Applied Microbiology and Biotechnology

, Volume 63, Issue 5, pp 578–583

Engineering of carbon catabolite repression in recombinant xylose fermenting Saccharomyces cerevisiae

Authors

  • C. Roca
    • Centre for Process Biotechnology, BioCentrum-DTUTechnical University of Denmark
  • M. B. Haack
    • Centre for Process Biotechnology, BioCentrum-DTUTechnical University of Denmark
    • Centre for Process Biotechnology, BioCentrum-DTUTechnical University of Denmark
Original Paper

DOI: 10.1007/s00253-003-1408-2

Cite this article as:
Roca, C., Haack, M.B. & Olsson, L. Appl Microbiol Biotechnol (2004) 63: 578. doi:10.1007/s00253-003-1408-2

Abstract

Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (Δmig1, XYL1, XYL2, XKS1) and CPB.MBH2 (Δmig1, Δmig2, XYL1, XYL2, XKS1) were analysed for changes in xylose consumption rate and ethanol production rate during anaerobic batch and chemostat cultivations on a mixture of 20 g l−1 glucose and 50 g l−1 xylose, and their characteristics were compared to the parental strain S. cerevisiae TMB3001 (XYL1, XYL2, XKS1). Improvement of xylose utilisation was limited during batch cultivations for the constructed strains compared to the parental strain. However, a 25% and 12% increased xylose consumption rate during chemostat cultivation was achieved for CPB.CR2 and CPB.MBH2, respectively. Furthermore, during chemostat cultivations of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting that xylose is a repressive sugar for S. cerevisiae.

Introduction

Carbon catabolite repression is a well documented and investigated regulatory mechanism in Saccharomyces cerevisiae (for review see Trumbly 1992; Gancedo 1998; Klein et al. 1998). Put simply, whenever glucose is present in a cultivation broth, alternative carbon sources such as galactose or sucrose are used only after the glucose is depleted. In addition to utilisation of alternative sugars, a large number of other known, or still to be identified, cell functions will be influenced by glucose repression. Industrial effluents such as lignocellulose hydrolysates are usually composed of a mixture of sugars, and utilisation of all the sugars present in industrial effluents, other than glucose and mannose, is delayed due to glucose repression. Use of carbon catabolite repression mutants for fermentation of sugar mixtures has already been demonstrated to be successful when using recombinant Escherichia coli for ethanol production from lignocellulosic hydrolysates, resulting in co-fermentation of hexoses and pentoses (Hernández-Montalvo et al. 2001; Nichols et al. 2001). S. cerevisiae strains have also been successfully engineered for co-consumption of galactose and glucose (Klein et al. 1998; Østergaard et al. 2000).

Xylose-fermenting S. cerevisiae TMB3001 has been constructed by integration of XYL1 and XYL2 from Pichia stipitis encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and by over-expression of the endogenous XKS1 gene encoding xylulokinase (XK) (Eliasson et al. 2000). However, strain efficiency is still not satisfactory and much effort is currently aimed at improving its performance on xylose. Alleviation of carbon catabolite repression might lead to a derepression of unidentified functions necessary for efficient xylose consumption.

We constructed different strains in which the MIG1 and MIG2 genes, known to be involved in glucose repression, were deleted. Binding sites for Mig1p, whose binding leads to repression of gene expression, have been found on a number of genes, for example, genes for alternative sugar utilisation (MAL, SUC, GAL genes) (Nehlin and Ronne 1990; Lundin et al. 1994; Klein et al. 1998) and respiratory metabolism (Gancedo 1992; de Winde and Grivell 1993). The action of Mig2p is similar to that of Mig1p as it can repress gene expression by binding to the promoters of the genes in question (Lutfiyya and Johnston 1996). Klein et al. (1999) have shown that co-deletion of MIG1 and MIG2 led to a higher relief of glucose repression as far as invertase expression is concerned, whereas the double deletion had a reduced effect on the improvement of galactose utilisation, compared to a single MIG1 deletion, suggesting that differences between the action of Mig1p and Mig2p still exist.

