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Applied and Environmental Microbiology, August 2000, p. 3381-3386, Vol. 66, No. 8
Department of Applied Microbiology, Lund
University, SE-221 00 Lund, Sweden
Received 10 December 1999/Accepted 2 June 2000
For ethanol production from lignocellulose, the fermentation of
xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have
not yet been achieved. To quantitatively analyze metabolic fluxes in
recombinant S. cerevisiae during metabolism of
xylose-glucose mixtures, we constructed a stable xylose-utilizing
recombinant strain, TMB 3001. The XYL1 and XYL2
genes from Pichia stipitis, encoding xylose reductase (XR)
and xylitol dehydrogenase (XDH), respectively, and the endogenous
XKS1 gene, encoding xylulokinase (XK), under control of the
PGK1 promoter were integrated into the chromosomal
HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation
from xylose by recombinant S. cerevisiae was demonstrated
for the first time. However, the strain grew on xylose only in the
presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C
(0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C
h To obtain an economically feasible
industrial process for ethanol production from lignocellulose, it is
necessary to ferment all sugars present with high yields and
productivities (53). The commonly used Saccharomyces
cerevisiae has many advantages as an ethanol producer, such as
fast sugar consumption, high ethanol yield from hexoses, and high
resistance to inhibitory compounds that are present in the
hydrolysates. However, a major drawback is that S. cerevisiae cannot utilize the pentose sugar xylose, only its
isomer xylulose. In xylose-utilizing yeasts, the conversion from xylose
to xylulose is a two-step process catalyzed by xylose reductase (XR)
and xylitol dehydrogenase (XDH) (10), whereas bacteria
perform the conversion in one step with xylose isomerase (XI)
(23).
Xylose fermentation by recombinant S. cerevisiae carrying
heterologous XYL1 and XYL2 genes from
Pichia stipitis, which encode XR and XDH, respectively, has
resulted mainly in xylitol formation (24, 44, 48).
Similarly, if xylA from Thermus thermophilus, which encodes XI, is introduced into S. cerevisiae, then
only limited xylose fermentation is observed (47). Limited
xylose fermentation by recombinant S. cerevisiae has been
ascribed to poor xylose uptake (9, 24, 25), a cofactor
imbalance generated by the discrepancy in cofactor usage by XR and XDH
(8, 24, 49), limitations in the pentose phosphate pathway
(12, 24, 38, 48), and insufficient induction or activation
of ethanologenic enzymes (5, 17, 20, 29). When homologous
XKS1, which encodes xylulokinase (XK), was overexpressed in
a Saccharomyces sp. strain carrying XYL1 and
XYL2, the ethanol yield and the xylose uptake rate increased
under oxygen-limited conditions, but xylitol was still a major
by-product (22).
Although the shortcomings of xylose fermentation by recombinant
S. cerevisiae have been investigated in several studies,
data from anaerobic fermentations do not exist and quantitative data are sparse. Chemostat cultivations in which growth rate and
concentrations of substrates and products are constant enable
quantitative determinations of metabolic fluxes. Analysis of xylose
fluxes is the first step towards identifying causes for by-product
formation and low productivity during xylose fermentation. A stable
recombinant strain was constructed by integration of the
XYL1 and XYL2 genes from P. stipitis and the homologous XKS1 gene under control of
the PGK1 promoter into the chromosomal HIS3 locus
of S. cerevisiae. Metabolic fluxes were determined for
different xylose-to-glucose ratios in chemostat cultivations under
anaerobic conditions. Glucose was used as a cosubstrate since the
recombinant strain was unable to grow on xylose in the absence of oxygen.
Strains and plasmids.
