Microorganisms
C. tenuis CBS4435 was obtained from the Centraalbureau voor Schimmelcultures (Baarn, The Netherlands) and maintained at 25 °C on YPD-agar containing yeast extract (1 % w/v), peptone from Casein (2 % w/v), glucose (2 % w/v) and agar (1.6 % w/v). The pH value of the YP portion of the medium was adjusted to 5.5 prior to sterilization. Cells from late-exponential phase grown in YPD medium at 25 °C were used for long-term storage in a glycerol (60 % v/v)—YPD medium at −70 °C. The recombinant S. cerevisiae strain BP000 was used that expresses chromosomally integrated XR from C. tenuis CBS4435 (CtXR), XDH from Galactocandida mastotermitis (GmXDH) and endogenous XK (ScXK), each under the control of a strong constitutive TDH promoter [20].
Biomass preparation
Biomass of CBS4435 used in conversion experiments was prepared at 25 °C by two consecutive cultivations. First, a loopful of cells was transferred aseptically from a freshly overgrown YPD-agar plate into a baffled 300 mL shake flask containing 50 mL of mineral (M-) medium (see later). Cells were cultivated overnight at 125 rpm. These cultures served as inoculum for the preparation of biomass which was carried out at 125 rpm in baffled 1000 mL shake flasks containing 300 mL of M-medium. The initial optical density at 600 nm (OD600) was 0.05. Cells from preparatory cultures were harvested at an OD600 of 3–4 by centrifugation (4400g, 15 min, 4 °C) and washed once with a cold physiological NaCl (0.9 % w/v) solution prior to xylose conversion experiments. The M-medium used contained 14.4 g/L KH2PO4, 5 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H2O, and 0.025 % (v/v) Antifoam 204 (Sigma-Aldrich, Vienna). The pH was adjusted to a value of 5.5 prior to sterilization. Vitamins as well as trace elements were added as described by [29]. Xylose (18 g/L) represented the sole carbon source to induce expression of enzymes which constitute the XA pathway in CBS4435 [22]. The same protocol for biomass preparation was applied for BP000 with the exception of glucose as the carbon source (instead of xylose), an initial pH of 5.5 (instead of 6.5) and a cultivation temperature of 30 °C (instead of 25 °C).
Anaerobic xylose conversions
Xylose conversions were carried out at 25 °C (CBS4435) or at 30 °C (BP000) in a Labfors bioreactor (Infors HT, Bottmingen, Switzerland, working volume: 2000 mL; stirrer: two six-bladed disc impellers; ratio of impeller to reactor diameter was 0.4; 200 rpm). The same M-medium as described above was used except for KH2PO4 (3 g/L), ergosterol (10 mg/L) and Tween-80 (0.42 g/L). The pH was maintained at values of 4.5 ± 0.1 (CBS4435) and 5.5 ± 0.1 (BP000). Conversions were carried out with 0.9 and 3.8 g/L (BP000) or with 1.1 g/L (CBS4435) of dry cells. Anaerobic conditions were maintained by sparging the culture continuously with nitrogen (purity 5.0, 0.16 vvm). All conversion experiments were carried out in duplicates.
Analytics of external metabolites
Sample work-up, monitoring of cell growth (recorded as OD600) and off-gas analysis (CO2 and ethanol) using an acoustic gas analyzer (IN1313, Innova AirTech Instruments, Ballerup, Denmark) were carried out as described by [29]. Cell dry weights were determined as described recently [30] and correlated with optical densities using an averaged factor of 0.41 g dry cells (gdc) per unit of OD600 for both strains. Concentrations of xylose and fermentation products were analyzed by HPLC–UV/RI using an Aminex HPX87-H column and 0.5 mM H2SO4 as eluent [20].
