Scholarly article on topic 'Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae'

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae Academic research paper on "Agricultural biotechnology"

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Academic research paper on topic "Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose-utilizing Saccharomyces cerevisiae"

Microbial Cell Factories

BioMed Central

Research

Increased expression of the oxidative pentose phosphate pathway and gluconeogenesis in anaerobically growing xylose utilizing Saccharomyces cerevisiae

David Runquist, Bärbel Hahn-Hägerdal and Maurizio Bettiga*

Address: Department of Applied Microbiology, Lund University, PO Box 124, SE-221 00 Lund, Sweden

Email: David Runquist - david.runquist@tmb.lth.se; Bärbel Hahn-Hägerdal - barbel.hahn-hagerdal@tmb.lth.se; Maurizio Bettiga* - maurizio.bettiga@tmb.lth.se * Corresponding author

Open Access

Published: 24 September 2009 Received: 20 August 2009

Accepted: 24 September 2009 Microbial Cell Factories 2009, 8:49 doi:10.1186/1475-2859-8-49 H H

This article is available from: http://www.microbialcellfactories.com/content/8/1/49

© 2009 Runquist et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: Fermentation of xylose to ethanol has been achieved in S. cerevisiae by genetic engineering. Xylose utilization is however slow compared to glucose, and during anaerobic conditions addition of glucose has been necessary for cellular growth. In the current study, the xylose-utilizing strain TMB 3415 was employed to investigate differences between anaerobic utilization of glucose and xylose. This strain carried a xylose reductase (XYLI K270R) engineered for increased NADH utilization and was capable of sustained anaerobic growth on xylose as sole carbon source. Metabolic and transcriptional characterization could thus for the first time be performed without addition of a co-substrate or oxygen.

Results: Analysis of metabolic fluxes showed that although the specific ethanol productivity was an order of magnitude lower on xylose than on glucose, product yields were similar for the two substrates. In addition, transcription analysis identified clear regulatory differences between glucose and xylose. Respiro-fermentative metabolism on glucose during aerobic conditions caused repression of cellular respiration, while metabolism on xylose under the same conditions was fully respiratory. During anaerobic conditions, xylose repressed respiratory pathways, although notably more weakly than glucose. It was also observed that anaerobic xylose growth caused up-regulation of the oxidative pentose phosphate pathway and gluconeogenesis, which may be driven by an increased demand for NADPH during anaerobic xylose catabolism.

Conclusion: Co-factor imbalance in the initial twp steps of xylose utilization may reduce ethanol productivity by increasing the need for NADP+ reduction and consequently increase reverse flux in glycolysis.

Introduction

Production of fuel ethanol has increased several fold during the last decade due to increasing oil prices and environmental concerns [1]. The vast majority of this production comes from fermentation of agricultural prod-

ucts, primarily sugar cane and corn, by baker's yeast S. cerevisiae. Lignocellulose biomass from forest and agricultural residues is an alternative to sucrose (sugar cane) and starch (corn) based ethanol production [2,3]. Next to glucose, the main component of lignocellulose is

xylose, and the use of this substrate by S. cerevisiae has been enabled through expression of heterologous enzymes [4-6]. Xylose utilizing S. cerevisiae strains have been constructed by expressing a reduction/oxidation pathway involving xylose reductase (XR) and xylitol dehydrogenase (XDH) [7,8] or a xylose isomerase (XI) pathway [9-11].

Successive cycles of metabolic engineering have improved xylose utilization in recombinant S. cerevisiae [12,13]. Compared to glucose however the ethanol productivity from xylose is still low. Poor xylose utilization has been ascribed to potentially rate-controlling metabolic steps including: low substrate affinity of the recombinant enzymes [8]; cofactor imbalance in the XR-XDH reactions [7,14]; low xylose transport capacity [15,16]; and failure to recognize xylose as a fermentable carbon source [17,18]. Among several experimental approaches, glucose and xylose metabolism have been investigated by tran-scriptional analysis to identify rate-controlling processes in xylose metabolism [17,19-22]. Growing cells are needed to establish (pseudo) steady-state conditions for transcription analysis and determination of metabolic fluxes [23,24]. The analysis of xylose utilizing strains has thus been hampered by poor anaerobic growth on xylose. Transcription analysis has consequently been conducted under aerobic conditions [17,19,20,22] and/or with addition of glucose as a co-substrate [21]. Transcriptional characterization of anaerobic xylose metabolism has however remained elusive, regardless of the importance of this particular condition in a production setting.

For S. cerevisiae expressing the oxidoreductive xylose assimilating pathway, a recent accomplishment has been alteration of the cofactor specificity of XR through site directed mutagenesis [25-27]. By increasing the affinity of the P. stipitis XR for NADH, the objective has been to improve cofactor recycling in the XR-XDH coupled reactions. The current study utilized a S. cerevisiae strain harboring a mutated XR (K270R) with significantly improved substrate uptake rate and ethanol productivity [26]. The strain grew anaerobically on xylose as a sole carbon source

Table 1: S. cerevisiae strains and plasmids used in this study.

which for the first time enabled quantitative metabolic flux determination and genome wide transcriptional analysis. The focus of the study was to compare metabolic fluxes during anaerobic glucose and xylose growth, and to analyze the observed differences on a transcriptional level.

