CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast
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CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast. / Strucko, Tomas; Buron, Line Due; Jarczynska, Zofia Dorota; Nødvig, Christina Spuur; Mølgaard, Louise; Halkier, Barbara Ann; Mortensen, Uffe Hasbro.
I: Scientific Reports, Bind 7, 41431, 2017.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast
AU - Strucko, Tomas
AU - Buron, Line Due
AU - Jarczynska, Zofia Dorota
AU - Nødvig, Christina Spuur
AU - Mølgaard, Louise
AU - Halkier, Barbara Ann
AU - Mortensen, Uffe Hasbro
PY - 2017
Y1 - 2017
N2 - Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside.
AB - Over-expression of a gene by increasing its copy number is often desirable in the model yeast Saccharomyces cerevisiae. It may facilitate elucidation of enzyme functions, and in cell factory design it is used to increase production of proteins and metabolites. Current methods are typically exploiting expression from the multicopy 2 μ-derived plasmid or by targeting genes repeatedly into sequences like Ty or rDNA; in both cases, high gene expression levels are often reached. However, with 2 μ-based plasmid expression, the population of cells is very heterogeneous with respect to protein production; and for integration into repeated sequences it is difficult to determine the genetic setup of the resulting strains and to achieve specific gene doses. For both types of systems, the strains often suffer from genetic instability if proper selection pressure is not applied. Here we present a gene amplification system, CASCADE, which enables construction of strains with defined gene copy numbers. One or more genes can be amplified simultaneously and the resulting strains can be stably propagated on selection-free medium. As proof-of-concept, we have successfully used CASCADE to increase heterologous production of two fluorescent proteins, the enzyme β-galactosidase the fungal polyketide 6-methyl salicylic acid and the plant metabolite vanillin glucoside.
KW - Journal Article
U2 - 10.1038/srep41431
DO - 10.1038/srep41431
M3 - Journal article
C2 - 28134264
VL - 7
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
M1 - 41431
ER -
ID: 180763420