Escherichia coli DH5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Anaerobic Xylose Fermentation by Recombinant Saccharomyces
cerevisiae Carrying XYL1, XYL2, and
XKS1 in Mineral Medium Chemostat Cultures
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
1 g (dry weight) of cells1 (0.24 to
0.30 g h1 g [dry weight] of cells1)
were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h1. The anaerobic
ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of
xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The
xylose uptake rate increased with increasing xylose concentration in
the feed, from 3.3 mmol of C h1 g (dry weight) of
cells1 when the xylose-to-glucose ratio in the feed was
1:3 to 6.8 mmol of C h1 g (dry weight) of
cells1 when the feed ratio was 3:1. With a feed content
of 15 g of xylose/liter and 5 g of glucose/liter, the xylose
flux was 2.2 times lower than the glucose flux, indicating that
transport limits the xylose flux.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
[F 
80 dlacZ
M15
(lacZYA-argF) U169 deoR recA1 endA1
hsdR17(rK, mK+)
supE44 
thi-1 gyrA96 relA1]
(GIBCO BRL, Gaithersburg, Md.) was used for subcloning. S. cerevisiae CEN.PK113-7A (MATa
his3-1 MAL2-8C SUC2) (18) was
used as the recipient yeast strain for the integrating plasmid
YIpXR/XDH/XK. All strains were stored frozen at 
80°C. Agar plates
streaked from the frozen stocks were used to inoculate the precultures.
Media. S. cerevisiae CEN.PK PK113-7A was grown in YPD medium (40) for transformation. In all other experiments, a defined medium including vitamins and trace elements was used (50). L-Histidine was added at 50 mg per liter for strain CEN.PK PK113-7A. For the continuous cultivations, the medium was also supplemented with ergosterol and unsaturated fatty acids in the form of Tween 80 (Sigma, St. Louis, Mo.) (1, 2). Ergosterol and Tween 80 were dissolved in boiling 96% (vol/vol) ethanol to final concentrations of 0.01 and 0.42 g/liter, respectively. Bacterial strains were grown in Luria-Bertani medium (35). Transformants were selected by adding ampicillin (50 mg/liter). For growth on solid media, 20 g of agar per liter was added.
Nucleic acid manipulations. Standard techniques for nucleic acid manipulations were used (35). Plasmids were prepared using the QIAGEN Maxi Plasmid Purification Kit (Qiagen GmbH, Hilden, Germany). Restriction enzymes and other modifying enzymes were purchased from Boehringer Mannheim Scandinavia AB (Bromma, Sweden). DNA fragments separated by agarose gel electrophoresis were purified with the QIAquick Gelextraction Kit (Qiagen GmbH).
Construction of integrating vector expressing XYL1,
XYL2, and XKS1.
The 2µ origin of replication
and the P-ribosyl-anthranilate isomerase (TRP1) gene were
removed by partial digestion with XmnI and PvuII
from plasmid pXks, a yeast episomal plasmid carrying the
XKS1 gene under the control of the phosphoglycerate kinase (PGK) promoter and terminator (Johansson et al., submitted) (Fig. 1a). This construct has two base
substitutions introduced in the XKS1 gene upstream from the
start codon to maximize translational efficiency; furthermore, the
codon for the N-terminal amino acid was altered to increase the protein
stability of the gene product. The remaining fragment of 6,465 bp was
recircularized by self-ligation and then partially digested with
HindIII and BamHI, creating a fragment of
6,435 bp (Fig. 1b). The genes XYL1 and XYL2 under the control of alcohol dehydrogenase (ADH) and PGK promoters, respectively, were excised from pY7 (46) by partial
digestion with HindIII and BamHI, yielding a
fragment of 6,153 bp (Fig. 1c). The 1,150-bp HIS3 cassette
was excised from YDp-H (3) by BamHI (Fig. 1d).
Finally, the plasmid carrying XKS1 was ligated to the
HIS3 cassette and the XYL1-XYL2 fragment,
yielding YIpXR/XDH/XK, an integrating vector carrying XYL1,
XYL2, and XKS1 and with HIS3 as a
selection marker (Fig. 1e). Restriction enzyme digestions verified the
map of the constructed vector.
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Yeast strain transformation. The lithium acetate method was used for transformation (37).