Materials and methods Strains and cultivation conditions

S. cerevisiae strains and plasmids used in this study are summarized in Table 1. Escherichia coli strain DH5a was used for sub-cloning and was grown on LB medium supplemented with 100 mg/L ampicillin. Defined mineral medium was used for S. cerevisiae cultivation and was composed of: xylose or glucose, 60 g/L; mineral salts ((NH4)2SO4, 5 g/L; KH2PO4, 3 g/L; MgSO4 -7H2O, 0.5 g/ L); buffer (potassium hydrogen phthalate, 50 mM pH 5.5); Tween 80, 0.4 g/L; ergosterol, 0.01 g/L [28]; vitamins and trace elements [29]. Identical medium was used for pre-cultures and batch fermentation in instrumented bio-reactors with the exception that buffering agent was omitted in the latter case. At the start of each experiment, yeast strains were streaked from 15% (v/v) glycerol stocks and grown two days on Yeast Nitrogen Base (YNB) glucose plates. Pre-cultures were inoculated in baffled shake-flasks (10% liquid volume) at a predetermined cell density, OD 62o nm = 0.5/0.025 (xylose/glucose), and grown for 20 hrs to yield cells in late exponential phase (OD620 nm~14). Cultivation of S. cerevisiae was performed at 30°C.

Anaerobic batch cultivation was performed in an instrumented bioreactor (Applikon Biotechnology, AC Schiedam, the Netherlands) with 1.5 L working volume and a starting OD620 nm of 0.2. The medium contained 60 g/L of glucose or xylose and was prepared as described above with antifoam (Dow Corning, Midland, USA) added to the reactor at a final concentration of 0.2 mL/L. Temperature was maintained at 30°C and the pH was controlled at 5.5 through addition of 3 M KOH. Cultures were grown under aerobic conditions until the cell density reached OD 620 nm = 10, upon which conditions were changed to anaerobiosis for the remainder of the experiment (Figure 1). During the aerobic phase, the culture was sparged with

Plasmids and Strains Relevant Features Reference

Plasmids YIpOB9 YIplacl28 S. cerevisiae strains TMB 3043

TMB 3662 TMB 3415

URA3 TDH3p-XYLI(K270R)-ADHIt, PGK.ip-XYL2-PGK.it LEU2

CEN.PK 2-lC AGRE3, his3::PGKIp-XKSI-PGKIt, TALI::PGKIp-TALI-PGKIt,

TKLI ::PGKI p-TKL I-PGKI t, RKII ::PGKIp-RKII -PGKI t, RPEI ::PGK Ip-RPEI -PGKI t, leu2,

TMB 3043, ura3::YIpOB9, leu2 TMB 3662, feu2::YIplacl28

This work

70 60 50

Time (hrs)

Time (hrs)

2.5 2 1.5 1

-0.5 -1 -1.5 -2

70 60 50

£ 30 X

80 100 Time (hrs)

120 140 160 180

80 100 120 Time (hrs)

Figure 1

Fermentation profiles of glucose and xylose growth. The dashed vertical line indicates switch between aerobic and anaerobic conditions. Samples for transcription analysis were collected at OD620 nm ~ 1 and OD620 nm ~ 4, which corresponds In OD ~ 0 and ln OD ~ 1.4 in the figure. A. Biomass production during glucose cultivation. B. Biomass production during xylose cultivation C. Substrate consumption and metabolite formation during glucose cultivation. D. Substrate consumption and metabolite formation during xylose cultivation. Symbols: glucose/xylose, "squares"; ethanol, "diamonds"; glycerol, "circles"; xylitol, "triangles" and biomass, "stars".

air at a flow rate of 0.4 L/min and the stirring was set to 500 rpm. During the anaerobic phase, oxygen free conditions were maintained by nitrogen (> 99.995%) sparging at a flow rate of 0.2 L/min and the stirring rate was reduced to 200 rpm. Dissolved oxygen (DO) was monitored using a DO probe and CO2 production was detected online by an INN OVA 1313 fermentation monitor (LumaSense Technologies, Ballerup, Denmark). Cultures were sampled for HPLC, OD620 nm and dry cell weight measurements. Samples were collected for transcriptome analysis during exponential aerobic (OD620 nm~1.0) and anaerobic growth (OD620 nm~4.0). Transcriptome samples (50 mL) were collected from the bioreactor into a pre-chilled (-80 °C) glass bottle. Samples were immediately centrifuged (5000*g, 2 min, 4°C) and the biomass pellet

was frozen in liquid nitrogen. Experiments were performed in biological duplicate.

Strain construction

S. cerevisiae strain TMB 3415 was constructed from the TMB 3043 (Table 1) parent strain [13]. This genetic background encompasses genetic changes previously identified as beneficial for xylose utilization. Genes for the non-oxidative pentose phosphate pathway [12] and xyluloki-nase XKS1 [30] have been over-expressed, and the nonspecific aldose reductase gene GRE3 has been deleted [31]. Genes encoding Pichia stipitis mutated xylose reductase (XYL1 K270R) and xylitol dehydrogenase (XYL2) were integrated in TMB 3043 by transformation with YIpOB9 (Table 1) linearized with EcoRV, using the Lithium Acetate

method [32]. The resulting strain, TMB 3662, was rendered prototrophic by integration of the linearized (EcoRV) vector YIplac128 (Table 1), yielding strain TMB 3415. Standard molecular biology techniques were employed [33] and Fermentas GeneJet plasmid miniprep kit (Fermentas, Vilnius, Lithuania) was used for plasmid extraction.

Calculation of metabolic fluxes

Exponential anaerobic growth was confirmed by linear regression of the natural logarithm of cell concentration against time. Specific rates of product formation and substrate consumption were calculated in Matlab (Matlab R2007b, The MathWorks Inc., MA, USA) using Equation 1 & 2 and measured metabolite and cell concentrations. A pseudo-steady state assumption was validated by observing constant specific production- and consumption rates within 2-3 cell duplications, as well as good agreement of measured values between biological replicates.

— = ux X dt ^

dMeti =

= rMet¡ x X

Where: X = biomass (g/L); Met = metabolite concentration (g/L); | = specific growth rate (h-1); r = specific production rate (g/hxgDW).

