Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae

Strains and culture conditions

E. coli strain NEB5α (New England Biolabs, USA) was used for sub-cloning of plasmid DNA. Transformants were selected on solid LB plates (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, 15 g/L agar, pH 7.0), supplemented with 100 mg/L of ampicillin, for 16 h at 37°C. Cultures of transformed E. coli were recovered from 25% glycerol stocks stored at −80°C and grown in liquid LB medium, supplemented with ampicillin, for 14–16 h at 37°C and 180 rpm in an orbital shaker.

Cloning procedures

Standard molecular biology techniques were used for all cloning procedures [37]. Plasmid purification was performed using GeneJet Plasmid Mini-prep Kit (Thermo Scientific, USA). DNA fragments digested with restriction enzymes were excised from agarose gel and purified using QIAquick Gel Extraction Kit (Qiagen, Germany) when needed. DNA fragments amplified by PCR were purified using GeneJet PCR Purification Kit (Thermo Scientific, USA). All enzymes for PCR amplifications, ligations and restriction digestions were obtained from Thermo Scientific unless stated otherwise. PCR products were amplified using the High Fidelity Phusion Hotstart II Polymerase or High Fidelity Taq Polymerase Mix (Thermo Scientific, USA). Competent yeast cells were prepared and transformed according to the PEG/LiAc method [38] and heat shock competent E. coli cells were prepared according to the Inoue method [37] and transformed according to the supplier’s instructions.

The gene FRD1 was amplified from yeast genomic DNA by PCR using primers FRD1-F1-SpeI (5′-GAC TAG TAA ATG TCT CTC TCT CCC GTT G-3′) and FRD1-R1-SalI (5′-GTT GTC GAC TTA CTT GCG GTC ATT GGC-3′). The gene OSM1 was amplified from yeast genomic DNA by PCR using primers OSM1-F1-SpeI (5′-GAC TAG TAA AAT GAT TAG ATC TGT GAG-3′) and OSM1-R1-SalI (5′-GTT GTC GAC TCA GTA CAA TTT TGC TAT G-3′). The amplified FRD1 and OSM1 genes were digested with SpeI and SalI and inserted between the TDH3 promoter and CYC1 terminator of the previously constructed vector YIpDR1, yielding the two plasmids YIpBB1 and YIpBB2 (Table 1). The cloned genes were verified by DNA sequencing.

The ERO1 gene, including a 300 bp downstream sequence, was amplified from yeast genomic DNA by PCR using primers ERO1-F1-BamHI (5′-GAG GAT CCA AAA TGA GAT TAA GAA CCG CCA TTG-3′) and ERO1-R1-XhoI (5′-GTC TCG AGC CCT TGA AGA TGG TAC C-3′). The amplified ERO1 ORF and terminator was digested with BamHI and XhoI and inserted between the TEF1 promoter and CYC1 terminator of the vector p426TEF (Table 1). The TEF1p-ERO1-ERO1t cassette was amplified from plasmid p426TEF-ERO1 using primers p426TEF-F1-SacI (5′-GGA GCT CAT AGC TTC AAA ATG-3′) and p426ERO1t-R1-NdeI (5′-CAT ATG CCC TTG AAG ATG GTA C-3′). The amplified cassette was digested with BamHI and XhoI and ligated into plasmids YIpBB1 and YIpBB2, digested with the same enzymes, yielding vectors YIpBB3 and YIpBB4, respectively (Table 1). The cloned cassette was verified by DNA sequencing.

