55NISR Research GRANT
2009 Young Investigator Research Grant

Generation of Saccharomyces cerevisiae Capable to Ferment
Hexoses and Pentoses Derived from Lignocellulosic Biomass
by Protein- and Metabolic-engineering
Seiya WATANABE
Graduate School of Agriculture, Kyoto University (Present address: Faculty of Agriculture, Ehime University)

Research aims
Generation of microorganism (s) with an increased ability to produce ethanol from lignocellulosic biomass is oneof most important technological problems for industrial
bioethanol production. Since lignocellulosic biomass contains a large amount of xylose, microorganisms capable ofefficiently fermenting xylose have been sought. Althoughstrains of Saccharomyces cerevisiae transformed withxylose-metabolic genes from other yeasts such as Pichia
stipitis is the most potent recombinant strains capable offermenting xylose, their ability to produce ethanol is not
sufficient for industrial applications because their consumption rate of xylose is much slower than that of glucose.
Therefore, in this study, I first sought P. stipitis genes involved in sugar transport and/or sensing by bioinformatic
genome analysis, then characterized the putative transporter
genes and finally introduced those genes into S. cerevisiae
to improve the pentose fermentation.
Methods
Protein-BLAST search was performed against the P.
stipitis genome sequence using the S. cerevisiae HXT1 protein as a query. Thirty-six genes with over 30% similarity
were individually subcloned between the phophoglycerate
kinase (PGK) promoter and terminator of the yeast multi-Fig. 1.

Identification of hexose transporters from P. stipitis.

copy vector YEpPGK. S. cerevisiae KY73 strain (hxt1-7gal2 ura3) was a kind gift from Prof. Toshiko Kasahara(Teikyo University, Japan). Metabolite analysis for fermentation experiments was carried out by using HPLC and gas
chromatography.
ResultsThe S. cerevisiae genome has the highest numbers ofsugar (hexose) transporter (HXT) genes in all sequenced
genomes, and a null mutant of S. cerevisiae deleted for allof the HXT genes, incapable to grow on any hexose, is now
available. Furthermore, since several HXT genes also play a(potential) role in pentose transport, the hxt-null mutantlacks the ability to transport not only hexose (s) but alsopentose (s) such as xylose and L-arabinose. P. stipitis canmetabolize (and ferment) pentoses much more efficiently
than S. cerevisiae, indicating the existence of an efficientpentose transport system (s) in P. stipitis, which is missing
in S. cerevisiae.
I first identified thirty-six putative sugar transporter
genes in P. stipitis by bioinformatic analysis and then evaluated physiological function of each gene products by expressing the identified genes in the S. cerevisiae hxt-nullmutant, KY73 and examining their phenotypes (Fig. 1). Although it was found that P. stipitis has a limited number oftransporters capable of transporting hexose, I could identify

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Fig. 2.

Identification of pentose transporters from P. stipitis.

Fig. 3.

Xylose fermentation by P. stipitis xylose transporters in S. cerevisiae KY73

several novel P. stipitis hexose transporters in addition tothe known transporters, SUT1-3 1). In particular, HGT2 wasshown to be the most abundantly expressed transporter
gene in P. stipitis cells grown on all the hexoses (and alsothe pentoses) tested in this study. Although RGT2 andSNF3 had been annotated as “sugar sensor”, this study revealed that RGT2 had the ability to at least transport mannose, indicating some difference (s) in sugar-sensing mechanism between P. stipitis and S. cerevisiae.
I next examined transformants expressing each of theputative P. stipitis transporter genes for their ability to transport pentose by measuring intercellular pentose concentration using HPLC (Fig. 2). In a previous study1), it was
shown that certain transporters of P. stipitis (SUT1, SUT2and SUT3) have an ability to transport xylose and that
SUT1, in particular, exhibits a relatively high ability for xylose transport. In addition, the previous observation that thesut1-disrupted strain exhibited a similar transport activity
for xylose compared to the wild-type strain suggested thatP. stipitis may possess additional major xylose transporter(s) other than SUT1. Transport activity for xylose observedwith HGT2, a newly identified transporter from P. stipitis,
was similar to that observed with SUT1, suggesting an important role of HGT2 for xylose metabolism in P. stipitis.
Significant transport activity for L-arabinose was foundonly with HGT2 and XUT1, but not with SUT1, SUT2 andSUT3. Interestingly, when HGT2 and XUT1 were co-expressed, an additive effect was observed. On the other hand,their L-arabinose uptake ability was found to be less thanthat observed for the S. cerevisiae GAL2. Furthermore,
HGT2 transported xylose 5-fold faster than L-arabinose.
Indeed, L-arabinose has previously been shown to be apoorer carbon source for P. stipitis than xylose. This isprobably due to low uptake ability of the P. stipitis transporters for L-arabinose.
The function of the P. stipitis xylose transporters wereevaluated in the S. cerevisiae KY73X strain, which wasconstructed by introducing the three xylose-metabolic
genes (XR, XDH and XK) into the hxt-null mutant, KY732)(Fig. 3). Significant xylose fermentation was found onlywith transformants expressing SUT1 or HGT2. Although
the xylose consumption rate observed for the SUT1expressing strain was 2-fold higher that observed for theHGT2-expressing strain, there was no difference in ethanol
production between the two strains, indicating that a part ofxylose incorporated by the SUT1-expressing strain wasaccumulated in cells as by-products such as xylitol andglycerol.
Conclusion
In this study, I carried out a systematic search for sugartransporters in P. stipitis, a pentose-metabolizing yeast, andidentified several novel pentose transporters. Based on theresults obtained in this study, I already had applied for national and international patents.
References
1.
2.

Weierstall, T., Hollenberg, C.P., and Boles, E. (1999) Mol. Microbiol.
31, 871–883.
Matsushika, A., Inoue, H., Watanabe, S., Kodaki, T., Makino, K., andSawayama, S. (2009) Appl. Environ. Microbiol. 75, 3818–3822.