Genetically Engineered SaccharomycesYeast

Capable of Effective Cofermentation of Glucose

and Xylose

Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
Nancy W. Y. Ho, Zhengdao Chen and Adam P. Brainard
Appl. Environ. Microbiol. 1998, 64(5):1852.

Updated information and services can be found at:

http://aem.asm.org/content/64/5/1852

These include:
REFERENCES This article cites 20 articles, 4 of which can be accessed free at:
http://aem.asm.org/content/64/5/1852#ref-list-1

CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles
cite this article), more»

Information about commercial reprint orders: http://aem.asm.org/site/misc/reprints.xhtml

To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1998, p. 1852–1859 Vol. 64, No. 5
0099-2240/98/$04.0010
Copyright © 1998, American Society for Microbiology

Genetically Engineered Saccharomyces Yeast Capable of

Effective Cofermentation of Glucose and Xylose

Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
NANCY W. Y. HO,* ZHENGDAO CHEN, AND ADAM P. BRAINARD
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana 47907-1295
Received 6 October 1997/Accepted 20 February 1998

Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However,
Saccharomyces spp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We devel-
oped recombinant plasmids that can transform Saccharomyces spp. into xylose-fermenting yeasts. These
plasmids, designated pLNH31, -32, -33, and -34, are 2mm-based high-copy-number yeast-E. coli shuttle
plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant
selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a
xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae).
These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae
glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the
conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can
effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the
resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the
xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the
presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These
properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently
coferment glucose and xylose present in the same medium to ethanol simultaneously.

Saccharomyces spp. are the safest and most effective micro-
organisms for fermenting sugars to ethanol and traditionally
have been used in industry to ferment glucose (or hexose
sugar)-based agricultural products to ethanol. Cellulosic bio-
mass, which includes agriculture residues, paper wastes, wood
chips, etc., is an ideal inexpensive, renewable, abundantly avail-
able source of sugars for fermentation to ethanol, particularly
ethanol used as a liquid fuel for transportation. However, Sac-
charomyces spp. have been found to be unsuitable for ferment-
ing sugars derived from cellulosic biomass. This is because
most of the hydrolysates of cellulosic biomass contain two
major fermentable sugars, glucose and xylose. Saccharomyces
spp., including Saccharomyces cerevisiae, are not able to fer-
ment xylose to ethanol or to use this pentose sugar for aerobic
growth.
Even though Saccharomyces spp. are not able to metabolize
xylose aerobically and anaerobically, there are other yeasts,
such as Pichia stipitis and Candida shehatae, that are able to
ferment xylose to ethanol and to use xylose for aerobic growth.
However, these naturally occurring xylose-fermenting yeasts
are not effective fermentative microorganisms, and they also
have a relatively low ethanol tolerance (18). Thus, they are not
suitable for large-scale commercial production of ethanol from
cellulosic biomass.
The naturally occurring xylose-fermenting yeasts metabolize
xylose by relying on xylose reductase (XR) to convert xylose to
xylitol, on xylitol dehydrogenase (XDH) to convert xylitol to
xylulose, and on xylulokinase (XK) to convert xylulose to xy-
lulose 5-phosphate (17). The synthesis of these xylose-metab-
olizing enzymes in these yeasts, such as P. stipitis, requires the
* Corresponding author. Mailing address: Laboratory of Renewable
Resources Engineering, Purdue University, 1295 Potter Center, West
Lafayette, IN 47907-1295. Phone and fax: (765) 494-7046. E-mail:
nwyho@ecn.purdue.edu.
presence of xylose for induction and is also totally or at least
partially repressed by the presence of glucose (5).
Although Saccharomyces spp. are not able to ferment xylose,
they are able to ferment xylulose (14). Furthermore, they also
are able to ferment xylose when xylose isomerase, a bacterial
enzyme that catalyzes the conversion of xylose to xylulose, is
present in the medium (10). Thus, previous studies have indi-
cated that Saccharomyces spp. lack only the enzymes for con-
version of xylose to xylulose. However, it has also been dem-
onstrated that at least some Saccharomyces spp., if not all of
them, contain very low levels of XK activity (12).
More than a decade ago, attempts were made to clone and
express various bacterial xylose isomerase genes in S. cerevi-
siae, but these attempts failed to produce any recombinant
Saccharomyces strains that were able to ferment xylose or uti-
lize this sugar for growth. In this paper, we describe the devel-
opment of a group of high-copy-number recombinant plas-
mids, collectively designated the pLNH plasmids (pLNH31,
pLNH32, pLNH33, and pLNH34), that can transform some of
the best glucose-fermenting Saccharomyces strains into effec-
tive xylose-fermenting yeasts which can also effectively utilize
xylose for aerobic growth. These plasmids all contain the 2mm
replication origin (2, 6), geneticin and ampicillin resistance
genes (as the dominant selectable markers), and three xylose-
metabolizing genes. The xylose-metabolizing genes cloned into
the pLNH plasmids include the XR gene (XR), the XDH gene
(XD), and the XK gene (XK) (collectively referred to below as
the XYL genes). In addition, the 59 control regions (both the
transcriptional and the translational signals) of these XYL
genes have been replaced by the effective 59 control regions of
the S. cerevisiae glycolytic genes. An ideal Saccharomyces strain
for the fermentation of the sugars present in cellulosic biomass
to ethanol not only should effectively ferment xylose to etha-
nol, but also should effectively coferment glucose and xylose
simultaneously to ethanol. In this paper, we also demonstrate
that each of the pLNH plasmids can transform a Saccharomy-

