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Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS6054.

Wahlbom, Fredrik LU ; van Zyl, W H; Jönsson, Leif J; Hahn-Hägerdal, Bärbel LU and Otero, R R (2003) In FEMS Yeast Research 3(3). p.319-326
Abstract
The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding Image-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on Image-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only Image-xylose as carbon and energy source. The... (More)
The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding Image-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on Image-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only Image-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h−1 for S. cerevisiae TMB 3399 to 0.14 h−1 for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h−1. All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose−1 and 0.001, 0.10, and 0.16 g ethanol g biomass−1 h−1 for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein−1, was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
FEMS Yeast Research
volume
3
issue
3
pages
319 - 326
publisher
Elsevier
external identifiers
  • pmid:12689639
  • wos:000182560000009
  • scopus:12444258773
ISSN
1567-1364
DOI
10.1016/S1567-1356(02)00206-4
language
English
LU publication?
yes
id
ddbee6b0-62af-4edb-a954-a0767384250e (old id 132859)
date added to LUP
2007-06-28 14:36:10
date last changed
2018-01-07 06:16:43
@article{ddbee6b0-62af-4edb-a954-a0767384250e,
  abstract     = {The recombinant xylose-utilizing Saccharomyces cerevisiae TMB 3399 was constructed by chromosomal integration of the genes encoding Image-xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK). S. cerevisiae TMB 3399 was subjected to chemical mutagenesis with ethyl methanesulfonate and, after enrichment, 33 mutants were selected for improved growth on Image-xylose and carbon dioxide formation in Durham tubes. The best-performing mutant was called S. cerevisiae TMB 3400. The novel, recombinant S. cerevisiae strains were compared with Pichia stipitis CBS 6054 through cultivation under aerobic, oxygen-limited, and anaerobic conditions in a defined mineral medium using only Image-xylose as carbon and energy source. The mutation led to a more than five-fold increase in maximum specific growth rate, from 0.0255 h−1 for S. cerevisiae TMB 3399 to 0.14 h−1 for S. cerevisiae TMB 3400, whereas P. stipitis grew at a maximum specific growth rate of 0.44 h−1. All yeast strains formed ethanol only under oxygen-limited and anaerobic conditions. The ethanol yields and maximum specific ethanol productivities during oxygen limitation were 0.21, 0.25, and 0.30 g ethanol g xylose−1 and 0.001, 0.10, and 0.16 g ethanol g biomass−1 h−1 for S. cerevisiae TMB 3399, TMB 3400, and P. stipitis CBS 6054, respectively. The xylitol yield under oxygen-limited and anaerobic conditions was two-fold higher for S. cerevisiae TMB 3399 than for TMB 3400, but the glycerol yield was higher for TMB 3400. The specific activity, in U mg protein−1, was higher for XDH than for XR in both S. cerevisiae TMB 3399 and TMB 3400, while P. stipitis CBS 6054 showed the opposite relation. S. cerevisiae TMB 3400 displayed higher specific XR, XDH and XK activities than TMB 3399. Hence, we have demonstrated that a combination of metabolic engineering and random mutagenesis was successful to generate a superior, xylose-utilizing S. cerevisiae, and uncovered distinctive physiological properties of the mutant.},
  author       = {Wahlbom, Fredrik and van Zyl, W H and Jönsson, Leif J and Hahn-Hägerdal, Bärbel and Otero, R R},
  issn         = {1567-1364},
  language     = {eng},
  number       = {3},
  pages        = {319--326},
  publisher    = {Elsevier},
  series       = {FEMS Yeast Research},
  title        = {Generation of the improved recombinant xylose-utilizing Saccharomyces cerevisiae TMB3400 by random mutagenesis and physiological comparison with Pichia stipitis CBS6054.},
  url          = {http://dx.doi.org/10.1016/S1567-1356(02)00206-4},
  volume       = {3},
  year         = {2003},
}