APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2009, p. 2304–2311 Vol. 75, No.

8
0099-2240/09/$08.00⫹0 doi:10.1128/AEM.02522-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Functional Expression of a Bacterial Xylose Isomerase in

Saccharomyces cerevisiae䌤
Dawid Brat, Eckhard Boles,* and Beate Wiedemann
Institute of Molecular Biosciences, Goethe-Universität Frankfurt am Main, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
Received 4 November 2008/Accepted 8 February 2009

In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol
production. However, it lacks the ability to ferment pentose sugars like D-xylose and L-arabinose. Heter-
ologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many
attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened

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nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully
express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S.
cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize
D-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities
to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously
functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are com-
parable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which
accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be
improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae.
Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and
to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improve-
ment of xylose fermentation in industrial yeast strains.

It is widely acknowledged that fuels from regenerative re- the XYL1 and XYL2 genes (encoding xylose reductase [XR]
sources are becoming increasingly important in times of a and xylitol dehydrogenase [XDH], respectively) from Pichia
dwindling crude oil supply and the growing environmental stipitis (8, 12, 14, 15) or by expression of a xylA gene (encoding
concern of the public. Plant biomass, particularly when accru- xylose isomerase [XI]) from Piromyces sp. strain E2 (17) or
ing as a waste product, is an attractive feedstock for bioethanol Thermus thermophilus (33). Both approaches resulted in strains
production. An important prerequisite for such an alternative growing on xylose and fermenting it into ethanol. Although
strategy would be the complete conversion of all available expression of XR and XDH resulted in rapid fermentation of
sugars in biomass hydrolysates into ethanol. While the hexose xylose, NADPH/NAD cofactor imbalance under anaerobic con-
sugars are easily fermentable, no suitable microorganism is ditions led to considerable accumulation of xylitol (6, 14, 15, 30,
available for fermenting pentose into ethanol. Calculations 32). However, employing XI instead of XR/XDH avoids cofactor
have resulted in an estimate that production of lignocellulosic imbalance and xylitol accumulation, as D-xylose is converted di-
ethanol would reduce the cost of producing ethanol by nearly rectly into D-xylulose without a redox reaction being involved.
20% (3). Therefore, ethanol production from pentose sugars Many attempts to express an active prokaryotic XI in S.
has received considerable attention (4, 9). cerevisiae have failed. None of the efforts to express XI from
Although some anaerobic fungi and bacteria are able to
Escherichia coli (25), Bacillus subtilis (2), Lactobacillus pentosus
metabolize xylose, they are not suitable for industrial bioetha-
(10), or Clostridium thermosulfurogenes (23) in S. cerevisiae
nol production due to low and inefficient production rates and
resulted in active XI, arguing for the inability of yeast either
the mixed acid fermentation life-style (28), which generates too
to express xylA or to synthesize active enzyme (25). The first
many by-products. The baker’s yeast Saccharomyces cerevisiae
successful attempt was made with the xylA gene from the ther-
remains the organism of choice for industrial production of
mophilic bacterium Thermus thermophilus. XI from T. ther-
ethanol. However, while hexoses are converted rapidly to high
yields of ethanol, wild-type S. cerevisiae strains are not able to mophilus could be expressed in S. cerevisiae in an active form,
ferment pentose sugars, such as D-xylose and L-arabinose, ef- but the activity of this thermophilic enzyme, with a tempera-
ficiently. Several different approaches in genetic engineering ture optimum at 85°C, was very low at 30°C (33). In subsequent
have been used to enable D-xylose fermentation in yeast. rounds of mutagenesis, the enzyme could be considerably im-
Successful xylose fermentation in recombinant S. cerevisiae proved but, however, still not enough for efficient xylose con-
strains was previously achieved by heterologous expression of version in yeast (22).
For the first time, Kuyper et al. (17) successfully expressed a
xylA gene from the anaerobic fungus Piromyces sp. strain E2 in
* Corresponding author. Mailing address: Institute of Molecular Bio- S. cerevisiae with high enzymatic activity. However, a drawback
sciences, Goethe-Universität Frankfurt, Max-von-Laue-Str. 9, D-60438
of this enzyme was its strong inhibition by xylitol. A laboratory
Frankfurt am Main, Germany. Phone: 49 69 798 29513. Fax: 49 798 29527.
E-mail: e.boles@bio.uni-frankfurt.de. haploid yeast strain which exhibited fast anaerobic growth on
䌤
Published ahead of print on 13 February 2009. D-xylose and also high ethanol production rates was con-