1852
VOL. 64, 1998 RECOMBINANT XYLOSE-FERMENTING SACCHAROMYCES SPP.
FIG. 1. Construction of high-copy-number yeast-E. coli shuttle plasmids
pLNH31, pLNH32, pLNH33, and pLNH34. containing the XYL three-gene
cassette KK-AR (or A*R)-KD. The XhoI DNA fragment containing KK-AR (or
A*R)-KD was inserted into pUCKm10 at its SalI site.
ces strain to a recombinant yeast that can efficiently coferment
glucose and xylose present in the same medium.
While our work was in progress, Kotter et al. (20), Walfrids-
son et al. (25), and Tantirungkij et al. (24) described the met-
abolic engineering of S. cerevisiae for xylose fermentation by
cloning of only the XR and XD genes. The major differences
between our recombinant Saccharomyces strains and the
strains described by Kotter et al. (20), Walfridsson et al. (25),
and Tantirungkij (24) are discussed below (see Discussion).
(Some of the results were presented at the Tenth Interna-
tional Symposium on Alcohol Fuel, Colorado Springs, Colo., 7
to 10 November 1993, and at the 16th Symposium on Biotech-
nology for Fuels and Chemicals, Gatlinburg, Tenn., 9 to 13
May 1994.)
MATERIALS AND METHODS
Strains and plasmids. S. cerevisiae AH22, Saccharomyces sp. strain 1400 (a
product of fusion between Saccharomyces diastaticus and Saccharomyces uvarum
(11), and recombinant strain 1400(pLNH32) were the Saccharomyces strains
used in this study. P. stipitis 5773 was used for comparison studies. Escherichia
coli DH5a was used as the host during development and characterization of
various recombinant E. coli plasmids or yeast-E. coli shuttle plasmids.
pUCKm10 (Fig. 1) is a high-copy-number yeast-E. coli plasmid which is iden-
tical to pUCKm8 (8) except that the two EcoRI restriction sites present in
pUCKm8 are not present. pUCKm8, pUCKm10, pUC19, and pBluescript KS(2)
(referred to below as pKS) were the plasmids used in this work.
Conversion of XR to AR or A*R, of XD to KD, and of XK to KK. XR, the XR gene
used in this study, was cloned from P. stipitis by PCR, and the 59 control region
of the XR gene was replaced by the 59 control region of the S. cerevisiae alcohol
dehydrogenase gene (ADC1) (1) as described by Chen and Ho (8). The resulting
gene was designated AR or A*R. The 59 control region of AR consisted of the
original 59 noncoding sequence of P. stipitis XR from positions 21 to 250,
followed by the fragment cloned in pMA56 (1). Since it is not known what effect
the nucleotide sequence (positions 21 to 250) from P. stipitis XR might have on
expression of the XR gene in the Saccharomyces strains, the A*R gene was also
constructed, in which the 59 control region of XR was totally replaced by the
intact 59 control region of the ADC1 gene (positions 21 to 21500, noncoding
sequence) (4).
The P. stipitis XDH gene, XD, was also amplified by PCR. The forward primer
59-TCTAGACCACCCTAAGTCG-39 and the reverse primer 59-GGATCCACT
ATAGTCGAAG-39 were used to amplify the intact XD gene (with its original 59
and 39 control sequences), and the forward primer 59-CACACAATTAAAATG
A-39 and the reverse primer 59-GGATCCACTATAGTCGAAG-39 were used to
amplify the promoterless XD gene (without the 59 noncoding sequence) from the
Pichia chromosomal DNA by using the previously published sequence of the
Pichia XD gene (20). The amplified XD gene was first cloned and stored in
pUC19. The original 59 control region of XD was subsequently replaced by the
intact 59 control region (positions 21 to 2911 of the noncoding sequence) of the
1853
S. cerevisiae pyruvate kinase gene (PYK) (7), and the resulting gene was desig-
nated KD. XD was fused to the 59 control region of PYK by cloning both the
fragment containing the 59 noncoding sequence of PYK and the promoterless XD
gene (with its original 39 noncoding sequences) on pKS and then removing the
extra nucleotides in the 59 noncoding sequences by site-specific mutagenesis (21).
The resulting plasmid was designated pKS-KD.
XK, the XK gene used in this study, was cloned from S. cerevisiae (15). The
Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
nucleotide sequence of the cloned XK gene containing 2,467 bp (bp 1 to 345
constitute the 59 control region; bp 346 to 2118 constitute the coding sequence;
and bp 2119 to 2467 constitute the 39 noncoding sequence) has been analyzed
and deposited in the EMBL data library (EMBL accession no. X61377). The
promoterless XK gene can be amplified from S. cerevisiae chromosomal DNA by
PCR with the following two primers: 59-ATGTTGTGTTCAGTAAT-39 and 59-
CAATAACCTAGCTCTTT-39. The promoterless XK gene was also fused to the
intact 59 control region (positions 21 to 2911 of the noncoding sequence) of
PYK (7), and the resulting gene was designated KK. XK was fused to the 59
control region of PYK by cloning both the fragment containing the intact 59
noncoding sequence of PYK and the structural gene of XK with its native 39
noncoding sequences on pKS and then removing the extra nucleotides in the 59
noncoding sequences by site-specific mutagenesis (21). The resulting plasmid was
designated pKS-KK.
Yeast transformation. The plasmids used for transformation of the Saccharo-
myces strains described in this paper were all pUCKm8 or pUCKm10 derivatives
which contain the geneticin resistance gene (the Kmr gene [also known as the
kanamycin resistance gene]) and the ampicillin resistance gene (the Apr gene).
The Kmr gene can be a dominant selectable marker for many yeasts, particularly
Saccharomyces species (27). Transformation of the Saccharomyces strains by the
pUCKm8 or pUCKm10 derivatives, such as pLNH31, pLNH32, pLNH33, or
pLNH34, was carried out by electroporation largely by using the procedure
described by Becker and Guarente (3), with some minor modifications. The yeast
cells were grown to a density of 120 to 160 Klett units (KU) (optical density at
600 nm [OD600], 12 to 16). A yeast cell suspension (60 ml containing 1 to 5 ml of
