Pichia stipitis xylose reductase helps detoxifying lignocellulosic hydrolysate by reducing 5-hydroxymethyl-furfural (HMF)

Abstract

Background: Pichia stipitis xylose reductase (Ps-XR) has been used to design Saccharomyces cerevisiae strains that are able to ferment xylose. One example is the industrial S. cerevisiae xylose-consuming strain TMB3400, which was constructed by expression of P. stipitis xylose reductase and xylitol dehydrogenase and overexpression of endogenous xylulose kinase in the industrial S. cerevisiae strain USM21.

Results: In this study, we demonstrate that strain TMB3400 not only converts xylose, but also displays higher tolerance to lignocellulosic hydrolysate during anaerobic batch fermentation as well as 3 times higher in vitro HMF and furfural reduction activity than the control strain USM21. Using laboratory strains producing various levels of Ps-XR, we confirm that Ps-XR is able to reduce HMF both in vitro and in vivo. Ps-XR overexpression increases the in vivo HMF conversion rate by approximately 20%, thereby improving yeast tolerance towards HMF. Further purification of Ps-XR shows that HMF is a substrate inhibitor of the enzyme.

Conclusion: We demonstrate for the first time that xylose reductase is also able to reduce the furaldehyde compounds that are present in undetoxified lignocellulosic hydrolysates. Possible implications of this newly characterized activity of Ps-XR on lignocellulosic hydrolysate fermentation are discussed.

Figure 1

Fermentation profile of the control strain USM21 (top) and the xylose-fermenting strain TMB3400 (down) in batch fermentation of dilute-acid spruce hydrolysate. Strains were grown in defined mineral medium until biomass concentration reached approximately 2.5 g/L. Then hydrolysate was added at approximately time 18 hours (dashed line). CER is represented by the continuous line. Furfural (▲), HMF (●), Glucose (○), Ethanol (△). Data for USM21 is taken from [16].

Figure 2

Xylose (left graph), HMF (middle graph) and furfural (right graph) reduction activity measured in crude cell extracts from cells of strains USM21 and TMB3400. Cells were grown aerobically overnight in defined mineral medium. The assays were performed using NADH (top) or NADPH (bottom) as cofactor. The values are averages of two independent measurements. The bars represent the deviation from the average.

Figure 3

Xylose (left graph), HMF (middle graph) and furfural (right graph) reduction activity measured in crude cell extracts from cells of strains TMB3290 (no Ps-XR), TMB3001 (low Ps-XR) and TMB3260 (high Ps-XR). Cells were grown aerobically overnight in defined mineral medium. The assays were performed using NADH (top) or NADPH (bottom) as cofactor. The values are averages of two independent measurements. The bars represent the deviation from the average.

Figure 4

Glucose batch fermentation with the S. cerevisiae strains TMB3290 (control – top), TMB3001 (low Ps-XR – middle) and TMB3260 (high Ps-XR – bottow) in the presence of 2 g/L HMF. Glucose (○), HMF (●), biomass (□) and ethanol (△). The experiments were done in duplicate and the figure shows a representative profile for each strain.

Figure 5

Measured (black boxes) and model (line) values of HMF conversion rate obtained with Pichia stipitis xylose reductase.