2304
VOL. 75, 2009 BACTERIAL XYLOSE ISOMERASE IN S. CEREVISIAE 2305

TABLE 1. S. cerevisiae strains and plasmids used in this study

Strain or plasmid Relevant genotype/phenotype Source or reference

S. cerevisiae strains
CEN.PK2-1C MATa leu2-3,112 ura3-52 trp1-289 his3-⌬1 MAL2-8c SUC2 K. D. Entian, Frankfurt,
Germany
MKY9 (MATa leu2-3,112 ura3-52 trp1-289 his3-⌬1MAL2-8c SUC2 Boles lab stock
PromTKL1::loxP-Prom-vkHXT7 PromRPE1::loxP-Prom-vkHXT7
PromRKI1::loxP-Prom-vkHXT7 PromGAL2::loxP-Prom-vkHXT7
PromXKS1::loxP-Prom-vkHXT7) ⫹ unknown beneficial mutations
for pentose growth
BarraGrande Industrial strain for bioethanol production Brazilian ethanol plant
BWY10Xyl BarraGrande with plasmid YEp-opt.XI-Clos-K evolved on xylose growth This work

Plasmids
p426H7 URA3 11
YEp-XI-Agro pHXT7; xylA from A. tumefaciens; tCYC1 URA3 This work

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YEp-XI-Arab pHXT7; xylA from A. tumefaciens; tCYC1 URA3 This work
YEp-XI-BaLi pHXT7; xylA from B. licheniformis; tCYC1 URA3 This work
YEp-XI-Burk pHXT7; xylA from B. xenovorans; tCYC1 URA3 This work
YEp-XI-Clos pHXT7; xylA from C. phytofermentans; tCYC1 URA3 This work
YEp-opt.XI-Clos pHXT7; codon-optimized xylA from C. phytofermentans; tCYC1 URA3 This work
YEp-XI-Lacto pHXT7; xylA from L. pentosus; tCYC1 URA3 This work
YEp-opt.XI-Piro pHXT7; codon-optimized xylA from Piromyces sp. strain E2; tCYC1 URA3 Boles lab stock
YEp-XI-Pseudo pHXT7; xylA from Pseudomonas syringae; tCYC1 URA3 This work
YEp-XI-Robi pHXT7; xylA from R. biformata; tCYC1 URA3 This work
YEp-XI-Saccha pHXT7; xylA from S. degradans; tCYC1 URA3 This work
YEp-XI-Salmo pHXT7; xylA from S. enterica serovar Typhimurium; tCYC1 URA3 This work
YEp-XI-Staph pHXT7; xylA from S. xylosus; tCYC1 URA3 This work
YEp-XI-Xantho pHXT7; xylA from X. campestris; tCYC1 URA3 This work
YEp-opt.XI-Clos-K pHXT7; codon-optimized xylA from C. phytofermentans; tCYC1 URA3 This work
loxP-kanMX-loxP FAA2 locus