plasmid DNA) in a 0.2-cm sterile Bio-Rad cuvette was electroporated with a
Gene Pulser and a Pulse Controller (both obtained from Bio-Rad) set at 1.5 to
2.0 kV (the value varied from strain to strain), 25 mF, and 200 V. Authentic yeast
transformants were selected as described below. The Kmr gene in a plasmid
served as the primary selectable marker which rendered the host cells carrying
the plasmid resistant to geneticin present in the medium. The concentration of
geneticin used varied somewhat for different Saccharomyces yeasts. For example,
fusion 1400 transformants were selected on YEPD (1% yeast extract, 2% pep-
tone, 2% glucose) plates containing 40 mg of geneticin per ml, and S. cerevisiae
AH22 transformants were selected on YEPD plates containing 50 mg of gene-
ticin per ml. However, some untransformed yeast cells can be induced to become
resistant to geneticin. Thus, geneticin is not ideal for direct selection of trans-
formants, and not every geneticin-resistant colony is a true transformant. It has
been reported that the Apr gene can be expressed in S. cerevisiae (19), and we
found that it can also be expressed in fusion yeast strain 1400, as well as most
other Saccharomyces strains. However, most Saccharomyces strains are resistant
to ampicillin, and therefore the Apr gene cannot be used directly as a dominant
selectable marker for selection of yeast transformants. Nevertheless, Saccharo-
myces strains exhibiting high levels of Apr gene expression produce penicillinase,
and it is possible to identify yeast colonies containing the cloned Apr gene on
special plates by the penicillinase test (9). The latter test provides a way to
identify true transformants from geneticin-resistant colonies.
Preparation of seed cultures. Yeast strains were grown on agar plates con-
taining the proper media. For example, the untransformed Saccharomyces strains
(such as strain 1400 or AH22) were grown on YEPD, and the transformed
recombinant Saccharomyces strains [such as 1400(pLNH32)] were grown on
YEPX (1% yeast extract, 2% peptone, 2% xylose). To prepare the seed culture
for a new strain, first yeast cells from the agar plates were inoculated directly into
test tubes, each containing 5 ml of medium [YEPD for strain 1400 or AH 22 and
YEPX for 1400(pLNH32) and AH22(pLNH32)], and then the cells were incu-
bated overnight in a shaker at 30°C and 200 rpm. Each of the 5-ml seed cultures
was then transferred to a 300-ml Erlenmeyer flask containing 50 ml of the same
sterile medium. Each flasks had a side arm (Bellco), which allowed us to directly
monitor the growth of the yeast cultures with a Klett-Summerson photoelectric
colorimeter. All of the seed cultures were incubated under the same conditions
until they reached the late log phase (400 to 450 KU [OD600, 80 to 90]) and then
were stored at 4°C. The established seed cultures were transferred once a month
by transferring 2 ml of each 1-month-old seed culture to another flask containing
50 ml of sterile medium and growing the cells to a density of 400 to 450 KU.
Enzyme assays. To analyze the XR, XDH, XK activities, strain AH22 or
fusion 1400 transformants carrying pLNH32, pUCKm10-AR, pUCKm10-A*R,
pUCKm10-KD, or pUCKm10-KK were cultured in YEPD containing the proper
concentration of geneticin until the density was 200 KU (20 OD600, 20). Cells (10
ml) were harvested by centrifugation, washed with 10 ml of sterile water, resus-
pended in 400 ml of buffer (0.1 M sodium phosphate, pH 7.2), and chilled in a
freezer for at least 30 min until they were frozen. The cells were then thawed and
transferred to a 1.5-ml Eppendorf tube. About 0.3 g of acid-treated glass beads
and 150 ml of 0.1 M 2-mercaptoethanol were added to the cells, which were then
disrupted by vortexing. The resulting cell extracts were assayed for XR and XDH
activities as described by Bolen and Detroy (5) and for XK activity as described
1854 HO ET AL.
TABLE 1. In vitro XR, XDH, and XK enzyme activities
Sp act (nmol/mg of protein21 min21)
Strain
XR (NADPH)a XDH (NAD)b XKc
1400(pLNH32) 411 839 100
1400 54 22 NDd
P. stipitis 5773 417 410 35.2
a
The specific activity of XR is expressed as the amount of NADPH oxidized
by the enzyme.
b
The specific activity of XDH is expressed as the amount of NAD reduced by
the enzyme.
c
The specific activity of XK is expressed as the amount of xylulose phosphor-
ylated by the enzyme.
d
ND, not detectable.
by Shamanna and Sanderson (23). For comparison, 10 ml of P. stipitis cells
cultured in YEPX until the density was 200 KU processed in the same way, and
cell extracts were used to determine the XR, XDH, and XK activities by the same
procedures.
Cofermentation of glucose and xylose or fermentation of xylose by yeasts. For
cofermentation studies, 2 ml of a seed culture of strain 1400 or 1400(pLNH32)
was inoculated into 100 ml of YEPD nonselective medium in a 300-ml Erlen-
meyer flask equipped with a side arm. The culture was incubated aerobically at
30°C in a floor shaker at 200 rpm until the density was 400 to 450 KU, and there
was no more than a 20-KU difference between the cultures used in the compar-
ison studies. Twenty milliliters of 50% glucose and 10 ml of 50% xylose were then
added to each culture, making the final glucose and xylose concentrations ap-
proximately 8 and 4%, respectively. After thorough mixing, 1 ml of the super-
natant broth (containing some cells) was removed from each flask to serve as the
zero time sample. The flasks were then sealed with two layers of Saran Wrap to
allow fermentation to occur anaerobically and were incubated in a shaker under
identical conditions. Samples (1 ml) of the fermentation broth were removed at
intervals and used for analyses of substrates and fermentation products, includ-
ing glucose, xylose, ethanol, xylitol, and glycerol. To analyze fermentation of
