CASCADE, a platform for controlled gene amplification for high, tunable and selection-free gene expression in yeast.

Sci Rep

Eukaryotic Molecular Cell Biology, Section for Eukaryotic Biotechnology, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 223, 2800 Kongens Lyngby, Denmark.

Published: January 2017

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.

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5278378PMC
http://dx.doi.org/10.1038/srep41431DOI Listing

Publication Analysis

Top Keywords

gene amplification
8
gene expression
8
gene
7
cascade platform
4
platform controlled
4
controlled gene
4
amplification high
4
high tunable
4
tunable selection-free
4
selection-free gene
4

Similar Publications

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!