Exploring the adaptive landscape of multigenerational inter-specific hybrids to improve iso-butanol tolerance.

Climate change has catalysed a worldwide search for renewable and sustainable products to replace oil-derivatives. One quarter of the global greenhouse gas emissions come from the transportation and chemicals industry, and there is a strong desire to find alternative fuels and chemicals, which can be produced with environmentally friendly processes. One possibility is the use of genetically modified microorganisms grown on renewable feedstocks to produce fuels and chemicals by fermentation. Of importance is the production of branched-chain alcohols, namely isobutanol, a desirable next-generation biofuel, suitable for a multiproduct biorefinery. Isobutanol is quite toxic to the production microorganism, and depending on the concentration, it can inhibit cell growth completely, or affect cell physiology in a way that lowers the yield and total amount of isobutanol produced. Yeasts belonging to the Saccharomyces genus can hybridise readily, creating first-generation (F1) hybrids with unique phenotypes enabling them to survive and proliferate in the stressful environments, and hence can be considered as potential microbial hosts for industrial processes. Hybridisation provides a novel source of variation for evolution to act upon, leading to adaptations that could not occur in either parental species. The problem with the development of inter-specific hybrids is that, although they are viable and sometimes fitter than the parents, they are sterile, and hence not genetically tractable. An example from Aristoteles's time is the hybrid between a female horse and a male donkey, namely the mule, which is a healthy animal employed in several human activities but cannot produce any offspring. Adaptive Laboratory Evolutionary (ALE) experiments, where a microbial population is evolved under a specific pressure to select for fitter progeny, has only been carried out on specific strains or sometimes on F1 hybrids. Although the F1 hybrids contain all the genetic diversity of the two parents, there is no recombination and random segregation of alleles, as it would happen after meiosis where specific parental traits are distributed in the offspring in different combinations and can give rise to different phenotypes. In our lab, we were able to overcome hybrid sterility by duplicating the genome content of the diploid F1 hybrids, making them tetraploids. Such tetraploid lines could undergo meiosis and so we were able to create F12 progenies with a large and diverse combination of traits. Here, we propose to use ALE to evolve F12 hybrid lines and their parents, for tolerance to isobutanol. The objective is to identify specific genotypes with high tolerance to this branched-chain alcohol, and to develop new potential production hosts. Furthermore, we will demonstrate that the higher genetic diversity, engrained in the ancestral F12 population, increases the microbial adaptation potential, and leads to a larger pool of extreme phenotypes in the evolved F12 hybrids compared to the evolved parents. We plan to sequence the genome of the best candidates and to study their gene expression to identify genes, promoters, and pathways that are responsible for the isobutanol tolerance trait. We will genetically re-construct in the parental strains a selection of genetic variants, identified in the evolved F12 population that are resistant to isobutanol, to validate their phenotypic effects. Lastly, we will grow the evolved high tolerant hybrids in the presence of other toxic compounds of industrial relevance to see whether they acquired some cross-protection to other branched-chain alcohols or whether, due to the acquired adaptation to isobutanol, they lost the ability to grow efficiently on other inexpensive renewable substrates. Ultimately, the more valuable hybrids will be those that have acquired isobutanol resistance with the least trade off in other relevant industrial conditions.