Contributions of Adaptive Laboratory Evolution towards the Enhancement of the Biotechnological Potential of Non-Conventional Yeast Species.

Changes in biological properties over several generations, induced by controlling short-term evolutionary processes in the laboratory through selective pressure, and whole-genome re-sequencing, help determine the genetic basis of microorganism's adaptive laboratory evolution (ALE). Due to the versatility of this technique and the imminent urgency for alternatives to petroleum-based strategies, ALE has been actively conducted for several yeasts, primarily using the conventional species Saccharomyces cerevisiae, but also non-conventional yeasts. As a hot topic at the moment since genetically modified organisms are a debatable subject and a global consensus on their employment has not yet been attained, a panoply of new studies employing ALE approaches have emerged and many different applications have been exploited in this context. In the present review, we gathered, for the first time, relevant studies showing the ALE of non-conventional yeast species towards their biotechnological improvement, cataloging them according to the aim of the study, and comparing them considering the species used, the outcome of the experiment, and the employed methodology. This review sheds light on the applicability of ALE as a powerful tool to enhance species features and improve their performance in biotechnology, with emphasis on the non-conventional yeast species, as an alternative or in combination with genome editing approaches.

Torulaspora delbrueckii Phenotypic and Metabolic Profiling towards Its Biotechnological Exploitation.

Wine is a particularly complex beverage resulting from the combination of several factors, with yeasts being highlighted due to their fundamental role in its development. For many years, non-Saccharomyces yeasts were believed to be sources of spoilage and contamination, but this idea was challenged, and many of these yeasts are starting to be explored for their beneficial input to wine character. Among this group, Torulaspora delbrueckii is gaining relevance within the wine industry, owing to its low volatile acidity production, increased release of aromatic compounds and enhanced color intensity. In addition, this yeast was also attracting interest in other biotechnological areas, such as bread and beer fermentation. In this work, a set of 40 T. delbrueckii strains, of varied geographical and technological origins, was gathered in order to characterize the phenotypic behavior of this species, focusing on different parameters of biotechnological interest. The fermentative performance of the strains was also evaluated through individual fermentations in synthetic grape must with the isolates' metabolic profile being assessed by HPLC. Data analysis revealed that T. delbrueckii growth is significantly affected by high temperature (37 °C) and ethanol concentrations (up to 18%), alongside 1.5 mM SO2, showing variable fermentative power and yields. Our computation models suggest that the technological origin of the strains seems to prevail over the geographical origin as regards the influence on yeast properties. The inter-strain variability and profile of the products through the fermentative processes reinforce the potential of T. delbrueckii from a biotechnological point of view.

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

Torulaspora delbrueckii has attracted interest in recent years, especially due to its biotechnological potential, arising from its flavor- and aroma-enhancing properties when used in wine, beer or bread dough fermentation, as well as from its remarkable resistance to osmotic and freezing stresses. In the present review, genomic, biochemical, and phenotypic features of T. delbrueckii are described, comparing them with other species, particularly with the biotechnologically well-established yeast, Saccharomyces cerevisiae. We conclude about the aspects that make this yeast a promising biotechnological model to be exploited in a wide range of industries, particularly in wine and bakery. A phylogenetic analysis was also performed, using the core proteome of T. delbrueckii, to compare the number of homologous proteins relative to the most closely related species, understanding the phylogenetic placement of this species with robust support. Lastly, the genetic tools available for T. delbrueckii improvement are discussed, focusing on adaptive laboratorial evolution and its potential.

Improvement of Torulaspora delbrueckii Genome Annotation: Towards the Exploitation of Genomic Features of a Biotechnologically Relevant Yeast.

Saccharomyces cerevisiae is the most commonly used yeast in wine, beer, and bread fermentations. However, Torulaspora delbrueckii has attracted interest in recent years due to its properties, ranging from its ability to produce flavor- and aroma-enhanced wine to its ability to survive longer in frozen dough. In this work, publicly available genomes of T. delbrueckii were explored and their annotation was improved. A total of 32 proteins were additionally annotated for the first time in the type strain CBS1146, in comparison with the previous annotation available. In addition, the annotation of the remaining three T. delbrueckii strains was performed for the first time. eggNOG-mapper was used to perform the functional annotation of the deduced T. delbrueckii coding genes, offering insights into its biological significance, and revealing 24 clusters of orthologous groups (COGs), which were gathered in three main functional categories: information storage and processing (28% of the proteins), cellular processing and signaling (27%), and metabolism (23%). Small intraspecies variability was found when considering the functional annotation of the four available T. delbrueckii genomes. A comparative study was also conducted between the T. delbrueckii genome and those from 386 fungal species, revealing a high number of homologous genes with species from the Zygotorulaspora and Zygosaccharomyces genera, but also with Lachancea and S. cerevisiae. Lastly, the phylogenetic placement of T. delbrueckii was clarified using the core homologs that were found across 204 common protein sequences of 386 fungal species and strains.

Learning from 80 years of studies: a comprehensive catalogue of non-Saccharomyces yeasts associated with viticulture and winemaking.

Non-Saccharomyces yeast species are nowadays recognized for their impact on wine´s chemical composition and sensorial properties. In addition, new interest has been given to the commercial exploitation of non-Saccharomyces starter cultures in the wine sector. However, over many years, these yeast species were considered sources of contamination in wine production and conservation, mainly due to the high levels of volatile acidity obtained. The present manuscript systematizes 80 years of literature describing non-Saccharomyces yeast species isolated from grapes and/or grape musts. A link between each reference, the accepted taxonomic name of each species and their geographical occurrence is presented, compiling information for 293 species, in a total of 231 citations. One major focus of this work relates to the isolation of non-Saccharomyces yeasts from grapevines usually ignored in most sampling studies, also as isolation from damaged grapes. These particular niches are sources of specific yeast species, which are not identified in most other explored environments. These yeasts have high potential to be explored for important and diversified biotechnological applications.

Population Analysis and Evolution of Saccharomyces cerevisiae Mitogenomes.

The study of mitogenomes allows the unraveling of some paths of yeast evolution that are often not exposed when analyzing the nuclear genome. Although both nuclear and mitochondrial genomes are known to determine phenotypic diversity and fitness, no concordance has yet established between the two, mainly regarding strains' technological uses and/or geographical distribution. In the current work, we proposed a new method to align and analyze yeast mitogenomes, overcoming current difficulties that make it impossible to obtain comparable mitogenomes for a large number of isolates. To this end, 12,016 mitogenomes were considered, and we developed a novel approach consisting of the design of a reference sequence intended to be comparable between all mitogenomes. Subsequently, the population structure of 6646 Saccharomyces cerevisiae mitogenomes was assessed. Results revealed the existence of particular clusters associated with the technological use of the strains, in particular regarding clinical isolates, laboratory strains, and yeasts used for wine-associated activities. As far as we know, this is the first time that a positive concordance between nuclear and mitogenomes has been reported for S. cerevisiae, in terms of strains' technological applications. The results obtained highlighted the importance of including the mtDNA genome in evolutionary analysis, in order to clarify the origin and history of yeast species.