xylose by 1400(pLNH32), the cells were cultured in YEPX.
Analysis of fermentation products. A high-performance liquid chromatograph
(Hitachi Ltd., Tokyo, Japan) equipped with an RI detector was used to analyze
the concentrations of glucose, xylose, xylitol, ethanol, and glycerol. A Bio-Rad
type HPX-87H ion-exclusion column was used. The mobile phase was 0.005 M
H2SO4 at a flow rate of 0.4 ml/min.
RESULTS
Expression of AR, A*R, KD, or KK in S. cerevisiae transfor-
mants cultured in medium containing glucose. Previously, we
described the cloning of a BamHI fragment containing AR or
A*R into the yeast-E. coli shuttle plasmid pUCKm8. The re-
sulting plasmids were designated pLXR10 and pLXR13, re-
spectively (8). These plasmids were used to transform S. cer-
evisiae AH22. The resulting transformants carrying pLXR10 or
pLXR13, cultured in rich medium containing glucose as the
sole carbon source (YEPD containing 50 mg of geneticin per
ml), were found to be able to synthesize high levels of XR, and
the enzyme activity was similar to that present in wild-type P.
stipitis cultured in YEPX (Table 1). In control experiments,
untransformed parent S. cerevisiae cells and P. stipitis cells
cultured in YEPD were found to contain practically no detect-
able XR activity.
The BamHI-XhoI fragment containing the cloned KD gene
was isolated from plasmid pKS-KD (Fig. 2) and subcloned into
pUCKm10 (Fig. 1) at its BamHI and SalI sites. The resulting
plasmid, pUCKm10-KD, was used to transform S. cerevisiae
AH22. When the transformants were cultured in YEPD sup-
plemented with 50 mg of geneticin per ml, they were able to
synthesize high levels of XDH, and the enzyme activity was
comparable to the activity present in 1400(pLNH 32) cultured
in YEPD (Table 1). Untransformed AH22 did not have any
detectable XDH activity.
The BamHI-XhoI fragment containing the cloned KK gene
was also isolated from pKS-KK (Fig. 2) and subcloned into
APPL. ENVIRON. MICROBIOL.
pUCKm10. The resulting plasmid, pUCKm10-KK, was used to
transform S. cerevisiae AH22. The resulting transformants
were able to synthesize high levels of XK, like 1400(pLNH32)
(Table 1), when they were cultured in YEPD supplemented
with the proper levels of geneticin. Untransformed AH22 did
not have any detectable XK activity when it was cultured in
Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
YEPD.
S. cerevisiae AH22 was used as the host to test the expression
of these cloned genes in a Saccharomyces strain because AH22
can be very effectively transformed by electroporation under
the conditions described in this paper. For example, more than
70% of geneticin-resistant AH22 colonies were true transfor-
mants.
Construction of E. coli plasmids containing the XYL gene
cassette, KK-AR (or A*R)-KD. Our strategy for developing the
desired recombinant yeast-E. coli shuttle plasmid containing
the XYL genes was first to subclone AR (or A*R), KD, and KK
into pKS in such a way that the three genes formed a cassette
which could be excised from the pKS plasmid together as a
fragment by a single restriction endonuclease digestion. As a
result, the three-gene cassette could be easily inserted (by a
single ligation and transformation) into various yeast-E. coli
shuttle plasmids for simultaneous expression of all three genes
in yeast cells. Figure 2 is a schematic diagram showing the
construction of four such recombinant plasmids, pKS-KK-
A*R-KD-1 and -2 and pKS-KK-AR-KD-3 and -4 containing
the XYL gene cassette (referred to below as pKRD-1, -2, -3,
and -4, respectively). The XYL gene cassette can be removed
from these plasmids by digestion with restriction endonuclease
XhoI. The four plasmids differed only in AR gene orientation
and in the promoter of the AR gene, as described in Materials
and Methods.
Construction of high-copy-number yeast-E. coli shuttle plas-
mid containing the three-gene cassette KK-AR-KD or KK-
A*R-KD. As shown in Fig. 1, pUCKm10 is a 2mm replicon-
based yeast-E. coli shuttle plasmid and contains the Kmr and
Apr genes as selectable markers. The pUCKm10 plasmid could
be introduced into most of the Saccharomyces strains that we
tested by using Kmr and Apr for selection of the putative
transformants, as described in Materials and Methods. The
XhoI fragments containing the three-gene cassette were iso-
lated from pKRD1, -2, -3, and -4 and inserted into the SalI site
of pUCKm10; this resulted in eight possible plasmids, which
were collectively designated the pLNH plasmids. Four such
plasmids, pLNH31, -32, -33, and -34 (containing the XYL gene
cassettes from pKRD1, -2, -3, and -4, respectively) were char-
acterized and have the general structures shown in Fig. 1.
Xylose as the ideal selective pressure in rich medium for
Saccharomyces transformants carrying the pLNH plasmids.
Although true pLNH transformants of yeast strain 1400 can be
obtained by selecting colonies that not only grow on plates
containing geneticin but also are positive for the penicillinase
test (see Materials and Methods), geneticin is not an ideal
selective pressure for culturing and maintaining strain 1400
transformants carrying the pLNH plasmids for at least two
reasons. One reason is that the transformed yeast cells can all
be gradually induced to become resistant to geneticin without
relying on the action of the Kmr gene product. This allows
plasmid-free yeast cells to grow in the presence of geneticin.
Therefore, geneticin can lose its effectiveness as the agent
which exerts selection pressure to maintain the pLNH plasmids
in the strain 1400 transformants. The second reason is that
geneticin is too costly and too great an environmental hazard
to be used to maintain cultures for large-scale ethanol produc-
tion. It is known that in minimal medium all microorganisms
require a carbon source, such as glucose or xylose, for growth.
VOL. 64, 1998 RECOMBINANT XYLOSE-FERMENTING SACCHAROMYCES SPP. 1855

Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
FIG. 2. Construction of E. coli plasmids containing the XYL gene cassette KK-AR (or A*R)-KD. KK, S. cerevisiae XK gene fused to the promoter of the S. cerevisiae
pyruvate kinase gene; AR (or A*R), P. stipitis XR gene fused to the promoter of the S. cerevisiae alcohol dehydrogenase gene; KD, P. stipitis XDH gene fused to the
promoter of the S. cerevisiae pyruvate kinase gene. The double dagger (‡) indicates that the XhoI site was regenerated after ligation, and the asterisks (p) indicate an
intact ADC1 promoter.

Thus, xylose could be used as the selection pressure to main-
tain the transformants in minimal medium supplemented with
xylose as the sole carbon source. However, yeast-based indus-
trial ethanol fermentation is usually carried out in an inexpen-
sive rich medium, such as corn steep liquor, and most micro-
organisms should be able to grow in rich medium without a
carbon source. Nevertheless, we discovered that most Saccha-
romyces strains (more than 10 Saccharomyces strains were
tested, and there were no exceptions) do require a carbon
source for growth even in rich medium, such as YEP (1% yeast
extract, 2% peptone), as shown in Fig. 3. This seems to be a
special characteristic of a few genera of yeasts but not all
yeasts. For example, it was found that P. stipitis and C. shehatae
do not require a carbon source to grow in rich medium, such as
YEP. The latter discovery made it possible to use xylose as the
ideal selection pressure to culture and maintain the pLNH
transformants in both rich and minimal media. Furthermore, it
also provided an alternative mechanism for selection of true
transformants carrying the pLNH plasmids in case certain
yeasts could not use the Kmr and Apr genes as the selection
markers.
Transformation of Saccharomyces sp. strain 1400 with the
pLNH plasmids and the ability of the resulting transformants
to utilize xylose for growth. Saccharomyces sp. strain 1400 (11),
which can tolerate high temperatures and high ethanol con-
centrations, should be an ideal strain for producing fuel etha-
nol. Thus, this yeast strain was used as the host to receive the
pLNH plasmids. Transformation of Saccharomyces sp. strain
1400 with the pLNH plasmids and selection of pLNH trans-
formants of strain 1400 were carried out as described in Ma-
terials and Methods. Each pLNH plasmid was able to transform
strain 1400 to a xylose-utilizing strain. The 1400 recombinant
1856 HO ET AL.
FIG. 3. Growth of S. cerevisiae in rich medium with or without a carbon
source (sugar). (A) S. cerevisiae AH22 cultured in YEP or YEPD. (B) S. cerevi-
siae AH22 cultured in YEP or YEPXylu (1% yeast extract, 2% peptone, 2%
xylulose). Symbols: E, YEP; F, YEPD; Œ, YEPXylu.
strains were initially identified by their ability to grow on
YEPD plates containing geneticin (40 mg per ml) and by their
ability to form halos on ampicillin test plates (9). Each one was
verified by its ability to transfer the specific plasmid from the
transformant back into E. coli (26). Most importantly, these
transformants were able to grow in YEPX. Table 2 clearly
shows that a pLNH transformant of strain 1400, such as
1400(pLNH32), can grow in rich medium containing xylose as
the sole carbon source (YEPX) but the untransformed parent
strain 1400 cannot. These results further demonstrate the use-
fulness of xylose or a carbon source as an effective selective
agent for selection and maintenance of recombinant Saccha-
romyces strains.
Comparison of the abilities of 1400(pLNH32) and parent
strain 1400 to cofermenting a mixture of glucose and xylose.
Because pLNH32 and related plasmids are 2mm-based high-
copy-number plasmids, the transformants were relatively sta-
ble and could be cultured for four or five generations in rich,
APPL. ENVIRON. MICROBIOL.
TABLE 2. Growth of Saccharomyces recombinant and parent yeast
strains in media containing glucose and xylose
Density (KU) ata:
Strain Medium
Zero time 2h 4h 6h 8h 11 h 15 h
1400(pLNH32) YEPD 21 22 44 120 289 455 472
Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
YEPX 18 21 37 68 122 450 550
1400 YEPD 17 17 39 120 290 433 460
YEPX 54 66 72 75 75 79 79
a
Density was determined with a Klett-Summerson photoelectric colorimeter;
1 KU was equivalent to 0.1 OD600 unit at densities between 20 and 200 KU and
to 0.15 to 0.2 OD600 unit at densities of more than 300 KU.
nonselective medium, such as YEPD, without losing much of
their xylose-fermenting ability. This allows the use of these
recombinant yeast strains to ferment xylose or to coferment
glucose and xylose in YEPD. To compare the abilities of
1400(pLNH32) and related transformants containing other
pLNH plasmids to coferment glucose and xylose with the abil-
ity of the parent strain 1400 to coferment glucose and xylose,
these strains were cultured in YEPD (50 ml) until the density
was 400 to 450 KU (initial density, 20 KU [OD600, 2]), and then
10 ml of 50% glucose and 5 ml of 50% xylose were added to
each culture to start the fermentation. The results obtained
with 1400(pLNH32) and parent strain 1400 are shown in Fig. 4.
The results obtained with strain 1400 transformants containing
other pLNH plasmids were similar to the results obtained with
1400(pLNH32). These results demonstrated that the geneti-
cally engineered strain 1400(pLNH32) and related transfor-
