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Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae

Johansson, Björn and Hahn-Hägerdal, Bärbel LU (2002) In Yeast 19(3). p.225-231
Abstract
Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active... (More)
Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Integrase/genetics, Pentosephosphate Pathway/genetics/*physiology, Plasmids/genetics, Polymerase Chain Reaction, Saccharomyces cerevisiae/*enzymology/*genetics, Non-U.S. Gov't, Support, Transaldolase/biosynthesis/genetics, Viral Proteins/genetics, Transketolase/biosynthesis/genetics, Genetic Vectors/*genetics, Genetic Markers, Fungal/genetics, Gene Expression Regulation
in
Yeast
volume
19
issue
3
pages
225 - 231
publisher
John Wiley & Sons
external identifiers
  • wos:000174109200004
  • pmid:11816030
  • scopus:0036187741
ISSN
1097-0061
DOI
10.1002/yea.833
language
English
LU publication?
yes
id
6aeaad44-f028-4670-99d0-1cc8e1caa3d3 (old id 106352)
date added to LUP
2007-06-28 14:02:42
date last changed
2017-05-28 04:22:24
@article{6aeaad44-f028-4670-99d0-1cc8e1caa3d3,
  abstract     = {Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.},
  author       = {Johansson, Björn and Hahn-Hägerdal, Bärbel},
  issn         = {1097-0061},
  keyword      = {Integrase/genetics,Pentosephosphate Pathway/genetics/*physiology,Plasmids/genetics,Polymerase Chain Reaction,Saccharomyces cerevisiae/*enzymology/*genetics,Non-U.S. Gov't,Support,Transaldolase/biosynthesis/genetics,Viral Proteins/genetics,Transketolase/biosynthesis/genetics,Genetic Vectors/*genetics,Genetic Markers,Fungal/genetics,Gene Expression Regulation},
  language     = {eng},
  number       = {3},
  pages        = {225--231},
  publisher    = {John Wiley & Sons},
  series       = {Yeast},
  title        = {Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae},
  url          = {http://dx.doi.org/10.1002/yea.833},
  volume       = {19},
  year         = {2002},
}