Development of CRI-SPA, a mating-based, CRISPR-Cas9 assisted method for high-throughput yeast strain construction, and its applications in yeast cell factory research

Abstract

The yeast Saccharomyces cerevisiae is a workhorse of industrial biotechnology and the use of yeast cell factories for the production of industrially important compounds from renewable biomass offers sustainable alternatives to climate-damaging and environmentally polluting oil-based production. Nevertheless, although technological advances in biological engineering is continuously increasing the number of products which can be produced by genetically engineered microbes, the majority of yeast cell factories are not yet commercially competitive with less costly petrochemical production routes. Improving the performance of cell factories in terms of titers, rates and yields is therefore a main goal of metabolic engineering and which is typically pursued by targeted modifications to the native metabolism of the producing organism. However, due to the inherent complexity of biological systems, less rational approaches employing genome-wide screens of large libraries to find genetic variants yielding improved production may be an attractive alternative requiring no a priori knowledge of genetics and metabolism, and enabling the identification of non-intuitive engineering targets. In regards to this, genome-wide mutant strain libraries such as the yeast deletion collection offers exiting possibilities to not only serve as tools in fundamental research but also as screening platforms for metabolic engineering studies. Their implementation does, however, require high-throughput methods for parallel introduction of exogenous genetic material to a large number of individual strains. The research conducted within the scope of this dissertation aimed at developing and establishing such a method, ultimately making strain library screening a more accessible tool in yeast fundamental research and yeast cell factory optimisation. This thesis first describes the conceptualisation and development of a HT mating-based method for transfer of DNA from a Universal Donor Strain into the mutant strains of the yeast deletion collection. The method, termed CRI-SPA, makes use of CRISPR-Cas9 for integrating the transferred DNA in the genome of the recipient strains, and of Selective Ploidy Ablation (SPA) for haploidising mated cells without the need for meiosis and complex sporulation procedures. The study further presents results of a proof-of-principle experiment in which CRI-SPA was used to transfer a genetic deletion to a subset of the deletion collection and which demonstrated the usefulness of the method in fundamental research uncovering genetic interactions and cellular network structures. Next, the use of CRI-SPA in cell factory optimisation was established as the HT capacity of the method enabled the yeast deletion collection to serve as a screening platform for metabolic engineering designs. By use of CRI-SPA, genes for synthesising the aromatic, yellow pigment betaxanthin – used here as a proxy molecule for the medicinally important benzylisoquinoline alkaloids - were transferred to the deletion library and mutants with improved production were identified and verified by reverse engineering. Moreover, the study took advantage of the systematic evaluation of phenotypes that the CRI-SPA method enables and an overrepresentation analysis of Gene Ontology terms amongst the top producing mutants yielded observations that could be of interest for further investigation of the translational regulation of the aromatic amino acid pathway. Finally, the work presented in this thesis also illustrates the possibility of using CRI-SPA for tapping into the great genetic diversity found between different strains of S. cerevisiae in order to identify superior hosts for the bio-based production of different compounds. However, strains used in the fermentation industry and strains that have been isolated from the wild are non-mating and prototrophic and are not compatible with the method. In the presented study, a CRISPR-Cas9 based strategy was used for restoring mating-competence and for ensuring CRI-SPA compatibility in a set of diploid strains of both industrial and environmental origin. The study confirmed the ability of the constructed strains to mate and further demonstrated their use together with the CRI-SPA method. This preliminary study proved the feasibility of the presented approach and encouraged generation of larger strain libraries encompassing greater genetic diversity and which could be a useful tool in the search for strains with improved production capacities. In summary, the work presented here describes the development of the novel method CRI-SPA for high throughput strain construction and modification of large strain libraries. The dissertation further demonstrates the applicability of CRI-SPA in fundamental science and cell factory optimisation as the method is used in genetic interaction screens, in the search for metabolic engineering targets amongst the yeast deletion library, and in the introduction of genetic material to genetically diverse, diploid strain backgrounds. These examples serve to establish the method and its usefulness and sets the stage for wider application of CRI-SPA in the continued research and optimisation of yeast cell factories.