Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein-engineered NADH-preferring xylose reductase from Pichia stipitis

Abstract

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis (PsXR and PsXDH, respectively) has the ability to convert xylose to ethanol together with the unfavourable excretion of xylitol, which may be due to intercellular redox imbalance caused by the different coenzyme specificity between NADPH-preferring XR and NAD + -dependent XDH. In this study, we focused on the effect(s) of mutated NADH-preferring PsXR in fermentation. The R276H and K270R/N272D mutants were improved 52-and 146-fold, respectively, in the ratio of NADH/NADPH in catalytic efficiency [(k cat /K m with NADH)/(k cat /K m with NADPH)] compared with the wild-type (WT), which was due to decrease of k cat with NADPH in the R276H mutant and increase of K m with NADPH in the K270R/N272D mutant. Furthermore, R276H mutation led to significant thermostabilization in PsXR. The most positive effect on xylose fermentation to ethanol was found by using the Y-R276H strain, expressing PsXR R276H mutant and PsXDH WT: 20 % increase of ethanol production and 52 % decrease of xylitol excretion, compared with the Y-WT strain expressing PsXR WT and PsXDH WT. Measurement of intracellular coenzyme concentrations suggested that maintenance of the of NADPH/NADP + and NADH/NAD + ratios is important for efficient ethanol fermentation from xylose by recombinant S. cerevisiae.

Characterization of xylose-utilizing yeasts isolated from herbivore faeces in Thailand

“…Comparison the obtained partial amino acid sequences of C. tropicalis A26 to those of other yeast strains in Gene Bank database revealed that C. tropicalis A26 xylose reductase possessed 98.4%, 78.5%, and 77.5% identity and 99.0%, 86.4%, and 85.9% similarity to xylose reductase of CtrXR of C. tropicalis (ABX60132C), CtXR of C. tenuis (AAC25601), and CsXR of C. shehatae (ABK35120), respectively (Table 4). Amino acid sequence alignment of the partial xylose reductase revealed tetra amino acid motif (Ile-Pro-Lys-Ser), an NAD(P)H species motif 14,17,53 ( Fig. 2).…”

“…Cocultivating of S. cerevisiae and S. stipitis strains to co-ferment glucose and xylose remains unsatisfactory, due to their difference in fermenting condition and ethanol tolerance 12,13 . S. stipitis strain prefers to ferment glucose more than xylose while it has lower ethanol tolerance than S. cerevisiae strain 14 . So xylose was not fermented.…”

“…So xylose was not fermented. Therefore, there has been an www.scienceasia.org attempt to construct recombinants S. cerevisiae strain capable of fermenting xylose by overexpressing the genes encoding enzymes in the xylose fermentation pathway of S. stipitis strain 4,14,15 . Three key step enzymes in S. stipitis xylose fermentation pathway are (1) xylose reductase (EC.1.1.1.21), which converts xylose to xylitol using NAD(P)H as a cofactor 16,17 (2) xylitol dehydrogenase (EC.1.1.1.9), which converts xylitol to xylulose using NAD as a cofactor 18 , and (3) xylulose kinase (EC 2.7.1.17), which converts xylulose to xylulose-5-phosphate 19 .…”

“…So ethanol production from xylose of the recombinant S. cerevisiae was low. Screening for NADH dependent xylose reductase which has high activity to replace the NAD(P)H dependent xylose reductase of S. stipitis will be useful in constructing a recombinant S. cerevisiae capable of fermenting xylose 14,20,21 . Digestive tract of herbivores contains microorganisms which can hydrolyse only cellulose in the lignocelluloses consumed to glucose 22 .…”

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.…”