Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae

Balancing the flux of a heterologous metabolic pathway by tuning the expression and properties of the pathway enzymes is difficult, but it is critical to realizing the full potential of microbial biotechnology. One prominent example is the metabolic engineering of a Saccharomyces cerevisiae strain harboring a heterologous xylose-utilizing pathway for cellulosic-biofuel production, which remains a challenge even after decades of research. Here, we developed a combinatorial pathway-engineering approach to rapidly create a highly efficient xylose-utilizing pathway for ethanol production by exploring various combinations of enzyme homologues with different properties. A library of more than 8,000 xylose utilization pathways was generated using DNA assembler, followed by multitiered screening, which led to the identification of a number of strain-specific combinations of the enzymes for efficient conversion of xylose to ethanol. The balancing of metabolic flux through the xylose utilization pathway was demonstrated by a complete reversal of the major product from xylitol to ethanol with a similar yield and total by-product formation as low as 0.06 g/g xylose without compromising cell growth. The results also suggested that an optimal enzyme combination depends on not only the genotype/phenotype of the host strain, but also the sugar composition of the fermentation medium. This combinatorial approach should be applicable to any heterologous pathway and will be instrumental in the optimization of industrial production of value-added products.

Section: Discussionmentioning

confidence: 99%

Combinatorial Design of a Highly Efficient Xylose-Utilizing Pathway in Saccharomyces cerevisiae for the Production of Cellulosic Biofuels

Kim

Eriksen

et al. 2013

“…The capacity to recycle NADH to NAD ϩ under anaerobic or oxygen-limiting conditions can reduce xylitol production and increase the capacity for xylose assimilation (3,17,19,22,24,29,32). Recently, Hou (15) reported that the crude XR activity of S. passalidarum has a 1.8-fold higher affinity for NADH than for NADPH.…”

Section: Figmentioning

confidence: 99%

Cofermentation of Glucose, Xylose, and Cellobiose by the Beetle-Associated Yeast Spathaspora passalidarum

Headman

et al. 2012

125

dFermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.

“…Thus, many studies attempted to optimize the expression levels of the genes coding for the XR/XDH pathway or to engineer XR and XDH proteins with balanced cofactor preferences to reduce xylitol accumulation and increase the ethanol yield (19,28,(54)(55)(56)(57)(58)(59)(60). While previous efforts to improve xylose fermentation mostly focused on manipulation of enzymatic reactions related to xylose metabolism, this study demonstrated an innovative approach that controls the carbon flux by accumulating the intermediate product (i.e., xylitol) inside the cell to facilitate the reaction toward the target direction.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Wei

Kim

et al. 2013

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD ؉ / NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.