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Yarrowia lipolyticaexcels in microbial lipid production, thriving across diverse conditions. Batch or fed-batch fermentation is the not only 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 engineeredY. lipolyticastrain 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 parameters 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 maintaining the lipid yield at around 0.1 g/g. The CF sample at 1008 hours 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 in biomanufacturing. 

Omega-3 fatty acids, including alpha-linolenic acids (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have shown major health benefits, but the human body’s inability to synthesize them has led to the necessity of dietary intake of the products. The omega-3 fatty acid market has grown significantly, with a global market from an estimated USD 2.10 billion in 2020 to a predicted nearly USD 3.61 billion in 2028. However, obtaining a sufficient supply of high-quality and stable omega-3 fatty acids can be challenging. Currently, fish oil serves as the primary source of omega-3 fatty acids in the market, but it has several drawbacks, including high cost, inconsistent product quality, and major uncertainties in its sustainability and ecological impact. Other significant sources of omega-3 fatty acids include plants and microalgae fermentation, but they face similar challenges in reducing manufacturing costs and improving product quality and sustainability. With the advances in synthetic biology, biotechnological production of omega-3 fatty acids via engineered microbial cell factories still offers the best solution to provide a more stable, sustainable, and affordable source of omega-3 fatty acids by overcoming the major issues associated with conventional sources. This review summarizes the current status, key challenges, and future perspectives for the biotechnological production of major omega-3 fatty acids.