Development of transformation systems for wine yeasts
Bauer, F F
University of Stellenbosch. Faculty of AgriSciences. Institute for Wine Biotechnology
Gey van Pittius, M H
Franken, C J
The aim of this project is to develop a reliable and inexpensive genetic system for the introduction of useful genes into wine yeasts. This might result in a patentable vector system or method for the transformation of wine yeasts. This should also considerably shorten the amount of time required to reproduce laboratory experiments in industrial yeasts.
In recent years, the genetic manipulation of wine yeast to produce superior new strains has gained increasing interest and much effort has gone into identifying and characterising genes which encode useful properties using laboratory strains of Saccharomyces cerevisiae. However, the lack of a reliable genetic system for constructing stable transgenic wine yeasts has prevented the use of these genes in the wine industry. This is mainly due to the fact that in contrast to laboratory strains of S. cerevisiae, which are genetically well characterised and easy to manipulate, industrial yeast strains are notoriously resistant to genetic manipulation. Industrial strains are often polyploid, which means that conventional selectable auxotrophic markers (e.g. URA3, TRP1 and LEU2) commonly used in laboratory strains are ineffective. Another major problem is that a genetically modified organism (GMO) which is to be released into the environment should not contain any other foreign DNA apart from the gene (e.g. BGL2) of interest. Conventional vector systems would therefore also be unsuitable as the recombinant strain often carries genetic material of bacterial origin (e.g. antibiotic resistance markers).
This project therefore confronts two major challenges: firstly, to identify suitable, dominant markers for the selection of transformed industrial yeast cells and secondly, to develop a vector system that will introduce only the gene of interest.
The proposed vector system will give the option to place the gene of interest under the control of different promoters (PGK1, ENO1, ADH2 and HSP30). The promoters are either constitutive (PGK1 & ENO1), repressed by glucose (ADH2) or induced by stress or during the stationary phase of growth (HSP30). A duplicate set of vectors will be constructed that will also contain the MFa1 secretion signal. This set of vectors will allow for the secretion of enzymes into the medium. The vector system consists of a transformation cassette that is composed of the desired regulatory control sequences, the G418 resistance marker gene that is flanked by loxP sequences (see cassette construction). The gene of interest is cloned into the cassette under the desired regulation. The entire cassette then undergoes PCR with primers containing homologous recombination sequences (sigma, LEU2 etc.) that are responsible for the stable integration of the cassette into the yeast genome (see transformation procedure). This occurs by means of homologous recombination with naturally occurring sigma/etc. sequences within the yeast genome. Integration of the cassette is confirmed by Southern blot. An episomal plasmid containing the cre gene (encoding a recombinase) under the control of the GAL1 promoter and the sulfometuron methyl resistance marker (SMR1) is then transformed into the yeast. By shifting the yeast to galactose medium the expression of the cre gene is induced. The recombinase targets the loxP sequences and causes homologous recombination between them, thereby removing the G418 marker (kanMX) gene. The desired gene remains integrated in the yeast genome. The episomal plasmid is lost by growing the yeast on a non-selective medium. This vector system does not incorporate bacterial sequences into the yeast and enables repeated use of the same marker gene.