Continuous cultivations. Continuous fermentations were conducted anaerobically in computer-controlled glass bioreactors (Belach Bioteknik AB, Stockholm, Sweden) at 30°C with a stirring speed of 200 rpm. The working volume of the reactors was 600 ml, and the pH was adjusted to 5.5 with 3 M KOH. Anaerobic conditions were maintained by sparging with nitrogen containing less than 5 ppm of O2 (ADR class2 1A; AGA, Sundbyberg, Sweden) at a constant gas flow of 0.3 liter/min controlled by mass flow meters (El-Flow, Insat, Switzerland). The feed reservoir was degassed before connection to the fermentor. During the continuous cultivation, a rubber ball filled with nitrogen connected to the reservoir prevented a vacuum buildup when medium was pumped out. The off-gas condensers were cooled to 4°C. Antifoam (Dow Corning Antifoam RD Emulsion; BDH Laboratory Supplies, Poole, United Kingdom) was included in the feed at a concentration of 0.5 ml/liter of feed.
Precultures, 200 ml of medium in 1-liter baffled shake-flasks, were incubated overnight at 30°C and were shaken at 140 rpm in an orbital incubator (INR-200; Gallenkamp, Leicester, United Kingdom). Cells were harvested by centrifugation (5,000 × g, 5 min, 4°C) and were washed once with a solution containing 9 g of NaCl/liter before inoculation into the fermentors. Fermentations were started as anaerobic batch cultures with glucose (5 g/liter) as the sole carbon source. When the batch fermentation was depleted of glucose, as detected by a rapid drop in the carbon dioxide evolution rate, a feed of fresh medium was started, yielding a dilution rate (D) of 0.06 h1. The volume was kept at
600 ml by continuous removal of broth through a siphon. The total
concentration of carbon source in the feed was 20 g/liter. Steady
states with various ratios of xylose and glucose were analyzed. One set
of fermentations was started with 20 g of glucose/liter, and after
a steady state was reached, the feed was switched to 5 g of
xylose/liter plus 15 g of glucose/liter, followed by 10 g of
xylose/liter plus 10 g of glucose/liter and, finally, 15 g of
xylose/liter plus 5 g of glucose/liter. A second set of
fermentations was operated in the opposite mode and started with
15 g of xylose/liter plus 5 g of glucose/liter so that each
steady state was performed in duplicate.
Analyses. Substrates consumed and products formed were analyzed by column liquid chromatography. A Gilson (Villiers-le-bel, France) CLC system equipped with an ion-moderated partition chromatography column, Aminex HPX-87H (Bio-Rad, Richmond, Calif.), was used together with a Shimadzu (Kyoto, Japan) refractive index detector, RID-6A, with a mobile phase of 5 mM H2SO4. The flow rate was 0.6 ml/min, and the separation temperature was 45°C.
For determination of ethanol evaporation, the experimental setup was identical to the one used for the continuous cultivations. Ethanol was added to non-cell-containing medium to a concentration of 6 g/liter, and the ethanol concentration in the fermentor was assayed over time. The composition of the outgoing gas was continuously monitored by a carbon dioxide and oxygen monitor (type 1308; Brüel&Kj
r, Copenhagen, Denmark) (11) using photoacoustic and
magnetoacoustic detection for CO2 and O2, respectively.
The dry weight of the cells was determined by filtering a known volume
of the culture broth through a 0.45-µm-pore-size Supor membrane
(Gelman Sciences, Ann Arbor, Mich.). After being washed with 3 volumes
of double-distilled water and dried in a microwave oven for 15 min, the
filter was weighed. The dry weight of the cells was determined in
triplicate for each steady state.
Enzymatic assays.
Cell extracts for enzyme assays were
prepared using glass beads (0.5 mm in diameter). Cells were harvested
by centrifugation and, after washing, were resuspended in a
disintegration buffer, 0.1 M triethanolamine buffer (pH 7.0) containing
1 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and 0.5 mM
EDTA. The suspension was vortexed for 5 min at 4°C, put on ice for 5 min, and then vortexed again. Cell debris and glass beads from the cell
extract were separated by centrifugation (20,000 × g,
5 min, 4°C), and the remaining supernatant was used for enzyme
determinations. Enzyme activities were measured with two different
sample concentrations (each in duplicate) by using a U-2000 model
spectrophotometer (Hitachi Ltd., Tokyo, Japan) operating at 340 nm and
30°C and with buffers and reagents according to Table
1. Assays were adapted from previously
reported assays (4, 7, 32, 33, 39, 51). Specific activities
are expressed as units per milligram of protein. Units are defined as
micromoles of NADH reduced or oxidized per minute. For the XK assay,
the reaction occurring before the addition of ATP (XDH in the reverse
direction) was subtracted from the reaction observed in the presence of
ATP. No reaction was observed in the absence of xylulose. Xylulose was
produced as previously described (31).