Strain construction

S. cerevisiae strain TMB3452 was constructed by transforming strain TMB3043 with YIpDR7, linearized with EcoRV. The integrative plasmids YIpBB1-4 were linearized with EcoRV, AflII, Eco91I and AflII, respectively, and used to transform strain TMB 3452, yielding strains TMB3456 (FRD1), TMB3457 (OSM1), TMB3458 (FRD1 ERO1) and TMB3459 (OSM1 ERO1) (Table 1). The control strain TMB3455 was constructed by transforming TMB3452 with YIplac128, linearized with EcoRV. Correct integration of the vectors was verified by PCR using primer pairs that annealed on the plasmid and on the chromosome outside the homologous regions of URA3 or LEU2 loci. The functionality of the over-expressed FRD1 and OSM1 genes was verified by measuring the specific fumarate reductase activity in cell-free protein extracts from strains TMB3455-59 grown anaerobically on xylose. Strains TMB3456-59 had between 9- and 30-fold higher activities than the control showing that the over-expressed genes generated increased levels of functional enzymes (Additional file 1: Figure S1). The functionality of the over-expressed ERO1 gene was verified by determining the sensitivity toward DTT. Single colonies of strains TMB3455-59 were inoculated in 5 mL YNB medium with 2% glucose and grown over-night. 200 μL of this culture was used to inoculate fresh medium and cultivated until the cells reached OD_{620 nm} ≈ 1. A volume containing 2 × 10⁶ cells (150–200 μL) was spread on solid YPD medium after which a filter disc was placed in the centre of the plate and soaked with 20 μL or 30 μL of 1 M DTT solution. The plates were incubated 48 h at 30°C before measuring the diameter of the halo formed around the disc. The halos formed with strains TMB3458 and TMB3459 were 20-30% smaller compared with the other strains showing that the over-expressed ERO1 gene generated increased level of a functional enzyme (Additional file 1: Figure S2).

Anaerobic batch fermentations

Yeast strains were pre-grown in YNB medium with 2% glucose until mid-exponential phase and inoculated into 50 mL medium with 2% glucose and 5% xylose in 250 mL crimp-sealed serum flasks at a concentration of 0.04 g CDW/L. Prior to inoculation the headspace was flushed with N₂-gas containing less than 5 ppm oxygen (AGA Gas AB, Sweden) for at least 30 min to ensure anaerobic conditions. Cultivations were carried out in a 30°C water bath with continuous stirring at 180 rpm using magnetic stirrers. To prevent diffusion of oxygen into the culture during sampling, the excess gas was released through a 0.2 μm filter connected to Norprene tubing submerged under water. Cell dry weight was calculated from OD measurements using a pre-determined relationship between OD and cell dry weight. Fermentation experiments were performed in biological duplicates.

Preparation of cell-free protein extracts

Yeast strains were cultivated anaerobically in crimp-sealed serum flasks as described above using 2X YNB medium with 2% glucose and 5% xylose. After 36 h, when xylose was the only carbon source, the cells were harvested by centrifugation at 4000 rpm and 4°C for 10 min. The resulting cell pellets were resuspended in 1X Lysis buffer (20 mM K,Na-PO₄, 20 mM EDTA, 1 mM DTT, pH 7.0) to obtain a final suspension of 1 g wet cells/mL. Protein extracts were prepared using a Precellys®24 homogenizer with cooling provided by the Cryolys® unit (Bertin Technologies, France) and Precellys Lysing Kit VK05 containing 0.5 mm glass beads. The unit was operated for 3 × 45 s at 5000 rpm with 30 s pauses between each cycle and at max 8°C. Cell debris was pelleted by centrifugation at 14,000 × g and 4°C for 15 min and the supernatant was transferred to a small glass vial. Before sealing the vial the headspace was replaced with N₂-gas in an anaerobic chamber. The protein extracts were stored at 4°C until the following day. Protein concentrations were determined with the Coomassie Protein Assay Reagent (Thermo Scientific, USA) using BSA as standard (Thermo Scientific, USA).

