Reverse Engineering Stress Tolerance in Yarrowia lipolytica. Bottom-up genetic-engineering approach to improve the yeast Yarrowia lipolytica’s tolerance to high temperature, high osmolarity, and low pH based on Adaptive Laboratory Evolution

Examensarbete för masterexamen
Gorter de Vries, Philip
With the goal of moving towards a sustainable production of organochemical compounds, the workhorses at the heart of biotech production processes, namely the microorganisms used as cell-factories, need to be optimized. While process and metabolic engineering aspects such as the yield, titer and production rate are often the main focus of these optimizations, the research presented in this thesis focuses on improving the organisms robustness in increased environmental stresses. Specifically, the aim was to improve the resistance of the yeast Yarrowia lipolytica to high temperature, acidity and osmolarity. To achieve this goal a bottom-up approach was used starting with adaptive laboratory evolution predating this thesis, where several populations of Y. lipolytica were grown for a three-month period under an increasing exposure to one of the three different stresses stated above. The genomes of these evolved strains were then sequenced and compared to identify mutations that have arisen and are possibly interesting targets. These found targets were reverse engineered into the wild-type parent strain using CRISPR-Cas9. Finally, growth of the reverse engineered strains was assessed to determine whether these mutations indeed improve the yeasts’ stress tolerance. Eight target genes have been found that are mutated in more than one population of the different stresses. These mutations have occurred independently and are likely to be involved in the increased stress tolerances. Of these eight mutations, one mutation that is possibly associated with heat tolerance has been reverse engineered in the parent strain to assess its growth. We were not able to show an increased thermotolerance in the engineered strains through the introduction of this mutation. However, we were able to verify the improved heat resistance of the laboratory evolved strains; At 32ºC they have a shorter lag-phase than the control and at 34ºC they are able to grow while the wild-type isn’t.
Yarrowia lipolytica , Adaptive laboratory evolution , stress , tolerance , temperature , osmolarity , salinity , pH , reverse engineering , CRISPR-Cas9
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