A strain of S. cerevisiae expressing the fungal d-xylose pathway can ferment d-xylose to ethanol but only at a low rate and yield. To improve the rate and yield of d-xylose fermentation to ethanol, an NADP+-utilizing GAPDH (GDP1) was expressed and the gene for G6PDH (ZWF1) was deleted in order to shut down the oxidative part of the pentose phosphate pathway. As a result of these genetic modifications, the strain was converted from a mainly xylitol- and CO2-producing strain to a mainly ethanol-producing strain.
d-Xylose fermentation to equimolar amounts of CO2 and ethanol is redox neutral; however, in this pathway NADPH is utilized, which has to be regenerated elsewhere. The expression of an NADP-GAPDH provides such a mechanism for the regeneration of NADPH within the pathway. For the conversion of 3 mol of d-xylose to d-xylulose, 3 mol of NADPH and NAD+ have to be regenerated. Three moles of d-xylulose can lead to up to 5 mol of GAP; i.e., 3 mol of GAP must go through the NADP-GAPDH and 2 mol must go through the NAD-GAPDH, leaving 5 mol of NADH for the alcohol dehydrogenase reaction to produce equimolar amounts of ethanol and CO2. The simultaneous presence of NAD- and NADP-GAPDH activities allows the cell to automatically adjust the flux through these enzymes according to the cofactor requirement.
The main path for the regeneration of NADPH is the oxidative part of the pentose phosphate pathway. This path, in which the regeneration is furthermore coupled to CO2
production, competes with the NADP-GAPDH. To block this part of the pathway, we deleted the gene for the G6PDH, ZWF1
. A haploid strain of S. cerevisiae
has only one gene coding for such an enzyme, but the deletion of this enzyme has no major detrimental effects except that the sensitivity to oxidizing agents is elevated (12
). The combination of overexpression of GDP1
and deletion of zwf1
had a bigger effect than expression of GDP1
alone, indicating that the oxidative part of the pentose phosphate pathway was indeed a pathway competing for NADP+
, and by closing this pathway more NADP+
was forced through the NADP-GAPDH.
Since NADPH regeneration through NAPD-GAPDH is not coupled to CO2 production, anaerobic d-xylose fermentation by only this route would yield equimolar amounts of CO2 and ethanol; i.e., the CO2/ethanol ratio is 1. A higher CO2/ethanol ratio (under anaerobic conditions) is an indication that other routes for NADPH regeneration, which are coupled to CO2 production, are used. In the control strain H2674, this CO2/ethanol ratio was 2.5 (Table ), indicating that NADPH regeneration is coupled mainly to CO2 production. Through expression of GDP1 in combination with deletion of zwf1, this ratio was lowered to about 1.3, indicating that the NADPH regeneration was redirected to a non-CO2-producing reaction.
A high CO2/ethanol ratio was correlated with a low ethanol/xylitol ratio. When the NADPH is regenerated in a reaction, which is directly coupled to CO2 production, d-xylose fermentation to equimolar amounts of CO2 and ethanol is no longer redox neutral; i.e., the reaction will not occur. In the control strain H2674, the CO2/ethanol ratio is about 2.5. One mole of CO2 per mole of ethanol is produced in the pyruvate decarboxylase reaction; the rest is produced in other reactions, such as the oxidative reaction of the pentose phosphate pathway, where through CO2 production the redox balance is shifted so that more of a reduced product, i.e., xylitol, is formed. In strain H2684, with overexpression of GDP1 and deletion of zwf1, the CO2/ethanol ratio is 1.3; i.e., much less NADPH is regenerated in reactions coupled to CO2 production, which is reflected by less xylitol formation or a higher ethanol/xylitol ratio than that of the control strain (Table ).
In the strain with a zwf1
deletion, H2723, the CO2
/ethanol ratio was lower than in the control strain. It is not clear whether in this strain the NADPH is regenerated in an alternative way or whether the P. stipitis
-xylose reductase, which can use NADH, now uses it as its major coenzyme. Alternative ways to regenerate NADPH, which are not directly coupled to CO2
production, are transhydrogenases or transhydrogenase cycles such as the d
-mannitol cycle (20
), a cycle with glutamate dehydrogenases with different cofactor specificities (4
), or a cycle around the malic enzyme (2
). However there is no experimental support for the existence of natural transhydrogenase activities or transhydrogenase cycles in yeasts or molds (5
The effect of the zwf1
deletion alone on anaerobic d
-xylose fermentation in S. cerevisiae
was previously described by Jeppsson et al. (8
). Those authors tested a d
-glucose and d
-xylose cofermentation in continuous culture, which is difficult to compare with our batch fermentations; however, those authors also observed a lowered xylitol production.