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Calculations. Carbon balances and yields were calculated using single carbon unit equivalents (expressed as moles of carbon) (13) to allow comparison of hexose and pentose sugar metabolism. The elemental formula CH1.745O0.627N0.129S0.0025 was used for calculation of assimilated carbon converted to biomass (16).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of a recombinant strain expressing XYL1, XYL2, and XKS1. An integrating vector, YIpXR/XDH/XK, carrying XYL1, XYL2, and XKS1 and with HIS3 as selectable marker, was constructed (Fig. 1). HIS3 in S. cerevisiae CEN.PK 113-7A is deleted between the HindIII sites (37). The constructed plasmid was cut with PstI within the HIS3 gene, but outside the HindIII deletion in the recipient strain, to generate homologous ends to initiate integration. The resulting recombinant, S. cerevisiae TMB 3001, grew in the absence of histidine and expressed XR, XDH, and XK activities. The specific activities of XR, XDH, and XK in cell lysates of the parental strain CEN.PK 113-7A and the recombinant strain TMB 3001 grown on a xylose-glucose mixture in shaken-flask cultures were 0.03, 0.01, and 0.02 U/mg of protein and 0.21, 1.78, and 0.93 U/mg of protein, respectively.
Anaerobic fermentations of xylose and glucose.
Product
formation by S. cerevisiae TMB 3001 was investigated in a
high-performance bioreactor under anaerobic conditions. The recombinant
strain carrying YIpXR/XDH/XK grew aerobically on xylose, but not
anaerobically. We included glucose in all fermentations to enable
anaerobic chemostat cultivation. The maximum growth rate anaerobically
on glucose was 0.35 h1 (data not shown). Four different
steady states were established, three with mixtures of xylose and
glucose, the xylose-to-glucose ratios in the feed being 3:1, 1:1, and
1:3, respectively, and one with glucose as the sole carbon source
(Table 2). For one set of fermentations,
the first steady state was established with the medium containing the
highest xylose concentration; the following three steady states were
conducted in the order of decreasing xylose concentration in the feed.
A second set was conducted in the opposite mode, i.e., started with a
feed containing glucose only. The total amount of carbon source in the
feed was 667 mmol of C, equivalent to 20 g/liter, and the D
was 0.06 h1. A post hoc analysis (Scheffe, = 0.05) showed no significant difference between replicates. Steady-state
concentrations were independent of the order in which steady states
were reached. The standard deviation was less than 5% of the
consumption and production for mean values of all substances (Table 2).
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1 g (dry weight) of cells1 when the
xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C
h1 g (dry weight) of cells1 when the
xylose-to-glucose ratio in the feed was 3:1 (Fig.
2; Table 2). However, even at the highest
xylose concentration, only 12% of the xylose was consumed (Table
3).
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1
h1 for steady states with increasing glucose
concentration. At an initial concentration of 6 g of
ethanol/liter, the experimentally determined evaporation rate was
0.06 g liter1 h1, which is in
agreement with the calculated values. The ethanol concentration
differed at the four steady states, which may have influenced the
evaporation rate. Furthermore, ethanol is highly volatile and the
experimental error is large. To obtain as accurate a determination of
the ethanol concentrations as possible, degree of reduction balances
were used throughout the study. The corrected ethanol yield on total
carbohydrates decreased with increasing xylose in the feed, from 0.53 to 0.45 mol of C/(mol of C of consumed carbohydrates) (0.41 to 0.35 g/g) (Table 2). However, the corrected ethanol yield calculated on
consumed glucose increased, showing that xylose is converted to ethanol
under anaerobic conditions. The corrected ethanol yield for the steady
state with only glucose in the feed was 0.53 mol of C/(mol of C of
glucose) (0.41 g/g), and with a xylose-to-glucose ratio in the feed of
3:1, the yield increased to 0.65 mol of C/(mol of C of glucose) (0.50 g/g). Hence, for the steady state with a xylose-to-glucose ratio in the
feed of 3:1, the ethanol yield on xylose was estimated to be 0.27 mol of C/(mol of C of xylose) [(0.65 0.53) × 15.1/6.8 (Table
2)] (0.21 g/g), assuming the ethanol yield on glucose to be constant for the four different steady states.
