Construction of low-ethanol\u2013wine yeasts through partial deletion of the Saccharomyces cerevisiae PDC2 gene

Raúl Andrés Cuello,Karina Johana Flores Montero,Laura Analía Mercado,Iván Francisco Ciklic,Mariana Combina

doi:10.1186/s13568-017-0369-2

Abstract

We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance. In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol content. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.

Highlights

Nowadays there is a growing demand for softer wines with reduced ethanol content
It is quite surprising that none of the mutants showed a growth defect, especially BY4741pdc2Δ519 that carries only the mutant version of Pdc2p with a deletion of 519 amino acids which accounts for 56% of the wild type protein
The phenotypic analysis showed that BY4743pdc2Δ519 is the most interesting mutant strain, displaying a consistent reduction of the ethanol concentration of up to 7.4%, as a result of a slight inefficiency for its production

Summary

Introduction

Nowadays there is a growing demand for softer wines with reduced ethanol content. during the last twenty years there has been an increment in the alcohol concentration of wine of about 2% (v/v) (Kutyna et al 2010; Tilloy et al 2014). The biosynthesis of polyphenols is usually delayed with respect to sugar production, leading to a harvest of grapes with high sugar amounts. This sugar excess in turn, produces wines with high ethanol. In recent works, ethanol reduction between 1.5 and 2.2% (v/v) has been achieved through different strategies like sequential fermentation with immobilized non-Saccharomyces yeasts (Canonico et al 2016) and the use of nonSaccharomyces yeasts combined with S. cerevisiae under controlled aeration conditions (Contreras et al 2015; Morales et al 2015). Like in previous works, the increase of glycerol formation was accompanied with high levels of oxidized by-products like acetic acid, acetoin and acetaldehyde, and this effect could not be completely neutralized with additional genetic modifications

Methods

Results

Conclusion