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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

Comparison of XR activities of BP000 and BP10001 at different cofactor concentrations. Cells were grown aerobically on 20 g/L glucose and 20 g/L xylose and disrupted with Y-Per reagent. One hundred % specific activity of strain BP000 corresponds to values of 0.15 U/mg with NADH and 0.18 U/mg with NADPH. In strain BP10001, the specific activities are 0.26 U/mg with NADH and 0.33 U/mg with NADPH.
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Figure 2: Comparison of XR activities of BP000 and BP10001 at different cofactor concentrations. Cells were grown aerobically on 20 g/L glucose and 20 g/L xylose and disrupted with Y-Per reagent. One hundred % specific activity of strain BP000 corresponds to values of 0.15 U/mg with NADH and 0.18 U/mg with NADPH. In strain BP10001, the specific activities are 0.26 U/mg with NADH and 0.33 U/mg with NADPH.

Mentions: Figure 2 compares XR activities of BP000 and BP10001 recorded at three concentrations of NADH and NADPH. The relative decrease in enzymatic rate in response to lowering the level of NADH from 350 μM to 7 μM was similar in the two strains. By contrast, the drop in activity caused by the same change in the concentration of NADPH was much more significant in BP10001 than in BP000. These results are in excellent agreement with expectations from the Km values of purified wild-type (NADH: 38 μM; NADPH: 3 μM) and K274R-N276D (NADH: 41 μM; NADPH: 128 μM) [40]. They also serve to verify functional expression of the double mutant in BP10001.


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)

Comparison of XR activities of BP000 and BP10001 at different cofactor concentrations. Cells were grown aerobically on 20 g/L glucose and 20 g/L xylose and disrupted with Y-Per reagent. One hundred % specific activity of strain BP000 corresponds to values of 0.15 U/mg with NADH and 0.18 U/mg with NADPH. In strain BP10001, the specific activities are 0.26 U/mg with NADH and 0.33 U/mg with NADPH.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Comparison of XR activities of BP000 and BP10001 at different cofactor concentrations. Cells were grown aerobically on 20 g/L glucose and 20 g/L xylose and disrupted with Y-Per reagent. One hundred % specific activity of strain BP000 corresponds to values of 0.15 U/mg with NADH and 0.18 U/mg with NADPH. In strain BP10001, the specific activities are 0.26 U/mg with NADH and 0.33 U/mg with NADPH.
Mentions: Figure 2 compares XR activities of BP000 and BP10001 recorded at three concentrations of NADH and NADPH. The relative decrease in enzymatic rate in response to lowering the level of NADH from 350 μM to 7 μM was similar in the two strains. By contrast, the drop in activity caused by the same change in the concentration of NADPH was much more significant in BP10001 than in BP000. These results are in excellent agreement with expectations from the Km values of purified wild-type (NADH: 38 μM; NADPH: 3 μM) and K274R-N276D (NADH: 41 μM; NADPH: 128 μM) [40]. They also serve to verify functional expression of the double mutant in BP10001.

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