The constructed strains were grown during anaerobic batch and chemostat cultivations on either glucose or a mixture of glucose and xylose to investigate if glucose derepression improved xylose consumption in the recombinant S. cerevisiae strains.

Materials and methods

Strains

All S. cerevisiae strains used in this study were generated from the CEN.PK 113-7D wild type strain (Table 1). The genes encoding XR and XDH from P. stipitis and the endogenous gene encoding XK have already been integrated in the chromosome of CEN.PK 113-7D, using the integrative plasmid YipXR/XDH/XK, leading to the stable construct TMB3001 (Eliasson et al. 2000). The MIG1 and MIG2 genes have been deleted using the loxP-kanMX-loxP disruption cassette (Güldener et al. 1996). Two different strains have been obtained: CEN.PK451-1D (Δmig1) and T475 (Δmig1 Δmig2) (Klein et al. 1999). Transformation of CEN.PK451-1D and T475 with the plasmid YipXR/XDH/XK was performed using the lithium acetate method as described by Gietz et al. (1992), leading to the xylose-fermenting strains CPB.CR2 and CPB.MBH2, respectively (see genotype in Table 1). The strains were stored at 4°C on YPD agar plates.
Table 1.

Yeast strains used in this study

Strain

Relevant genotype

Origin/reference

CEN.PK 113-7D

MATa SUC2 MAL2-8C

SRDa

TMB3001

MATa SUC2 MAL2-8C

pADH-XYL1 pPGK-XYL2 pPGK-XKS1

Eliasson et al. 2000

CPB.CR2

MATa SUC2 MAL2-8C

pADH-XYL1 pPGK-XYL2 pPGK-XKS1

Δmig1

This study

CPB.MBH2

MATa SUC2 MAL2-8C

pADH-XYL1 pPGK-XYL2 pPGK-XKS1

Δmig1 Δmig2

This study

aScientific Research and Development GmbH, Oberusel, Germany

Medium preparation

A defined medium containing trace metal elements and vitamins was used in all cultivations. Fatty acids in the form of Tween 80 and Ergosterol were added to the cultivations to sustain anaerobic growth of S. cerevisiae. The medium used for batch and chemostat cultivations was prepared according to Verduyn et al. (1990). For cultivations on glucose, the concentration was 20 g l−1; for mixed sugar cultivations, 20 g l−1 glucose and 50 g l−1 xylose were used.

Batch cultivations

Cultivations were carried out in well-controlled four-baffled 5-l in-house-manufactured bioreactors with a working volume of 4 l; the temperature was controlled at 30°C. The bioreactors were equipped with two disk-turbine impellers rotating at 500 rpm. The pH was kept constant at 5.0 by automatic addition of 2 M NaOH. Nitrogen containing less than 5 ppm O2 (AGA, Copenhagen, Denmark) was used for sparging of the fermentor at a flow rate of 0.8 l min−1 to maintain anaerobic conditions. Off-gas passed through a condenser to minimise evaporation of ethanol from the bioreactor. Cultivations were performed in duplicate or triplicate. A maximum of 5% was found between the measurements.

Continuous cultivations

Carbon-limited cultivations were carried out at 30°C in 2 l Applikon bioreactors (Schiedam, The Netherlands) with a constant working volume of 1 l and with a stirring speed of 500 rpm. The pH was kept constant at 5.0 by automatic addition of 1 M NaOH. Nitrogen containing less than 5 ppm O2 was sparged in the fermentor at a constant flow rate of 0.2 l min−1. Cultivations were performed in duplicate or triplicate. A maximum of 5% was found between the measurements.

Off-gas analysis

Carbon dioxide and oxygen concentrations in the exhaust gas were determined by a Brüel and Kjaer 1308 acoustic gas analyser (Brüel and Kjaer, Naerum, Denmark; Christensen et al. 1995).

Cell mass determination

The dry weight was determined as previously described (Roca and Olsson 2003). Biomass composition used in yield calculation was CH1.8O0.5N0.2.