Fumarate reductase activity assay

Fumarate reductase activity was determined in a 1.2 mL reaction mixture containing: 50 mM K,Na-PO₄ buffer (pH 7.3), 10 mM fumaric acid (pH 7.3), 0.4 mM FMN-Na, 2% (w/v) sodium hydrosulfite. All reagents, except protein extract, were added to a rubber-sealed cuvette inside an anaerobic chamber. The assay buffer was prepared by dissolving 2.762 g of KH₂PO₄ and 2.837 g of Na₂HPO₄·2H₂O in 50 mL anaerobic water (separately) inside the anaerobic chamber, creating one 10X solution and one 2X solution, respectively. A 200 mM stock solution at the correct pH was made by mixing 5 mL 10X with 25 mL 2X and completing the volume to 50 mL with anaerobic water. A 40 mM stock solution of fumaric acid was prepared by dissolving 0.464 g fumaric acid in 50 mL of 5 mM K,Na-PO₄ buffer at pH 7.3. The pH was adjusted to 7.3 using 5 M NaOH and the volume was completed to 100 mL with water. Solutions of the cofactor and reducing agent were prepared fresh before each assay. Both compounds were weighed and brought into the anaerobic chamber. The FMN cofactor was dissolved in anaerobic water and sodium hydrosulfite was dissolved in 100 mM K,Na-PO₄ buffer, but only after all other reagents had been added to the cuvette to ensure that FMN becomes completely reduced to FMNH₂. The reaction was started by addition of protein extract using a gas-tight syringe. The formation of FMN was measured at 445 nm in cuvettes with light path length of 1 cm using an Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences Corp., USA) controlled by computer software SWIFT Reaction Kinetics v. 2.05 (Biochrom Ltd., Cambridge, UK). The absorbance was converted to concentration units using the molar extinction coefficient of FMN (ϵ_{445 nm} = 12,500 L/mol/cm). The slope was determined within an interval of at least 1 min, using at least three different dilutions of the protein extract. Only the slopes within the range 0.045-0.40 A/min were considered acceptable. One unit is defined as the conversion of 1 μmol of fumarate per minute.

Biomass determination and analysis of metabolites

Optical density was measured at 620 nm using an Ultrospec 2100 Pro spectrophotometer (Amersham Biosciences Corp., USA). Cell dry weight was measured in triplicate by filtering a known volume of the culture through a pre-weighed nitrocellulose filter with 0.45 μm pore size. The filters were washed with three volumes of water, dried in a microwave oven and weighed after equilibrating to room temperature in a desiccator.

Concentrations of glucose, xylose, xylitol, glycerol, succinate, acetate and ethanol were analysed by HPLC (Waters, USA). Aminex HPX-87H ion exchange column (Bio-Rad, USA) was used at 45°C with a mobile phase of 5 mM H₂SO₄ at a flow rate of 0.6 mL/min. All compounds were detected with a RID-10A refractive index detector (Shimadzu, Japan).

Calculation of metabolic rates

In complex medium all calculations were made as described above except for the maximum specific consumption rate of glucose which could not be determined accurately enough. Hence, the μ_max under these conditions was determined as the slope in a linear regression of ln(OD) vs. time and r _glc was calculated using Equation 7. The significance of a difference between two mean values was calculated using a two-tailed t-test assuming equal variance of the two samples with two degrees of freedom (n₁ = n₂ = 2) unless stated otherwise. Balances of carbon and degree of reduction, determined for the glucose and xylose phases using calculated yield coefficients, are given as supplementary information in Additional file 2: Table S1.

Transcriptional analysis of UPR related genes

The list of genes reported as targets of the UPR were taken from [26]. Transcript data from cultivations of a XR/XDH-strain using glucose or xylose in aerobic and anaerobic conditions was taken from [27]. UPR target genes that had a significant change in expression (P ≤ 0.1) during the transition from aerobic to anaerobic conditions with xylose as carbon source were included in the analysis. Two genes commonly used as targets of the UPR (KAR2 and INO1) [27] did not pass the statistical requirement and were thus not included in the analysis (Additional file 2: Table S5). Hierarchical clustering was performed using the clustergram function in MATLAB R2010b with log₂ values of transcriptional changes. Euclidean distance was used to calculate the distance between the data points and average linkage was used to generate the dendrogram. Data used for the hierarchical clustering is given as supplementary information in Additional file 2: Table S2.

