Abstract:BackgroundThe sugar sensing and carbon catabolite repression in Baker’s yeast Saccharomyces cerevisiae is governed by three major signaling pathways that connect carbon source recognition with transcriptional regulation. Here we present a screening method based on a non-invasive in vivo reporter system for real-time, single-cell screening of the sugar signaling state in S. cerevisiae in response to changing carbon conditions, with a main focus on the response to glucose and xylose.ResultsThe artificial reporte… Show more
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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).…”
Bioconversion of xylose—the second most abundant sugar in nature—into high‐value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long‐term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially‐targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed‐batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value‐added products.
“…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.…”
Section: Discussionmentioning
confidence: 99%
“…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).…”
Bioconversion of xylose—the second most abundant sugar in nature—into high‐value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long‐term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially‐targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed‐batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value‐added products.
“…5). Lastly, as extracellular xylose cannot sufficiently interact with Snf1p [134], a sensor protein that downregulates the expression of ACC1 and upregulates β-oxidation [133], xylose culture could prevent inhibition of fatty acid synthesis and degradation of acyl-CoA.Fig. 5Schematic comparison between glucose ( orange ) and xylose ( blue ) metabolisms in engineered S. cerevisiae .…”
Section: Production Of Advanced Biofuels and Chemicals From Xylose Bymentioning
Efficient xylose utilization is one of the most important pre-requisites for developing an economic microbial conversion process of terrestrial lignocellulosic biomass into biofuels and biochemicals. A robust ethanol producing yeast Saccharomyces cerevisiae has been engineered with heterologous xylose assimilation pathways. A two-step oxidoreductase pathway consisting of NAD(P)H-linked xylose reductase and NAD+-linked xylitol dehydrogenase, and one-step isomerase pathway using xylose isomerase have been employed to enable xylose assimilation in engineered S. cerevisiae. However, the resulting engineered yeast exhibited inefficient and slow xylose fermentation. In order to improve the yield and productivity of xylose fermentation, expression levels of xylose assimilation pathway enzymes and their kinetic properties have been optimized, and additional optimizations of endogenous or heterologous metabolisms have been achieved. These efforts have led to the development of engineered yeast strains ready for the commercialization of cellulosic bioethanol. Interestingly, xylose metabolism by engineered yeast was preferably respiratory rather than fermentative as in glucose metabolism, suggesting that xylose can serve as a desirable carbon source capable of bypassing metabolic barriers exerted by glucose repression. Accordingly, engineered yeasts showed superior production of valuable metabolites derived from cytosolic acetyl-CoA and pyruvate, such as 1-hexadecanol and lactic acid, when the xylose assimilation pathway and target synthetic pathways were optimized in an adequate manner. While xylose has been regarded as a sugar to be utilized because it is present in cellulosic hydrolysates, potential benefits of using xylose instead of glucose for yeast-based biotechnological processes need to be realized.
“…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 .…”
Extending the host substrate range of industrially relevant microbes, such as Saccharomyces cerevisiae, has been a highly-active area of research since the conception of metabolic engineering. Yet, rational strategies that enable non-native substrate utilization in this yeast without the need for combinatorial and/or evolutionary techniques are underdeveloped. Herein, this review focuses on pentose metabolism in S. cerevisiae as a case study to highlight the challenges in this field. In the last three decades, work has focused on expressing exogenous pentose metabolizing enzymes as well as endogenous enzymes for effective pentose assimilation, growth, and biofuel production. The engineering strategies that are employed for pentose assimilation in this yeast are reviewed, and compared with metabolism and regulation of native sugar, galactose. In the case of galactose metabolism, multiple signals regulate and aid growth in the presence of the sugar. However, for pentoses that are non-native, it is unclear if similar growth and regulatory signals are activated. Such a comparative analysis aids in identifying missing links in xylose and arabinose utilization. While research on pentose metabolism have mostly concentrated on pathway level optimization, recent transcriptomics analyses highlight the need to consider more global regulatory, structural, and signaling components.
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