Factors influencing heterologous gene expression in the yeast Saccharomyces cerevisiae.
thesisposted on 19.11.2015, 08:53 by Bernadette. Jordan
The yeast Saccharomyces cerevisiae is widely employed as a host for the production of recombinant proteins. In the work presented here, expression of recombinant reporter proteins has been analysed in yeast, with the aim of determining factors that contribute to the expression performance of recombinant genes. Initially, expression of bacterial aminoglycoside phosphotransferase was analysed in yeast. This heterologous gene showed very low expression levels, resulting from a small decrease in mRNA abundance, plus a large decrease in translation efficiency. This appeared to be due to an unfavourable codon usage confirmed with an alcohol dehydrogenase reporter gene having highly favourable codon usage. Addition of a PGKl, ADHl, or CYCl promoter to a promoterless aminoglycoside phosphotransferase gene on a 2mum-based plasmid resulted in a reduced transformation efficiency and copy number, despite the product being non-toxic to yeast. This was not manifest in ARS plasmids. The decrease in copy number, represented a loss of potential product-encoding genes. Further investigation implicated a number of factors; regulatory functions, transcription and codon usage/translation. The promoter-inhibition did not correlate with promoter strength, nor did it result from transcriptional bypass of terminators. It was increased by a PGK promoter without a coupled coding sequence, but was reduced by promoter deletions in heterologous gene constructions, although not homologous PGK. The copy number reduction was also accompanied by reduced background transcription which was not apparent in deleted promoters that had increased copy number. A high codon bias gene lacking a promoter, showed both lower plasmid copy number and lower background transcription than one with low codon bias. Plasmid-borne PGK promoters were found to cause transactivation of chromosomal ADH expression, which did not occur with the ADHl promoter. This was not influenced by expression of ADH protein, nor other promoters that were tested, but could be mediated by single additional PGK promoters. The effect was influenced by some promoter deletions.