Genes targeted by the unfolded protein response are differentially expressed on xylose compared to glucose

A previously performed transcripome study in a recombinant XR/XDH-strain [27] was used to investigate the expression level of genes associated with the UPR. It revealed that IRE1 and HAC1 levels were ~65% and ~50% higher (P = 0.02 and P = 0.05, respectively) in cells growing anaerobically on xylose compared to cells growing aerobically (Figure 2). However, the transcript level of TRL1, encoding the ligase, was ~50% lower (P = 0.02) (Figure 2), suggesting that HAC1 is mainly present as fragments rather than mature mRNA molecules. The transcript levels of these genes did not change significantly during the same transition (i.e. from aerobic to anaerobic conditions) on glucose (Figure 2) indicating that the response was associated with the type of carbon source available. The cluster analysis illustrated in Figure 3 indeed shows that a majority (61%) of the included UPR target genes were down-regulated on xylose when the conditions changed from aerobic to anaerobic (Group A). The genes belonging to this group represent functions taking place in all steps of the maturation process (translocation, glycosylation, disulphide bond formation, protein folding, vesicle budding and vesicle transport to the cell membrane) (Additional file 2: Table S2) and do not change during the same transition on glucose. The smallest group (Group B) contained those genes which are regulated equally on glucose and xylose, with only small differences in abundance between the two carbon sources. A small number of genes were up-regulated on xylose while remaining largely unchanged on glucose (Group C). These genes are related to peroxisome biogenesis (PEX4), ubiquitin recycling (DOA4) and the cell integrity signalling pathway (TUS1). The three genes ECM3, ECM8 and YET2 encode proteins with unknown functions. RIB1 encodes GTP cyclohydrolase II which catalyses the first step of the riboflavin (FAD and FMN) biosynthesis pathway. The fourth group contains genes which are strongly up-regulated in response to anaerobiosis in both conditions (Group D). However, in contrast to the genes in Group B, the transcript levels of these genes were in general lower on xylose compared to glucose. This analysis shows that the UPR responded differently to xylose compared to glucose. Furthermore, the repression of a majority of the target genes suggests that the capacity of the secretory pathway could be limited during xylose fermentation.

Consistent with previously reported expression patterns on non-fermentable carbon sources and entry into stationary phase [39, 40], the expression of ERO1 was reduced by ~30% (P < 0.02) on xylose compared to glucose, both under anaerobic and aerobic conditions (Additional file 2: Table S5) [27]. Hence, a reduced capacity to form disulphide bonds in nascent polypeptides translocated to the ER could be one underlying cause for the low anaerobic growth rate on xylose. The previously reported accumulation of intracellular fumarate during xylose fermentation [31] suggested that recombinant XR/XDH strains have a limited FR activity under such conditions, which would lead to insufficient recycling of the FAD co-factor required by Ero1p. This hypothesis was investigated by evaluating the physiological effect of over-expressing the cytosolic and mitochondrial FR enzymes, alone and in combination with ERO1, during fermentation of a mixture of glucose and xylose.