Analysis of extracellular metabolites

Samples taken from the cultivation broth were immediately filtered through a 0.45 μm pore size cellulose acetate filter (Osmonics, Westborough, Mass.) and stored at −20°C until analysis. Glucose, xylose, glycerol, ethanol, succinate and acetate concentrations were determined as previously described (Roca and Olsson 2003).

Invertase activity

Determination of invertase activity was performed as described in Dynesen et al. (1998). Specific activity was expressed in units per milligram, corresponding to the amount of glucose (in micromoles) produced during 1 min per milligram of protein.

Results

Batch cultivations of the constructed glucose derepressed strains CPB.CR2 (Δmig1), CPB.MBH2 (Δmig1 Δmig2) and the parental (glucose repressed) strain TMB3001 were performed anaerobically on 20 g l−1 glucose and on a mixture of 20 g l−1 glucose and 50 g l−1 xylose. Such a high concentration of xylose was used to facilitate the uptake and overcome the low affinity of XR for xylose (68–97 mM; Rizzi et al. 1988).

All the strains were able to utilise xylose during batch growth. After glucose depletion, complete xylose utilisation took more than 150 h for all strains. This is illustrated in Fig. 1, which shows the fermentation of CPB.MBH2 (Δmig1 Δmig2). During the first 30 h, glucose was rapidly consumed together with a small fraction of xylose (Figs. 1, 2) and glycerol, ethanol and biomass were formed (Fig. 1). Once glucose was depleted, xylose, the only remaining carbon source available for the strains, was used more slowly than glucose. During this second phase, the specific growth rate decreased dramatically (Fig. 1). The main by-product was xylitol, which started to be produced immediately after glucose depletion (Fig. 1). Very little glycerol was formed during xylose consumption (less than 0.6 g l−1). Calculation of sugar consumption rates during batch cultivations on glucose or on mixture of glucose and xylose revealed that glucose consumption rates were lower for the glucose-derepressed strains (Table 2).
Fig. 1.

Sugar consumption, product formation and xylose consumption rate during anaerobic batch cultivation of Saccharomyces cerevisiae strain CPB.MBH2 (Δmig1 Δmig2) on a mixture of 20 g l−1 glucose and 50 g l−1 xylose

Fig. 2.

Co-consumption of glucose (filled symbols) and xylose (opened symbols) during batch cultivation of TMB3001 (●, ○), CPB.MBH2 (■, □) and CPB.CR2 (▲, △). Time of glucose depletion was taken as reference time in order to be able to compare the three strains

Table 2.

Specific uptake rates, specific growth rates and product yield coefficients for anaerobic batch fermentations. The yields were calculated at the end of the cultivation

Strain

Carbon source

μa

rglub

rxylc

YSXd

YSGe

YSEf

YSxolg

YXylxolh

g/l

h−1

Cmmol gDW−1·h−1

Cmol/Cmol

TMB3001

20 glucose

0.34

150

0.10

0.11

0.53

20 glucose +50 xylose

0.27a

136

13

0.04

0.07

0.43

0.29

0.47

CBP.CR2 (Δmig1)

20 glucose

0.34

125

0.11

0.09

0.44

20 glucose +50 xylose

0.28a

116

13

0.05

0.07

0.31

0.35

0.55

CPB.MBH2 (Δmig1 Δmig2)

20 glucose

0.29

120

0.10

0.10

0.51

20 glucose +50 xylose

0.20a

96

13

0.04

0.05

0.27

0.36

0.55

aSpecific growth rate during first phase of growth on glucose

bSpecific uptake rate of glucose

cSpecific uptake rate of xylose at the time when glucose was depleted

dBiomass,

eGlycerol,

fEthanol, and

gXylitol yields based on total consumed sugars

hXylitol yield based on consumed xylose only

The Δmig1 strain presented the highest consumption of xylose during the first growth phase on glucose (Fig. 2), consuming 20 g l−1 glucose together with 8 g l−1 xylose (0.4 g xylose per gram glucose) compared to the other strains (0.17–0.21 g g−1). Once glucose was depleted, maximum xylose consumption rate was 13 Cmmol gDW−1 h−1 (Table 2). Xylose consumption rate decreased throughout the xylose consumption phase (Fig. 1). The constructed strains produced more xylitol than the parental strain at the expense of ethanol (Table 2).

