AbstractThe oleaginous yeast Metshnikowia pulcherrima has previously been investigated as a potential platform organism for microbial oil production. This is due to several key phenotypes in addition to efficient oil production, including its ability to be grown in non-sterile conditions whilst metabolising a range of oligo- and monosaccharide carbon sources within lignocellulosic hydrolysates. To further improve the industrial potential of this organism in the absence of an appropriate genetic toolkit, adaptive laboratory evolution was applied to selected strains.
First, strains were adapted to lignocellulosic fermentation inhibitors in two strategies; either a single inhibitor, formic acid, or an inhibitor cocktail containing formic and acetic acid, 5-HMF and furfural. Phenotypic analysis of evolved cell lines reveals improved tolerance versus the progenitor across all strains, including cross adaptation of single-inhibitor evolved strains to conditions where all four inhibitors are present. Interestingly, the lipid production of the inhibitor cocktail evolved strains markedly increases, with one strain, named ‘4x3’, producing 41% lipid by dry weight compared to 22% by the progenitor. Following this, 4x3 was taken forward for further characterisation within 2L bioreactors and whole genome sequencing was performed to prospect for causative genetic modifications underpinning improved inhibitor tolerance and lipid production.
Strain 4x3 was then subject to a second round of adaptive laboratory evolution to improve its poor utilisation of xylose. Here, though all evolved cell lines had improved xylose metabolism versus the progenitor, a large degree of variation was observed suggesting multiple evolutionary routes to phenotypic improvement. One cell line in particular showed a drastic improvement, producing over 17x the biomass of the progenitor when grown on xylose as the sole carbon source. Whole genome sequencing was again performed on all strains, revealing both highly modified and dissimilar genomic structures, characterised by a high frequency of copy number variants. From this data, causative mutations are suggested and a general theory explaining the novel genome complexity following adaptive laboratory evolution is presented and discussed.
To support this study and the wider M. pulcherrima project, a simple, image based high-throughput lipid quantification method was developed after the commonly applied method of lipid straining was found to be inconsistent for this organism. The developed method allows for population dynamics to be tracked which was applied to overcome a potential production bottleneck within a semi-continuous bioprocess.
|Date of Award||19 Jun 2019|
|Supervisor||Chris Chuck (Supervisor) & Daniel Henk (Supervisor)|