Creation of new Saccharomyces yeast hybrids useful to agro-industrial purposes, through innovative genetic engineering strategies

Yeasts, particularly those belonging to the Saccharomyces genus, are widely used in industrial and food biotechnology. They are employed in a wide range of fermentation processes, including the production of wine, beer, and distilled beverages, as well as in the generation of biofuels such as bioethanol. In addition, yeasts can be powerful cell factories for the synthesis of valuable biomolecules. Thus, the improvement of yeast strains for specific applications is of great interest in the industrial sector. Among the available methods for strain improvement, hybridization is the most widely applied, offering several advantages. It enables the combination of distinct genetic backgrounds to generate novel phenotypes, the enhancement of desirable parental traits while reducing their limitations, and the improvement of complex or multifactorial characteristics. Furthermore, hybrid strains often display increased fitness under industrial conditions, a phenomenon called “hybrid vigour” (heterosis). However, the targeted crossing of natural strains is not trivial: most industrial yeasts are diploid and homothallic, which makes experimentally difficult to isolate haploid spores from the strains to be crossed. In fact, unlike heterothallic yeasts, homothallic strains can switch their mating type thanks to the action of the HO endonuclease, and undergo self-diploidization in a short time. The objective of this Thesis was to develop a faster and efficient strategy for the generation and evaluation of yeast hybrids, with a focus on Saccharomyces cerevisiae. To this end, CRISPR/Cas9 system was employed to inactivate the HO gene in diploid homothallic parental strains, replacing it with two different selectable markers in the two parental strains. This genetic modification allowed the generation of stable heterothallic haploids after sporulation, which could then be easily crossed to produce novel hybrids. Several natural strains with different properties and industrial application, sourced from the collection of the project partner company Italiana Biotecnologie Srl (Trecate, Novara, Italy), were used as parental strains for implement the strategy. Yeast crosses were guided by the company’s specific requirements. In particular, three crosses were performed with oenological strains and one with a whisky strain. Moreover, the strategy was optimized also for performing an interspecific cross between a S. cerevisiae strain and S. uvarum strain. The resulting hybrids were characterized primarily through micro-fermentation assays, to evaluate their phenotypic traits of interest. Each crossbreeding experiment aimed to assess how the desired characteristics of the parental strains were combined in the hybrids, with particular attention to the improvement of specific traits relative to the parents. Data obtained demonstrated the power and efficacy of the CRISPR/Cas9-assisted breeding strategy to obtain and fully characterize yeast hybrids. Importantly, within only six months, representing a significant reduction compared to the 30 months typically required by conventional hybridization methods, it was possible to provide the company with meaningful insights into the phenotypic outcomes of the selected crosses and the extent to which desirable traits could be combined or enhanced in the hybrids. In addition, the selectable marker transgenes present in the hybrid strains were subsequently removed and the HO gene was restored by CRISPR/Cas9, allowing the hybrids to be reconverted from heterothallic to homothallic. This approach generated cisgenic hybrids, which cannot be genetically and phenotypically distinguished from naturally occurring hybrids. The implications of using genetic engineering in food-related organisms will be also discussed.