RU-2861648-C1 - GENETIC CONSTRUCT FOR INTEGRATING MALOLACTIC FERMENTATION GENES INSTEAD OF ARGINASE GENE INTO GENOMES OF WINE YEAST STRAINS OF SACCHAROMYCES CEREVISIAE

RU2861648C1RU 2861648 C1RU2861648 C1RU 2861648C1RU-2861648-C1

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to a genetic construct for integrating malolactic fermentation genes into the genomes of wine yeast strains of Saccharomyces cerevisiae , intended for creating new wine yeast strains and containing the genes of malolactic enzyme (mleA) under the control of the PGK1 gene promoter and malate permease (mae1) under the control of the TDH1 gene promoter, encoding enzymes that provide malolactic fermentation, for integration into the genome of Saccharomyces cerevisiae yeast instead of the CAR1 gene encoding arginase. EFFECT: possibility of obtaining genetically modified Saccharomyces cerevisiae strains capable of carrying out malolactic fermentation and at the same time providing reduced urea content. 1 cl, 13 dwg, 4 tbl, 4 ex

Inventors

Urakov Valerii Nikolaevich
Kushnirov Vitalii Vladimirovich
Vasiagin Egor Arkadevich
Mardanov Andrei Vladimirovich
ALEKSANDROV ALEKSANDR IVANOVICH
Ravin Nikolai Viktorovich
Tanashchuk Tatiana Nikolaevna
IVANOVA ELENA VLADIMIROVNA
Shalamitskii Maksim Iurevich

Dates

Publication Date: 20260506
Application Date: 20250303

Claims (1)

A genetic construct for integrating malolactic fermentation genes instead of the arginase gene into the genomes of wine yeast strains Saccharomyces cerevisiae , designed to create new wine yeast strains and representing a nucleotide sequence containing the genes of malolactic enzyme ( mleA ) under the control of the PGK1 gene promoter and malate permease ( mae1 ) under the control of the TDH1 gene promoter, encoding enzymes that ensure malolactic fermentation, for integration into the genome of the yeast Saccharomyces cerevisiae instead of the CAR1 gene encoding arginase.

Description

The invention relates to biotechnology and molecular biology, specifically genetic engineering. Its use, through genome editing, will enable the production of genetically modified Saccharomyces cerevisiae strains capable of malolactic fermentation while simultaneously producing reduced urea levels. The use of these modified strains will improve the flavor and other consumer properties of wine and can be used in the production of wine and other fermented foods. The invention provides a platform for the development of new promising S. cerevisiae strains for the food industry, and especially for winemakers. Saccharomyces cerevisiae yeast is a traditional microorganism used in winemaking. Various approaches are used to improve the flavor and other properties of wine materials and fermentation products. One such technique is malolactic fermentation. Malolactic fermentation The vast majority of red wines, as well as some white and sparkling wines, are produced through two sequential fermentation stages: vinifera fermentation, carried out by yeast, and malolactic fermentation (the conversion of malic acid to lactic acid with the release of carbon dioxide and water, abbreviated MLF). Including the MLF stage in the fermentation process reduces the acidity of the wine and improves its bouquet and microbiological stability. MLF is typically carried out by lactic acid bacteria found in wine must, such as Oeonococcus oeni . However, these bacteria are sensitive to the inhibitory conditions of wine fermentation. Therefore, a wine yeast strain capable of simultaneously carrying out MLF would be of great interest to winemakers. The ML01 strain (Husnik et al., 2006) was previously described as capable of simultaneously carrying out both vinifera fermentation and malolactic fermentation. This strain, derived from the wine strain S92, contains the mae1 malate permease gene from S. pombe and the mleA malolactic enzyme gene from O. oeni under the control of a strong constitutive promoter of the PGK1 gene from S. cerevisiae . The strain is capable of completely fermenting malic acid at a concentration of 5 g/L in must within 5 days without adversely affecting the sensory qualities of the wine. Further phenotypic, transcriptomic, and proteomic analysis showed that the ML01 strain is equivalent to the original parental wine strain. Ethyl carbamate in winemaking. It is well known that ethyl carbamate (EC) accumulates in wine must during the winemaking process (Larcher et al., 2013; Ryu et al., 2015; Leca et al., 2021). This substance is a Group 2A carcinogen. EC (urethane) is formed in wine must as a result of the interaction of ethanol with urea excreted from yeast cells. In turn, urea and L-ornithine are products of arginine breakdown by Car1 arginase, encoded by the CAR1 gene. The source of arginine is wine must, in which this amino acid is present in high concentrations. EC can accumulate in wine and wine products over time. Legislation in many countries strictly regulates the maximum content of this substance in food products. The problem is that removing this substance accumulated in wine distillate is a complex and expensive procedure. Therefore, reducing the level of EC formation represents an important biotechnological challenge for winemakers. One promising approach to addressing this challenge is the targeted modification of wine yeast by reducing the level of urea synthesis during growth (Shalamitskiy et al., 2023). Of greatest practical interest in this regard is the CAR1 gene, which encodes arginase . It is well known that disruption of this gene leads to reduced urea accumulation. CAR1 is not essential, so one way to reduce arginase activity is to deleting this gene (Weber et al., 2008; Wu et al., 2014; Guo et al., 2016; Chin et al., 2016; Chadani et al., 2021; Chin et al., 2021; Jung et al., 2022; Klinkaewboonwong et al., 2023, Urakov et al., 2023). Polyploid strains One of the features of yeast application in the food industry is the predominant use of polyploid strains. Such strains generally possess favorable technological properties. However, the presence of multiple genome copies can pose certain difficulties in producing mutant variants of polyploid strains, as it is often necessary to introduce the desired genetic changes into all copies of the gene being modified. Various methods can be used to create such variants of industrial strains. One of the most promising approaches for efficient yeast genome editing is the use of CRISPR/Cas9 technology (Giersch & Finnigan, 2017; Rainha J, 2020). The desired genome modification can be achieved by targeted introduction of a double-strand break into a specific chromosomal region and its repair in vivo using an artificially produced fragment of donor DNA with a specified sequence. For example, for the polyploid industrial yeast strain ATCC 4124, single-gene deletion efficiencies of 15% to 60% were reported, compared to nearly 100% efficiency for laboratory haploid analo