Microarray analysis

Microarray analysis was performed on cell samples collected from aerobic and anaerobic batch cultivation on glucose and xylose as described above. RNA from two independent biological cultivations was analyzed. Total RNA was extracted from frozen cell pellets using a bead-beater (Biospecs products, Bartlesville, OK, USA) and Tri-zol reagent (Invitrogen, CA, USA). All extractions where performed on the same total amount of cells (approximately 10 mg dry weight). RNA was further purified using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA quality and concentration were measured using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and Nanodrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) respectively. Total RNA was processed using the GeneChip® Expression 3'-Amplification Reagents One-cycle cDNA synthesis kit (Affymetrix Inc, Santa Clara, CA, USA) to produce double-stranded cDNA. This was used as a template to generate biotin-targeted cRNA following the manufacturer's specifications. Fifteen Ig of the biotin labeled cRNA was fragmented to strands between 35 and 200 bases in length, 10 |g of which was hybridised onto the GeneChip® genome array overnight in the GeneChip® Hybridisation oven6400 using standard procedures. The arrays were washed and then stained in a

GeneChip® Fluidics Station 450. Scanning was performed with the GeneChip® Scanner 3000 and image analysis was performed using GeneChip® Operating Software. The RMA algorithm [34] was used for normalization and scaling of the raw signal data. Student's t-test was used to identify genes with significantly (p < 0.05) altered gene expression. All array data is presented as fold changes, i.e. the log2 ratio of expression signals.

Analysis of substrate and products

Metabolite concentrations were determined by HPLC using a Waters HPLC system (Milford, MA, USA). An Aminex HPX-87H ion-exchange column (Bio-Rad, Hercules, CA, USA) and a refractive index detector (RID-6a, Shimadzu, Kyoto, Japan) were used for separation and detection, respectively. The column temperature was 45°C and 5 mM H2SO4 was used as a mobile phase at a flow rate of 0.6 mL/min. Cell dry weight was determined by filtering a known sample volume through a dried and pre-weighed 0.45-^m pore membrane (Pall Corporation, New York, USA), washing with distilled water and drying in a microwave oven for 8 min at 350 W.

Results

Aerobic and anaerobic batch cultivation

TMB 3415 was cultivated in synthetic medium containing either glucose or xylose as the sole carbon source, under aerobic and anaerobic conditions. Aerobic growth was maintained for approximately 2 cell divisions, after which anaerobiosis was established by switching the sparging gas from air to nitrogen. The fermentation profiles of glucose and xylose growth in this setup are presented in Figure 1. Balanced exponential cell growth was seen for glucose and xylose under both aerobic and anaerobic conditions, allowing for the assumption of pseudo steady-state conditions [24]. Growth rates and metabolic fluxes were calculated for the anaerobic growth phase (Table 2).

In glucose medium, growth rates were similar regardless of oxygenation (0.43 h-1 vs. 0.33 h-1) (Figure 1A) and comparable to previous reports [35]. During xylose consumption on the other hand, the growth rate decreased from 0.20 h-1 to 0.025 h-1 under anaerobic conditions (Figure 1C). Specific rates of substrate uptake and metabolite production were also different during anaerobic glucose and xylose fermentation (Table 2). The substrate uptake rate and the ethanol production rate were approximately an order of magnitude lower during xylose utilization than during glucose utilization (Table 2). The productivity, 0.13 g/gDWxh, and yield, 0.43 g/g, of etha-nol from xylose (Table 2) was however significantly higher compared to several recently investigated xylose-utilizing strains [20,26,36,37]. It is reasonable to assume that anaerobic growth of strain TMB 3415 results from increased ethanol productivity and low xylitol yield,

Table 2: Metabolite production rates and yields during anaerobic growth on glucose and xylose.

rs rxol rg ra re Yxe Yxg Yse Ysxol

Glucose 0.334 ± 2.6 ± 0 0.33 ± 0.01 ± 1.2 ± 3.3 0.98 0.43 0

anaerobic 0.006 0.5 0.01 0.01 0.2

Xylose 0.0246 ± 0.285 ± 0.058 ± 0.015 ± 0 0.126 ± 5.1 0.59 0.44 0.20

anaerobic 0.0003 0.005 0.003 0.004 0.005

Symbols: specific growth rate (h-1); r, specific consumption/production rates (g/gDWxh): rs, specific substrate consumption rate; rxo|, specific xylitol production rate; rg, specific glycerol production rate; ra, specific acetate production rate; re, specific ethanol production rate; Y, yields (g/g): Yxe, ethanol yield on biomass; Yxg, glycerol yield on biomass; Yse, ethanol yield on substrate; Ysxol, xylitol yield on substrate.

which in turn can be ascribed to the K270R mutation in XR [26]. Compared to strains expressing a xylose isomer-ase based pathway, the ethanol productivity in TMB 3415 was higher than in non-growing strains [10,11,38], whereas it was similar to a strain growing at | = 0.03 h-1 [39] and lower than a strain growing at | = 0.09 h-1 [40].

Topography of microarray data

The present study aimed at highlighting regulatory differences between anaerobic glucose and xylose growth by xylose-utilizing S. cerevisiae. Specifically, differences in growth rate, substrate consumption and ethanol productivity were analyzed on a transcriptional level. Transcrip-tional characterization have not previously been performed under anaerobic conditions due to the inability of recombinant S. cerevisiae strains to grow on xylose in the absence of oxygen [17,20-22]. A few strains expressing Piromyces XI are able to grow anaerobically on xylose [39,40], however to the best of our knowledge transcription analysis of these strains has not been reported.

The overall structure of the microarray data was examined using Principle Component Analysis (PCA) [41]. PCA has been used to reduce the dimensionality of microarray data and to identify features of experimental conditions that best explain the observed variance in gene expression [42]. The projection of the two principle components, oxygen availability and carbon source, segregated the individual samples in a two dimensional space (Figure 2A). The four conditions, glucose aerobic (GA), xylose aerobic (XA), glucose anaerobic (GAnA) and xylose anaerobic (XAnA), were separated along the axis of the first and second principle component. The first principal component, which was responsible for the most variance in the data set, separated samples according to oxygen availability (aerobic or anaerobic) (Figure 2A). The second principal component separated samples according to carbon source (glucose or xylose). The PCA projection of the transcription data singled out anaerobic xylose growth as the most unique group in the data set (Figure 2A).

