Genetic modification and adaptive laboratory evolution of Yarrowia lipolytica to utilize acetate as a primary carbon source
Author
Leifsdóttir, Steinunn
Term
4. semester
Publication year
2026
Submitted on
2026-06-01
Abstract
Acetate is a low-cost carbon source that can be produced from renewable feedstocks, making it attractive for more sustainable bioprocessing. The yeast Yarrowia lipolytica can grow on acetate and store large amounts of lipids, which makes it a promising production host. We asked whether two strategies could help this yeast use acetate even better: (1) overexpressing ACS1 (increasing the amount of the ACS1 gene product), and (2) adaptive laboratory evolution (ALE), where cells are gradually challenged with higher acetate levels to select for better performers. We engineered a strain that overexpresses ACS1 and then evolved it by increasing acetate from 2 g/L to 50 g/L over 51 generations. We compared the adapted strain (T-ALE) with the unevolved transformed strain (T) and the original strain (OG) in shake-flask cultures using a standard yeast medium (YPA) with 50 g/L acetate. We measured biomass, lipid content, and acetate consumption. Across all three strains, we observed no statistically significant differences in final biomass, lipid accumulation, or acetate use. The acetate consumption profiles were highly similar, and the original strain performed as well as the engineered and evolved strains. Under the tested conditions, neither ACS1 overexpression nor ALE improved performance. These results highlight the natural ability of Y. lipolytica to grow at high acetate concentrations and suggest that additional metabolic targets will be needed to further improve acetate-based bioprocesses.
Acetat er en billig kulstofkilde, der kan fremstilles af fornybare råstoffer, og er derfor attraktiv i mere bæredygtige bioprocesser. Gærarten Yarrowia lipolytica kan vokse på acetat og lagre store mængder lipider, hvilket gør den lovende som produktionsvært. Vi undersøgte, om to strategier kunne få gæren til at udnytte acetat endnu bedre: (1) overekspression af ACS1 (øget produktion af det tilhørende genprodukt) og (2) adaptiv laboratorieevolution (ALE), hvor celler gradvist udsættes for højere acetatniveauer for at selektere bedre præstation. Vi konstruerede en stamme, der overekspresserer ACS1, og udviklede den ved at øge acetat fra 2 g/L til 50 g/L over 51 generationer. Den tilpassede stamme (T-ALE) blev sammenlignet med den ikke-evolverede transformerede stamme (T) og den oprindelige stamme (OG) i rystekolber med et standard gærmedie (YPA) indeholdende 50 g/L acetat. Vi målte biomasse, lipidindhold og acetatforbrug. Vi fandt ingen statistisk signifikante forskelle mellem de tre stammer i slut-biomasse, lipidakkumulering eller acetatudnyttelse. Acetatforbrugsprofilerne var meget ens, og den oprindelige stamme klarede sig lige så godt som den konstruerede og evolverede stamme. Under de undersøgte betingelser gav hverken ACS1-overekspression eller ALE forbedringer. Resultaterne peger på, at Y. lipolytica allerede kan vokse effektivt ved høje acetatkoncentrationer, og at yderligere metaboliske mål sandsynligvis er nødvendige for at forbedre acetatbaserede bioprocesser.
[This abstract has been rewritten with the help of AI based on the project's original abstract]