Novel and generally relevant findings for the xylose-fermenting S. cerevisiae strain BP10001 are: a direct correlation showing that q
xylitol decreases in response to an increase in q
xylose; high tolerance of a genome-scale metabolic flux model of S. cerevisiae to large variations in the usage of NADPH and NADH for xylose reduction; strong evidence that the mutated XR (from C. tenuis) works as a NADH-dependent reductase under the physiological reaction conditions. Furthermore, a detailed analysis of glucose-xylose co-fermentation by BP10001 is presented.
Fermentation of mixed glucose-xylose substrates by BP000 and BP10001
The largely sequential utilization of substrates, glucose prior to xylose, by BP000 and BP10001 is in agreement with previous studies of xylose-fermenting strains of S. cerevisiae and is thought to reflect, among other effects, the substrate selectivity of the transport systems involved in uptake of the two sugars [17, 19, 27, 28]. A specific xylose transport rate (q
TRxylose) of about 0.8 - 0.9 g/g CDW/h was previously determined for S. cerevisiae at 20 g/L xylose [18, 29]. This q
TRxylose surpasses q
xylose for BP000 and BP10001 by one order of magnitude, suggesting that xylose transport is not a limiting factor for the overall xylose conversion rate in the two strains under conditions where xylose is the sole carbon source. This notion is fully corroborated by findings of others, showing for recombinant yeast strains having either PUA or CEN.PK genetic background that xylose transport has little control over the xylose utilization rate unless there is substantial improvement in the rate of xylose metabolic steps located downstream of xylose uptake [18, 28–30]. Positive effects on the distribution of fermentation products from xylose (increase in Y
ethanol, decrease in Y
xylitol; see Table 1) that result from use of the mutated, NADH-preferring XR as compared to the NADPH-preferring wild-type enzyme were retained upon changing the reaction conditions from xylose (20 g/L) as the sole source of carbon  to a mixed glucose-xylose substrate (10 g/L each; this work). However, one must exercise caution in comparing the two fermentations directly, especially in terms of Y
xylitol because the ~2-fold enhancement of q
xylose resulting from a doubling of the xylose concentration from 10 g/L to 20 g/L caused a decrease in Y
xylitol by 27% from 0.26 g/g to 0.19 g/g (Table 1 and ). The clear correlation between Y
xylitol and q
xylose established for BP10001 (Figure 1) implies that xylitol by-product formation is controlled not only by the extent to which XR is matched with XDH in respect to coenzyme usage (see later). Moreover, the results (Table 1, Figure 2) validate BP10001 as a useful strain for ethanol production from mixed glucose-xylose substrates.
Is coenzyme recycling between XR and XDH still a limiting factor for xylose fermentation by BP10001?
Despite the fact that results of FBA were inconclusive regarding the coenzyme preference of the mutated XR under physiological reaction conditions, a number of indirect experimental observations suggest that mainly NADH is used for xylose reduction. Engineered strains of S. cerevisiae expressing the genes for Pichia stiptis XR and XDH formed less xylitol when glucose-xylose was offered instead of xylose alone [17, 31]. The lowering of Y
xylitol was plausibly explained as a consequence of enhanced coenzyme recycling that results because of the increased glycolytic flux when glucose is present . For BP10001, however, the xylitol yield in fed-batch co-fermentation of glucose and xylose was identical to Y
xylitol of the corresponding batch reaction in which the same concentration (50 g/L) of xylose was employed as sole source of carbon. These findings would be consistent with balanced coenzyme usage by XR and XDH in BP10001.
Comparison of fed-batch fermentations using glucose and glucose-xylose as the substrate reveals a lowered yield coefficient for glycerol under conditions of the mixed sugar carbon source. Interestingly, even the total amount of "redox sink" products, that is glycerol + xylitol, was smaller during utilization of glucose-xylose (~0.11 mol/mol total sugar consumed) than the glycerol produced from glucose alone (~0.14 mol/mol). The low value of Y
acetate (< 0.001 g/g) in either fed-batch fermentation indicates that production of NADPH via the acetate pathway was negligible. Release of CO2 was similar in both fermentations, suggesting that formation of NADPH in the oxidative pentose phosphate pathway cannot have been significantly elevated in the presence of glucose-xylose as compared to glucose alone. There is, therefore, no evidence of formation of excess NADH in the conversion of xylose to xylulose by BP10001, supporting the notion that the XR used functions as an NADH-dependent enzyme in vivo.
