Towards tools for 'seamless' modification of genes in industrial yeast

http://www.awitc.com.au/past_conferences/Twelfth

PCR-based gene disruption and recombinatory marker excision to produce modified industrial Saccharomyces cerevisiae without added sequences

The dominant selectable Kanr marker, which confers geneticin resistance in yeast, is extensively used for PCR based disruption of genes in functional analysis studies in laboratory strains of Saccharomyces cerevisiae. We have developed a gene disruption cassette, which incorporates the Kanr marker, and direct repeat sequences designed from the target gene to enable the deletion of the gene without the introduction of added DNA sequences. We report on the disruption of the HO gene as a test case, using the hodr-Kanr-hodr cassette. The cassette was shown to integrate at the HO locus and the Kanr marker excised by recombination between the two direct repeat sequences. The disruption/excision event resulted in the removal of one direct repeat and the coding sequence of the gene, and hence in this case loss of HO function, with the introduction of no foreign or additional sequences, including the Kanr marker. Having been derived from the target site, the remaining direct repeat sequence is native sequence in its native location. This design template has the potential to be adapted to other genes, and as such will be of advantage in instances such as the optimization of strains by recombinant DNA technology where the retention of minimal or no foreign sequences is desired.Michelle Walker, Andrea Vystavelova, Scott Pedler, Jeff Eglinton and Vladimir Jirane

Application of the reuseable, KanMX selectable marker to industrial yeast: construction and evaluation of heterothallic wine strains of Saccharomyces cerevisiae, possessing minimal foreign DNA sequences

The characterisation of wine yeasts and the complex metabolic processes influencing wine fermentation and the quality of wine might best be achieved by exploiting the standard classical and recombinant genetic techniques which have been successfully used with laboratory strains. However, application of these techniques to industrial strains has been restricted because such strains are typically prototrophic and often polyploid. To overcome this problem, we have identified commercial wine strains with good mating and sporulation properties from which heterothallic derivatives were constructed by disruption of the HO gene. Consequently, these haploids are amenable to genetic analysis, whilst retaining desirable wine-making properties. The approach used was an adaptation of a previously published gene disruption procedure for laboratory yeast and is based on the acquisition of geneticin resistance from a removable KanMX marker. The present work is the first report of the application of a construct of this type to the disruption of the HO gene in wine yeasts that are in common commercial use. Most of the 4.9-kb disruption construct was successfully removed from the genome of the haploid derivative strains by loop-out of the KanMX marker through meiotic recombination. Sequencing of the HO region confirmed the reduction of foreign sequences to a 582-bp fragment comprised largely of a single direct repeat at the target gene. The removal of the active foreign gene (conferring antibiotic resistance) allows the application of other constructs based on the KanMX module without the need to resort to other selectable marker systems. Laboratory-scale fermentation trials typically showed minimal differences between the HO disruptants and the parental wine strains in terms of fermentation kinetics and formation of key metabolites.Michelle E. Walker, Jennie M. Gardner, Andrea Vystavelova, Colin McBryde, Miguel de Barros Lopes and Vladimir Jirane

Identification of genes affecting glucose catabolism in nitrogen-limited fermentation

The definitive version is available at www.blackwell-synergy.comIn recognition of the importance of assimilable nitrogen in the successful completion of several fermentation processes, we have sought to develop yeast strains that utilise this typically limited nutrient group more efficiently. With the aid of transposon mutagenesis together with a high-throughput method for analysis of multiple fermentations, we have identified nitrogen-efficient mutants that catabolise more sugar for a given amount of nitrogen utilised. In this way we have identified two genes, NGR1 and GID7, whose disruption leads to an enhanced catabolism of sugar in an industrial strain and/or a laboratory strain, during growth in a chemically defined grape juice medium with limiting nitrogen. Deletion of NGR1 or GID7 also resulted in minor changes in metabolites produced, and biomass yield, measured as dry weight, was also decreased in NGR1 mutant strains.Jennifer M. Gardner, Colin McBryde, Andrea Vystavelova, Miguel De Barros Lopes and Vladimir Jirane