Improved Xylose Metabolism by a CYC8 Mutant of Saccharomyces cerevisiae

Abstract

Engineering Saccharomyces cerevisiae for the utilization of pentose sugars is an important goal for the production of second-generation bioethanol and biochemicals. However, S. cerevisiae lacks specific pentose transporters, and in the presence of glucose, pentoses enter the cell inefficiently via endogenous hexose transporters (HXTs). By means of in vivo engineering, we have developed a quadruple hexokinase deletion mutant of S. cerevisiae that evolved into a strain that efficiently utilizes D-xylose in the presence of high D-glucose concentrations. A genome sequence analysis revealed a mutation (Y353C) in the general corepressor CYC8, or SSN6, which was found to be responsible for the phenotype when introduced individually in the nonevolved strain. A transcriptome analysis revealed altered expression of 95 genes in total, including genes involved in (i) hexose transport, (ii) maltose metabolism, (iii) cell wall function (mannoprotein family), and (iv) unknown functions (seripauperin multigene family). Of the 18 known HXTs, genes for 9 were upregulated, especially the low or nonexpressed HXT10, HXT13, HXT15, and HXT16. Mutant cells showed increased uptake rates of D-xylose in the presence of D-glucose, as well as elevated maximum rates of metabolism (V max ) for both D-glucose and D-xylose transport. The data suggest that the increased expression of multiple hexose transporters renders D-xylose metabolism less sensitive to D-glucose inhibition due to an elevated transport rate of D-xylose into the cell. IMPORTANCEThe yeast Saccharomyces cerevisiae is used for second-generation bioethanol formation. However, growth on xylose is limited by pentose transport through the endogenous hexose transporters (HXTs), as uptake is outcompeted by the preferred substrate, glucose. Mutant strains were obtained with improved growth characteristics on xylose in the presence of glucose, and the mutations mapped to the regulator Cyc8. The inactivation of Cyc8 caused increased expression of HXTs, thereby providing more capacity for the transport of xylose, presenting a further step toward a more robust process of industrial fermentation of lignocellulosic biomass using yeast.KEYWORDS sugar transporter, xylose transport, evolutionary engineering, transcriptome, yeast A n increasing energy demand and concerns of obtaining this energy from fossil fuels have stimulated the development of liquid fuels from renewable feedstock. Bioethanol, mostly used as a fuel additive, produced from readily fermentable agricultural feedstocks, such as sugar cane and corn, is less desired because the production of these feedstocks requires large amounts of arable land and competes with the food supply (1). A more sustainable source of feedstock is lignocellulosic biomass from hardwood, softwood, and agricultural residues (2). However, a major drawback of lignocellulosic feedstocks is the inability of the most commonly used yeast in industry,

Engineering of Pentose Transport in Saccharomyces cerevisiae for Biotechnological Applications

“…Evolutionary engineering aimed at improving growth on D-xylose led to the upregulation of Hxt2 (de Vilela et al, 2015). Similar results were obtained via a mutation of the glucose sensitive co-repressor Cyc8 causing the generic upregulation of virtually all hxt genes concomitantly with improved D-xylose transport (Nijland et al, 2017). Importantly, all of the abovementioned approaches indeed elevated the D-xylose flux into the cells but, as expected, do not result in sugar co-consumption as D-glucose remains to be the preferred substrate.…”

“…In these experimental approaches two different pentose utilizing yeast strains were employed: (1) a multiple hexokinase deletion strain which is unable to phosphorylate D-glucose and thus cannot grow on D-glucose. Such strains can be used to select for improved D-xylose transport in the presence of high concentrations of D-glucose (Farwick et al, 2014;Nijland et al, 2014Nijland et al, , 2017Reznicek et al, 2015;Shin et al, 2015;Li et al, 2016) and (2) a hexose transporter deletion strain in which the main, or even all, Hxt transporters have been deleted. This strain cannot grow on D-xylose because of an inability to transport this pentose.…”

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

“…While initial engineering attempts identified flux limitations in xylose isomerase and PPP shunt, further directed evolution and metabolic engineering have iteratively removed rate limitations in several of these metabolic reactions. Adaptive laboratory evolution (ALE) studies on extensively engineered strains have been identifying mutations in proteins that are involved in regulation, signaling, etc., insinuating the need to look at limitations beyond metabolic fluxes . Similarly, mutations or variations in expression of signaling pathway genes or regulatory genes have been shown to further increase growth rates in galactose…”

Structure-based directed evolution improves S. cerevisiae growth on xylose by influencing in vivo enzyme performance

“…Improving d-xylose metabolism is an ongoing challenge for optimizing second-generation bioethanol production by engineered strains of S. cerevisiae. Important improvements in yeast performance have been achieved by evolutionary and metabolic engineering, which can influence different steps in central metabolism [16,43], and by enhancing the xylose uptake system [44,45]. [12,13], but very high expression levels are needed for optimal performance, as indicated by gene amplification up to over 10 copies during adaptation [15,21], leading to production of xylose isomerase at up to 25% of the cellular protein (Fig.…”