Next, the microarray data was organized into four relevant pairwise comparisons: GA vs. GAnA (Comparison 1 = C1); GA vs. XA (Comparison 2 = C2); XA vs. XAnA (Com-

parison 3 = C3) and GAnA vs. XAnA (Comparison 4 = C4) (Figure 2B). Comparisons C1 and C2 have previously been reported in slightly different experimental setups [17,20,23]. However, comparisons C3 and C4 allowed for the first time analysis of transcription during anaerobic growth on xylose as a sole carbon source. Globally, the transition from aerobic to anaerobic growth changed the expression level of more genes on xylose (C3, 809 genes) than on glucose (C1, 546 genes) (Figure 2B). This difference is presumably due to that aerobic metabolism on xylose is fully respiratory, while it is respiro-fermentative on glucose. Likewise a higher number of genes displayed different expression levels between glucose and xylose utilization under aerobic conditions (C2, 1059 genes) than under anaerobic conditions (C4, 641 genes) (Figure 2B).

Finally, subsets of genes were isolated according to the following criteria: (i) genes that changed expression levels between aerobic and anaerobic conditions regardless of carbon source (C1&C3, Figure 3A) and (ii) genes that changed expression levels on glucose and xylose regardless of oxygenation level (C2&C4, Figure 3B). Relatively few genes, 113, changed expression on both glucose and xylose during transition from aerobic to anaerobic conditions (Figure 3A). Likewise, only 130 genes changed expression level on xylose compared to glucose irrespective of oxygenation level (Figure 3B).

Gene ontology (GO) terms

Within a group of genes, up- and down-regulated pathways and processes can be identified by searching for over-represented gene ontology (GO) terms http:// www.geneontology.org/. Each annotated gene in the S. cerevisiae genome is associated with one or several GO terms that describe the corresponding biological process, e.g. amino acid synthesis. Significantly enriched gene ontology terms (p < 0.01) were identified within the previously described groups (C1, C2, C3, C4, C1&C3 and C2&C4) (Table 3). If more than one GO term in the same "family" were identified, only the most significant term was listed.

Comparing aerobic and anaerobic metabolism on glucose (C1, Figure 2B), unilateral down-regulation of respiratory

с CD С

"О с

о о CD СЯ

First Principal Component

GA *-► GAnA

C2 1059

lation of ribosome biogenesis and amino acid synthesis [42,44] reflects the ten fold reduction of growth rate between aerobic and anaerobic conditions on xylose (Figure 1B). In addition, the GO terms "alcohol metabolism" and "hexose catabolism" were up-regulated under anaerobic conditions on xylose (Table 3). In the group of genes that changed expression in response to anaerobiosis irrespective of carbon source (C1&C3, Figure 3A), repression of cellular respiration was identified (Table 3).

Aerobic glucose metabolism was respiro-fermentative, while aerobic xylose metabolism was completely respiratory with absent ethanol production (Figure 1). On a transcription level, this difference was visible in higher expression of several respiration related GO terms on xylose compared to glucose during aerobic conditions (C2, Table 3) [17,20]. During anaerobic conditions, protein synthesis was down-regulated on xylose compared to glucose, while expression of respiratory processes and the GO term "hexose metabolism" was up-regulated (C4, Table 3). The lower expression of amino acid biosynthesis [42,44] is related to the lower growth rate on xylose compared to glucose under anaerobic conditions. Oxidative phosphorylation was up-regulated on xylose in the group of genes that were differently expressed on glucose and xylose under both aerobic and anaerobic conditions (C4&C2, Table 3).

XA «-► XAnA

Figure 2

A PCA projection of individual microarray samples.

The dashed lines separate samples in 4 quadrants depending on the experimental condition. Symbols: Aerobic glucose, "empty circle"; Anaerobic glucose, "filled circle"; Aerobic xylose, "empty square"; Anaerobic xylose, "filled square". B The number of differently expressed genes (95% confidence interval) is indicated for pairwise comparisons of experimental conditions. Abreviations: GA, glucose aerobic; GAnA, glucose anaerobic; XA, xylose aerobic; XAnA, xylose anaerobic. CI-C4 designate specific pairwise comparisons, e.g. CI = glucose aerobic-glucose anaerobic.

processes was identified (Table 3). Down-regulation of respiratory genes in response to anaerobiosis has previously been reported for glucose grown cells in batch culture [17] and C- and N-limited chemostat culture [42,43]. In contrast, respiratory pathways were not down-regulated between aerobic and anaerobic xylose metabolism (C3, Figure 2B), whereas processes related to protein synthesis were significantly repressed (Table 3). Down-regu-

Respiration was not repressed between aerobic and anaerobic growth on xylose (C3, Table 3), and higher expression of respiratory processes was observed during both aerobic and anaerobic conditions compared to glucose (C4&C2, Table3). Certain processes in cellular respiration were however down-regulated both on glucose and xylose in response to anaerobiosis (C1&C3, Table 3). This indicates that although oxidative metabolism was not unilaterally down-regulated under anaerobic conditions, some processes were still repressed.