The xylitol yield, calculated with total consumed carbohydrates,
increased with increasing xylose uptake rate, from 0.03 to 0.12 mol of
C/(mol of C of carbohydrates) (0.03 to 0.12 g/g), when the xylose
concentration in the feed increased from 5 to 15 g/liter (Table 2) so
that the xylose fraction excreted as xylitol increased from 24 to 40%.
The glycerol yield increased slightly at the lowest xylose
concentration. At the highest xylose concentration, the lowest glycerol
yield and highest acetate yield were observed.
The biomass yield on consumed sugar was rather constant (Table 2), but
the concentration of biomass increased from 0.64 to 1.78 g/liter with
increasing glucose concentration (Table 3).
Enzyme activities and strain stability.
The specific
activities of the enzymes XR, XDH, XK, PGK, and ADH were measured
throughout the fermentations (Table 4).
The recombinant S. cerevisiae strain TMB 3001 expressed XR,
XDH, and XK with activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg of protein, respectively, corresponding to an approximate
XR-to-XDH-to-XK ratio of 1:6:4. Furthermore, the strain exhibited
stable recombinant enzyme activities throughout more than 4 weeks of
continuous cultivation, equivalent to more than 40 generations. The PGK
and ADH activities were 15 to 23 and 21 to 28 U/mg of protein,
respectively (Table 4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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For the first time, anaerobic ethanol formation from xylose has
been demonstrated for a recombinant xylose-utilizing strain of S. cerevisiae. The corrected ethanol yields (0.45 to 0.50 mmol of
C/mmol of C; 0.35 to 0.38 g/g) and productivities (9.7 to 13.2 mmol of
C h1 g [dry weight] of cells1; 0.24 to
0.30 g h1 g [dry weight] of cells1)
obtained for cofermentation of xylose and glucose by TMB 3001 were
slightly lower than those previously reported for
Saccharomyces sp. strain 1400(LNH-ST) in oxygen-limited
batch fermentation, 0.56 mmol of C/mmol of C and 14.3 mmol of C
h1 g [dry weight] of cells1, respectively
(21). The discrepancy could arise from differences in strain
background as well as in the fermentation setup, i.e., absence or
presence of oxygen and continuous or batch mode. For P. stipitis, the most efficient natural xylose-fermenting
yeast, the highest ethanol yield on xylose, 0.63 mmol of C/mmol of C (0.48 g/g), was obtained in oxygen-limited continuous culture with a
D of 0.06 h1 (42). The productivity
obtained under the same conditions was 8.7 mmol of C h1 g
(dry weight) of cells1 (0.20 g h1 g [dry
weight] of cells1). In contrast, both ethanol yield and
productivity under anaerobic conditions were considerably lower
(42). One reason for choosing S. cerevisiae as
the host strain for development of a xylose-fermenting yeast is its
rapid anaerobic growth on glucose (30, 52). However, anaerobic growth on xylose has not yet been demonstrated for
recombinant xylose-utilizing S. cerevisiae. This lack of
growth has been attributed to slow xylose metabolism (24),
resulting in too little ATP formation to maintain growth. P. stipitis has recently been metabolically engineered for anaerobic
growth (41). When S. cerevisiae URA1 was
expressed in P. stipitis, the yeast grew anaerobically on glucose, but not on xylose, supporting the suggestion that anaerobic growth on xylose is limited by slow xylose metabolism.
