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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

Cloning strategy employed for construction of plasmids YCtXRWt/GmXHD/XKS1 and YCtXRWDm/GmXHD/XKS1. Further details are given in Table 4 and text. The TDH3 promoter and the CYC1 terminator are labeled G and C, respectively.
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Figure 5: Cloning strategy employed for construction of plasmids YCtXRWt/GmXHD/XKS1 and YCtXRWDm/GmXHD/XKS1. Further details are given in Table 4 and text. The TDH3 promoter and the CYC1 terminator are labeled G and C, respectively.

Mentions: In a first step, the promoterless genes for native or K274R-N276D CtXR, GmXDH, and the endogenous yeast XK1 were amplified from pET11-CtXRWt or pET11-CtXRDm, pBTac1, and genomic S. cerevisiae DNA, respectively. Polymerase chain reactions were performed using forward and reverse oligonucleotide primers whose 5'-ends contained a BamHI and SalI restriction site, respectively (see Table 4). Amplification products were digested with BamHI and SalI and inserted into the multiple cloning site of pRS416GPD, situated between the TDH3, formerly glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter and the cytochrome-c-oxidase (CYC1) terminator. Gene cassettes were constructed where each of the target genes (XRWt, XRDm, XDH, and XK) was integrated separately between a TDH3 promoter and CYC1 terminator. Correct insertion was verified by sequencing. In a second step, the gene cassettes were amplified by PCR using oligonucleotide primers containing restriction sites at their respective 5'-end that are unique for YIp5 (see Table 4, Figure 5). The XK gene cassette was cloned into the AatII site, resulting in vector YXKS1. Third, the XDH gene cassette was inserted into the ClaI site of YXKS1, resulting in YGmXDH/XKS1. Fourth and finally, the gene cassette for either XRWt or XRDm was cloned into the EcoRI site of YGmXDH/XKS1, resulting in YCtXRWt/GmXDH/XKS1 and YCtXRDm/GmXDH/XKS1. Correct orientation of the inserted gene cassettes was verified after each step of integration using PCR screening with a pair of oligonucleotide primers matching a sequence upstream of the cloning site in the target vector and a sequence of the inserted gene.


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)

Cloning strategy employed for construction of plasmids YCtXRWt/GmXHD/XKS1 and YCtXRWDm/GmXHD/XKS1. Further details are given in Table 4 and text. The TDH3 promoter and the CYC1 terminator are labeled G and C, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cloning strategy employed for construction of plasmids YCtXRWt/GmXHD/XKS1 and YCtXRWDm/GmXHD/XKS1. Further details are given in Table 4 and text. The TDH3 promoter and the CYC1 terminator are labeled G and C, respectively.
Mentions: In a first step, the promoterless genes for native or K274R-N276D CtXR, GmXDH, and the endogenous yeast XK1 were amplified from pET11-CtXRWt or pET11-CtXRDm, pBTac1, and genomic S. cerevisiae DNA, respectively. Polymerase chain reactions were performed using forward and reverse oligonucleotide primers whose 5'-ends contained a BamHI and SalI restriction site, respectively (see Table 4). Amplification products were digested with BamHI and SalI and inserted into the multiple cloning site of pRS416GPD, situated between the TDH3, formerly glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter and the cytochrome-c-oxidase (CYC1) terminator. Gene cassettes were constructed where each of the target genes (XRWt, XRDm, XDH, and XK) was integrated separately between a TDH3 promoter and CYC1 terminator. Correct insertion was verified by sequencing. In a second step, the gene cassettes were amplified by PCR using oligonucleotide primers containing restriction sites at their respective 5'-end that are unique for YIp5 (see Table 4, Figure 5). The XK gene cassette was cloned into the AatII site, resulting in vector YXKS1. Third, the XDH gene cassette was inserted into the ClaI site of YXKS1, resulting in YGmXDH/XKS1. Fourth and finally, the gene cassette for either XRWt or XRDm was cloned into the EcoRI site of YGmXDH/XKS1, resulting in YCtXRWt/GmXDH/XKS1 and YCtXRDm/GmXDH/XKS1. Correct orientation of the inserted gene cassettes was verified after each step of integration using PCR screening with a pair of oligonucleotide primers matching a sequence upstream of the cloning site in the target vector and a sequence of the inserted gene.

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