Over-expression of cytosolic FR has limited impact on xylose fermentation

Due to the higher contribution of the cytosolic FR to the total cellular reductase activity the over-expression of FRD1, alone and in combination with ERO1, was evaluated first. When cultivated in defined medium (2X YNB) with 20 g/L glucose and 50 g/L xylose, TMB3456 (FRD1) and TMB3458 (FRD1 ERO1) consumed the glucose within 20 h and ca. 70% of the xylose within 90 h (Figure 4B and D). The two strains behaved very similar to the control strain (Figure 4A) and no significant physiological differences from this strain were observed (Additional file 2: Table S3). These results suggest that the implemented genetic modifications had no positive effect on the anaerobic growth on xylose in defined medium. However, the modifications only ensured that the genes required for regeneration of FAD co-factors were present (Figure 1). If substrates are not available in sufficient amounts (in this case unfolded polypeptides) these modifications are not effective in regenerating the co-factor. The poor repressive capability of xylose leads to an up-regulation of MDH2[27] (Additional file 2: Table S5), encoding cytosolic malate dehydrogenase, which has previously been suggested as an unbeneficial reaction that directs carbon away from amino acid synthesis by converting oxaloacetate into malate [31]. A reduced synthesis of amino acids could thus hamper the formation of polypeptides and mask the effect of the genetic modifications. To investigate whether the strains were limited by amino acid synthesis they were cultivated in a complex medium containing yeast extract. Changing from a defined medium to a complex medium led to several significant changes in the physiology of all strains, e.g. increased maximum growth rate, increased biomass yield and reduced by-product yields (Additional file 2: Table S3 and Table S4). Hence, the glucose was consumed within 15 h and ca. 80% of the xylose was consumed within 65 h (Additional file 1: Figure S3). However, despite the increased nutrient availability no significant differences were observed between the control strain and the FRD1-overexpressing strains TMB3456 and TMB3458 (Additional file 2: Table S4).

Over-expression of mitochondrial FR has a detrimental effect on xylose fermentation

Although the change from a defined medium to a complex medium led to a general increase in the growth rate on xylose, the over-expression of FRD1, alone or in combination with ERO1, had little effect on the cellular physiology. One explanation could be that the previously observed accumulation of fumarate [31] does not occur in the cytosol, but is restricted to the mitochondria. To investigate this possibility we over-expressed the OSM1 gene, alone and in combination with ERO1, and evaluated the fermentation performance in both defined and complex media with 20 g/L glucose and 50 g/L xylose.

These results indicate that the reactions taking place in the mitochondria have a significant role in xylose metabolism and that they are more sensitive to changes in metabolite levels (leading to altered regulatory signals, degree of allosteric inhibition of enzymes etc.) on xylose compared to glucose. Hence, the over-expression of OSM1 caused physiological responses that were not observed in strains over-expressing FRD1. In strain TMB3457 (OSM1) these responses were: i) a 20-40% reduction in glycerol yield, ii) a 63-68% increase in acetate yield and iii) a 27-38% reduction in growth rate during xylose fermentation (Table 2 and Table 3). A possible explanation for this phenotype could be that Osm1p interacts with Gut2p and Ach1p which are both located specifically inside the mitochondria (Additional file 1: Figure S4). GUT2 encodes a FAD-dependent glycerol 3-phosphate dehydrogenase located in the mitochondrial inner membrane [41] and expression of the gene is repressed in the presence of glucose and induced during growth on non-fermentable carbon sources [42]. An induction of the GUT2 gene has also been shown to occur during xylose fermentation with transcript levels more than 7-fold higher compared to glucose [27] (Additional file 2: Table S5). Recently, the protein encoded by ACH1 was shown to function as a Coenzyme A (CoA) transferase rather than an acetyl-CoA hydrolase [43]. Hence, this enzyme catalyses the following reaction: acetyl-CoA + succinate → succinyl-CoA + acetate (Δ_rG’° = −8.1 kJ/mol) and is also induced nearly 5-fold during xylose fermentation compared to glucose [27] (Additional file 2: Table S5). If both Gut2p and Ach1p are active on xylose, the pathway formed together with Osm1p could explain the diversion of carbon from glycerol to acetate as well as the reduced growth rate due to a reduced acetyl-CoA availability (Additional file 1: Figure S4). However, this hypothesis needs to be validated through further investigations.

