Deletion of the GRE3 Aldose Reductase Gene and Its Influence on Xylose Metabolism in Recombinant Strains of Saccharomyces cerevisiae Expressing the xylA and XKS1 Genes

Abstract

Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified in S. cerevisiae. The GRE3 gene, encoding the AR enzyme, was deleted in S. cerevisiae CEN.PK2-1C, yielding YUSM1009a. XI from T. thermophilus was produced, and endogenous xylulokinase from S. cerevisiae was overproduced in S. cerevisiae CEN.PK2-1C and YUSM1009a. In recombinant strains from which the GRE3 gene was deleted, xylitol formation decreased twofold. Deletion of the GRE3 gene combined with expression of the xylA gene from T. thermophilus on a replicative plasmid generated recombinant xylose utilizing S. cerevisiae strain TMB3102, which produced ethanol from xylose with a yield of 0.28 mmol of C from ethanol/mmol of C from xylose. None of the recombinant strains grew on xylose.

Endogenous NADPH‐dependent aldose reductase activity influences product formation during xylose consumption in recombinant Saccharomyces cerevisiae

“…XR, XDH and XK activities were measured in the reference strain CEN.PK 113-7A and in the two constructed strains TMB3265(XYL2, XKS1 ) and TMB3267(XYL2, XKS1, GRE3 ) ( Table 2). XR activity with NADPH as co-factor was 5-6 mU/mg protein in CEN.PK 113-7A and TMB3265(XYL2, XKS1 ), similar to previously reported (Kuhn et al, 1995;Träff et al, 2001), whereas no XR activity was detected with NADH as co-factor. In TMB3267, overexpressing GRE3, the XR activity with NADPH was increased four times.…”

Development of Efficient Xylose Fermentation in Saccharomyces cerevisiae: Xylose Isomerase as a Key Component

“…At a specific d-xylose consumption rate of 0.73 mmol (g biomass) -1 h -1 this yeast excreted d-xylulose at a rate of 0.20 mmol (g biomass) -1 h -1 (corresponding to 30% of consumed d-xylose), which suggested that reactions downstream of d-xylulose were rate-controlling. Moreover, small amounts of xylitol were produced in these cultivations, suggesting involvement of a non-specific aldose reductase such as encoded by GRE3 [66]. This information on d-xylulose and xylitol production was used in subsequent metabolic engineering attempts to improve the d-xylose consumption rate and to minimise xylitol formation.…”

“…E2 XylA gene [43]. Since the non-specific aldose reductase encoded by GRE3 had previously been implicated in xylitol formation by S. cerevisiae, this gene was also deleted in the engineered strain [45,66].…”

“…Despite the inherent redox constraints of S. cerevisiae strains based on the xylose reductase/xylitol dehydrogenase strategy, this strategy has resulted in many important insights into the kinetics of d-xylose metabolism by engineered S. cerevisiae strains. These findings include the benefits of overexpression of xylulokinase [29,56], the side role of the S. cerevisiae aldose reductase (Gre3) (besides the heterologous dual specificity xylose reductases) in xylitol formation [66], the role of the enzymes of the non-oxidative part of the pentose phosphate pathway [34,43], characterisation of d-xylose transport [27,62] and many studies on the inhibitor tolerance/sensitivity of d-xylose-consuming strains [54]. The latter will be especially crucial for successful application of d-xylose-consuming S. cerevisiae strains for ethanol production from lignocellulosic hydrolysates (see Sect.…”

Deletion of FPS1 , Encoding Aquaglyceroporin Fps1p, Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

“…Expressing the XI pathway can avoid the cofactor imbalance problem under anaerobic conditions, but xylitol accumulation has also been observed in strains expressing XI (17,18,20), because the nonspecific aldose reductase encoded by the GRE3 gene can produce xylitol from xylose (27). Various rational approaches have been used to reduce xylitol accumulation and improve xylose utilization, such as optimizing the expression levels of xylose-assimilating reactions (26), engineering the cofactor preference of XR/XDH enzymes (28)(29)(30)(31)(32)(33), perturbing the pentose phosphate pathway by gene knockout or overexpression (34)(35)(36)(37)(38)(39), or deleting GRE3 in strains expressing the XI pathway (21,40,41). While extensive previous efforts focused on manipulating intracellular metabolic reactions to improve xylose utilization and reduce by-product (e.g., xylitol) accumulation, controlling the xylitol export process might also be a meaningful strategy for reducing its formation and increasing carbon flux toward target products.…”