1. Introduction

Modern industrial winemaking is based on the use of starter cultures of specialized wine strains of Saccharomyces cerevisiae yeast. Commercial wine strains have a number of advantages over natural isolates, and it is their use that guarantees the stability and reproducibility of industrial winemaking technologies. For the highly competitive wine market with new demands for improved wine quality, it has become increasingly critical to develop new wine strains and winemaking technologies. Novel opportunities for precise wine strain engineering based on detailed knowledge of the molecular nature of a particular trait or phenotype have recently emerged due to the rapid progress in genomic and “postgenomic” studies with wine yeast strains.

2. CRISPR-Cas for Wine Yeast

For laboratory strains of S. cerevisiae, an extensive and diverse set of tools for genetic engineering and directed modification of the genome has been developed quite a long time ago and are widely used for research in the fields of functional genomics, synthetic biology, biotechnology, and metabolic engineering [102]. At the same time, the application of such approaches for industrial strains faces a number of difficulties. These strains are usually polyploids and aneupoloids, poorly sporulate, there are no convenient auxotrophic markers for them, etc. [103].

The use of CRISPR-Cas genome editing systems can successfully overcome these limitations. The first work on the application of the CRISPR-Cas system for S. cerevisiae was published back in 2013 [104] and the advantages of this approach for yeast, in which the system of homologous recombination was already well developed, were at first not obvious. However, after overcoming a number of technical difficulties aimed at optimizing the expression and delivery of CRISPR-Cas system components, the system quickly gained popularity and is now successfully used in areas such as multiplex genome engineering, reprogramming transcription, creating synthetic genomes, etc. [105].

Examples of the successful application of CRISPR-Cas systems for industrial yeast strains relate to such aspects as the production of bioethanol from lignocellulosic raw materials, metabolic engineering for the production of vitamins and antibiotics, the improvement of aromatic and taste properties of beer, and a number of others [106–108]. From the point of view of food safety, it is fundamentally important that the use of CRISPR-Cas genome editing methods does not carry the risk of introducing foreign genes and genetic elements, markers of antibiotic resistance into the genomes of food yeast strains, i.e., the resulting strains are safe according to regulatory restrictions adopted in some countries.

One recent study describes the use of the CRISPR-Cas system for producing wine strains with reduced urea production. A group of scientists from Canada and Italy constructed derivatives of wine strains EC1118 and AWRI1796 defective in both alleles of the CAN1 gene [84]. The CAN1 gene encodes arginine permease, which along with GAP1 amino acids permease is responsible for the transport of arginine to yeast cells from the culture medium. During the subsequent stages of catabolism, arginine is cleaved by Car1p arginase to ornithine and urea, which is either excreted by Dur4p permease or converted to carbon dioxide and ammonia by Dur1p/Dur2p urea amidolyase. The resulting recombinant strains were characterized by reduced urea production (18–36% compared to the initial ones) under experimental micro-winemaking with the ability to ferment a synthetic substrate, although at a slightly reduced growth rate. The authors believe that further verification of the strains is necessary under the conditions of industrial winemaking. The advantage of introducing a mutation into the CAN1 gene compared to other methods of modifying arginine utilization pathways is that this technique is less sensitive to fluctuations in the content of nitrogen sources in the wort and less affects the growth parameters of yeast strains [108].

A promising area of application of genome editing methods is the directed change in the pathways of biosynthesis of aromatic compounds. Thus, in a recent work, yeast strains with increased production of phenylethyl acetate (PEA) were obtained using the CRISPR-Cas system [109]. PEA is an important aromatic compound that provides alcoholic drinks a pink and honey flavor. Genetic mapping methods first identified unique alleles of the FAS2 genes (encodes the α subunit of fatty acid synthase) and TOR1 (a growth regulator in response to the availability of a nitrogen source), linked to the trait of increased PEA production. Then, using CRISPR-Cas in commercial wine strains, wild alleles were replaced with mutant ones. As a result, the production of PEA increased by 70% [110].

In another work, the CRISPR-Cas system was used to reduce the production of 4-vinyl guaiacol (4VG) in a hybrid S. pastorianus/bayanus beer yeast strain [111]. It is known that 4VG is a sharp-tasting phenolic compound that spoils the organoleptic characteristics of beer. Formed from ferulic acid, 4VG is present in beer wort under the influence of yeast decarboxylase Fdc1p. Ale beer yeast strains do not produce 4VG due to the nonsense mutation in the FDC1 gene. Using the CRISPR-Cas system, the authors introduced a mutation characteristic of ale strains into all four copies of the FDC1 gene in the lager strain. The result was a strain containing a cis-gene mutation that lacks the ability to produce 4VG and has significant potential for use in the beer industry.

The CRISPR-Cas system is an extremely convenient tool for research in the field of functional genomics of wine strains. Until recently, the vast majority of experiments in the field of functional genomics of yeast were performed using laboratory strains. Nevertheless, according to the latest information from the SGD database (27 June 2020), when classified in terms of gene ontology, a significant number of yeast genes remain “unknown” (in the category “Biological Process”-1768 genes, 2548 genes in the category “Molecular Function” and 1298 genes in the cell compartment category). Such uncertainty is partly determined by the lack of specific conditions in which these genes are important. At the same time, these unknown genes experience regular changes in expression during many technological processes, including at different stages of wine fermentation (see, for example, [112]).

Characteristic changes in the expression pattern of a number of “unknown” genes were revealed in our recent work during the transcriptome analysis of the sherry strain at different stages of film formation [113]. CRISPR-Cas mediated genetic inactivation of “unknown” genes, allele replacement in wine strains of yeast can significantly clarify their role in various winemaking processes, and will help to create strains with improved characteristics(Table 1).

Table 1. Selected metabolically-engineered yeast strains and their oenology-related phenotypes.

* other modifications had non-significant effects.