Analytics of intracellular metabolites
For the analysis of intracellular metabolites, xylose fermentations for BP000 and CBS4435 were carried out in a bioreactor (working volume 1000 mL) as described above and by applying 0.25 g/L KH2PO4 and 1.3–1.4 gdc/L (BP000) or 0.9–1.0gdc/L (CBS4435). Low concentrations of KH2PO4 in the medium were necessary to reduce the effect of ion suppression during LC/MS analysis. The applied KH2PO4 concentrations did not affect phenotypes of BP000 and CBS4435 (data not shown). Samples were withdrawn after 24 h of xylose fermentation which represented the pseudo-steady state of xylose-to-ethanol conversion. A protocol for sample preparation as described by [21] was used with slight modifications (see Additional file 3). Metabolite samples containing 13C-labeled metabolite yeast extract as internal standard (ISTD) were analyzed by LC–MS. A Dionex Ultimate 3000 HPLC setup (Thermo Fisher Scientific, USA) equipped with a reversed-phase Atlantis T3 C18 pre- and analytical column (Waters, USA) was used [31]. For metabolite separation, a 40-min gradient was applied: 2-propanol was used as eluent A and aqueous methanol solution [(5 % methanol v/v), tributylamine (10 mM), acetic acid (15 mM), pH 4.95] was used as eluent B. The injection volume per sample was 10 µL. Mass spectrometric detection was carried out with an Exactive™ Orbitrap system (Thermo Fisher Scientific, USA). Heated electrospray ionization (HESI) was used for negative ionization and masses between 70 and 1100 m/z were detected with a resolution of 50,000 (at m/z 200) in full scan mode. Identical compounds (Sigma-Aldrich, USA, of highest purity available) containing ISTD were used as standards to cover a concentration range of 0.4–100 µM. MS data were analyzed with TraceFinder™-software Version 3.1. (Thermo Fisher Scientific, USA). Eleven intracellular metabolites [ATP, ADP, AMP, ribulose-5P (Ru5P), ribose-5P (Ri5P), 6-phosphogluconate (6PG), fructose 1,6-bisP (F1,6BP), DHAP, glycerol-3P (Glyc3P), phosphoenolpyruvate (PEP) and pyruvate (PYR)] and three metabolite pools [sum of hexose phosphates (HXP), sum of 2PG and 3PG (XPG; PG, phosphoglycerate), sum of 1,3-BPG and 2,3BPG (X,3BPG)] were quantified for each strain and respective concentrations were calculated as averages from a total of eight samples obtained from two individual fermentations (four replicates per fermentation).
Determination of specific enzyme activities in cell-free extracts
Enzyme activities were analyzed at 25 °C (CBS4435, BP000) and at 30 °C (BP000) from cell-free extracts by means of the initial rate method. Cell-free extracts were prepared at 4 °C from cells metabolizing xylose at the pseudo-steady state using a French Press (1500 psi, SLM Aminco, Silver Spring, MD, USA). Cells were washed once with ice-cold physiological sodium chloride solution and re-suspended in 50 mM potassium phosphate buffer (PBB) pH 7.0 prior to cell disruption. The amount of soluble protein was analyzed using the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA, USA) referenced against bovine serum albumin. Time-dependent utilization of NADPH (XR) or NADH [XR, alcohol dehydrogenase (ADH), XK, phosphoglucose isomerase (PGI), 6-phosphofructokinase (PFK), F1,6BP aldolase (FBA), pyruvate kinase (PYK)] as well as formation of NADH [XDH, aldehyde dehydrogenase (ALDH)] or NADPH (ALDH, G6PDH and GND) was recorded at 340 nm using a DU800 spectrophotometer (Beckman Coulter, Fullerton, CA, USA) equipped with a Peltier temperature controller. A molar extinction coefficient of 6.22/cm/mM was used for NAD(P)H. Release of phosphate from Glyc3P over time was analyzed for Glyc3P phosphatase (GPP) activity. Phosphate concentrations were determined from cleared samples treated at 99 °C for 5 min as described by [32]. Compositions of reaction mixtures are presented in the Additional file 1: Table S4. Appropriate reference measurements were carried out and considered as required.
