Yi Qin* and Qing Du
*College of Enology, Northwest A&F University, 22 Xinong Rd, Yangling, Shaanxi, 712100, China (qinyi@nwsuaf.edu.cn)

With global warming, over recent decades the alcohol concentrations of wines from warm regions around the world has increased by ~2% (v/v). There is significant interest in the wine industry to develop methods to reduce the ethanol content of wine. Generally, microbiological strategies to isolate and/or generate the yeast strains used to make wine, including Saccharomyces cerevisiae and non-conventional yeast species, have proved the simplest and most economic methods. We used global transcriptional machinery engineering (gTME) technology, based on the mutation of the SPT15 gene, to weaken the capacity of S. cerevisiae to produce ethanol and ultimately created a new strain of S. cerevisiae, YS59-409, with ethanol-production capacity reduced by 34.9% below that of the control strain. Sequence analysis was performed on the mutated Spt15p, demonstrating that the five mutation sites (Ile 46 Met, Asp 56 Gly, Ser 118 Pro, Tyr 195 His, and Leu 205 Ser) may work collectively, or at least in part, to create the specific characteristics of S. cerevisiae YS59-409, including greater CO2 release, biomass, and glycerol formation. Integration of RNA-Seq and metabolomics analysis showed that the specific phenotype of the S. cerevisiae YS59-409 featured changes in ribosome biogenesis, nucleotide metabolism, glycolysis flux, the Crabtree effect, NAD+/NADH homeostasis, and energy metabolism. Furthermore, two genes related to energy metabolism, RGI1 and RGI2, were associated with the weakened ethanol production capacity, although the precise mechanisms involved are still unclear. This study highlights the potential of gTME technology to reduce the ethanol content of yeast for the winemaking industry.

Funding Support: National Key Research and Development Project (2019YFD1002500), National Natural Science Foundation of China (31301541 and 31960470), the Fundamental Research Funds for the Central Universities
(2452020177), and China Agriculture Research System (grant no. CARS-29-jg-3).