mants could effectively coferment most of the 8% glucose and
4% xylose to ethanol in 48 h. In contrast, parent strain 1400
could ferment glucose to ethanol but could not ferment xylose.
XR, XDH, and XK activities in Saccharomyces transformant
1400(pLNH32). To further establish that the ability of the
strain 1400 transformants containing the pLNH plasmids to
effectively metabolize xylose was due to the presence of the
cloned XYL genes, the strain 1400 transformant containing
plasmid pLNH32 was cultured in YEPD to a density of 200 KU
(OD600, 20), and the resulting cells were used to determine
XR, XDH, and XK activities, which were compared with the
activities present in parent strain 1400 and P. stipitis, as shown
in Table 1.
DISCUSSION
This paper describes the strategies used to develop recom-
binant Saccharomyces strains that can effectively ferment xy-
lose and, in particular, can coferment glucose and xylose
present in the same medium. Using Saccharomyces sp. strain
1400 as an example, we successfully demonstrated that by clon-
ing XR, XD, and XK, each of which was fused to an efficient
glycolytic promoter, a superior glucose-fermenting Saccharo-
myces strain can be transformed to a recombinant yeast strain
that is able to effectively metabolize xylose aerobically for
growth and anaerobically for the production of ethanol as the
overwhelmingly major product. Furthermore, the resulting re-
combinant xylose-fermenting Saccharomyces strains can also
effectively coferment glucose and xylose present in the same
medium. This unique property should make the recombinant
Saccharomyces strains particularly effective in large-scale pro-
duction of ethanol from fermenting mixed sugars present in
cellulosic biomass hydrolysates.
Although only the results obtained with strain 1400(pLNH32)
are presented here, the transformants containing pLNH31,
pLNH33, or pLNH34 do not differ significantly in their ability
VOL. 64, 1998 RECOMBINANT XYLOSE-FERMENTING SACCHAROMYCES SPP.
FIG. 4. Comparison of the abilities of recombinant Saccharomyces sp. strain
1400(pLNH32) and parent strain 1400 to coferment glucose and xylose. (A)
Strain 1400(pLNH32). (B) Strain 1400. Utilization of glucose and xylose and
production of ethanol, xylitol, and glycerol were determined. Symbols: ■, glu-
cose; F, xylose; Œ, ethanol; h, xylitol; ‚, glycerol.
to coferment glucose and xylose or to utilize xylose for growth.
The presence of either AR or A*R in the transformants also
does not significantly affect their ability to ferment xylose or
utilize it for growth.
Our recombinant Saccharomyces strains can effectively fer-
ment xylose in the presence of glucose, as shown in Fig. 4.
However, they do not depend on the presence of glucose to
ferment xylose. They can effectively ferment xylose in the ab-
sence of glucose when they are cultured in xylose medium, as
shown in Fig. 5.
As mentioned above, while our work was in progress, Kotter
et al. (20), Walfridsson et al. (25), and Tantirungkij et al. (24)
described the development of their recombinant S. cerevisiae
strains, which was accomplished by cloning only the P. stipitis
XR gene and XDH gene. However the recombinant yeast
1857
Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
FIG. 5. Fermentation of xylose by recombinant Saccharomyces sp. strain
1400(pLNH32) cultured in the xylose-containing medium YEPX. Utilization of
xylose and production of ethanol, xylitol, and glycerol were determined. Symbols:
F, xylose; Œ, ethanol; h, xylitol; ‚, glycerol.
strains of these groups ferment xylose extremely slowly and
produce little ethanol. Furthermore, the major fermentation
product produced by these recombinant yeast strains is not
ethanol but xylitol. Most importantly, there are fundamental
differences between our strategy and the strategies used by the
other three groups to develop recombinant yeast strains. For
example, as mentioned above, our recombinant Saccharomyces
strains not only contain the cloned XR gene and the XDH
gene but also contain the cloned XK gene. As a result, our
recombinant yeast strains can effectively ferment high concen-
trations of xylose almost completely to ethanol, and very little
xylitol is produced as a by-product (Fig. 4A and 5).
Our strategy to fuse the cloned XR, XD, and XK genes to
highly effective glycolytic promoters (or expression signals)
made our recombinant yeast strains better than the recombi-
nant yeast strains developed by others, as well as naturally
occurring microorganisms in fermenting substrates containing
both glucose and xylose. The other groups did not include this
strategy in their designs. As a result, the expression of the
cloned XR, XD, and XK genes in our recombinant yeast strains
does not require the presence of xylose for induction, and also
the expression of the cloned genes is not repressed by glucose
in the cultural medium. This allowed our recombinant Saccha-
romyces strains to effectively coferment glucose and xylose
without a lag period, as shown in Fig. 4A. However, for the
conversion of cellulosic biomass to ethanol, the fact that our
yeast strains do not require xylose for induction and the fact
that they are able to maintain expression of the xylose-metab-
olizing genes in the presence of glucose provide a much greater
benefit than metabolizing xylose without a lag period. The
naturally occurring xylose-fermenting yeasts, such as P. stipitis
