An improved method of xylose utilization by recombinant Saccharomyces cerevisiae

Abstract

The aim of this study was to develop a method to optimize expression levels of xylose-metabolizing enzymes to improve xylose utilization capacity of Saccharomyces cerevisiae. A xylose-utilizing recombinant S. cerevisiae strain YY2KL, able to express nicotinamide adenine dinucleotide phosphate, reduced (NADPH)-dependent xylose reductase (XR), nicotinamide adenine dinucleotide (NAD(+))-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK), showed a low ethanol yield and sugar consumption rate. To optimize xylose utilization by YY2KL, a recombinant expression plasmid containing the XR gene was transformed and integrated into the aur1 site of YY2KL. Two recombinant expression plasmids containing an nicotinamide adenine dinucleotide phosphate (NADP(+))-dependent XDH mutant and XK genes were dually transformed and integrated into the 5S ribosomal DNA (rDNA) sites of YY2KL. This procedure allowed systematic construction of an S. cerevisiae library with different ratios of genes for xylose-metabolizing enzymes, and well-grown colonies with different xylose fermentation capacities could be further selected in yeast protein extract (YPX) medium (1 % yeast extract, 2 % peptone, and 2 % xylose). We successfully isolated a recombinant strain with a superior xylose fermentation capacity and designated it as strain YY5A. The xylose consumption rate for strain YY5A was estimated to be 2.32 g/gDCW/h (g xylose/g dry cell weight/h), which was 2.34 times higher than that for the parent strain YY2KL (0.99 g/gDCW/h). The ethanol yield was also enhanced 1.83 times by this novel method. Optimal ratio and expression levels of xylose-metabolizing enzymes are important for efficient conversion of xylose to ethanol. This study provides a novel method that allows rapid and effective selection of ratio-optimized xylose-utilizing yeast strains. This method may be applicable to other multienzyme systems in yeast.

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

“…Hence, a 1:1:1 ratio of the three classes of enzymes may not yield an optimum hydrolysis synergy. Yamada and colleagues performed repeated rounds of integration at delta sites to generate an S. cerevisiae strain with 24 cellulase genes integrated into the genome in a ratio of 16:6:2 for egl2, cbh2 , and bgl 1, respectively (Tien-Yang et al, 2012). Cellulose degradation activity of this strain was lower than in a strain which had the cellulase genes egl2, cbh2 , and bgl 1 in a ratio of 13:6:1, respectively.…”

“…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.…”

Xylitol production from non-detoxified Napiergrass hydrolysate using a recombinant flocculating yeast strain

“…S. cerevisiae strain YYA1 (ADH2::PGKlp-CXYLl-ADHlt, XKSlp::PGKp, HO::PGKIp-XYL2-ADHlt, Migl::TEFp-TAL1-TEFt) was used for cloning of the xylose reductase gene (XYL1) from C. guilliermondii (hereafter "CXYL1") (Ma et al 2012). The flocculent strain WLP 550 was purchased from White Labs (San Diego, CA) and used as a host for xylitol production.…”

“…In a previous study (Ma et al 2012), a genetically engineered a strain of xyloseutilizing Saccharomyces cerevisiae was developed with improved capacity for converting xylose into ethanol. Numerous efforts have focused on the initial xylose metabolic pathway in S. cerevisiae for its critical role in xylose utilization.…”

Heterologous secretory expression of β-glucosidase from Thermoascus aurantiacus in industrial Saccharomyces cerevisiae strains

“…Conversion of L-arabinose into D-xylulose-5-phosphate is based on heterologous expression of bacterial genes encoding L-arabinose isomerase (AraA), L-ribulokinase (AraB), and L-ribulose-5-phosphate-4epimerase (AraD). Further improvements on alcoholic fermentation of these most abundant pentoses have been obtained through directed-evolution strategies aimed to accumulate spontaneous beneficial mutation [5][6][7]. A redox engineering study has revealed that deletion of S. cerevisiae genes encoding glycerol-3-phosphate dehydrogenase and expression of an acetylating acetaldehyde dehydrogenase from Escherichia coli (A-ALD) allow researchers to achieve conversion of inhibitory acetic acid to ethanol and to eliminate glycerol formation in anaerobic cultures of yeast [3].…”