In the present work we show that by increasing the intracellular concentration of glutathione, strain robustness could be improved in industrially relevant conditions posed by pretreated spruce in an SSF process. As a mechanism for the improved robustness, we hypothesize a redox buffering capacity potentiating effect, originating from the increased availability of reduced glutathione. The enhanced redox buffering capacity would confer higher inherent capability of the intracellular environment to titrate cofactor consuming exogenous molecules. Indeed, at the time of writing this article, it was reported that furan aldehydes, and in particular furfural, depleted the intracellular pool of glutathione. This fact further corroborates the hypothesis of the redox potentiating effects of increasing the intracellular concentration of GSH
]. In the same paper, it was shown that overexpression of GSH1
resulted in a decreased lag-phase of growth in the presence of furfural, whereas no effect was observed in the case of HMF. In fact, similar strategies, such as overproduction of the intracellular protective metabolite and ROS scavenger ascorbic acid, have been shown to increase robustness to various environmental stresses
]. It is known that inhibitors such as HMF and furfural not only drain microorganisms of reducing power, but have also been shown to induce the generation of reactive oxygen species in S. cerevisiae
Quantification of the [GSH]:[GSSG] ratio and the half-cell redox potential Ehc
were used to assess the magnitude of the changes in intracellular redox environment upon overexpressing genes involved in the glutathione biosynthesis pathway. Ehc
, which is calculated from the Nernst equation (see Methods section), has been reported to be a more reliable method, as the stoichiometry of the glutathione redox couple (GSSG + 2H+
2GSH) is taken into account
is thus dependent on both the [GSH]:[GSSG] ratio and the absolute concentration of GSH, which means that cells having the same [GSH]:[GSSG] ratios can have different redox potentials depending on the concentration of GSH. Consequently, cells with a high concentration of intracellular reduced glutathione have a higher buffering capacity against oxidative insults than cells with a lower concentration of reduced glutathione.
Although overexpression of GSH1
increased the intracellular levels of reduced glutathione, unexpectedly none of the recombinant strains had a more reducing intracellular environment compared to the wild type when grown in defined mineral medium (as it would be indicated by a higher [GSH]:[GSSG] ratio and/or a lower Ehc
). In fact, the ultimate reason for the higher estimates of Ehc
in these strains was indeed the higher intracellular concentration of GSSG in all recombinant strains harboring the GSH1
overexpression construct. However, it has been shown in a recent study
] that the cytosolic glutathione redox potential was independent of changes in whole-cell GSSG levels due to compartmentalization of GSSG. The GSSG concentration was maintained at very low levels in the cytosol through the action of Ycf1p pumping GSSG to the vacuole. Hence, calculating the Ehc
using GSSG levels determined from whole cell-extracts as in the present study in fact overestimates the cytosolic redox potential. Moreover, it has recently been demonstrated that significant differences exist in the redox potential between different cellular compartments
]. At a purely speculative level, assuming an oxidation level of 0.03% of the cytosolic glutathione pool (as estimated by Østergaard et al.
]) for all strains, the estimated Ehc
values for all recombinant strains overexpressing GSH1
would in fact be lower than those in the wild type (-277 mV versus -273 mV), which then would indicate that GSH1
overexpression increases the redox buffering capacity and consequently strain robustness.
The increased redox buffering capacity of the strains overexpressing GSH1
was evident in SSF of pretreated spruce at 10% (w/w) water insoluble solids (WIS) concentration, which contains a spectrum of inhibitory compounds and in particular significant amounts of HMF and furfural (Table
]. All strains carrying the GSH1
overexpression construct were able to consume glucose and convert HMF and furfural for a longer period of time resulting in higher ethanol yields for these strains than the wild type. As overexpression of CYS3
in combination with GSH1
did not result in higher intracellular concentrations of glutathione than overexpression of GSH1
alone, the performance of this strain did not differ form the latter. An explanation could be that the high GSH levels obtained by GSH1
overexpression prevent a further increase of the intracellular glutathione concentration, since it is known that Gsh1p is feedback inhibited by GSH
]. Unexpectedly, overexpression of GLR1
in combination with GSH1
did not result in decreased levels of GSSG, possibly due to limitation of NADPH. Consequently, the GSH1/GLR1
strain performance was equivalent to that of the strain overexpressing GSH1
. In contrast, GLR1
overexpression alone was found to be a burden for the cells when cultivated in the spruce slurry, which was manifested in an earlier cessation of ethanol production than the wild type. Competition for NADPH between Glr1p and furan aldehyde detoxifying oxidoreductases could be a possible explanation for this observation.
Although integration of the overexpression constructs influenced the maximum specific growth rate, the difference between the strains was not severe. All recombinant strains carrying the GSH1
overexpression construct exhibited a lower specific growth rate than the wild type, and the strain overexpressing both GSH1
grew at the lowest specific growth rate. The lower specific growth rates can be explained by a combination of metabolic burden on the cell imposed by the overexpression of the introduced genetic constructs, and uncharacterized effects on strain growth behavior commonly encountered as a consequence of using auxotrophic markers. There is also a possibility that the decrease in specific growth rate in the strains overexpressing GSH1
is connected to partial depletion of cysteine due to overproduction of glutathione. In fact, overexpression of CYS3
somewhat restored the specific growth rate in strains overexpressing GSH1
). On the other hand, these small changes in specific growth rate may be of minor importance for industrial bioethanol production, since the microorganisms are thought to reside in a non-growing state in a process such as SSF
]. Therefore, an engineering strategy that results in a small decrease in specific growth rate may not be unfavorable as long as the microorganism is more tolerant to the process conditions.
Overall, the results from this study show that engineering of S. cerevisiae with increased intracellular glutathione production has a beneficial effect on strain robustness by extending the time of survival in an SSF process using a challenging substrate. The mechanism for the increased tolerance supposedly originates from an increased redox buffering capacity resulting from the increased pool of reduced glutathione in the recombinant strains.