Novel lessons from FBA using a genome-scale metabolic model
It is interesting to compare the results of FBA for upper and lower boundary conditions with respect to the consumption of NADPH for xylose reduction (Figure 6). In the batch fermentation of xylose, usage of 36% NADPH by XR resulted in a high flux (0.3 mol/mol xylose) from pyruvate to oxalacetate. In the fed-batch co-fermentation of glucose and xylose, the assumption of a solely NADPH-dependent reaction of XR was reflected by a similarly high flux (0.33 mol/mol sugar) towards oxalacetate. The flux pyruvate→ oxalacetate was decreased when it was assumed that q
xylitol equaled q
NADPH in the XR reaction. The lowest flux towards oxalacetate (< 0.10 mol/mol sugar) was calculated for the condition of an NADH-specific XR. Wahlbom et al. used S. cerevisiae strain TMB 3001, which is similar to our strain BP000 in that it overexpresses genes (from P. stipitis) encoding NAD(P)H-dependent XR and NAD+-dependent XDH, and applied data from chemostat fermentations of glucose (20 g/L) and glucose-xylose (5 and 15 g/L; 10 g/L each) to FBA using a condensed metabolic model . It is unfortunately not clear how these authors handled the issue of XR coenzyme preference in the FBA. However, the flux pyruvate→ oxalacetate was low (< 0.10 mol/mol sugar) for strain TMB 3001 irrespective of the substrate conditions used () and corresponded to the flux calculated for BP10001 with the assumption of an NADH-dependent XR. Pitkänen et al. applied FBA to S. cerevisiae strain H2490 which like TMB 3001 overexpresses wild-type genes for P. stipitis XR and XDH . Using a fixed 1:1 ratio for NADPH and NADH usage by XR, these authors calculated a similarly low flux pyruvate→ oxalacetate (0.02 mol/mol) . In agreement with Wahlbom et al. , we find that the relative flux towards oxalacetate was identical for fed-batch fermentations using glucose or glucose-xylose (NADH-dependent XR).
Strains TMB 3001  and H2490  displayed enhanced flux through the oxidative pentose phosphate pathway when xylose was present in the medium, an effect ascribed to the requirement for regeneration of the NADPH used up in the XR reaction. Consistent with this notion, application of a mutated XR (from P. stipitis) that showed a higher preference for NADH than the wild-type enzyme , resulted in a comparatively lowered flux from glucose 6-phosphate to ribulose 5-phosphate. However, the FBA shown in Figure 6 predicts that only 2 - 5 mol% of total sugar is metabolized by BP10001 via the oxidative pentose phosphate pathway when it is assumed that XR utilizes NADH only. The relative flux through the oxidative pentose phosphate pathway increases dramatically to 40% under conditions of the fed-batch co-fermentation of glucose and xylose, assuming XR to be dependent on NADPH. The relevant figure is 14% given that q
xylitol equaled q
NADPH in the XR reaction. A positive correlation between the predicted fluxes glucose 6-phosphate→ ribulose 5-phosphate and pyruvate→ oxalacetate was noted, probably indicating that the CO2 lost in the oxidative pentose phosphate pathway is formally re-incorporated through synthesis of oxalacetate. This suggestion from FBA is very unlikely to reflect the true in vivo situation, and we conclude therefore that results in Figure 6 are most consistent with an XR reaction that depends on NADH.
Beyond coenzyme recycling: the role of q
Figure 1 implies that in BP10001, the distribution of fermentation products from xylose is favourably affected by an increase in q
xylose. We have shown in a recent paper that S. cerevisiae strain BP11001 expressing an engineered pair of XR (from C. tenuis) and XDH (from G. mastotermitis) having almost completely matched in vitro coenzyme specificities fermented xylose less efficiently in terms of both yield and productivity than BP10001 . The tentative explanation, now corroborated by Figure 1, was that the mutated XDH, which was just ~1/10 as active as the wild-type enzyme, introduced an extra kinetic bottleneck that irrespective of the presumed near-perfect recycling of NAD(P)H during conversion of xylose into xylulose caused Y
xylitol to increase as compared to strain BP10001 . Like coenzyme recycling, kinetic "pull" to remove xylitol, the thermodynamically favoured intermediate product of the two-step oxidoreductive isomerization of xylose into xylulose, appears to be an additional critical factor that controls Y
xylitol. The importance for XDH to be present in excess (≥ 10-fold) over XR was recognized by Hahn-Hägerdal and co-workers before .
We observed herein and in previous works that q
xylose decreased slowly during the course of conversion of xylose [8, 9]. Loss of cell viability and inactivation of xylose pathway enzymes (XR, XDH, XK) were ruled out as possible causes for the drop in xylose consumption rate (this work). Xylose transport could be an issue although there is currently no clear evidence suggesting its importance as a rate-determining factor in BP10001. A plausible, yet speculative explanation is that because of its high K
m for xylose (~100 mM) , the XR is difficult to saturate with substrate and therefore becomes an increasingly less efficient catalyst for xylose reduction as the fermentation progresses. However, despite supporting findings from the work of other groups, a quantitative relationship between the level of XR activity and q
xylose remains to be demonstrated [29, 35]. Notwithstanding, further optimization of xylose-fermenting strains of S. cerevisiae should consider q
xylose (see below). Moreover, interpretation of experimental yield coefficients (e.g. Y
xylitol) should not disregard the possibility that observations may be complex manifestations of the combined effects of the intracellular redox balance and the substrate consumption rate.