and C. shehatae, which ferment xylose as effectively as our
recombinant xylose-fermenting Saccharomyces strains but do
not have the properties mentioned above, are not able to
ferment xylose at all when glucose is present in the medium,
even though they effectively ferment xylose in the absence of
glucose. Data comparing the ability of our recombinant yeast
strains to ferment xylose and to coferment glucose and xylose
with the ability of the naturally occurring xylose-fermenting
1858 HO ET AL.
yeasts P. stipitis and C. shehatae to ferment xylose and to
coferment glucose and xylose will be published elsewhere.
The fact that we developed a set of broad-host-range pLNH
plasmids by using the Kmr and Apr genes as the primary
selection markers also contributed greatly to the development
of effective recombinant xylose-fermenting Saccharomyces
strains, such as 1400(pLNH32). The development of such plas-
mids was intended to provide a means by which wild-type yeast
strains, particularly diploid or polyploid industrial strains (such
as strain 1400) that may have superior properties desirable for
ethanol fermentation, can easily be transformed by these vec-
tors. Furthermore, since there are other factors besides the
enzymes encoded by the cloned XYL genes that may seriously
affect the effectiveness of yeast xylose fermentation, we do not
expect that all Saccharomyces strains that receive the same
cloned XYL genes will ferment xylose with the same efficiency.
Thus, the pLNH plasmids can also be excellent tools for
screening new Saccharomyces strains that may be superior for
cofermenting glucose and xylose. Nevertheless, since we did
not have to screen many yeast strains to obtain strain 1400,
which is an effective host for the expression of the XYL genes,
there may be other Saccharomyces strains that are as effective
as or more effective than strain 1400 as hosts for expression of
the XYL genes for efficient fermentation of xylose to ethanol.
It is desirable to design a broad-host-range selective mech-
anism that requires only inexpensive and nontoxic chemicals as
the selective pressure to select and maintain Saccharomyces
transformants, and our discovery that Saccharomyces strains
require a carbon source to grow even in rich medium provided
an answer. This discovery provided an ideal and effective
mechanism that could be used to screen and maintain true
transformants containing the cloned XYL genes. Furthermore,
it also provided a rapid and effective method for differentiating
xylose-fermenting Saccharomyces transformants from other xy-
lose-metabolizing microorganisms, including the naturally oc-
curring xylose-fermenting yeasts, such as P. stipitis and C. she-
hatae. This is because there are very few microorganisms that
require the presence of a carbon source in rich medium to
grow.
The fact that xylose can serve as a selective agent to maintain
the pLNH plasmids even in rich medium, such as YEPX,
coupled with the fact that the pLNH plasmids are stable, high-
copy-number plasmids in Saccharomyces strains, allows the
pLNH transformants of Saccharomyces strains to effectively
ferment xylose to ethanol in the absence of selection for at
least four or five generations, as shown in Fig. 4. This makes it
possible for industry to use these recombinant yeast strains,
such as 1400(pLNH32), to carry out large-scale fermentation
of the glucose and xylose present in cellulosic biomass to eth-
anol in the absence of selection. To achieve this, the only
requirements are that the process is a batch fermentation pro-
cess and that the yeast cells are propagated in the early stages
in medium containing xylose as the major or sole carbon
source.
Several groups in the United States and Canada are using
our xylose-fermenting recombinant strain 1400 yeasts to per-
form various studies, and they have all verified what we report
here. One group that used 1400(pLNH32) to ferment corn
fiber sugars to ethanol has published their results (22).
Nevertheless, additional improved xylose-fermenting Sac-
charomyces strains can still be developed. For example, even
though the Saccharomyces transformants carrying the pLNH
plasmids are very stable and can be maintained by an ideal
selection mechanism (in which xylose is used as the selection
pressure), an ideal transformant should be completely stable
APPL. ENVIRON. MICROBIOL.
and not require the use of selection pressure at any stage of
growth or fermentation.
Recently, we have been very successful in developing stable
derivatives of xylose-fermenting recombinant yeast strain 1400,
designated 1400(LNH-ST) strains (or LNH-ST strains). The
latter Saccharomyces strains are completely stable and do not
Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
require any selection pressure to maintain their ability to fer-
ment xylose to ethanol or their ability to utilize this sugar for
growth. Furthermore, these stable xylose-fermenting recombi-
nant yeast strains also ferment xylose to ethanol slightly more
efficiently than 1400(pLNH32) or any other pLNH transfor-
mant of strain 1400. The stable strains were developed by
integrating multiple copies of the XYL genes into the yeast
chromosome. Brief descriptions of the ability of these stable
xylose-fermenting Saccharomyces strains to ferment mixed sug-
ars in the absence of any selection pressure have been pre-
sented previously (16). The details concerning the develop-