Anaerobic continuous cultivations were performed on 20 g l−1 glucose and on a mixture of 20 g l−1 glucose and 50 g l−1 xylose. Batch cultivations revealed the specific growth rate on xylose to be very low (Table 2). The dilution rate (D) was therefore fixed at 0.05 h−1 so that the cell could take up a significant fraction of xylose. During chemostat cultivations on glucose, the strains presented less than 5% difference in their glucose consumption rate compared to the parental strain TMB3001 and no major differences were observed in product formation (Table 3). During continuous cultivations on glucose and xylose, xylose consumption rate was increased by 25% and 12% in CPB.CR2 (Δmig1) and CPB.MBH2 (Δmig1 Δmig2), respectively, compared to the parental strain (10.2 Cmmol g DW−1 h−1). The ethanol yield increased by 11% in CPB.CR2 (Δmig1) compared to the parental strain.
Table 3.

Specific uptake rates and product yield coefficients for anaerobic carbon limited chemostat fermentations at dilution rate D=0.05 h−1. The yields were calculated at the end of the cultivation

Strain

Carbon source

rglua

rxylb

YSXc

YSGd

YSEe

YSxolf

YXylxolg

g/l

Cmmol gDW−1·h−1

Cmol/Cmol

TMB3001

20 glucose

19.6

0.11

0.07

0.51

20 glucose +50 xylose

13.9

10.2

0.08

0.06

0.37

0.23

0.55

CPB.CR2 (Δmig1)

20 glucose

18.5

0.11

0.06

0.52

20 glucose +50 xylose

15.2

13.0

0.07

0.07

0.42

0.20

0.44

CPB.MBH2 (Δmig1Δmig2)

20 glucose

19.5

0.10

0.07

0.53

20 glucose +50 xylose

14.5

11.4

0.08

0.04

0.38

0.25

0.58

aSpecific uptake rate of glucose

bSpecific uptake rate of xylose

cBiomass,

dGlycerol,

eEthanol, and

fXylitol yields based on total consumed sugars

gXylitol yield based on consumed xylose

Invertase activity was measured during continuous cultivations of TMB3001 (parental) and CPB.CR2 (Δmig1). During growth on glucose, activity was higher in the glucose-derepressed strain (22.6 U mg−1) than in the parental strain (18 U mg−1). During growth on glucose and xylose, activity was still higher in the glucose-derepressed strain CPB.CR2 (10 U mg−1) than in the parental strain (5.1 U mg−1).

Discussion

Co-utilisation of all the sugars, such as glucose, galactose, mannose or xylose present in industrial effluents is essential for an economically feasible process with high ethanol productivity. Alleviation of glucose repression was used as a global approach to attempt improved xylose fermentation by derepression of putative factors favouring xylose utilisation, as well as co-utilisation of several sugars. However, co-utilisation of xylose and glucose was poor during batch cultivations, and the improvement of the xylose consumption was not as good as that reported for galactose consumption (Østergaard et al. 2000). Interestingly, a single deletion of MIG1 seems to relieve glucose repression on xylose consumption more efficiently than a MIG1 MIG2 double deletion, as has been already shown for galactose (Klein et al. 1999).

During batch cultivations, low affinity sugar transporters are used for transport, whereas the expression of high affinity sugar transporters is repressed by glucose. Hamacher et al. (2002) showed that xylose is transported by high affinity transporters such as HXT4, HXT5, HXT7 or GAL2 that are normally repressed under the conditions prevailing in batch fermentations. Furthermore, glucose has a higher affinity for the transporters than does xylose (Kotyk 1967; Kötter and Ciriacy 1993), resulting in competition for the transporter. Consequently, xylose uptake during batch growth will be slow even if the high affinity transporters are present due to derepression in the glucose repression engineered strains.