Enhancement of q
xylose at low levels of glucose: observations and process-related opportunities
Results for BP10001 confirm the notion from a number of prior studies on xylose-fermenting strains of S. cerevisiae that glucose inhibits the utilization of xylose (e.g. [17, 19, 27]). Fewer studies, however, have so far addressed the role of a low glucose level on enhancing q
xylose [15, 17, 20]. Measurement of xylose consumption in the presence of a small concentration of glucose presents a challenge to both the experimental set-up and the analytical tools used. Despite notable efforts (e.g. ), therefore, the q
xylose-stimulating effect of glucose has not been fully analyzed and its occurrence is sometimes related to a glucose concentration "greater than zero". Suggestions for its molecular interpretation include the induction of relevant sugar transport proteins in S. cerevisiae at low glucose and the proposal that in order to drive xylose assimilation via the pentose phosphate pathway the cell needs to maintain a certain amount of glycolytic flux (see later) [17, 36].
It was determined herein from results of a controlled fed-batch fermentation in which glucose was available in a q
xylose-enhancing concentration of below 0.3 g/L that xylose uptake by BP10001 was accelerated about twofold as compared to reference reaction using xylose alone. The value of 0.30 ± 0.04 g/g CDW/h obtained for q
xylose under the fed-batch conditions was identical with limits of error to the xylose uptake rate of 0.29 g/g CDW/h reported for strain TMB 3415 in a batch fermentation of 60 g/L xylose . Unlike BP10001, TMB 3415 incorporates a substantial history of strain optimization including overexpression of genes encoding all enzymes of the non-oxidative pentose phosphate pathway and deletion of GRE3 (a non-specific NADPH-dependent aldose reductase that reduces xylose) . Therefore, design of process conditions could complement genetic approaches of strain engineering that aim at optimizing q
xylose. It is also worth noting that conditions used in the fed-batch process may not be too different from the situation encountered during SSF of pretreated lignocellulose [21, 38]. The often used high-temperature pretreatment at mildly acidic conditions liberates most of the xylan fraction as xylose while leaving the cellulose unhydrolysed. The relatively slow action of subsequently added cellulases provides the "glucose feed" for glucose-xylose co-fermentation by the ethanologenic yeast. Innovative strategies for controlling the release of glucose in SSF include pulsed addition of substrate or feeding of cellulases [39, 40]. Maintenance of a constant glucose release rate is expected to ensure constant glucose uptake by the yeast cells, which normally do not grow in lignocellulose hydrolysates used. The fed-batch scheme developed herein presents a novel and significant addition to the overall concept of enhancing q
xylose by a low concentration of glucose. It is conducive to the accurate determination of q
xylose at a constant q
glucose under conditions in which yeast cells are growing. We expect that for obvious practical reasons, an initial evaluation of novel yeast strains will always be done in synthetic media based on soluble substrates. We hope therefore that others will find the results in Figure 5 useful with respect to an application-oriented physiological characterization of their yeast strains. An interesting finding for BP10001 is that the molar ratio (2.6 : 1) of glucose and xylose utilized in the fed-batch fermentation nicely matches the relative content of these sugars in common lignocellulosic feedstocks (e.g. corn stover, 2.2 : 1; rice straw, 2.5 : 1 ).
The results of FBA (Figure 6; NADH-dependent XR) provide a useful picture about the flux changes in BP10001 that may result upon switch from xylose fermentation in batch to glucose-xylose co-fermentation in the fed-batch. The presence of a low glucose concentration is predicted to bring about substantial enhancement of flux through different steps of the pentose phosphate pathway (non-oxidative: ~2-fold; oxidative: ~10-fold) and glycolysis (~10-fold) as compared to xylose-only reaction conditions. Furthermore, it prevents a small "back-flux" from fructose 6-phosphate to glucose 6-phosphate, occurring when only xylose is present, from taking place. Figure 6 is in line with the idea that accumulation of glycolytic and pentose phosphate intermediates facilitates "pull" of xylose into the metabolism, through the law of mass action as well as by inducing a global cellular response that affects both the level of transcription of key metabolic genes (e.g. hexose transporters , glycolytic and ethanologenic enzymes [17, 42]) and the protein level . Studies employing various "omics" techniques have demonstrated that S. cerevisiae recognizes glucose very differently from xylose as substrate for alcoholic fermentation [17, 18, 20, 36, 37, 43, 44]. However, the major rate-limiting factors in xylose fermentation are unfortunately still elusive.