ment and characterization of such stable xylose-fermenting
Saccharomyces strains will be described elsewhere.
ACKNOWLEDGMENTS
This work was supported in part by the U.S. Department of Energy,
Amoco Corporation, and Swan Biomass Company.
REFERENCES
1. Ammerer, G. 1983. Expression of genes in yeast using the ADC1 promoter.
Methods Enzymol. 101:192–201.
2. Armstrong, K. A., T. Som, F. C. Volkert, A. Rose, and J. R. Broach. 1989.
Propagation and expression of genes in yeast using 2-micron circle vectors, p.
165–192. In P. J. Barr, A. J. Brake, and P. Valenzuela (ed.), Yeast genetic
engineering. Butterworths, Boston, Mass.
3. Becker, D., and L. Guarente. 1991. High efficiency transformation of yeast by
electroporation. Methods Enzymol. 194:182–186.
4. Bennetzen, J. L., and B. D. Hall. 1982. The primary structure of the Sac-
charomyces cerevisiae gene for alcohol dehydrogenase I. J. Biol. Chem. 257:
3018–3025.
5. Bolen, P. L., and R. W. Detroy. 1985. Induction of NADPH-linked D-xylose
reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen
tannophilus by D-xylose, L-arabinose, or D-galactose. Biotechnol. Bioeng.
27:302–307.
6. Broach, J. R. 1983. Construction of high copy number yeast vectors using
2mm circle sequences. Methods Enzymol. 101:307–325.
7. Burke, R. L., P. Tekamp-Olson, and R. Najarian. 1983. The isolation, char-
acterization, and sequence of the pyruvate kinase gene of Saccharomyces
cerevisiae. J. Biol. Chem. 258:2193–2201.
8. Chen, Z. D., and N. W. Y. Ho. 1993. Cloning and improving the expression
of P. stipitis xylose reductase gene in Saccharomyces cerevisiae. Appl. Bio-
chem. Biotechnol. 39/40:135–147.
9. Chevallier, M. R., and M. Aigle. 1979. Qualitative detection of penicillinase
produced by yeast strains carrying chimeric yeast-coli plasmids. FEBS Lett.
108:179–184.
10. Chiang, L. C., H. Y. Hsiao, P. P. Veng, L. F. Chen, and G. T. Tsao. 1981.
Ethanol production from xylose by enzymic isomerization and yeast fermen-
tation. Biol. Bioeng. Symp. 11:263–274.
11. D’Amore, T., G. Celotto, I. Russell, and G. G. Stewart. 1989. Selection and
optimization of yeast suitable for ethanol production at 40°C. Enzyme Mi-
crob. Technol. 11:411–416.
12. Deng, X. X., and N. W. Y. Ho. 1990. Xylulokinase activity in various yeasts
including Saccharomyces cerevisiae containing the cloned xylulokinase gene.
Appl. Biochem. Biotechnol. 24/25:193–199.
13. Fink, G. R. 1970. The biochemical genetics of yeast. Methods Enzymol.
17A:59–78.
14. Gong, C. S., L. F. Chen, M. C. Flickinger, L. C. Chiang, and G. T. Tsao. 1981.
Production of ethanol from D-xylose by using D-xylose isomerase and yeasts.
Appl. Environ. Microbiol. 41:430–436.
15. Ho, N. W. Y., and S. F. Chang. 1989. Cloning of yeast xylulokinase gene by
complementation of E. coli and yeast mutations. Enzyme Microb. Technol.
11:417–421.
16. Ho, N. W. Y., Z. D. Chen, and A. P. Brainard. 1998. Stable xylose-fermenting
Saccharomyces yeasts for the conversion of cellulosic biomass to ethanol by
using a new method for gene integration, abstr. 228D. In Abstracts of the
American Institute of Chemical Engineers 1997 annual meeting. American
Institute of Chemical Engineers, New York, N.Y.
17. Jeffries, T. W. 1983. Utilization of xylose by bacteria, yeasts, and fungi. Adv.
Biochem. Eng. Biotechnol. 27:1–32.
18. Jeffries, T. W. 1985. Emerging technology for fermenting D-xylose. Trends
Biotechnol. 3:208–212.
VOL. 64, 1998 RECOMBINANT XYLOSE-FERMENTING SACCHAROMYCES SPP.
19. Jimenez, A., and J. Davies. 1980. Expression of a transposable antibiotic
resistance element in Saccharomyces. Nature 287:869–871.
20. Kotter, P., R. Amore, C. P. Hollenberg, and M. Ciriacy. 1990. Isolation and
characterization of the P. stipitis xylitol dehydrogenase gene, XYL2, and
construction of a xylose-utilizing Saccharomyces cerevisiae transformant.
Curr. Genet. 18:493–500.
21. Kunkel, T. A., J. D. Roberts, and R. A. Zakour. 1987. Rapid and efficient
site-specific mutagenesis without phenotypic selection. Methods Enzymol.
154:367–382.
22. Moniruzzaman, M., B. S. Dien, C. D. Skory, Z. D. Chen, R. B. Hespell,
N. W. Y. Ho, B. E. Dale, and R. J. Bothast. 1997. Fermentation of corn fibre
sugars by an engineered xylose utilizing Saccharomyces yeast strain. World J.
Microbiol. Biotechnol. 13:341–346.
23. Shamanna, D. K., and K. E. Sanderson. 1979. Uptake and catabolism of
D-xylose in Salmonella typhimurium LT2. J. Bacteriol. 139:64–70.
1859
24. Tantirungkij, M., N. Nakashima, T. Seki, and T. Yoshida. 1993. Construc-
tion of xylose-assimilating Saccharomyces cerevisiae. J. Ferment. Bioeng.
75:83–88.
25. Walfridsson, M., M. Anderlund, X. Bao, and B. Hahn-Hagerdal. 1997. Ex-
pression of different levels of enzymes from the Pichia stipitis XYL1 and
XYL2 genes in Saccharomyces cerevisiae and its effects on product formation
during xylose utilization. Appl. Microbiol. Biotechnol. 48:218–224.
Downloaded from http://aem.asm.org/ on April 12, 2012 by INDIAN INSTITUTE OF TECHNOLOGY NEW DELHI
26. Ward, A. C. 1990. Single-step purification of shuttle vectors from yeast for
high frequency back-transformation into E. coli. Nucleic Acids Res. 18:5318–
5319.
27. Webster, T. D., and R. C. Dickson. 1983. Direct selection of Saccharomyces
cerevisiae resistant to the antibiotic G418 following transformation with a
DNA vector carrying the kanamycin-resistance gene of Tn903. Gene 26:243–
252.