Controlled Gene Amplification Enables High, Stable, Selection Free Gene Expression in Saccharomyces Cerevisiae
Synthetic Biology Engineering Evolution Design SEED
2014
2014 Synthetic Biology: Engineering, Evolution & Design (SEED)
Poster Session
Poster Session
Yeast cell factories can be used to produce a wide range of products, from drugs and vaccines, biofuels, chemical building blocks etc. A widely used strategy to increase the production of a compound of choice in a yeast cell factory is by manipulating the gene copy number. A common way to obtain high gene copy numbers is by the expression from the yeast 2 micron (2μ) multi copy plasmid. The 2μ plasmid enables high expression levels, but comes with inherent genetic instability, since plasmid copies are lost over time, even under selective pressure, and without selection pressure, which is common for industrially relevant media, the plasmid loss is only exacerbated. Furthermore while 2μ often yield high average expression levels, the variance between individual cells in the populations are differs in several orders of magnitude. Genomic integration provides an attractive alternative to plasmid based expression, but brings other concerns, for instance not all chromosomal regions might be able to support high expression due silencing effects, while another concern might be that multiple integrations in the form of tandem repeats risk excision by direct repeat recombination.
To solve some of these issues we have developed a novel gene amplification system, which allows for the construction of stable yeast strains with up to ten integrated gene copies. The gene copy number can be precisely controlled and integration happens at predefined locations, capable of supporting high levels of expression. It is a simple procedure to use the system, only requiring transformation of the genes to be amplified followed by induction and transfer to selective media, which can be done in a timeframe of 15 days. Gene copy number is decided in advance, by choosing one of several strains we have created. Using CFP or β-galactosidase as model proteins a linear correlation between gene copy number and expression levels could be observed, when testing copy numbers between one and seven. Furthermore expression levels of a seven copy strain with CFP equaled that of a strain expressing CFP from a 2μ plasmid. To test the stability and consistency of the system, FACS (Fluorescence-activated cell sorting) experiments were setup, measuring the fluorescence on a single cell level. Results showed that CFP and RFP when integrated in our system, were expressed far more consistently when grown without selection, compared to when RFP and CFP were expressed from a 2μ plasmid and grown under selection pressure. Furthermore the system has successfully been applied to increase the production of both vanillin-glucoside and 6-Methyl Salicylic Acid. Finally it is possible to amplify large pieces of DNA, giving the opportunity to amplify whole pathways, greatly expanding the applications of the system.