structed (18, 20). Furthermore, mixed sugar utilization of D- 515, 514a, 53, 65, and 1, respectively. Salmonella enterica serovar Typhimurium
glucose and D-xylose could recently be achieved by evolution- (71-098L) (obtained from Molecular Toxicology Incorporated) was cultivated in
Luria-Bertani (LB) medium.
ary engineering of recombinant yeast strains (19).
Plasmids were propagated in Escherichia coli SURE (Stratagene, La Jolla,
In this paper, we report the cloning and successful expres- CA) grown on LB medium with 40 ␮g ml⫺1 ampicillin. E. coli was transformed
sion of the first XI of prokaryotic origin with high activity in S. via electroporation according to the methods of Dower et al. (7) and Wirth (36).
cerevisiae. As an advantage, the new enzyme is far less suscep- Preparation of genomic DNA. Bacterial DNA was prepared as described in
tible to inhibition by xylitol than is the enzyme from the Piro- reference 24; alternatively, PCRs were performed using broken cells as tem-
myces strain. plates. Genomic DNA from Agrobacterium tumefaciens and cDNA from Arabi-
dopsis thaliana were kind gifts from C. Weber, Frankfurt, Germany.
Plasmid construction. The coding regions of the xylA genes encoding XIs from
MATERIALS AND METHODS various organisms were amplified by PCR from genomic DNA or cDNA from
Strains and media. Yeast strains and plasmids used in this work are listed in the strains listed under “Strains and media” and cloned into the EcoRI/BamHI-
Table 1. S. cerevisiae was grown aerobically in synthetic complete (SC) medium linearized vector p426H7 (URA3) by recombination cloning employing the meth-
(6.7 g liter⫺1 Difco yeast nitrogen base without amino acids), supplemented with ods described by Wieczorke et al. (34) but omitting the six histidine codons. The
amino acids as described previously (38), supplemented with 20 g liter⫺1 D- open reading frames were amplified by using the specific primer pairs x-for and
glucose or 20 g liter⫺1 D-xylose as a carbon source, and buffered at pH 5.5 with x-rev (where x is specific for the organism [Table 2]). Vector YEp-opt.XI-Clos-K
20 mM KH2PO4. In case the sugar concentration exceeded 20 g liter⫺1 in the is based on vector p426H7 and was generated by cloning the dominant selection
medium, the concentration of yeast nitrogen base was doubled. For maintenance marker gene kanMX, amplified by PCR using primer pair FAA2-kanMX-f/
of plasmids, media lacked uracil or contained 200 mg/liter G418 (Calbiochem) FAA2-kanMX-r, into SacI/KpnI-linearized vector p426H7. The kanMX gene was
for selection in industrial strains. Screening for functional XIs was conducted on flanked by sequences homologous to sequences in the genome of yeast near the
the same solid medium with 18 g liter⫺1 agar but with 20 g liter⫺1 D-xylose FAA2 gene obtained by PCR using primer pair FAA2-1-f/FAA2-1-r and FAA2-
instead of glucose as the sole carbon source. In anaerobic fermentations, defined
2-f/FAA2-2-r. Homologous regions should enable later genomic integration of
medium (31) was used, supplemented with amino acids as described previously
the construct. 5⬘ of the kanMX gene, the codon-optimized gene version of xylA
(38) (30 g liter⫺1 D-xylose, 200 mg/liter G418, and 150 ␮l liter⫺1 of silicone
from C. phytofermentans, obtained by PCR with primers FAA2-optXI-Clos-f and
antifoam [Sigma]) as well as with the anaerobic growth factors ergosterol (0.01
FAA2-optXI-Clos-r, was inserted into PmeI-linearized vector, resulting in YEp-
g liter⫺1) and Tween 80 (0.40 g liter⫺1) dissolved in ethanol (resulting in 2.5 g
opt.XI-Clos-K.
liter⫺1 ethanol in the medium).
Bacillus licheniformis (DSM 13), Burkholderia xenovorans (DSM 17367), Clos- Codon-optimized gene versions were obtained from Geneart AG (Regens-
tridium phytofermentans (DSM 18823), Lactobacillus pentosus (DSM 20314), burg, Germany) after changing the original codons of the respective genes to
Leifsonia xyli subsp. cynodontis (DSM 46306), Pseudomonas savastanoi pv. phase- those used in the genes encoding glycolytic enzymes in S. cerevisiae as described
olicola (DSM 50282), Robiginitalea biformata (DSM 15991), Saccharophagus in the work of Wiedemann and Boles (35) and cloned into the vector p426H7 by
degradans (DSM 17024), Staphylococcus xylosus (DSM 20266), Streptomyces dia- recombination cloning as described above. Using the coding region of C. phyto-
staticus subsp. diastaticus (DSM 40496) and Xanthomonas campestris pv. campes- fermentans xylA and Piromyces sp. xylA resulted in YEp-opt.XI-Clos and YEp-
tris (DSM 3586) were cultivated according to the recommendations of the opt.XI-Piro, respectively.
DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braun- Phylogenetic analysis. Amino acid sequences of the XIs were obtained from
schweig, Germany) (http://www.dsmz.de/) in DSM media 1, 220, 520, 11, 751, 54, GenBank and compared using the BLAST algorithm (National Center for Bio-
2306 BRAT ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Primers used in this work

Primer Sequence

XI-Agro-for ...........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAAATTTTCAAACACCACCC
XI-Agro-rev...........................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAAACGTAACGATTGACCACG
XI-Arab-for ...........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAAGAAAGTTGAGTTTTTTATGC
XI-Arab-2rev.........................GTAAGCGTGACATAACTAATTACATGACTCGAGTTACATTGCAGATTGGAAAATC
XI-BaLi-for ...........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGTTTTTTAGAAATATCGGAATG
XI-BaLi-rev ...........................GTAAGCGTGACATAACTAATTACATGACTCGAGCTAGTGTGTCTCCTTTCCTGCCG
XI-Burk-for ...........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGTCCTATTTCGAACACATTCCCG
XI-Burk-rev ...........................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAGCGTCCGCTGTAAATAGCC
XI-Clos-for ............................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAAAAATTACTTTCCAAATG
XI-Clos-rev ............................GTAAGCGTGACATAACTAATTACATGACTCGAGTTATCTAAATAAAATATTATTTACG
XI-Lacto-for ..........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGACAAACGAATATTGGCAAGG
XI-Lacto-rev..........................GTAAGCGTGACATAACTAATTACATGACTCGAGTTATTTACTTAACGTCTCGATAAT
XI-Leif-for.............................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGCTGGCTCGAGCGCAGGCTCC
XI-Leif-rev.............................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAGCGCGCGCCGAGCAGATGC

