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Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase

Runquist, David LU ; Hahn-Hägerdal, Bärbel LU and Bettiga, Maurizio LU (2010) In Applied and Environmental Microbiology 76(23). p.7796-7802
Abstract
Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic... (More)
Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V-max of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased similar to 40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] x h versus 0.007 g/g [DW] x h), and the anaerobic growth rate was increased from similar to 0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
in
Applied and Environmental Microbiology
volume
76
issue
23
pages
7796 - 7802
publisher
American Society for Microbiology
external identifiers
  • wos:000284310500014
  • scopus:78650327471
  • pmid:20889775
ISSN
0099-2240
DOI
10.1128/AEM.01505-10
language
English
LU publication?
yes
id
7b04ed2d-5a68-4ad9-8a44-72af7893f547 (old id 1752662)
date added to LUP
2010-12-30 08:08:54
date last changed
2018-05-29 09:59:15
@article{7b04ed2d-5a68-4ad9-8a44-72af7893f547,
  abstract     = {Baker's yeast (Saccharomyces cerevisiae) has been genetically engineered to ferment the pentose sugar xylose present in lignocellulose biomass. One of the reactions controlling the rate of xylose utilization is catalyzed by xylose reductase (XR). In particular, the cofactor specificity of XR is not optimized with respect to the downstream pathway, and the reaction rate is insufficient for high xylose utilization in S. cerevisiae. The current study describes a novel approach to improve XR for ethanol production in S. cerevisiae. The cofactor binding region of XR was mutated by error-prone PCR, and the resulting library was expressed in S. cerevisiae. The S. cerevisiae library expressing the mutant XR was selected in sequential anaerobic batch cultivation. At the end of the selection process, a strain (TMB 3420) harboring the XR mutations N272D and P275Q was enriched from the library. The V-max of the mutated enzyme was increased by an order of magnitude compared to that of the native enzyme, and the NADH/NADPH utilization ratio was increased significantly. The ethanol productivity from xylose in TMB 3420 was increased similar to 40 times compared to that of the parent strain (0.32 g/g [dry weight {DW}] x h versus 0.007 g/g [DW] x h), and the anaerobic growth rate was increased from similar to 0 h(-1) to 0.08 h(-1). The improved traits of TMB 3420 were readily transferred to the parent strain by reverse engineering of the mutated XR gene. Since integrative vectors were employed in the construction of the library, transfer of the improved phenotype does not require multicopy expression from episomal plasmids.},
  author       = {Runquist, David and Hahn-Hägerdal, Bärbel and Bettiga, Maurizio},
  issn         = {0099-2240},
  language     = {eng},
  number       = {23},
  pages        = {7796--7802},
  publisher    = {American Society for Microbiology},
  series       = {Applied and Environmental Microbiology},
  title        = {Increased Ethanol Productivity in Xylose-Utilizing Saccharomyces cerevisiae via a Randomly Mutagenized Xylose Reductase},
  url          = {http://dx.doi.org/10.1128/AEM.01505-10},
  volume       = {76},
  year         = {2010},
}