Regulation of the central carbon metabolism

Expression of the GO term "hexose metabolism" increased specifically under anaerobiosis on xylose, both in comparison to anaerobic glucose utilization (C4) and to xylose under aerobic conditions (C3) (Table 3). This observation was dependent on anaerobic xylose growth and has thus not previously been reported. The GO term "hexose metabolism" encompasses several aspects of the central carbon metabolism (glycolysis, pentose phosphate pathway, gluconeogenesis etc.), which is responsible for the cell's energy metabolism. As such, regulation of central carbon metabolism is linked to growth rate, protein synthesis and ethanol production rate [44,45]. Several reaction steps in the central carbon metabolism are catalyzed by more than one isozyme, which enable the cell to regulate the direction/flux of the pathway in

Aerobic - Anaerobic

Glucose

Xylose

Glucose - Xylose

Aerobic

Anaerobic

Figure 3

Venn diagram showing the fraction of genes common for: (a) transition between aerobic-anaerobic conditions regardless of carbon source; or (b) transition between glucose and xylose regardless of oxygenation level. The total number inside each circle represents the number of genes with significantly changed expression levels in that particular comparison, e.g. 546 genes for the glucose aerobic - glucose anaerobic transition (CI, Figure 2B).

response to the energetic state of the cell. Therefore, expression of genes in the central carbon metabolism was investigated in greater detail to pinpoint differences between glucose and xylose anaerobic growth (Figure 4).

Increased reversed flux in glycolysis during anaerobic xylose utilization was indicated by expression of several isozymes specific for gluconeogenesis. The hexokinase gene HXK1 was expressed higher on xylose than on glucose irrespective of aeration (Figure 4). Previously, high HXK1 expression has been reported under aerobic xylose growth [17,20] and during metabolism of non-ferm entable carbon sources [46]. Further down the pathway, the exclusively gluconeogenetic enzyme fructose-1,6-bisphos-phatase FBP1 was up-regulated specifically during anaerobic xylose utilization (Figure 4). Expression of glyceraldehyde-3-phosphate dehydrogenase isozyme

TDH1 is linked to stationary growth and gluconeogenesis [47] and was similarly up-regulated on xylose during anaerobic conditions (Figure 4). Also the minor isoform of phosphoglycerate mutase GPM2 was up-regulated on xylose during anaerobic conditions (Figure 4) but the function of this enzyme is largely unknown [48].

While reversed flux in glycolysis was indicated during anaerobic xylose growth, increased activity of the oxida-tive pentose phosphate pathway was observed through expression of 6-phosphogluconate dehydroge-nase(GND2) and glucose-6-phosphate dehydrogenase (ZWF1) (Figure 4). Both GND2 and ZWF1 catalyze the reduction of NADP+ and were specifically up-regulated under anaerobic conditions on xylose (Figure 4). During anaerobic conditions, NADP+ reduction in the oxidative pentose phosphate pathway controls the rate of xylose utilization as a consequence of the cofactor imbalance in the XR-XDH reaction [49]. Increased flux in the oxidative pentose phosphate pathway (PPP) must however be supported by proportionally increased reversed flux in glycolysis, which indeed was seen in the current study.

Discussion

In the present study, metabolic fluxes and genome-wide transcription analysis were investigated in recombinant S. cerevisiae under strictly anaerobic conditions with xylose as a sole carbon source. The currently employed strain is the first strain utilizing an oxidoreductive xylose-assimilating pathway capable of sustained anaerobic xylose growth. Anaerobic growth on xylose has previously been described for strains expressing an isomerase based pathway [39,40], however to the best of our knowledge, transcription analysis have not been reported for these strains. In previous transcription studies, steady state condition on xylose has been achieved by addition of oxygen or glucose to support growth [17,20-22]. The difference between aerobic utilization of glucose and xylose is thus well described [17,20], whereas the current study represents the first complete characterization of anaerobic xylose growth.

Calculated metabolic fluxes showed that substrate uptake rate (2.6 g/gDWxh vs. 0.29 g/gDWxh) and ethanol productivity (1.2 g/gDWxh vs. 0.13 g/gDWxh) on glucose and xylose were proportional to the growth rates (0.33 h-1 vs. 0.025 h-1) (Table 2). Transcription data likewise verified that the low anaerobic growth rate on xylose correlated directly to reduced expression of genes for amino acid synthesis and protein synthesis (Table 3), which has previously been shown for glucose grown cultures [42]. The ethanol yield from consumed substrate (0.43 g/g) on the other hand was identical in anaerobic glucose and xylose fermentation. On xylose, 20% substrate was lost as xylitol, however on glucose this was approximately bal-

Table 3: Gene ontology (GO) terms.

Comparison

Regulation

Glucose Aerobic-Anaerobic (CI)

G0:0055ll4

oxidation reduction

G0:00l5980

energy derivation by oxidation of organic compounds

G0:0006ll9

oxidative phosphorylation

Xylose Aerobic-Anaerobic (C3)

G0:0006066

cellular alcohol metabolic process

G0:00l9320 G0:0007047

hexose catabolic process cell wall organization

G0:0042254

ribosome biogenesis

G0:0008652 G0:00l6072

amino acid biosynthetic process rRNA metabolic process

G0:0043l02

amino acid salvage

G0:0045333

cellular respiration

Aerobic Glucose - Xylose (C2)

G0:0007005

mitochondrion organization

G0:0045333

cellular respiration

G0:0022900

electron transport chain

G0:0032268

regulation of cellular protein metabolic process

G0:0006066

cellular alcohol metabolic process

Anaerobic Glucose - Xylose (C4)

G0:00l5980

energy derivation by oxidation of organic compounds

G0:00l93l8 G0:0042254

hexose metabolic process ribosome biogenesis

G0:0008652

amino acid biosynthetic process

G0:00l6072 G0:0006ll9

rRNA metabolic process oxidative phosphorylation

Up- and down-regulated gene ontology (GO) terms identified within comparisons CI-C4 (Figure 2B). If no significantly over-represented GO terms were found, this was indicated by "ND" for "not detected".

