Yeast expression booster

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BACKGROUND Yeast-based expression systems are commonly used to produce both recombinant proteins and small molecules. Overexpression of a gene by increasing its copy number is generally desirable, but that copy number (and therefore yield) is often traded for other important factors such as growth efficiency and/or unstable modifications. THE TECHNOLOGY Researchers at The University of Queensland (UQ) have developed a technology for targeted and stable integration of a gene of interest in a manner that affords control of copy number and – when tested on a range of target genes in S. cerevisiae – increased protein expression compared to standard yeast systems. As part of normal yeast processes, the fitness cost associated with loss/reduction of many genes is overcome by switching to alternative pathways that perform equivalent biological processes. The UQ innovation leverages the response of yeast to loss/reduction of genes that cannot be compensated for if impaired — haploinsufficient genes. The UQ technology comprises an insertion construct that includes a weak promoter and a gene of interest, which through homologous recombination, is integrated in place of the natural promoter of a haploinsufficient gene. In response to reduced expression of the haploinsufficient gene, the cell should amplify the region – including the gene of interest – to maintain cell fitness. The expected result is a stable strain with multiple copies of a gene encoding a target protein. The integration can be achieved by commonly used transformation protocols without requiring harsh selection conditions, thereby carrying key potential advantages over current yeast technologies. PROOF OF CONCEPT The technology has been used to produce a range of different products in Saccharomyces cerevisiae, with yields substantially improved over plasmid-integration controls (Figure 1). Additionally, when compared to literature reports, the UQ technology outcompetes E. coli expression systems for production of natural products, including the difficult to produce toxic monoterpene limonene. Proof-of-concept data includes production of: • Lycopene, through introduction of gene encoding 3 enzymes of the lycopene production pathway (farnesyl pyrophophase, phytoene synthase, and phytoene desaturase), • Trans-nerolidol, through introduction of gene encoding nerolidol synthase, • Limonene, through the gene encoding limonene synthase. This titer is approximately 6-fold that of the highest titer previously reported in yeast, and higher than the titer achieved in E. coli; and • HPV capsid protein.