Enhancing Lipid Production in Yarrowia lipolytica through Continuous Fermentation and Adaptive Laboratory Evolution in Bioreactors

Yarrowia lipolytica excels in microbial lipid production, thriving across diverse conditions. Batch or fed-batch fermentation is the common practice to achieve higher lipid titer and yield, but it is also subject to lower lipid productivity. Single-stage continuous fermentation (CF) provides a great potential for significantly higher productivity, but genetic instability is often seen and challenges strain performance over the long-period CF. This study harnesses single-stage CF to not only improve lipid productivity, but also evolve high-lipid mutants from a previously engineered Y. lipolytica strain E26 via adaptive laboratory evolution (ALE) in a continuous bioreactor, guided by a predictive kinetic model. The single-stage CF was run for 1128 hours (47 days) with key process parameter adjusted in a 1-L bioreactor to produce over 150 g/L yeast biomass, exceeding the targeted 113 g/L that is predicted by the model. Compared with the fed-batch fermentation process, the single-stage CF successfully improved lipid productivity from 0.3~0.5 g/L/h to about 1 g/L/h while maintained the lipid yield at around 0.1 g/g. The CF sample at 1008 h was used to isolate mutants with higher lipid production after ALE in the continuous bioreactor. A mutant E26E03 was identified which demonstrated improvements in biomass, lipid content, and lipid yield by 43%, 30%, and 51%, respectively, over the original strain E26 in fed-batch fermentation. Our study indicated that using model-guided CF with ALE in a continuous bioreactor provides a great potential for significantly higher product titer, rate, and yield (TRY) in biomanufacturing.