AbstractThe potential of biotechnological processes has an untapped potential compare the small amount of species used nowadays to the natural variation observed. One of these non-conventional yeasts is Metschnikowia pulcherrima, a potential platform organism for microbial oil production. It has interesting phenotypes for industry such as an ability to grow on non-sterile conditions and a wide range of oligo- and monosaccharides within lignocellulosic material.
One aspect that makes these unravelled yeasts less appealing to industry is the lack of information and research done with them. The first work done in this thesis was an attempt to develop genetic tools that can be used to genetically manipulate M. pulcherrima. A reliable transformation method was established, although the low rate of homologous recombination (HR) in all the tested strains, and the variable levels of expression of inserted gene depending on its genome location makes genetic manipulation a challenging task. Nevertheless, a modest increase in lipid accumulation was attained by the insertion of a homologous gene. This served as a prove of concept to try multiple other modifications as a team project. Furthermore, a homologous inducible promoter was tested using GFP as a reporter which can be used in gene expression.
An interesting phenotype of M. pulcherrima is its ability to compete with microorganism. M. pulcherrima has been used in winemaking to avoid spoilage during fermentation. One known molecule that M. pulcherrima produces related to this ability is pulcherrimin, which binds to iron and it’s responsible for the characteristic maroon colour of its colonies. We investigated the ecological role of this secondary metabolite. It was found that even though having a metabolic cost, its production is an advantageous trait when competing for resources with other microorganisms. Then, adaptive laboratory evolution (ALE) was used to co-culture M. pulcherrima with bacterial agents of disease in farm animals in order to improve its ability to compete with them. Strains with an improved competition phenotype were obtained. Their genomes were sequenced finding a pattern in copy number variation depending on the bacteria they were exposed to during the ALE.
|Date of Award||16 Jun 2021|
|Supervisor||Daniel Henk (Supervisor) & Roderick Scott (Supervisor)|