Chemical and Synthetic Genetic Array Analysis Identifies Genes that Suppress Xylose Utilization and Fermentation in Saccharomyces cerevisiae

Abstract

Though highly efficient at fermenting hexose sugars, Saccharomyces cerevisiae has limited ability to ferment five-carbon sugars. As a significant portion of sugars found in cellulosic biomass is the five-carbon sugar xylose, S. cerevisiae must be engineered to metabolize pentose sugars, commonly by the addition of exogenous genes from xylose fermenting fungi. However, these recombinant strains grow poorly on xylose and require further improvement through rational engineering or evolutionary adaptation. To identify unknown genes that contribute to improved xylose fermentation in these recombinant S. cerevisiae, we performed genome-wide synthetic interaction screens to identify deletion mutants that impact xylose utilization of strains expressing the xylose isomerase gene XYLA from Piromyces sp. E2 alone or with an additional copy of the endogenous xylulokinase gene XKS1. We also screened the deletion mutant array to identify mutants whose growth is affected by xylose. Our genetic network reveals that more than 80 nonessential genes from a diverse range of cellular processes impact xylose utilization. Surprisingly, we identified four genes, ALP1, ISC1, RPL20B, and BUD21, that when individually deleted improved xylose utilization of both S. cerevisiae S288C and CEN.PK strains. We further characterized BUD21 deletion mutant cells in batch fermentations and found that they produce ethanol even the absence of exogenous XYLA. We have demonstrated that the ability of laboratory strains of S. cerevisiae to utilize xylose as a sole carbon source is suppressed, which implies that S. cerevisiae may not require the addition of exogenous genes for efficient xylose fermentation.

Xylose Fermentation by Saccharomyces cerevisiae: Challenges and Prospects

“…Furthermore, these genetic modifications more directly linked to xylose consumption pathway, and there are some others genes detected through synthetic genomic array (SGA) whose modification affected xylose consumption. SGA identified four genes ( ALP1 , arginine transporter; ISC1 , mitochondrial membrane-localized inositol phosphosphingolipid phospholipase; RPL20B , component of 60S larger ribosomal subunit; BUD21 , a component of small ribosomal subunit) that, when individually deleted, improved xylose consumption to levels similar to those of recombinant XI/XK yeasts [ 59 ]. Although it is not clear how these genes act on xylose metabolism this result shows that adjustments on these unpredicted targets are important in order to obtain improvements in xylose consumption.…”

Metabolic engineering of yeasts by heterologous enzyme production for degradation of cellulose and hemicellulose from biomass: a perspective

“…Deletion of YLR042C, MNI1, and RPA49, resulted in an improvement in growth rates on xylose (Bengtsson et al, 2008), while deletion of PHO13, ALP1, ISC1 RPL20B, BUD21, NQM1, TKL2 led to increased ethanol yields from xylose (Van Vleet et al, 2008; Usher et al, 2011). …”

“…To facilitate the incorporation of xylose utilization into CBP, efforts have focussed on introducing xylose metabolic pathways from other species into natural ethanologenic Saccharomyces sp. (Karhumaa et al, 2007 ; Matsushika et al, 2009a , b ; Fernandes and Murray, 2010 ; Bera et al, 2011 ; Hasunuma et al, 2011 ; Hector et al, 2011 ; Usher et al, 2011 ; Xiong et al, 2011 ; Cai et al, 2012 ; Fujitomi et al, 2012 ; Kim et al, 2012 ; Tien-Yang et al, 2012 ; De Figueiredo Vilela et al, 2013 ; Demeke et al, 2013 ; Hector et al, 2013 ; Ismail et al, 2013 ; Kato et al, 2013 ; Kim et al, 2013 ). This topic has been reviewed recently (Matsushika et al, 2009a ; Fernandes and Murray, 2010 ; Cai et al, 2012 ) and so will not be extensively covered here.…”

Rational and Evolutionary Engineering Approaches Uncover a Small Set of Genetic Changes Efficient for Rapid Xylose Fermentation in Saccharomyces cerevisiae

“…Numerous studies developed strains with improved xylose utilization through laboratory evolution [33], [34], [35], [36]. Genome library screening, random transposon mutagenesis, and transcriptome analysis identified a number of candidate genes that could be deleted or overexpressed for improved xylose metabolism [37], [38], [39], [40], [41]. For example, overexpression of XYL2 , XKS1 , or TAL1 was sufficient to improve xylose fermentation [38], [39], [41].…”