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XI-Pseudo-for .......................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGCCGTACTTCCCCGCCGTCG
XI-Pseudo-rev .......................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAGAAGTAGATAAAGCGGTTGACC
XI-Robi-for ...........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGATTACCACCGGAGACAAAG
XI-Robi-rev ...........................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAGATAAATTGGTTGATAATG
XI-Saccha-for ........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAGCGTAGTACTTGGCGATAAAG
XI-Saccha-rev........................GTAAGCGTGACATAACTAATTACATGACTCGAGTTAACGAATATATTGGTTAATAATG
XI-Salmo-for .........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGCAGGCTTATTTTGACCAACTCG
XI-Salmo-rev.........................GTAAGCGTGACATAACTAATTACATGACTCGAGTTATTTATCAAACAGATAACGGTTAAC
XI-Staph-for ..........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGTCTTATTTTGATATCAATAAAG
XI-Staph-rev..........................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAATCCTTATTATTAATATTTAAG
XI-Strep-for...........................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAGCTACCAGCCCACCCCCG
XI-Strep-rev ..........................GTAAGCGTGACATAACTAATTACATGACTCGAGCTAGCCCCGCGCGCCCAGCAGG
XI-Xantho-for .......................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAGCAACACCGTTTTCATCGGC
XI-Xantho-rev.......................GTAAGCGTGACATAACTAATTACATGACTCGAGTCAACGCGTCAGGTACTGATTGATC
opt.XI-Clos-for......................CAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGAAGAACTACTTCCCAAACG
opt.XI-Clos-rev .....................GTAAGCGTGACATAACTAATTACATGACTCGAGTTATCTGAACAAAATGTTGTTAAC
opt.XI-Piro-for ......................AACACAAAAACAAAAAGTTTTTTTAATTTTAATCAAAAAATGGCTAAGGAATACTTCCCA
opt.XI-Piro-rev......................GAATGTAAGCGTGACATAACTAATTACATGACTCGAGTTATTGGTACATAGCAACAAT
FAA2-1-f................................CAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGTACTTATGACGATTTGGAAC
FAA2-1-r ...............................ATATCAGGGCTCACTACATG
FAA2-2-f................................CGGGGCGTAATCACCTAACTC
FAA2-2-r ...............................CAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTCTAATGGCGATTTAGCATATG
FAA2-kanMX-f ....................GATGGAGGTGCAGTGTTTTCCATGTAGTGAGCCCTGATATGTTTAAACTTCGTACGCTGCAGGTCGAC
FAA2-kanMX-r ....................GAGAAAGGTTTACCTTTGCTGAGTTAGGTGATTACGCCCCGGCATAGGCCACTAGTGGATCTG
FAA2-optXI-Clos-f ..............CAGATGGAGGTGCAGTGTTTTCCATGTAGTGAGCCCTGATATGTTTAAACGAGCTCGTAGGAACAA
TTTC
FAA2-optXI-Clos-r ..............ATTAAGGGTTGTCGACCTGCAGCGTACGAAGCTTCAGCGGCGAATTGGGTACCGGCCG

