Expression of protein engineered NADP+-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Abstract

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

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

“…The error bars represent standard deviations (n ϭ 3). Statistical significance: *, n ϭ 3, P Ͻ 0.05; **, n ϭ 3, P Ͻ 0.005. and activities of XR and XDH (22,28,29), and glycerol is a common by-product in anaerobic glucose fermentation and is related to the regeneration of NAD ϩ (30). Although the limited oxygen supply and growth in complex media can alleviate the cofactor imbalance reducing xylitol production, the significant reduction of the by-products and higher ethanol yield among the selected recombinants demonstrates the effects of pathway balancing.…”

“…The crude lysate was used for enzyme activity analysis in a 96-well plate format using a Biotek Synergy2 microplate reader (Winooski, VT). The activities were measured by monitoring the absorbance at 340 nm for depletion or generation of NAD(P)H as described previously (19)(20)(21)(22). A negative control was included, consisting of the yeast with an empty plasmid.…”

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

“…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.…”

Comparison of the Performance of Eight Recombinant Strains of Xylose-FermentingSaccharomyces cerevisiaeas to Bioethanol Production from Rice Straw Enzymatic Hydrolyzate

“…They are characterized by the ability to ferment xylose to ethanol in synthetic medium. [12][13][14][15][16][17] Among these strains, MA-R4, engineered by chromosomal integration from industrial diploid S. cerevisiae strain IR-2 to express the XYL1, XYL2, and XKS1 genes, converted xylose to ethanol faster than recombinant haploid laboratory strains.…”

“…The laboratory yeast strains used in this study were MA-D4 15) and MA-N4, 13) and the industrial recombinant strains were MA-R4, 15) MA-S4, 15) MA-T4, 15) MA-K74 (Kyokai no. 7, AUR1::pAUR-XKXDHXR 11) ), MA-K94 (Kyokai no.…”