The strategy for chromosomal integration of the genes encoding XR, XDH, and XK resulted in a stable recombinant strain which retained its physiological characteristics through more than 4 weeks of continuous cultivation without selection pressure. Translated to generation time, strain TMB 3001 was stable for more than 40 generations, which is considerably longer than the four to five generations of stability reported for strain 1400 (pLNH32), which carries the same genes on a 2µm-derived vector (22). Recombinant xylose-utilizing Saccharomyces strains carrying 2µm-based vectors have been stably maintained in batch cultivation (22, 24, 27, 44, 48), but in continuous cultivation they tend to be unstable (27, 28). The instability of strains carrying 2µm-based vectors may result from genetic instability at the plasmid level, i.e., spontaneous loss of the transformed phenotype and the plasmid (19, 26, 28) or high frequency of recombination, resulting in cells that still carry the selectable marker but have lost the cloned gene (15, 28). Consequently, strains carrying less foreign DNA usually dominate the culture since the high-copy-number plasmids and the high expression of the heterologous genes can reduce the growth rate and glycolytic flux (26-28, 43). For the construction of TMB 3001, a single target sequence rather than multiple integrations in ribosomal DNA or transposons was chosen to diminish the burden on the cells caused by expression of foreign DNA. The resulting XR, XDH, and XK activities for TMB 3001 were lower than those for a CEN.PK strain carrying the same genes on two multicopy plasmids (pY7 [46] and pXks [Johansson et al., submitted]) cultivated in batch, 0.4, 3, and 1.6 U/mg compared to 0.7, 18, and 36 U/mg, respectively (Johansson et al., submitted). Still, the ethanol yields of the two strains were similar, 0.45 and 0.42 mol of C/mol of C (0.35 and 0.32 g/g), respectively, implying that higher-level expression of XR, XDH, and XK does not increase the flux to ethanol.
The XYL1 gene, which encodes XR, that was expressed in S. cerevisiae originated from P. stipitis. This enzyme uses either NADPH or NADH as a cofactor, while XDH exclusively uses NAD+. Under anaerobic conditions, the fraction of xylose converted to xylitol by NADPH-dependent XR activity is not further converted to xylulose. This was demonstrated by increased xylitol excretion following the addition of the respiratory inhibitor antimycin A to an oxygen-limited culture of a recombinant xylose-utilizing strain of S. cerevisiae (24). Under the conditions applied in the present study, less than 50% of the consumed xylose was excreted as xylitol. The difference could result from the overexpression of XK, which increases ethanol production and lowers xylitol excretion (14, 17, 44), or from the simultaneous utilization of glucose, which may supply NAD+ by glycerol formation. The latter hypothesis is supported by the high glycerol yield at the steady state with the lowest xylose-to-glucose ratio, 1:3 (Table 2). The supplementary NAD+ might be used by XDH and reduce xylitol excretion. ATP is used in glycerol formation, but not in xylitol formation, and this result indicates that only when a surplus of glucose is available can the yeast use carbon for glycerol formation to supply XDH with NAD+.
In addition to redox constraints, xylose transport also may limit xylose utilization by recombinant S. cerevisiae. Xylose is transported by the facilitated glucose transport system in S. cerevisiae cells, which have a 200-fold lower affinity for xylose than for glucose (9, 24, 25). Increasing the xylose concentration in the feed enhanced the xylose flux. However, the xylose flux was still 2.2 times lower than the glucose flux when a feed consisting of 15 g of xylose/liter and 5 g of glucose/liter was utilized, suggesting that xylose transport to a large extent determines the xylose flux in recombinant S. cerevisiae TMB 3001.
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ACKNOWLEDGMENTS |
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We thank Dace Leveika for technical assistance and Jörg Hauf for supplying the vector YDp-H.
This work was financially supported by EC contract BIO-CT95-0107, Energimyndigheten (Swedish National Energy Administration), and TEMPUS contract JEP-09273-95.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. Phone: 46 46 222 8428. Fax: 46 46 222 4203. E-mail: Barbel.Hahn-Hagerdal{at}tmb.lth.se.
Present address: Institute of Molecular BioSciences, Massey
University, 11 222 Palmerston North, New Zealand.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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