technology Information). Sequences were aligned to plot the phylogenetic tree, medium. From these plates, single colonies were taken and used for further
using MEGA version 4 (27). growth and fermentation experiments.
Growth assays (shake flasks). Cultures of laboratory strains (50 ml) were Metabolite analysis. The concentrations of glucose, D-xylose, xylitol, glycerol,
grown in 500-ml shake flasks (Erlenmeyer flasks) at 30°C in a shaker. Precultures acetic acid, and ethanol were determined by high-performance liquid chroma-
were grown into the stationary phase in SC medium lacking uracil and containing tography (Dionex) using a Nugleogel Sugar 810 H exchange column (Macherey-
20 g liter⫺1 D-xylose as the sole carbon and energy source. Cells were washed Nagel GmbH & Co, Germany). The column was eluted with 5 mM H2SO4 as
with sterile water and inoculated to an optical density at 600 nm (OD600) of 0.5 mobile phase and a flow rate of 0.6 ml min⫺1 at the temperature of 65°C.
in the same medium. Growth experiments were performed in triplicate with the Detection was by means of a Shodex RI-101 refractive index detector. For data
given standard deviations, but cultures were started from the same precultures. evaluation, Chromeleon software (version 6.50) was used. Rates of D-xylose
Cultures of industrial strain BWY10Xyl (20 ml) were grown in 200-ml Erlen- consumption were determined in the phase of D-xylose growth.
meyer flasks at 30°C in a shaker. Precultures were grown to stationary phase in
Determination of culture dry weight. Dry weight was determined (in duplicate)
SC medium containing 20 g liter⫺1 D-xylose as the sole carbon and energy source.
by filtering 10 ml of the culture through a preweighed nitrocellulose filter
For maintenance of plasmids, media were made with 200 mg/liter G418 (21).
(0.45-␮m pore size; Roth, Germany). The filters were washed with demineralized
Cells were washed with sterile water and used to inoculate the same medium to
water, dried in a microwave oven for 20 min at 140 W, and weighed again.
an OD600 of 0.2. Growth experiments were performed in triplicate with the given
Anaerobic batch fermentations. Anaerobic batch fermentations were per-
standard deviations.
Selection of industrial strain growing on D-xylose. Mutants of BWY10Xyl able formed in Minifors bioreactors with a working volume of 2 liters (Infors AG,
to grow on D-xylose were selected by serial transfer in shake flasks. For serial Bottmingen, Switzerland). Shake-flask precultures were grown until late expo-
transfer experiments, a 200-ml shake flask containing 20 ml of SC medium nential phase in SC medium supplemented with 200 mg/liter G418 and with 20 g
supplemented with 20 g liter⫺1 D-xylose, 1 g liter⫺1 yeast extract, 2 g liter⫺1 liter⫺1 D-xylose. Cells were washed with sterile water. Cultures were inoculated
peptone, and 200 mg/liter G418 was inoculated with strain BarraGrande con- at an OD600 of about 0.6 and incubated at 30°C with 250-rpm stirring and at pH
taining plasmid YEp-opt.XI-Clos-K. This transfer procedure was repeated four 5.5, maintained by addition of 4 M KOH. The synthetic medium contained 30 g
times, covering a period of 28 days. Another two transfers in medium containing liter⫺1 D-xylose. Cells were grown under aerobic conditions until about 5 g liter⫺1
only 20 g liter⫺1 D-xylose as the sole carbon and energy source resulted in strain D-xylose was consumed and then shifted to anaerobic conditions by sparging with
BWY10Xyl. From the final culture, a sample was streaked out on mineral nitrogen gas (containing less than 5 ppm of O2; Air Liquide, Düsseldorf, Ger-
medium with xylose. Single colonies were picked and restreaked on identical many) for 30 min with a flow rate of 1 liter min⫺1. Evaporation of ethanol was
VOL. 75, 2009 BACTERIAL XYLOSE ISOMERASE IN S. CEREVISIAE 2307

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FIG. 1. Phylogenetic tree of the amino acid sequences of the tested XIs reported in the GenBank database.

