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Overproduction of pentose phosphate pathway enzymes using a new CRE– lox P expression vector for repeated genomic integration in Saccharomyces cerevisiae
Author(s) -
Johansson Björn,
HahnHägerdal Bärbel
Publication year - 2002
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.833
Subject(s) - selectable marker , biology , expression cassette , marker gene , ura3 , terminator (solar) , gene , transformation (genetics) , cre recombinase , expression vector , shuttle vector , genetics , microbiology and biotechnology , vector (molecular biology) , saccharomyces cerevisiae , transgene , recombinant dna , astronomy , ionosphere , physics , genetically modified mouse
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 lox P 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 lox P–zeocin– lox P cassette using pCRE3. The 2µ‐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/ lox P system proved to be a practical strategy to overexpress multiple genes without exhausting available markers. Copyright © 2002 John Wiley & Sons, Ltd.