Kinetic modeling
The reaction model as shown in Fig. 4 was used as the basis for in silico analysis. To reduce the degree of complexity, stoichiometry of carbon conversion was used for XK (R
4), the PP pathway (R
5), the upper part of glycolysis (UG) (R
9; combining PFK and FBA) and for the lower part of glycolysis (LG) up to ethanol formation (R
10). We did not consider ATP in the model as under the fermentation conditions used the reaction catalyzed by XK should be well saturated by ATP [compare K
ATP of 0.17 mM and 0.28 mM reported for XKs of S. cerevisiae [33] and S. stipitis [34], respectively, with intracellular concentrations of ATP estimated for BP000 and other xylose-metabolizing recombinant S. cerevisiae strains (3–4 mM) [21]]. As both strains investigated in this study were not able to grow on xylose under the conditions applied, biomass formation was not included in the model. Respective rate equations and corresponding kinetic parameters are shown in the Additional file 1: Table S2 and Table 2, respectively. Known rate equations including reversibility were used for XDH (R
3) and PGI (R
6). The complete rate equation for CtXR involving reversibility as well as alternate binding of NAD(P)(H) (R
1, R
2) was derived in this study and verified (see Additional files 2: Scheme S1; Fig. S3 and 3). Simplified rate equations were applied for XK (R
4), PP pathway (R
5), UG (R
9) and LG (R
10), while representative rate expressions were used for G6PDH (R
7) and GND (R
8) which take competitive product inhibition by NADPH into account.
Selection of known and determination of so far unknown model-relevant kinetic data are described in detail in the Additional file 3. Briefly, all 24 kinetic parameters required for simulating the XR reaction were determined. Values for \(K_{\text{m,NADP}}^{+}\) and K
i,NADPH were measured for Sc(Ct)G6PDH and Sc(Ct)GND. The set of kinetic parameters describing a reversible ordered Bi Bi mechanism reported for GmXDH was applied to both XDHs. Similarly, a reported K
m,xylulose for ScXK and the set of kinetic parameters reported for ScPGI were used. For lumped reactions R
5, R
9 and R
10, a Michaelis constant of 0.1 mM was assumed.
Parameter estimation analysis
Time courses of xylose utilization and xylitol production obtained for CBS4435 (this study) and BP000 (this study and from [20]) were used as a basis to fit the kinetic model by parameter estimation. Both time courses which represent mutually independent parameters were fitted simultaneously. The software package Copasi 4.8.35 (http://www.copasi.org [35, 36]) was used. Only the pseudo-steady-state phase of xylose conversion which is free of substrate limitation and ethanol or other unspecific inhibitions was subjected to parameter estimation by evolutionary programming. After transformation, upper and lower bounds of specific enzyme activities measured at 25 °C (CBS4435) and 30 °C (BP000) were applied to the respective cell-specific molar rates (mmol/h/gdc) by assuming that on a mass basis the protein content in dry yeast cells accounts for 40 % [37]. Intracellular activity levels of R
5, R
9 and R
10 were used in a range of 0.1–10, 1–100 and 1–100 mmol/h/gdc, respectively. Initial concentrations of coenzymes were constrained to ranges reported for BP000 [21]. A complete summary of input parameters is presented in the Additional file 1: Table S3.
Flux balance analysis
A metabolic reaction network was designed consisting of 32 reactions involving xylose assimilation, PP pathway, oxidative PP pathway (oPP) and glycolysis as well as glycerol, acetate and ethanol production. For XR, one reaction was used for each coenzyme. Upper and lower bounds of experimentally obtained molar production rates of ethanol, xylitol, glycerol and acetate were applied. To capture network flexibility and experimental variations, simulations were carried out at upper and lower bounds and at the average value of q
xylose. Two different objective functions (CO2 and ATP formation) were individually applied for linear optimization by linprog (Optimization Toolbox, Matlab R2013b, Mathworks, Inc., USA).