Identification of yeast genes enabling efficient oenological fermentation under nitrogen-limited conditions

Abstract

Nitrogen deficiency can often lead to slow or sluggish fermentation, resulting in wine out of specification and at risk of oxidation and microbial contamination. Problems due to nitrogen deficiency can be rectified by optimising grape chemistry (through vineyard fertilization), or more commonly supplementing the fermentation with ammonium salts. An alternative is to use wine yeast that can utilize nitrogen efficiently and complete fermentation more reliably. However, to develop ‘nitrogen efficient’ yeast, it is important to understand how such yeast can utilize nitrogen effectively by identifying genes that influence fermentation performance over a range of nitrogen concentrations. Past research related to the identification of genes influencing nitrogen efficiency under fermentative conditions is largely confined to laboratory yeast. Investigation of the ~5,000 non-essential genes in yeast is possible through research tools such as deletion libraries (collections of strains, each with a single gene deletion). Several genomewide studies have successfully used deletion libraries in the auxotrophic background of laboratory yeast to investigate phenotypes in response to exposure to single stress factors associated with fermentation. However, the need to supplement with amino acids to overcome auxotrophies makes quantitative physiological studies in nitrogen limiting conditions impractical. Therefore, in this study, we have used a prototrophic deletion collection in both laboratory and wine yeast backgrounds to identify genes influencing fermentation performance. Screening (micro-fermentation; 600 μL) of the prototrophic laboratory yeast deletion library (BY4741; 5,372 deletants) and the partial wine yeast library (AWRI1631; 1,844 deletants) for growth and consumption of sugar and nitrogen under limiting (75 mg FAN L⁻¹) and non-limiting nitrogen (450 mg FAN L⁻¹) conditions identified deletants with improved fermentation. To better understand the role of individual genes in fermentation, candidate gene sets from each screen were compared to each other and to other published data sets from genome wide transcriptomic analyses related to fermentation. Wine yeast deletants that enabled shortened micro-fermentation duration in low nitrogen conditions were further investigated, since the experiment best represented nitrogen deficient grape must associated with problematic fermentation. Fifteen deletants completed fermentation quicker than the wildtype (c.a. a 15-59% time reduction) when tested in larger (100 mL) fermentations. This group of genes were annotated to biological processes including protein modification, transport, metabolism and ubiquitination (UBC13, MMS2, UBP7, UBI4, BRO1, TPK2, EAR1, MRP17, MFA2 and MVB12), signalling (MFA2) and amino acid metabolism (AAT2). Among the genes identified, MFA2 (mating a-factor), which conferred a 34% decrease in fermentation duration, was further investigated. We were interested to understand how deletion of this mating type gene affected fermentation since a link between these metabolic pathways would be novel. The 15 strains identified in this study, which were fermentation proficient in a ‘wine-like', limited nitrogen condition, provide a basis to better understand how yeast adapt to nitrogen limitation during fermentation. Furthermore, the corresponding genes can be targeted in second generation strain improvement programs, using tools such as CRISPR (yet to be approved by relevant regulatory bodies) to generate nitrogen efficient yeast to reduce the need to supplement low nitrogen fermentations.