2.6 5.7

4.0 2.1

4.8 1.9

xylose XYL1 k" NAD(p)H

U- NAD(P)H ,£>NAD(P)+

xylitol £

NAD+ NADH

glucose GLK1 HXK1 HXK2

glu-6-P PGI1

fru-6P< PFK1 PFK2 I I FBP1 fru-1,6-bisP FBA1

DHAP-►GAP

NADH NAD+

TPI1 IAP-►GAI

GPD1 T GPD2 ' -D

1.6 1.0 SOL4 3.1 2.7 GND1 -0.6

xylulose XKS1

GPD2 G3P 1,3-DPG PGK1

G3P GPM1 GPM2

ENO1ENO2

PEP PYK2 CDC19

TDH1 TDH3

fru-6P

erythrose-4P

PDC6 PDC1 PDC2

ADH2 ADH3

0.1 -0.4

pyruvate -

PDA1 PDB1 LAT1 LPD1

-►acetaldehyde-

ethanol

ALD5 ALD6 ALD4

acetyl-CoA oxaloacetate MDH2

-1.0 2.4

-0.6 0.8

-0.8 0.7

-1.0 2.5

malate

CIT1 citrate CIT2 \ACO1

acetate

-1.0 1.5

fumarate SDH4 SDH3 SDH2 SDH1

succinate LSC1

cis-aconitate ACO1

succinyl-CoA

isocitrate IDH1 IDH2 2-oxoglutarate

succinyl -lipoate

Figure 4

Regulation of central metabolism under aerobic/anaerobic growth on glucose/xylose. The fold change (log2) of expression is presented for the CI, C2, C3 and C4 comparisons. Cofactor utilization is only depicted for relevant reactions. Standard three letter code is used for all genes names http://www.yeastgenome.org/.

anced by almost two times higher glycerol yield compared to xylose (Table 2). The lower glycerol yield during xylose utilization is most likely due to that xylitol acts as a redox sink for anabolic reactions analogously to glycerol [50]. Supporting this argument, it has previously been seen that addition of an external redox acceptor reduced both glycerol and xylitol formation in cultivation of xylose utilizing S. cerevisiae [51,52].

Transcription analysis showed that oxidative phosphor-ylation was de-repressed on xylose compared to glucose under aerobic conditions, which correlates with the absence of respiro-fermentative metabolism on xylose (Table 3) [17,18,20]. During anaerobic conditions however, most respiratory genes continued to be highly expressed on xylose while they were unilaterally repressed on glucose (Table 3). The maintenance of unrepressed oxidative metabolism during anaerobic xylose growth can not be completely explained by lack of catabolite repression [53] since exclusively oxygen dependent repression was seen on glucose. It is however possible that on xylose, expression of "oxidative metabolism" was partly maintained as compensatory response to the cofactor imbalance during anaerobic conditions. Altered redox metabolism and up-regulation of genes for NADPH formation and NADH oxidation was previously seen during oxygen-limited xylose growth [17] and in an evolutionary engineered xylose-utilizing strain [21]. In the current study, the NAD+/NADH dehydrogenase shuttle (NDI1, NDE1 and NDE2) and NADP+ linked glutamate dehydrogenase (GDH3) were down-regulated on glucose under anaerobic conditions but up-regulated on xylose (data not shown).

During xylose utilization, up-regulation of gluconeogenesis and the oxidative pentose phosphate pathway coincided with anaerobiosis (Figure 4). On xylose, increased flux in the oxidative PPP is explained by need for NADP+ reduction in anerobic co-factor recycling [49]. Increased reversed flux in upper glycolysis follows consequently from mass balance at the glucose-6-phosphate node (Figure 4). As such, it has previously been seen that high flux in the oxidative PPP, and consequently gluconeogenesis, lowered net glycolytic flux and ethanol productivity [54]. The connection between cofactor recycling, gene expression and metabolic flux offers an explanation to the low ethanol productivity on xylose compared to glucose, despite similar ethanol yields (Table 2). Similarly it is possible that reduced back-flow in glycolysis is the primary reason for the increased ethanol productivity and anaerobic growth rate in the mutant XR (K270R) utilized in the current study [26]. Further improvement of cofactor imbalance in the initial two steps of xylose utilization by protein engineering is expected to improve performance of S. cerevisiae strains utilizing an oxido-reductive xylose assimilating pathway.

Conclusion

The present work describes the metabolic and transcrip-tional characterization of the xylose utilizing strain TMB 3415 under aerobic and anaerobic conditions. Under anaerobic conditions, metabolism and growth rate on xylose were proportionally reduced compared to glucose, which was reflected in terms of repressed protein synthesis. In addition, cofactor imbalance during anaerobic xylose growth may have caused up-regulation of oxidore-ductive metabolism, pentose phosphate pathway and glu-coneogenesis. To further investigate regulation of xylose metabolism in anaerobic conditions, strains using oxi-doreductive and isomerase based xylose assimilating pathways should be compared at the transcriptional level. The regulatory effect of cofactor imbalance could thus be assayed.

Abbreviations

1,3-DPG: 1,3-bisphosphoglycerate; CoA: coenzyme A; DHAP: dihydroxyacetone-phosphate; G3P: 3-phos-phoglycerate; G2P: 2-phosphoglycerate; GAP: glyceralde-hyde 3-phosphate; fru-6P: fructose 6-phosphate; fru-1,6-bisP: fructose 1,6-bisphosphate; glu-6P: glucose 6-phos-phate; HPLC: high performance liquid chromatography; NADH: nicotinamide adenine dinucleotide; NADPH: nicotinamide adenine dinucleotide phosphate; OD: optical density; - P: phosphate; PEP: phosphoenolpyruvate; PPP: pentose phosphate pathway; XDH: xylitol dehydro-genase; XI: xylose isomerase; XK: xylulokinase; XR: xylose reductase; YNB: yeast nitrogen base.