minimized by using a reflux condenser at 4°C and was not calculated. The sion vector p426H7 (11), placing the respective genes under
experiment was performed in duplicate. the control of a strong and constitutive HXT7 promoter frag-
Enzyme assays. Yeast transformants expressing xylA from C. phytofermentans
and codon-optimized xylA from Piromyces sp. strain E2 and C. phytofermentans
ment and the CYC1 terminator. The coding sequences from L.
(carried on multicopy vectors) were cultivated until early exponential growth xyli subsp. cynodontis and S. diastaticus subsp. diasticus could
phase in selective medium. Cells were harvested and disrupted with glass beads not be amplified and were thus not analyzed in the screen. A
(diameter, 0.45 mm) using a Vibrax cell disrupter (Janke & Kunkel, Staufen, codon-optimized xylA gene version from Piromyces sp. strain
Germany). Protein concentration was determined with the method of Bradford
(5) by using bovine serum albumin as a standard. Enzyme assays were performed
E2 (YEp-opt.XI-Piro) was used as a positive control in the
immediately after preparation of crude extracts. screening system. The activity of the recombinant XIs was
XI activity in cell extracts of recombinant yeast strains was determined at 30°C. assessed by conferring growth on the yeast strain MKY9 on a
Assays were carried out in reaction mixtures containing 0.23 mM NADH, 10 mM synthetic medium with xylose as the only carbon source. In
MgCl2, 2 U sorbitol dehydrogenase in 100 mM Tris-HCl (pH 7.5), and crude cell
extracts, as described previously (17). The reaction was started by addition of
strain MKY9 all the enzymes of the nonoxidative part of the
D-xylose to a final concentration of 500 mM and monitored by measuring oxi- pentose phosphate pathway, the xylulokinase, and the GAL2
dation of NADH (during conversion of D-xylulose to xylitol by sorbitol dehydro- permease are overexpressed due to the replacement of their
genase) spectrophotometrically at 340 nm. For determination of the kinetic native promoters by the strong HXT71–392 promoter fragment.
parameters, 6.25 to 500 mM D-xylose was used.
Xylitol inhibition of the XI was measured by adding various concentrations
A similar approach was shown previously to improve growth of
of xylitol (10 to 50 mM) in the presence of 6.25 to 500 mM D-xylose (37). The the yeast cells on a xylose medium (18).
inhibition constant Ki was calculated from Km⬘ ⫽ Km ⫻ (1 ⫹ i/Ki) with i as the The 12 different expression plasmids, the empty vector control
xylitol concentration used and Km⬘ as the apparent Km for D-xylose at the re- (p426H7), and the Piromyces xylA positive control (p426H7-
spective xylitol concentration. All enzyme assays were carried out at least in
opt.XI-Piro) were transformed into strain MKY9, first select-
triplicate.
ing for growth on a medium with 20 g liter⫺1 glucose but
without uracil as the plasmid selection marker. Transformants
RESULTS were replica plated on a synthetic medium without uracil and
Screen for novel functionally expressed XIs in S. cerevisiae. with 20 g liter⫺1 xylose as the sole carbon source. The test was
The yeast S. cerevisiae is able to metabolize xylose only after scored as positive if yeast colonies could be detected after 4 to
heterologous expression of an XI or an XR/XDH enzyme pair. 5 days of incubation at 30°C. Nonfunctional XIs did not confer
However, all attempts to express nonfungal, nonthermophilic the ability to grow on D-xylose even after 2 weeks of incubation
XIs with high activity in S. cerevisiae have failed so far. and were thus scored as negative. Yeast transformants express-
Therefore, our aim was to screen for a new kind of heter- ing the XI from C. phytofermentans and the positive control
ologous XI with high activity in S. cerevisiae cells and not expressing XI from the Piromyces strain could grow on the
subject to, or less subject to, xylitol inhibition. To this end, we xylose medium; all other XIs tested in the screen did not
selected XIs from 14 organisms of different phylogenetic affil- enable yeast transformants to grow on D-xlyose.
iations which exhibited identities from 17% to 60% to the XI Analysis of the codon usage of the xylA gene from C. phyto-
from the Piromyces strain (Fig. 1). The coding sequences of the fermentans using CODONW (http://mobyle.pasteur.fr/cgi-bin
selected genes were amplified by PCR and cloned via homol- /MobylePortal/portal.py?form_codonw) revealed that, com-
ogous recombination into the high-copy-number yeast expres- pared to S. cerevisiae, its codon adaptation index is very low
2308 BRAT ET AL. APPL. ENVIRON. MICROBIOL.

(0.136), which may result in rather inefficient gene expression

in S. cerevisiae. Therefore, the codon usage of xylA was adapted
to that of the genes encoding glycolytic enzymes in S. cerevisiae
(35) to further improve xylose conversion in yeast. This ap-
proach has previously been reported to improve L-arabinose
conversion via heterologously expressed genes (35). The codon-
optimized gene exhibited a codon adaptation index of 0.991
and, when provided on plasmid YEp-opt.XI-Clos, enabled
growth of S. cerevisiae with D-xylose, as expected. The codon-
optimized gene version of xylA from C. phytofermentans was
further examined and compared to the codon-optimized xylA
variant from Piromyces sp. strain E2.
Characterization of the kinetic properties of the C. phyto-
fermentans XI. The kinetic properties of XI of C. phytofermen-

