Exploring the adaptive landscape of multigenerational inter-specific hybrids to improve iso-butanol tolerance.
Lead Research Organisation:
The University of Manchester
Department Name: School of Biological Sciences
Abstract
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.
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.
Technical Summary
In this proposal, we aim to evolve new promising hosts for isobutanol production exploiting the biodiversity of the Saccharomyces species and their multigenerational inter-specific hybrids. Adaptive Laboratory Evolution (ALE) has so far been applied to specific yeast strain, hence the starting population had generally a low genetic diversity limiting the phenotypic outcomes. Hybridisation enhances genetic diversity and hence the adaptation ability of the hybrid. In fact, compared to the parents, hybrids often show transgressive phenotypes that can readily be adaptive in new extreme environments. The problem with developing hybrids is that they cannot be bred further. We overcame hybrid sterility by creating tetraploid hybrids encompassing the genome of four different strains belonging to two different species. Meiotic progenies of such tetraploid lines have a large and diverse combination of traits and represent an untapped source of evolvable biodiversity. Here, we proposed to carry out ALE on the 12th meiotic generation of interspecific hybrids (F12) and their parental strains, under increased amount of isobutanol to identify genotypes with high tolerance to this branched-chain alcohol. We will characterize, the main genomics changes affecting isobutanol tolerance in the evolved F12 offspring, identify convergent transcriptional signatures in adapted cultures, disentangle the relative contribution of alleles and cis/trans expressional changes to isobutanol resistance, and offer an initial system-level insight on adaptation to this alcohol. We also intend to determine the extent of cross-protection and trade-offs present in adapted hybrids grown on other toxic compounds of industrial importance. Ultimately, we aim to select new exploitable hosts with an increased performance in presence of isobutanol and the least trade-offs under other stresses.