In biotechnological applications, it is often a mixture of d-glucose and d-xylose and not pure d-xylose which has to be fermented. Under our conditions, all the d-glucose and parts of the d-xylose were fermented. By using radiolabeled d-xylose, we were able to distinguish between the ethanol produced from d-xylose and that produced from d-glucose. In these experiments the overexpression of GDP1 increased the ethanol yield. The yield was further improved by the combination of deleting zwf1 and overexpressing GDP1. The zwf1 deletion alone led to a lower ethanol production.
We overexpressed the genes of the d
-xylose pathway from P. stipitis
because P. stipitis
is one of the best d
-xylose-fermenting yeasts. One reason for this might be that the P. stipitis d
-xylose reductase can use NADH as a cofactor; however, it has a preference for NADPH (16
). In a strain where NADPH is efficiently regenerated, the ability of the P. stipitis
xylose reductase to use NADH as a cofactor would not be an advantage. This fact is of relevance since the P. stipitis
xylose reductase has a relatively low affinity for d
-xylose. Other d
-xylose reductases which are strictly NADPH dependent, such as the d
-xylose reductase from S. cerevisiae
, have a higher affinity for d
s, 27.9 mM for S. cerevisiae
and 42 mM for P. stipitis
). For an accelerated fermentation, especially of low concentrations of d
-xylose, it might be of advantage to use a d
-xylose reductase with a higher affinity for d
The ethanol production rate was about 0.2 and 0.4 mmol per g (dry mass) and h in the control strain H2674 and in strain H2684 (GDP1
), respectively (Fig. ). The volumetric productivities were not compared since the biomasses were different in the different fermentations. Through our modifications, which affected the redox reactions, the rate of ethanol production was not greatly improved. The specific ethanol production rates under anaerobic conditions from d
-xylose are still about 2 orders of magnitude lower than the corresponding rate on d
-glucose. There are several possible reasons for the low fermentation rate on d
-xylose. One is d
-xylose transport. Kötter and Ciriacy estimated the initial d
-xylose uptake rate under comparable conditions to be 100 nmol mg (fresh weight)−1
, corresponding to about 20 mmol g (dry mass)−1
). The d
-xylose utilization during fermentation was about 0.4 mmol g (dry mass)−1
; i.e., the d
-xylose transport capacity seems not to limit the d
-xylose fermentation rate, at least not at high d
-xylose concentrations. At a low d
-xylose concentration it can be different. For d
-xylose transport, a high-affinity system with a Km
of 190 mM and a low-affinity system with a Km
of 1.5 M were reported for S. cerevisiae
Other factors limiting the rate of fermentation might be a nonoptimal xylulokinase activity (9
) and a low capacity of reactions of the pentose phosphate pathway, such as a limiting activity of transaldolase (18
). Also, NADPH regeneration can still be limiting. Under our enzyme assay conditions, i.e., in the reverse reaction, the NADP-GAPDH activity of this enzyme was 0.3 nkat/mg of protein in the recombinant yeast, which compares to an endogenous NAD-GAPDH activity of 15 nkat/mg under similar conditions (23
). Three-tenths nanokatal per milligram corresponds to 2.7 mmol g (dry mass)−1
, assuming that 40% of the dry weight is extractable protein. The NADP-GAPDH activity was measured in the reverse direction, the thermodynamically favorable direction, with 1,3-bisphosphoglycerate as a substrate. The velocity in the forward direction with GAP as a substrate might be slower under in vivo conditions, and despite the improvements seen in this work, this step might indeed be rate limiting.
Although the rate of ethanol production was not greatly improved, there was a significant effect on the ethanol yield. Through redox engineering we were able to modify a strain that produced mainly xylitol and CO2 from d-xylose to a strain that produced mainly ethanol.