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tans were determined in a crude extract of yeast cells contain-
ing plasmids YEp-opt.XI-Clos and YEp-XI-Clos. Yeast cells
were grown in minimal medium with 20 g liter⫺1 glucose into
the exponential growth phase and harvested, and crude ex-
tracts were prepared. As the XI amino acid sequences encoded
by the two plasmids are the same, the XI reactions exhibited
comparable apparent Km values for xylose (66.01 ⫾ 1.00 mM
for codon-optimized XI and 61.85 ⫾ 3.41 mM for native XI).
Cells expressing XI from Piromyces sp. strain E2 showed an
apparent Km for D-xylose of 49.85 ⫾ 2.82 mM.
Next, in order to compare the performances of the two
isomerases within the yeast cells, the reaction velocities (Vmax;
␮mol min⫺1 mg protein⫺1) of the XI from Piromyces sp. strain
E2 and from C. phytofermentans, respectively, were determined
in crude extracts. Extracts from cells containing the native
clostridial XI gene from C. phytofermentans catalyzed conver-
sion of D-xylose to xylulose at a maximal rate of 0.0076 ␮mol
min⫺1 mg protein⫺1 whereas the reaction in extracts derived FIG. 2. Inhibition of XI from C. phytofermentans (A) and Piromyces
from cells containing the codon-optimized gene version pro- sp. strain E2 (B) by xylitol. Strains carrying the gene for XI from C.
ceeded at a rate of 0.0344 ␮mol min⫺1 mg protein⫺1. Thus, phytofermentans or Piromyces sp. strain E2, respectively, on a multicopy
vector were grown as shake-flask cultures at 30°C into the exponential
codon adaptation resulted in a Vmax increased by 450% on growth phase in synthetic medium with 20 g liter⫺1 glucose and with-
average with a deviation of no more than 10% for every mea- out uracil. Crude extracts were prepared, and quantitative enzyme
sured value. For the codon-optimized xylA variant from Piro- activity tests were performed. Symbols: F, 0 mM xylitol; f, 10 mM
myces sp. strain E2, a Vmax of 0.0538 ␮mol min⫺1 mg protein⫺1 xylitol; Œ, 30 mM xylitol;
, 50 mM xylitol.
was determined.
An important feature of XIs is their inhibition by xylitol, a
side product during xylose fermentations which negatively af- Piromyces from plasmid YEp-opt.XI-Piro and that of a strain
fects the efficiency of the overall xylose fermentation process containing the empty vector p426H7 were also examined. Pre-
(13). To characterize the influence of xylitol inhibition on XIs cultures of the strains were grown aerobically in the same
from C. phytofermentans and Piromyces sp. strain E2, their medium, harvested in the stationary phase, and used to inoc-
apparent Ki values for xylitol were determined. Inhibition ki- ulate fresh medium to an OD600 of 0.5.
netics turned out to follow a competitive mechanism, as also S. cerevisiae cells expressing the native XI gene from C.
previously reported for XI from Lactobacillus brevis (37). How- phytofermentans grew slowly with D-xylose at a maximal rate
ever, the apparent inhibition constant Ki of the XI from C. of 0.039 ⫾ 0.0017 h⫺1. Recombinant strains expressing the
phytofermentans was only 14.51 ⫾ 1.08 mM, whereas that of the codon-optimized gene version from C. phytofermentans grew
enzyme from Piromyces sp. strain E2 was 4.6 ⫾ 1.777 mM (Fig. slightly faster (maximal growth rate, 0.057 ⫾ 0.0029 h⫺1) but
2). Thus, the enzyme from C. phytofermentans is three times exhibited a somewhat longer lag phase. Their growth rate was
less inhibited by xylitol than is that from Piromyces. nearly the same as that of the control strain expressing the
Growth performance of yeast transformants expressing the codon-optimized xylA gene from Piromyces (maximal growth
XI of C. phytofermentans. To test the performance of yeast cells rate, 0.056 ⫾ 0.0030 h⫺1). The differing lag phases probably
expressing the native and the codon-optimized versions of the depend on subtle differences in the time of harvest of the
XI gene from C. phytofermentans, growth on synthetic medium precultures. The strain carrying the empty vector could not
with 20 g liter⫺1 xylose of strain MKY9 carrying plasmids grow at all on D-xylose (Fig. 3).
YEp-XI-Clos and YEp-opt.XI-Clos, respectively, was analyzed Growth on xylose of an industrial S. cerevisiae strain. Indus-
under aerobic conditions in shake-flask cultures. As controls, trial S. cerevisiae strain BarraGrande expressing the xylA gene
growth of strains expressing the codon-optimized XI gene from from plasmid YEp-opt.XI-Clos-K could initially not grow on
VOL. 75, 2009 BACTERIAL XYLOSE ISOMERASE IN S. CEREVISIAE 2309

