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Yeast Biotechnology Doctoral Training Programme

Periodic Reporting for period 2 - YEASTDOC (Yeast Biotechnology Doctoral Training Programme)

Reporting period: 2019-09-01 to 2022-08-31

Yeasts have been used in the production of fermented foods and beverages for thousands of years, and have many traits that make them very attractive and useful for modern biotechnology. Modern yeast biotechnology products include fermented foods and beverages, feeds, biofuels, industrial chemicals, flavours, enzymes, and pharmaceuticals. high sugar in grapes). The overall research objectives of YEASTDOC were to apply modern genetics and develop methodology for improvement of yeast strains for novel applications in the fermented beverage industry and to enhance the robustness and performance of yeasts for industrial fermentation. For improving yeasts in fermented beverages, the aims were to better understand nutrient update and the links between fermentation conditions and flavour and aroma development. For industrial biotechnology the main questions centred on improving the capacity of yeast to grow on alternative feedstocks that would arise as industrial by or co-products. The use of waste products from other industries as the growth substrate for yeast cell factories is important to help achieve the European vision of a circular economy and reduce production costs across industries. From a training dimension, the project aimed to train a cohort of researchers that would have the skills and knowledge to play an active role in the development if the European bio-based economy. As well as technical skills, effective researchers need a suite of complementary skills that enable them apply their scientific know-how most productively to benefit society.
The YEASTDOC research programme is now completed with the original research goals successfully achieved. The work has led to the award of six double doctorates already, with six more theses due for submission in the coming months. There have been over forty conference proceedings and seventeen peer-reviewed papers in high quality journals published to date, with other research outcomes in preparation for publication. Several patents have or will be filed and other research outputs are technical know-how that will be applied in industrial research settings.
In a context of yeast-based fermented beverages, nitrogen source, availability and uptake is of utmost importance. Different projects in YEASTDOC focused on the study of nitrogen assimilation and catabolism in Saccharomyces and non-Saccharomyces yeasts from genetic, evolutionary, and biotechnological perspectives.The results allowed describing genotypic and environmental settings that are beneficial for the final aroma of fermented beverages, and the most promising combinations have been tested under industrially relevant conditions with real grape must.
Throughout the food and beverage industries that utilise yeasts in fermentation and production, hybrids are found that exhibit beneficial characteristics of each parental species (or strains within a species). In addition, non-conventional yeasts are less well characterised in terms of sexual reproduction, and most yeasts (conventional and non-conventional) generally have mating type switching mechanisms that make controlled breeding difficult. In YEASTDOC, we successfully screened many isolates of all the extant Saccharomyces species to identify potential partners for new hybrids that have desired traits for brewing. With Zygosaccharomyces yeasts, Z. balii is the main species found in food spoilage, however there are various hybrids used in beverage fermentation including Z. parabailii. Here, we developed tools for physical analysis of chromosome content as well as molecular tools for genetic engineering. Finally, genetic and molecular tools were developed for K. marxianus which until now has been resistant to attempts at crossing and breeding. In the project, several methods of yeast cell factory strain development and improvement were applied. Over the course of the project, the results contributed to significant advances in the capacity to develop different yeast species using engineering strategies such as genome engineering, ALE, reverse engineering, and construction of hybrids. The results show that these methods have now reached an advanced stage of maturity and are highly effective for a range of different yeast species.
In addition to their roles in the production of fermented beverages, yeast have features that make them very useful for cell factories to make commercial products such as bioethanol, organic acids, bioflavours and enzymes. One of the objectives of this project is to develop strains with new traits that can be exploited for the generation of beverages with new properties as well as more robust strains that cope with both substrate and product-induced cellular stress and that have the ability to grow on inexpensive substrates. To this end, we identified genes and alleles that are relevant to the improvement performance of yeast used in fermentation process. First, we identified genes that contribute to the improvement of aroma and flavour of fermented beverages by exploring the metabolism and characteristics of non-conventional yeasts opening possibilities of finding interesting traits with potential industrial applications. Second, we gained insight into molecular mechanisms involved in the robustness and performance of industrial yeasts growing on sustainable substrates like pentoses, glycerol, and succinic acid. Finally, we described genes that contribute to enhance organic acid stress tolerance that represents a major stress in industrial bio-based microbial processes.
The project substantial progress beyond the state of the art in multiple ways. The fact that several patents have or are being filed shows that new knowledge with commercial use was generated. In addition, the high number of publications reflects a large corpus of new knowledge that benefits the research community. The specific areas where research advances were made were described above and are not repeated here. In an overall sense, the broad topics where we have advanced the state of the art are
1. Technologies such as construction of hybrids, ALE, CRISPR-engineering, and QTL mapping were significantly advanced for yeasts where these techniques were not routinely, or in some cases ever, applied.
2. New transporters and enzymes that have short-term biotechnology applications to improve cell factory strains for biotechnology were identified or engineered.
3. New knowledge on the influence of different nitrogen sources on flavour and aroma production in traditional and new wine yeasts was generated
4. New strains with improved capacity to grow robustly on industrial substrates was generated
5. New knowledge on the physiology and genetics of traditional and non-traditional yeast species was generated and this will accelerate their use for a wide range of applications in the bioeconomy.
The ESRs that carried out the work were highly engaged and had many interactions with stakeholders and the public. Several of them have already progressed to industry positions, demonstrating the relevance of the training and the research area. The transition from a fossil-based to a bio-based society requires highly skilled researchers who have the skills and the awareness to apply these to benefit society. The research outputs from, and the personnel trained in, YEASDOC can make a very positive contribution to the development of a sustainable, responsible bioeconomy in Europe.