In contrast to batch cultivations, where sugar concentration is high, carbon-limited chemostat cultivations resulted in a residual glucose concentration below 0.2 g l−1 (Table 3). The xylose consumption rate was increased by 25% in CPB.CR2 (Δmig1) and 12% in CPB.MBH2 (Δmig1Δmig2) compared to the parental strain. This increase during continuous cultivations differs significantly from the unchanged xylose consumption rate in batch fermentations. At the low sugar concentrations that prevail during carbon-limited continuous cultivations at low D, S. cerevisiae can use high affinity hexose transporters such as HXT2 (Kruckeber and Bisson 1990), HXT4 (Theodoris et al. 1994), HXT5 or HXT7 (Diderich et al. 1999) or the permease GAL2 when galactose is present (Reifenberger et al. 1997). Xylose is also taken up via some of these high affinity transporters. The observed increased xylose consumption rate suggests that, at a low glucose concentration of 0.2 g l−1 in CEN.PK background strains, increased expression of high affinity transporters expression could be obtained by MIG1 deletion and MIG1 MIG2 double deletion. Under the conditions tested, Hxt7p is the most plausible target as HXT7 has been shown to be the only transporter gene expressed during anaerobic continuous cultivation on glucose at D=0.1 h−1 (Diderich et al. 1999).

Measurement of invertase activity can be used as an indication of the strength of glucose repression, as SUC2 coding for invertase is subject to glucose repression and no other regulation (Trumbly 1992). The specific activity of invertase was decreased 3-fold for the parental strain and around 2-fold for the mig1 deleted strain when they were grown on a glucose-xylose mixture, compared to growth solely on glucose. The invertase activity was higher in the Δmig1 strain, pointing to some relief of glucose repression. Özcan et al. (1997) have shown that expression of the SUC2 gene of S. cerevisiae was induced by low levels of glucose (1 g l−1) whereas Herwig et al. (2001) determined a threshold concentration of 0.5 g l−1 for derepression. The residual glucose concentration during continuous cultivations was 0.2 g l−1, below repressing concentration, i.e. the change in invertase expression cannot then be attributed to glucose. This may allow us to think that xylose in itself is a repressing sugar. The fact that invertase is less expressed on the sugar mixture revealed the direct influence of xylose. Richard et al. (1999) showed that S. cerevisiae has in reality the genetic prerequisites for xylose metabolism, suggesting that xylose could be utilised if the endogenous XYL genes were properly expressed, and that xylose might have been a natural sugar substrate for S. cerevisiae. Consequently, xylose could act as a repressing sugar in "recombinant" metabolism, much like glucose in natural metabolism. Xylose metabolism might lead to the formation of an intermediate that acts as a triggering molecule for a carbon catabolite repression cascade, in a manner similar to that of some glucose intermediates.

In conclusion, deletion of genes involved in glucose repression in xylose-fermenting S. cerevisiae strains resulted in a 25% increase in the specific xylose consumption rate during continuous cultivations, and the volumetric ethanol productivity could be increased from 0.475 g l−1 h−1 in the parental strain to 0.6 g l−1 h−1 in the mig1 deleted strain. Nevertheless, glucose repression does not seem to be the main obstacle to efficient xylose utilisation as xylose consumption per se is restricted and, therefore, glucose repression becomes a secondary obstacle. The problem of glucose repression might nevertheless appear important, once xylose consumption has been increased.

Acknowledgements

Professor Bärbel Hahn-Hägerdal, Department of Applied Microbiology, Lund University is sincerely thanked for kindly providing the plasmid carrying the XYL1, XYL2 and XKS1 genes and the S. cerevisiae strain TMB3001. We are also indebted to Juana M. Gancedo for communicating her unpublished results on the capacity of xylose to cause catabolite repression. The work on carbon catabolite repression at the Centre for Process Biotechnology at the Technical University of Denmark is supported under the European Commission Framework V, contract no. QLK3-CT-1999-00080.

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© Springer-Verlag 2004