Competing interests

The authors declare that they have no competing interests. Authors' contributions

DR participated in the design of the study, performed the experimental work and wrote the manuscript. BHH participated in the design of the study and commented on the manuscript. MB participated in the design of the study, performed the experimental work and commented on the manuscript. All the authors have read and approved the final manuscript.

Acknowledgements

This work was supported by the Swedish Energy Agency (Energimyndigheten).

References

1. Otero JM, Panagiotou G, Olsson L: Fueling industrial biotechnology growth with bioethanol. Adv Biochem Eng/Biotechnol 2007, 108:1-40.

2. Hahn-Hagerdal B, Galbe M, Gorwa-Grauslund MF, Liden G, Zacchi G: Bio-ethanol--the fuel of tomorrow from the residues of today. Trends Biotechnol 2006, 24:549-556.

3. Galbe M, Sassner P, Wingren A, Zacchi G: Process engineering economics of bioethanol production. Adv Biochem Eng Biotechnol 2007, 108:303-327.

4. Hahn-Hagerdal B, Karhumaa K, Jeppsson M, Gorwa-Grauslund MF: Metabolic engineering for pentose utilization in Saccharomy-ces cerevisiae. Adv Biochem Eng Biotechnol 2007, 108:147-177.

5. Jeffries TW: Engineering yeasts for xylose metabolism. Curr Opin Biotechnol 2006, 17:320-326.

6. van Maris AJ, Winkler AA, Kuyper M, de Laat WT, van Dijken JP, Pronk JT: Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component. Adv Biochem Eng Biotechnol 2007, 108:179-204.

7. Eliasson A, Christensson C, Wahlbom CF, Hahn-Hägerdal B: Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures. Appl Environ Microbiol 2000, 66:3381-3386.

8. Kötter P, Ciriacy M: Xylose fermentation by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 1993, 38:776-783.

9. Kuyper M, Harhangi HR, Stave AK, Winkler AA, Jetten MS, de Laat W, den Ridder JJ, Op den Camp HJ, van Dijken Jp, Pronk JT: Highlevel functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharo-myces cerevisiae? FEMS Yeast Res 2003, 4:69-78.

10. Brat D, Boles E, Wiedemann B: Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae. Appl Environ Microbiol 2009, 75:2304-231 I.

11. Walfridsson M, Bao X, Anderlund M, Lilius G, Bulow L, Hahn-Häger-dal B: Ethanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene, which expresses an active xylose (glucose) isomerase. Appl Environ Microbiol 1996, 62:4648-465I.

12. Johansson B, Hahn-Hägerdal B: The non-oxidative pentose phosphate pathway controls the fermentation rate of xylulose but not of xylose in Saccharomyces cerevisiae TMB3001. FEMS Yeast Res 2002, 2:277-282.

13. Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF: Investigation of limiting metabolic steps in the utilization of xylose by recombinant Saccharomyces cerevisiae using metabolic engineering. Yeast 2005, 22:359-368.

14. Bruinenberg PM, Debot PH, van Dijken JP, Scheffers WA: The role of redox balances in the anaerobic fermentation of xylose by yeasts. Eur J Appl Microbiol Biotechnol 1983, 18:287-292.

15. Runquist D, Fonseca C, Radstrom P, Spencer-Martins I, Hahn-Häger-dal B: Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae. Appl Microbiol Biotechnol 2009, 82:123-130.

16. Saloheimo A, Rauta J, Stasyk OV, Sibirny AA, Penttila M, Ruohonen L: Xylose transport studies with xylose-utilizing Saccharomyces cerevisiae strains expressing heterologous and homologous permeases. Appl Microbiol Biotechnol 2007, 74: I04I-1052.

17. Jin YS, Laplaza JM, Jeffries TW: Saccharomyces cerevisiae engineered for xylose metabolism exhibits a respiratory response. Appl Environ Microbiol 2004, 70:6816-6825.

18. Souto-Maior AM, Runquist D, Hahn-Hägerdal B: Crabtree-nega-tive characteristics of recombinant xylose-utilizing Saccharo-myces cerevisiae. J Biotechnol 2009, 143: II9-I23.

19. Bengtsson O, Jeppsson M, Sonderegger M, Parachin NS, Sauer U, Hahn-Hägerdal B, Gorwa-Grauslund MF: Identification of common traits in improved xylose-growing Saccharomyces cere-visiae for inverse metabolic engineering. Yeast 2008, 25:835-847.

20. Salusjärvi L, Kankainen M, Soliymani R, Pitkänen JP, Penttilä M, Ruo-honen L: Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae. Microb Cell Fact 2008, 7: I6.

21. Sonderegger M, Jeppsson M, Hahn-Hägerdal B, Sauer U: Molecular basis for anaerobic growth of Saccharomyces cerevisiae on xylose, investigated by global gene expression and metabolic flux analysis. Appl Environ Microbiol 2004, 70:2307-2317.

22. Wahlbom CF, Otero RRC, van Zyl WH, Hahn-Hägerdal B, Jonsson LJ: Molecular analysis of a Saccharomyces cerevisiae mutant with improved ability to utilize xylose shows enhanced expression of proteins involved in transport, initial xylose metabolism, and the pentose phosphate pathway. Appl Environ Microbiol 2003, 69:740-746.

23. DeRisi JL, Iyer VR, Brown PO: Exploring the metabolic and genetic control of gene expression on a genomic scale. Science I997, 278:680-686.

24. Nielsen J, Villadsen J, Liden G: Bioreaction engineering principles. 2nd edition. New York: Kluwer Academic/Plenum Publishers; 2003.

25. Kostrzynska M, Sopher CR, Lee H: Mutational analysis of the role of the conserved lysine-270 in the Pichia stipitis xylose reductase. FEMS Microbiol Lett 1998, 159:107-112.

26. Bengtsson O, Hahn-Hägerdal B, Gorwa-Grauslund MF: Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae. Biotechnol Biofuels 2009, 2:9.

