Assessing the effect of d-xylose on the sugar signaling pathways of Saccharomyces cerevisiae in strains engineered for xylose transport and assimilation

Abstract

One of the challenges of establishing an industrially competitive process to ferment lignocellulose to value-added products using Saccharomyces cerevisiae is to get efficient mixed sugar fermentations. Despite successful metabolic engineering strategies, the xylose assimilation rates of recombinant S. cerevisiae remain significantly lower than for the preferred carbon source, glucose. Previously, we established a panel of in vivo biosensor strains (TMB371X) where different promoters (HXT1/2/4p; SUC2p, CAT8p; TPS1p/2p, TEF4p) from the main sugar signaling pathways were coupled with the yEGFP3 gene, and observed that wild-type S. cerevisiae cannot sense extracellular xylose. Here, we expand upon these strains by adding a mutated galactose transporter (GAL2-N376F) with improved xylose affinity (TMB372X), and both the transporter and an oxidoreductase xylose pathway (TMB375X). On xylose, the TMB372X strains displayed population heterogeneities, which disappeared when carbon starvation was relieved by the addition of the xylose assimilation pathway (TMB375X). Furthermore, the signal in the TMB375X strains on high xylose (50 g/L) was very similar to the signal recorded on low glucose (≤5 g/L). This suggests that intracellular xylose triggers a similar signal to carbon limitation in cells that are actively metabolizing xylose, in turn causing the low assimilation rates.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

“…Glucose induces the Crabtree effect in S. cerevisiae (Diaz‐Ruiz, Rigoulet, & Devin, ) and is known to repress mitochondria biogenesis compared with nonfermentative carbon sources such as glycerol (Egner et al, ). In contrast, xylose is not recognized as a fermentable carbon source and induces a respiratory response in yeast engineered to consume it (Belinchón & Gancedo, ; Brink, Borgström, Tueros, & Gorwa‐Grauslund, ; Jin et al, ; Osiro, Borgström, Brink, Fjölnisdóttir, & Gorwa‐Grauslund, ; Osiro et al, ). Considering that the isobutanol production pathway in SR8‐Iso is compartmentalized in the mitochondria, it is likely that increased mitochondrial biogenesis is a major contributor to the enhanced isobutanol yields from xylose we observe.…”

“…Glucose induces the Crabtree effect in S. cerevisiae (Diaz-Ruiz, Rigoulet, & Devin, 2011) and is known to repress mitochondria biogenesis compared with nonfermentative carbon sources such as glycerol (Egner et al, 2002). In contrast, xylose is not recognized as a fermentable carbon source and induces a respiratory response in yeast engineered to consume it (Belinchón & Gancedo, 2003;Brink, Borgström, Tueros, & Gorwa-Grauslund, 2016;Jin et al, 2004;Osiro, Borgström, Brink, Fjölnisdóttir, & Gorwa-Grauslund, 2019;Osiro et al, 2018).…”

Reassessment of requirements for anaerobic xylose fermentation by engineered, non-evolved Saccharomyces cerevisiae strains

“…In contrast to glucose, xylose does not fully activate the cAMP/PKA pathway (Osiro et al. 2018 ). In comparison with other S. cerevisiae genetic backgrounds, CEN.PK strains carry many sequence differences in genes involved in this signal-transduction pathway (Vanhalewyn et al.…”

Pentose Metabolism in Saccharomyces cerevisiae: The Need to Engineer Global Regulatory Systems

“…Recently, the Gorwa‐Grauslund lab conducted several studies on xylose‐sensing. They assessed the effect of xylose when GFP was expressed under various promoters in strains that can or cannot metabolize xylose . Their results demonstrated that extracellular xylose was not detected by S. cerevisiae .…”