially in the case of the Gal2-overproducing transformants.
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DISCUSSION |
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The results were obtained after constitutive overexpression of the hexose-transporter genes. Normally, these genes are regulated by the presence and concentration of glucose or galactose (reviewed by Boles & Hollenberg, 1997 ; Özcan & Johnston, 1999
). Whereas expression of HXT1 to HXT4 requires the presence of glucose in the medium, GAL2 is only expressed in the presence of galactose and absence of glucose. In contrast, HXT5 and HXT7 are also expressed in the absence of glucose (Schulte et al., 2000
; Diderich et al., 1999
, 2001
). This means that in recombinant xylose-utilizing S. cerevisiae cells during growth on xylose mainly Hxt5 and Hxt7 may contribute to the uptake of the sugar.
None of the heterologous sugar transporters was able to support growth on xylose. In the case of E. coli XylE and the predicted A. thaliana xyloseproton symporter, we cannot exclude that these transporters are not correctly targeted to the plasma membrane in a functional form, as has been observed for several heterologous membrane proteins in yeast (Villalba et al., 1992 ; Jahn et al., 2002
; our own unpublished results). However, C. kessleri Hup1 and A. thaliana Stp2 and Stp3 were functionally expressed in the plasma membrane, as they supported growth of the hexose-transport-deficient yeast strain on glucose. Growth of these strains on glucose was significantly lower than parental growth, indicating that the capacity of the heterologous transporters was very low. For all three transporters, xylose-uptake activity has been demonstrated (Büttner & Sauer, 2000
). Therefore, we propose that the xylose-uptake activity mediated by Hup1, Stp2 and Stp3 is too low to support growth on xylose.
Overexpression of the xylose-transporting hexose transporters in a xylose-utilizing S. cerevisiae PUA strain did not result in faster growth on 2% xylose medium under aerobic conditions, nor did it result in an increase of the ethanol production rate under (semi-)anaerobic conditions. This is in contrast to the recent observation that under anaerobic conditions in chemostat cultures with mineral medium increasing the xylose concentration in the feed enhanced the xylose flux, suggesting that transport limits flux (Eliasson et al., 2000 ). However, in that study a different, CEN.PK-derived yeast strain was used. Indeed, the PUA strain that we have used has been selected for faster growth on xylulose medium (Porep, 1987
; Kötter & Ciriacy, 1993
). One of its new properties is a higher endogenous level of xylulose kinase activity. The growth rate on xylose was more than twofold higher for the PUA-derived recombinant strain than for the CEN.PK-derived strain. Interestingly, the xylose-uptake activity was also about twofold higher in PUA cells (data not shown). This could mean that in the PUA strain during selection for faster growth on xylulose the sugar-uptake system was one of the steps that were optimized. Unfortunately, the parental strain is no longer available to confirm this assumption. Nevertheless, it seems that in different yeast strains different factors might contribute to limited xylose fermentation, with xylose uptake being one of them.
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ACKNOWLEDGEMENTS |
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Received 29 April 2002;
revised 7 June 2002;
accepted 13 June 2002.