27. Petschacher B, Leitgeb S, Kavanagh KL, Wilson DK, Nidetzky B: The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography. Biochem J 2005, 385:75-83.

28. Andreasen AA, Stier TJ: Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. J Cell Physiol 1953, 41:23-36.

29. Verduyn C, Postma E, Scheffers WA, van Dijken JP: Effect of ben-zoic acid on metabolic fluxes in yeasts - a continuous culture study on the regulation of respiration and alcoholic fermentation. Yeast 1992, 8:501-517.

30. Xue XD, Ho NWY: Xylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase gene. Appl Biochem Biotechnol 1990, 24-5:193-199.

3 1. Träff KL, Cordero RR, van Zyl WH, Hahn-Hägerdal B: Deletion of the GRE3 aldose reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevi-siae expressing the xylA and XKS1 genes. Appl Environ Microbiol 2001, 67:5668-5674.

32. Gietz RD, Schiestl RH, Willems AR, Woods RA: Studies on the transformation of intact yeast cells by the Liac/S-DNA/PEG procedure. Yeast 1995, 11:355-360.

33. Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1989.

34. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP: Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003, 4:249-264.

35. Nissen TL, Hamann CW, Kielland-Brandt MC, Nielsen J, Villadsen J: Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis. Yeast 2000, 16:463-474.

36. Ruohonen L, Aristidou A, Frey AD, Penttilä M, Kallio PT: Expression of Vitreoscilla hemoglobin improves the metabolism of xylose in recombinant yeast Saccharomyces cerevisiae under low oxygen conditions. Enzyme Microb Technol 2006, 39:6-14.

37. Karhumaa K, Fromanger R, Hahn-Hägerdal B, Gorwa-Grauslund MF: High activity of xylose reductase and xylitol dehydrogenase improves xylose fermentation by recombinant Saccharomy-ces cerevisiae. Appl Microbiol Biotechnol 2007, 73:1039-1046.

38. Karhumaa K, Garcia Sanchez R, Hahn-Hägerdal B, Gorwa-Grauslund MF: Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant Saccharomyces cerevisiae. Microb Cell Fact 2007, 6:5.

39. Kuyper M, Winkler AA, van Dijken JP, Pronk JT: Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle. FEMS Yeast Res 2004, 4:655-664.

40. Kuyper M, Hartog MM, Toirkens MJ, Almering MJ, Winkler AA, van Dijken JP, Pronk JT: Metabolic engineering of a xylose-isomer-ase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation. FEMS Yeast Res 2005, 5:399-409.

41. Raychaudhuri S, Stuart JM, Altman RB: Principal components analysis to summarize microarray experiments: application to sporulation time series. Pac Symp Biocomput 2000:455-466.

42. Fazio A, Jewett MC, Daran-Lapujade P, Mustacchi R, Usaite R, Pronk JT, Workman CT, Nielsen J: Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: A three factor design. BMC Genomics 2008, 9:14.

43. Tai SL, Boer VM, Daran-Lapujade P, Walsh MC, de Winde JH, Daran JM, Pronk JT: Two-dimensional transcriptome analysis in chemostat cultures. Combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae. J Biol Chem 2005, 280:437-447.

44. Cipollina C, Brink J van den, Daran-Lapujade P, Pronk JT, Porro D, de Winde JH: Saccharomyces cerevisiae SFP1: at the crossroads of

central metabolism and ribosome biogenesis. Microbiology 2008, 154:1686-1699.

45. Moss T: At the crossroads of growth control; making ribos-omal RNA. Curr Opin Genet Dev 2004, 14:210-217.

46. Rodriguez A, de la Cera T, Herrero P, Moreno F: The hexokinase 2 protein regulates the expression of the GLK1, HXK1 and HXK2 genes of Saccharomyces cerevisiae. Biochem J 2001, 355:625-631.

47. McAlister L, Holland MJ: Differential expression of the three yeast glyceraldehyde-3-phosphate dehydrogenase genes. J Biol Chem 1985, 260:15019-15027.

48. Heinisch JJ, Muller S, Schluter E, Jacoby J, Rodicio R: Investigation of two yeast genes encoding putative isoenzymes of phos-phoglycerate mutase. Yeast 1998, 14:203-213.

49. Jeppsson M, Johansson B, Hahn-Hagerdal B, Gorwa-Grauslund MF: Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose. Appl Environ Microbiol 2002, 68:1604-1609.

50. Vemuri GN, Eiteman MA, McEwen JE, Olsson L, Nielsen J: Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc Nat Acad Sci USA 2007, 104:2402-2407.

51. Almeida JR, Bertilsson M, Hahn-Hagerdal B, Liden G, Gorwa-Graus-lund MF: Carbon fluxes of xylose-consuming Saccharomyces cerevisiae strains are affected differently by NADH and NADPH usage in HMF reduction. Appl Microbiol Biotechnol 2009.

52. Wahlbom CF, Hahn-Hagerdal B: Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae. Biotechnol Bioeng 2002, 78:172-178.

53. Gancedo JM: Yeast carbon catabolite repression. Microbiol Mol Biol Rev 1998, 62(2):334-6I.

54. Eliasson A, Boles E, Johansson B, Osterberg M, Thevelein JM, SpencerMartins I, Juhnke H, Hahn-Hagerdal B: Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Sac-charomyces cerevisiae. Appl Microbiol Biotechnol 2000, 53:376-382.

55. Bettiga M, Bengtsson O, Hahn-Hagerdal B, Gorwa-Grauslund MF: Arabinose and xylose fermentation by recombinant Saccha-romyces cerevisiae expressing a fungal pentose utilization pathway. Microb Cell Fact 2009, 8:40.

56. Gietz RD, Sugino A: New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking 6-base pair restriction sites. Gene I988, 74:527-534.

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