Adaptive Laboratory Evolution of Native Torulaspora delbrueckii YCPUC10 With Enhanced Ethanol Resistance and Evaluation in Co-inoculated Fermentation

Abstract

Torulaspora delbrueckii is a yeast species typically present in the early stages of the fermentation process. T. delbrueckii positively modifies the aromatic properties of wines. However, its contribution to the final quality of the wine is restricted by its low tolerance to ethanol. T. delbrueckii is capable of fermenting and tolerating an ethanol concentration ranging from 7.4% (v/v) to slightly higher than 9% (v/v). For this reason, it cannot complete fermentation, when alcohol reach levels higher than 12% (v/v), limiting their use in the industry. The objective of this work was to obtain new variants of T. delbrueckii with improved resistance to ethanol through adaptive laboratory evolution. Variants capable of tolerating ethanol levels of 11.5% (v/v) were obtained. These presented improved kinetic parameters, and additionally showed an increase in resistance to SO2 in ethanol compared to the original strain. Co-inoculated fermentations were performed with the original strain (FTd/Sc) and with the evolved strain (FTdF/Sc), in addition to a control fermentation using only Saccharomyces cerevisiae EC1118 (FSc). The results obtained show that FTdF/Sc present higher levels of 2-Ethylhexanol, compared to FTd/Sc and FSc. Furthermore, FTdF/Sc presents higher levels of total alcohols, total aldehydes, total phenolic derivatives, and total sulfur compounds with significant differences with FSc. These results provide a T. delbrueckii YCPUC10-F yeast with higher resistance to ethanol, which can be present throughout the fermentation process and be used in co-inoculated fermentations. This would positively impact the performance of T. delbrueckii by allowing it to be present not only in the early stages of fermentation but to remain until the end of fermentation.

Native Yeasts and Lactic Acid Bacteria Isolated from Spontaneous Fermentation of Seven Grape Cultivars from the Maule Region (Chile)

“…Although non-Saccharomyces yeasts have low tolerance to ethanol, our results show that some can be isolated once the alcoholic fermentation was completed (Table 2). Catrileo, Acuña-Fontecilla, and Godoy (2020) reported that T. delbrueckii YCPUC10, isolated from C. Sauvignon grape juice, was able to persist with ethanol levels of about 11% as the result of an adaptative metabolism [79]. Furthermore, certain non-Saccharomyces are able to increase their ethanol tolerance by producing succinic acid [80] and this might be the case of the Metschnikowia, Torulospora, Hanseniospora, and Candida spp.…”

Torulaspora delbrueckii Phenotypic and Metabolic Profiling towards Its Biotechnological Exploitation

“…The T. delbrueckii species is usually defined as a low ethanol producer [ 2 , 25 , 76 ], however in the work of Escribano et al [ 33 ], T. delbrueckii was also one of the highest ethanol producers, among the non- Saccharomyces species tested. Additionally, Canonico et al [ 77 ] reported ethanol production in values higher than 12% ( v / v ) by T. delbrueckii strains, and Catrileo et al [ 78 ], using adaptive laboratory evolution, were able to obtain a strain capable of producing ethanol levels of 11.5% ( v / v ). In this way, we concluded that this characteristic is strain-dependent, and should not be attributed as a feature associated to the species.…”

Biotechnological Importance of Torulaspora delbrueckii: From the Obscurity to the Spotlight

“…When considering K. marxianus, strains with higher growth rates were obtained under stressful high ethanol environments [116], within the range of 7-10% (v/v) of ethanol [117]. In a similar way, a recent study and, to the extent of our knowledge, the only report applying this technique to the generation of T. delbrueckii evolved strains, was conducted by Catrielo et al [96]. The authors efficiently obtained evolved clones of T. delbrueckii with improved growth kinetic parameters, increased ethanol resistance, variants capable of tolerating ethanol concentrations of 11.5% (v/v), and additional higher resistance to SO 2 , in comparison with the original strain.…”

“…In recent years, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/associated protein 9) revolutionized research as a gene-based editing mechanism allowing the selective manipulation of DNA [92,93]. Although there is yet no current employment of CRISPR towards T. delbrueckii, this engineering tool has been extensively used for other non-conventional yeasts such as Yarrowia lipolytica, Pichia pastoris, Komagataella phaffii, Kluyveromyces lactis, Kluyveromyces marxianus [94], Schizosaccharomyces pombe, Candida albicans and Candida glabrata, and for the conventional yeast S. cerevisiae [92][93][94][95][96][97].…”

“…Besides the versatility of T. delbrueckii species, some limitations have been hampering its wide biotechnological use, such as the limited ethanol resistance of some strains during wine fermentation, which ranges from 7.4% (v/v) [14] to 9% (v/v) [36], in addition to the fact that Belda et al [27] indicate decreases of cells viability already when ethanol levels exceed 8% (v/v). In this context, some strategies have been proposed in this species in order to improve phenotypes with a biotechnological impact, such as random mutagenesis, sexual hybridization, bioprospecting and metabolic engineering [67,96] to expand the boundaries of T. delbrueckii's biotechnological use. The use of Genetically Modified Organisms (GMOs) involves a certain ambiguity and divergence of opinions, and their implementation relies on the legislation in force in each country [97,98].…”