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

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 nextgeneration biofuel production.

Comparison of xylose fermentation by two high-performance engineered strains of Saccharomyces cerevisiae

“…It is worth noting that the XR/XDH pathway has been shown to produce much less xylitol or no xylitol when xylodextrins are used rather than xylose in fermentations [13], or when undetoxified lignocellulose hydrolysate is used [6]. Thus, xylodextrin consumption by means of the XR/XDH pathway could result in yeast strains with both high ethanol productivity and yield, without the drawback of xylitol byproduct formation.…”

Screening and directed evolution of transporters to improve xylodextrin utilization in the yeastSaccharomyces cerevisiae

“…which has led to many efforts to relieve this inhibition [21,22]. We had previously observed that cellobiose inhibits xylobiose transport by CDT-2 [7]. We therefore tested xylobiose transport activity in the presence of increasing molar concentrations of cellobiose, up to 10 times the concentration of X2.…”

“…We therefore chose ST16, which is derived from Trichoderma virens, as the starting point for setting up a directed evolution experiment to improve XD consumption. Based on experiments published previously, in which XD consumption was rapidly turned on and off in anaerobic conditions [7], we hypothesized that XD transport is limiting in terms of consumption rate. Notably, when we inserted the transporter ST16 under control of PGK1 promoter and TPS3 terminator into the yeast genome at the LEU2 locus, there was a dramatic decrease in the growth rate compared with overexpression ST16 from a plasmid.…”

“…CDT-1 and CDT-2 are two important cellodextrin transporters used by N. crassa in cellulose degradation [25], of which only CDT-2 is also involved in hemicellulose utilization [7,26]. In previous results, the discovery of the xylodextrin consumption pathway consisting of transporter CDT-2 along with two β-xylosidases GH43-2 and GH43-7 provided new modes for utilization of xylose derived from hemicellulose [7].…”

“…However, the xylan degrading and xylose utilization abilities of this recombinant S. cerevisiae strain requires further optimization to be industrially useful. Recently, a XD utilization pathway from N. crassa, which requires a transporter CDT-2 along with two intracellular β-xylosidases GH43-2 and GH43-7, was identified and subsequently engineered into S. cerevisiae, enabling the yeast to grow aerobically on XD, or co-ferment XDs with xylose or other hexose sugars [7]. However, the consumption rate of XDs remains quite slow and thus needs to be improved for future industrial applications.…”