Expanding xylose metabolism in yeast for plant cell wall conversion to biofuels.

Xin Li,Elizabeth A Znameroski,Kulika Chomvong,N Louise Glass,Jamie Hd Cate,Soo Rin Kim,Joanna Feehan,Julie M Liang,Raíssa Estrela,Annsea Park,Vivian Yaci Yu,Yong-Su Jin,Yuping Lin

doi:10.7554/elife.05896

Abstract

Sustainable biofuel production from renewable biomass will require the efficient and complete use of all abundant sugars in the plant cell wall. Using the cellulolytic fungus Neurospora crassa as a model, we identified a xylodextrin transport and consumption pathway required for its growth on hemicellulose. Reconstitution of this xylodextrin utilization pathway in Saccharomyces cerevisiae revealed that fungal xylose reductases act as xylodextrin reductases, producing xylosyl-xylitol oligomers as metabolic intermediates. These xylosyl-xylitol intermediates are generated by diverse fungi and bacteria, indicating that xylodextrin reduction is widespread in nature. Xylodextrins and xylosyl-xylitol oligomers are then hydrolyzed by two hydrolases to generate intracellular xylose and xylitol. Xylodextrin consumption using a xylodextrin transporter, xylodextrin reductases and tandem intracellular hydrolases in cofermentations with sucrose and glucose greatly expands the capacity of yeast to use plant cell wall-derived sugars and has the potential to increase the efficiency of both first-generation and next-generation biofuel production.

Highlights

Cellodextrins and xylodextrins derived from plant cell walls are not catabolized by wild-type S. cerevisiae (Matsushika, Inoue et al 2009, Galazka, Tian et al 2010, Young, Lee et al 2010)
By using transcription profiling data (Tian, Beeson et al 2009) and by analyzing growth phenotypes of N. crassa knockout strains, we identified separate pathways used by N. crassa to consume cellodextrins (Galazka, Tian et al 2010) and xylodextrins released by its secreted enzymes (Figure 1A and Figure 1—figure supplement 1) (Method 2014)
To test whether S. cerevisiae could utilize xylodextrins, a S. cerevisiae strain was engineered with the xylose reductase (XR)/xylitol dehydrogenase (XDH) pathway derived from Scheffersomyces stipitis–similar to the one utilized by N. crassa (Sun, Tian et al 2012)–and a xylodextrin transport and consumption pathway from N. crassa

Summary

Introduction

Cellodextrins and xylodextrins derived from plant cell walls are not catabolized by wild-type S. cerevisiae (Matsushika, Inoue et al 2009, Galazka, Tian et al 2010, Young, Lee et al 2010). Analyses of the supernatants from cultures with xylodextrins surprisingly revealed that the xylodextrins were converted into xylosyl-xylitol oligomers, a set of previously unknown compounds, by the engineered yeast, rather than hydrolyzed to xylose and consumed (Figure 2A and Figure 2—figure supplement 1). To test whether the xylosyl-xylitol oligomers resulted from side reactions of xylodextrins with endogenous S. cerevisiae enzymes, we used two separate yeast strains in a combined culture, one containing the xylodextrin hydrolysis pathway composed of CDT-2 and GH43-2, and the second engineered with the XR/XDH xylose consumption pathway.

Results

Conclusion