Overexpression studies – an approach to understand the mechanisms behind successful alcoholic fermentation.

Abstract

Predictable and reliable alcoholic fermentation by wine yeast Saccharomyces cerevisiae at specified temperatures as well as production of desirable flavour and aroma compounds, although not essential to the yeast, are sought-after winemaking attributes. The cellular mechanisms that allow yeast to grow in and respond to the harsh wine fermentation environment and successfully adapt to changing chemical stresses incurred during fermentation are largely unclear. A genome-wide screening approach utilising collections of yeast mutants with individual known gene deletions, is one paradigm being used to address this gap.

Our research has focused on screening approaches collections of yeast with individual gene deletants or gene overexpressers under fermentation conditions, to identify genes that are required for fermentation and which can modulate fermentation outcome. Such genes are representative of what we term the ‘fermentome’. We have previously reported on the identification of 93 genes which lead to fermentation protraction upon deletion, a dataset referred to as the Fermentation Essential Genes (FEG).

In this study we report on the construction of an over-expression library in a haploid wine yeast background, which is suitable for fermentation studies in high sugar medium, specifically chemically defined grape juice (CDGJM). This library was screened on a micro-scale (1.8 mL) with the aim of identifying those genes whose over-expression resulted in enhanced or protracted fermentation performance, and lastly, affected the fermentation profile of 36 known yeast-derived aroma compounds. This presentation will focus on preliminary findings on a subset of overexpression strains which are currently being evaluated for fermentation performance at a laboratory (100 mL) scale. Aroma data will not be discussed. It is envisaged that data collated from this study will expand on the FEG dataset as well as other datasets which together, comprise of the yeast ‘Fermentome’.

Through collation of gene datasets relevant to fermentation, we are able to identify and understand which genes (and their products) and cellular processes enable yeast to adapt and grow in grape juice and undergo reliable alcoholic fermentation as in winemaking. We have already constructed a number of gene modifications in haploid wine yeast, with the view of producing robust yeast which exhibit robust fermentation under nitrogen limited or high sugar containing CDGJM under laboratory scale conditions. We are also using the knowledge gained from our research to guide selection strategies in directed evolution approaches to generate new ‘industry ready’ wine yeast with improved fermentation attributes.