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FIG. 3. Growth of recombinant S. cerevisiae strains expressing dif- FIG. 5. Anaerobic batch fermentation by recombinant S. cerevisiae.
ferent XIs. SC medium (without uracil) contained 20 g liter⫺1 D-xylose Shown is a graph of anaerobic batch fermentation of strain BWY10Xyl.
as the sole carbon source. Yeast strains were grown aerobically as The strain was grown in mineral medium supplemented with amino
shake-flask cultures at 30°C. Yeast strain MKY9 contained the differ- acids and with 30 g liter⫺1 D-xylose as the sole carbon source. The
ent XI genes. Symbols: f, opt-XI-Clos; Œ, opt-XI-Piro; }, XI-Clos; 䡺, strain was pregrown in the fermentor under aerobic conditions until
empty vector. Shown are results of a typical experiment. about 5 g liter⫺1 D-xylose was consumed and then shifted to anaerobic
conditions (indicated by the arrow). Symbols: }, D-xylose; f, ethanol;
䡺, xylitol; Œ, biomass. Shown are results of a typical experiment.
D-xylose as the sole carbon and energy source. To select for
spontaneous mutants growing on xylose, the strain was sub-
mized XI from C. phytofermentans, two anaerobic batch fer-
jected to serial transfer in shake flasks containing a synthetic
mentor cultivations were performed in synthetic medium with
medium with 20 g liter⫺1 xylose, 1 g liter⫺1 yeast extract, and
30 g liter⫺1 of D-xylose. Precultures of strain BWY10Xyl con-
2 g liter⫺1 peptone. After only four transfers, covering a period
taining the codon-optimized gene were pregrown aerobically in
of 28 days, the strain showed growth on xylose. Another two
shaking flasks containing 100 ml of SC medium with 20 g
transfers on xylose medium resulted in a strain growing well on
liter⫺1 D-xylose. Cells were harvested, washed, and inoculated
xylose medium.
into batch fermentors. To generate enough biomass, cells were
First, growth on D-xylose medium was tested under aerobic
first grown under aerobic conditions until 5 g liter⫺1 of D-
conditions using shake-flask cultures (Fig. 4). It turned out that
xylose was consumed (Fig. 5). Anaerobic conditions were
strain BWY10Xyl, containing the codon-optimized xylA gene,
maintained by sparging with nitrogen gas until no oxygen was
could grow on xylose medium with a maximal specific growth
left in the medium (see Materials and Methods).
rate of 0.04 ⫾ 0.004 h⫺1. The strain reached a final OD of 10
D-Xylose consumption and ethanol production rates were
in less than 120 h. The wild-type strain could not grow at all on
determined for both cultures in the anaerobic phase of the
D-xylose.
fermentation, i.e., starting with approximately 25 g liter⫺1 D-
Fermentation characteristics of an industrial yeast strain
xylose in the medium. D-Xylose was consumed at a rate of 0.07
expressing XI. To analyze D-xylose consumption and ethanol
and 0.06 g D-xylose h⫺1 g (dry weight)⫺1, respectively. A
production of yeast transformants containing the codon-opti-
residual of ca. 7 g liter⫺1 D-xylose was still left in the medium after
170 h. The ethanol production rate was 0.03 (0.03) g ethanol
h⫺1 g (dry weight)⫺1, and the ethanol yield was 0.43 (0.42) g
ethanol g D-xylose consumed⫺1. As by-products, xylitol, glyc-
erol, and acetate were produced (0.03 g glycerol g D-xylose
consumed⫺1, 0.02 g acetate g D-xylose consumed⫺1, and 0.18 g
xylitol g D-xylose consumed⫺1).

DISCUSSION
The yeast S. cerevisiae is not able to metabolize the pentose
sugar D-xylose as it lacks the enzyme activities to convert xylose
into xylulose. One possible approach for efficient D-xylose fer-
mentation in S. cerevisiae would be the heterologous expres-
sion of an XI. However, most of the XIs expressed in S. cer-
evisiae before now were not active. Also, in our screening only
expression of 1 out of 12 tested XI genes conferred on the
FIG. 4. Aerobic growth of industrial S. cerevisiae strain expressing yeast cells the ability to grow on D-xylose medium as the sole
the codon-optimized XI from C. phytofermentans. SC medium con-
tained 20 g liter⫺1 D-xylose as the sole carbon source. Yeast strains
carbon and energy source, indicating that the others were not
were grown aerobically as shake-flask cultures at 30°C. Symbols: }, functional. The reasons for the absence of XI activity in S.
BWY10Xyl; f, BarraGrande (wild type). cerevisiae were discussed previously (25). The isomerases
2310 BRAT ET AL. APPL. ENVIRON. MICROBIOL.

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