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Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae.

Petschacher B, Nidetzky B - Microb. Cell Fact. (2008)

Bottom Line: Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose.Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274-->Arg Asn276-->Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH.This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. bernd.nidetzky@tugraz.at.

ABSTRACT

Background: Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274-->Arg Asn276-->Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.

Results: An isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 +/- 0.02 h-1) in both strains.

Conclusion: Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae.

No MeSH data available.


Related in: MedlinePlus

Pathways for utilization of D-xylose and L-arabinose in fungi. 1, aldose reductase (EC 1.1.1.21); 2, xylitol dehydrogenase (EC 1.1.1.9); 3, xylulose kinase (EC 2.7.1.17); 4, L-arabinitol 4-dehydrogenase (EC 1.1.1.12); 5, L-xylulose reductase (EC 1.1.1.10).
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Figure 1: Pathways for utilization of D-xylose and L-arabinose in fungi. 1, aldose reductase (EC 1.1.1.21); 2, xylitol dehydrogenase (EC 1.1.1.9); 3, xylulose kinase (EC 2.7.1.17); 4, L-arabinitol 4-dehydrogenase (EC 1.1.1.12); 5, L-xylulose reductase (EC 1.1.1.10).

Mentions: Rising oil prices and a growing awareness of a possible climate change caused by greenhouse gas emission have recently led to rekindled interest in bioethanol as a CO2-neutral liquid fuel. Lignocellulose will be the prime choice of feedstock for the production of bioethanol if major technical problems in its conversion can be overcome [1,2]. One notable difficulty has been in the development of robust microbial strains capable of fermenting efficiently all types of sugars present in the cellulose and hemicellulose fractions of the raw material [3-6]. While Saccharomyces cerevisiae is a top candidate to be used in the fermentation of D-glucose and the hemicellulose-derived hexoses D-galactose and D-mannose, the organism in its wild-type form cannot utilize the pentoses D-xylose and L-arabinose [3,4,6-11] which constitute ≥ 80% of the total sugar contained in typical hemicellulose hydrolyzates [12]. The particular deficiency of S. cerevisiae is caused by an insufficient expression of pathways which in other yeasts deliver either of the two sugars as D-xylulose 5-phosphate into the central metabolism (Figure 1).


Altering the coenzyme preference of xylose reductase to favor utilization of NADH enhances ethanol yield from xylose in a metabolically engineered strain of Saccharomyces cerevisiae.

Petschacher B, Nidetzky B - Microb. Cell Fact. (2008)

Pathways for utilization of D-xylose and L-arabinose in fungi. 1, aldose reductase (EC 1.1.1.21); 2, xylitol dehydrogenase (EC 1.1.1.9); 3, xylulose kinase (EC 2.7.1.17); 4, L-arabinitol 4-dehydrogenase (EC 1.1.1.12); 5, L-xylulose reductase (EC 1.1.1.10).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2315639&req=5

Figure 1: Pathways for utilization of D-xylose and L-arabinose in fungi. 1, aldose reductase (EC 1.1.1.21); 2, xylitol dehydrogenase (EC 1.1.1.9); 3, xylulose kinase (EC 2.7.1.17); 4, L-arabinitol 4-dehydrogenase (EC 1.1.1.12); 5, L-xylulose reductase (EC 1.1.1.10).
Mentions: Rising oil prices and a growing awareness of a possible climate change caused by greenhouse gas emission have recently led to rekindled interest in bioethanol as a CO2-neutral liquid fuel. Lignocellulose will be the prime choice of feedstock for the production of bioethanol if major technical problems in its conversion can be overcome [1,2]. One notable difficulty has been in the development of robust microbial strains capable of fermenting efficiently all types of sugars present in the cellulose and hemicellulose fractions of the raw material [3-6]. While Saccharomyces cerevisiae is a top candidate to be used in the fermentation of D-glucose and the hemicellulose-derived hexoses D-galactose and D-mannose, the organism in its wild-type form cannot utilize the pentoses D-xylose and L-arabinose [3,4,6-11] which constitute ≥ 80% of the total sugar contained in typical hemicellulose hydrolyzates [12]. The particular deficiency of S. cerevisiae is caused by an insufficient expression of pathways which in other yeasts deliver either of the two sugars as D-xylulose 5-phosphate into the central metabolism (Figure 1).

Bottom Line: Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose.Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274-->Arg Asn276-->Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH.This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria. bernd.nidetzky@tugraz.at.

ABSTRACT

Background: Metabolic engineering of Saccharomyces cerevisiae for xylose fermentation into fuel ethanol has oftentimes relied on insertion of a heterologous pathway that consists of xylose reductase (XR) and xylitol dehydrogenase (XDH) and brings about isomerization of xylose into xylulose via xylitol. Incomplete recycling of redox cosubstrates in the catalytic steps of the NADPH-preferring XR and the NAD+-dependent XDH results in formation of xylitol by-product and hence in lowering of the overall yield of ethanol on xylose. Structure-guided site-directed mutagenesis was previously employed to change the coenzyme preference of Candida tenuis XR about 170-fold from NADPH in the wild-type to NADH in a Lys274-->Arg Asn276-->Asp double mutant which in spite of the structural modifications introduced had retained the original catalytic efficiency for reduction of xylose by NADH. This work was carried out to assess physiological consequences in xylose-fermenting S. cerevisiae resulting from a well defined alteration of XR cosubstrate specificity.

Results: An isogenic pair of yeast strains was derived from S. cerevisiae Cen.PK 113-7D through chromosomal integration of a three-gene cassette that carried a single copy for C. tenuis XR in wild-type or double mutant form, XDH from Galactocandida mastotermitis, and the endogenous xylulose kinase (XK). Overexpression of each gene was under control of the constitutive TDH3 promoter. Measurement of intracellular levels of XR, XDH, and XK activities confirmed the expected phenotypes. The strain harboring the XR double mutant showed 42% enhanced ethanol yield (0.34 g/g) compared to the reference strain harboring wild-type XR during anaerobic bioreactor conversions of xylose (20 g/L). Likewise, the yields of xylitol (0.19 g/g) and glycerol (0.02 g/g) were decreased 52% and 57% respectively in the XR mutant strain. The xylose uptake rate per gram of cell dry weight was identical (0.07 +/- 0.02 h-1) in both strains.

Conclusion: Integration of enzyme and strain engineering to enhance utilization of NADH in the XR-catalyzed conversion of xylose results in notably improved fermentation capabilities of recombinant S. cerevisiae.

No MeSH data available.


Related in: MedlinePlus