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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

Xylose utilization and product formation during oxygen-limited shake flask cultivation of BP000 (panel A) and BP10001 (panel B). Xylose (full squares), ethanol (triangles), xylitol (circles), glycerol (stars) and acetate (empty squares) were analyzed by HPLC. The biomass concentration was constant at 1.4 ± 0.1 g/L for BP000 and 1.5 ± 0.1 g/L for BP10001. Error bars show the S.D. of triplicate fermentation experiments.
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Figure 3: Xylose utilization and product formation during oxygen-limited shake flask cultivation of BP000 (panel A) and BP10001 (panel B). Xylose (full squares), ethanol (triangles), xylitol (circles), glycerol (stars) and acetate (empty squares) were analyzed by HPLC. The biomass concentration was constant at 1.4 ± 0.1 g/L for BP000 and 1.5 ± 0.1 g/L for BP10001. Error bars show the S.D. of triplicate fermentation experiments.

Mentions: Batch conversions of xylose by glucose-grown resting cells of BP000 and BP10001 were carried out under oxygen-limited reaction conditions ([O2] ≤ 20 μM) in shake flasks using a mineral medium that contained 20 g/L sugar. Typical fermentation time courses are shown in Figure 3 and parameters derived from their analysis are summarized in Table 3. No biomass was formed under these conditions. In a carbon balance calculated from the data in Figure 3 whereby CO2 was inferred from the ethanol and acetate values, only ≤ 7% of the carbon from xylose remained unaccounted for. In comparison with the reference strain BP000, the XR double mutant strain BP10001 showed 40% enhanced ethanol yield. Its production of xylitol and glycerol was decreased by 53% and 30%, respectively. The yield of acetate was generally low in both strains, however, enhanced by about 50% 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)

Xylose utilization and product formation during oxygen-limited shake flask cultivation of BP000 (panel A) and BP10001 (panel B). Xylose (full squares), ethanol (triangles), xylitol (circles), glycerol (stars) and acetate (empty squares) were analyzed by HPLC. The biomass concentration was constant at 1.4 ± 0.1 g/L for BP000 and 1.5 ± 0.1 g/L for BP10001. Error bars show the S.D. of triplicate fermentation experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Xylose utilization and product formation during oxygen-limited shake flask cultivation of BP000 (panel A) and BP10001 (panel B). Xylose (full squares), ethanol (triangles), xylitol (circles), glycerol (stars) and acetate (empty squares) were analyzed by HPLC. The biomass concentration was constant at 1.4 ± 0.1 g/L for BP000 and 1.5 ± 0.1 g/L for BP10001. Error bars show the S.D. of triplicate fermentation experiments.
Mentions: Batch conversions of xylose by glucose-grown resting cells of BP000 and BP10001 were carried out under oxygen-limited reaction conditions ([O2] ≤ 20 μM) in shake flasks using a mineral medium that contained 20 g/L sugar. Typical fermentation time courses are shown in Figure 3 and parameters derived from their analysis are summarized in Table 3. No biomass was formed under these conditions. In a carbon balance calculated from the data in Figure 3 whereby CO2 was inferred from the ethanol and acetate values, only ≤ 7% of the carbon from xylose remained unaccounted for. In comparison with the reference strain BP000, the XR double mutant strain BP10001 showed 40% enhanced ethanol yield. Its production of xylitol and glycerol was decreased by 53% and 30%, respectively. The yield of acetate was generally low in both strains, however, enhanced by about 50% 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