Frd1p and Osm1p do not influence regeneration of cytosolic NAD

An early study on the role of FR showed that the addition of oxidised methylene blue or phenazine methosulfate, which chemically oxidises NADH to NAD, to cultures of a frd1Δ osm1Δ double mutant rescued the non-growing phenotype under anaerobic conditions [17]. It was thus proposed that the two FR enzymes are involved in the oxidation of excess NADH formed in the cytosol and the mitochondria under anaerobic conditions. The formation of xylitol during xylose fermentation has been ascribed to an inability of the cells to regenerate the NAD needed for the conversion of xylitol to xylulose by XDH [44]. The addition of acetoin, a substrate which can be reduced by yeast to 2,3-butanediol using NADH, to cultures fermenting xylose reduces the xylitol yield significantly [45, 46]. If the FR enzymes participate in the conversion of excess NADH a reduction of the xylitol yield during xylose fermentation would be expected when the FR activity is increased. However, the over-expression of FRD1 or OSM1 did not affect the amount of xylitol produced from xylose (Table 2, Table 3, Additional file 2: Table S3 and Table S4). This is in agreement with other studies showing that addition of acetoin to a culture of a frd1Δ osm1Δ double mutant does not rescue the non-growing phenotype under anaerobic conditions [11]. In addition, the proposed mechanism of NADH oxidation requires the presence of an enzyme capable of transferring electrons from NADH to FAD to generate the reduced cofactor used as substrate by fumarate reductase [11, 17]. As of yet, no such enzyme has been identified in S. cerevisiae that is expressed under anaerobic conditions. There are, however, FR enzymes that use NADH as co-factor instead of FADH₂. The NADH-dependent FR enzyme from Trypanosoma brucei has recently been expressed in a xylose-utilizing S. cerevisiae strain which resulted in increased ethanol yield and decreased xylitol yield when fermenting xylose [47]. Based on these results we conclude that the FR enzymes from S. cerevisiae do not influence the regeneration of cytosolic NAD.

Other potential limitations in the secretory pathway in S. cerevisiaeduring xylose fermentation

The results obtained in this study indicate that the activities of FR and Ero1p enzymes are not limiting during xylose fermentation. Measurements of the FR activity in strain TMB3455 when growing on glucose and when metabolizing xylose indeed showed equal activities around 3 mU/mg protein in both conditions (Additional file 1: Figure S5). This is contradicting previous studies showing that the activity and expression of FRD1 and OSM1 decrease upon entry into stationary phase [11, 39] and during carbon starvation [48]. It is, however, possible that the FR activity is maintained high on xylose in response to cellular stress. The up-stream regions of the FRD1 and OSM1 genes contain binding sites for stress-related transcription factors Yap1p and, in the case of FRD1, Msn2p/Msn4p [49]. Due to the involvement of Yap1p, an oxidative stress would be most likely in this case [50]. This possibility is supported by much higher intracellular concentrations of both reduced and oxidized glutathione [31] and higher transcript levels of MSN4 and YAP1[27] in recombinant S. cerevisiae strains during xylose fermentation compared to glucose fermentation (Additional file 2: Table S5).

The up-regulation of RIB1 (Figure 3) could be an indication that the need for flavin-containing complexes and/or the free co-factor is increased on xylose. Ero1p has been shown to use free FAD as electron acceptor in vitro under anaerobic conditions [51] which is in line with the ability of the FR enzymes to use free FADH₂ as cofactor and their essential nature under such conditions. As S. cerevisiae possesses one gene encoding a mitochondrial FAD transporter (FLX1) [52] and three genes encoding putative transporters associated with the ER (FLC1-3) [53], FAD-exchange between compartments should indeed be possible. This highlights free FAD as a potentially crucial metabolite for sustaining anaerobic growth.

Pdi1p is, together with Ero1p, essential for disulphide bond formation in the ER [54]. Although transcriptional analysis did not show any difference in expression of PDI1 between glucose and xylose fermentation [27] (Additional file 2: Table S5), proteome analysis of a mutant strain with improved xylose-fermenting capability identified Pdi1p as significantly more abundant in the improved strain compared with the wild-type [55]. This strongly indicates that there indeed is a need for a higher capacity in the protein folding mechanism for efficient xylose fermentation. Whether this capacity is for regulation of disulphide bond formation [56], reduction of non-native disulphide bonds [57] or targeting unfolded glycoproteins for degradation [58] remains to be investigated.