Developing engineering strategies to enhance the genetic stability of fatty alcohol-producing strains for production scale-up

Robust strain stability is essential for industrial-scale fatty alcohol production, as metabolic burden and toxicity not only constrain productivity but also create selective pressure for low-producing or non-producing subpopulations. This genetic instability leads to genetic heterogeneity and compromises strain performance during large-scale fermentation. In this study, we investigated the production stability of fatty alcohol-producing Yarrowia lipolytica strains and developed systematic strategies to improve the genetic stability of engineered strains for scale-up production. In a mock fermentation scale-up, a fatty acyl-CoA reductase (FAR)-expressing strain lost fatty alcohol production after five consecutive passages. To address this, we fused FAR to phosphoglycerate kinase I (PGK1), a gene essential to cell growth, to promote the stability of FAR expression and prevent production loss. This strategy extended fatty alcohol production by one passage. Additionally, FAR was fused to GFP and extended production stability by four additional passages. In parallel, competitive co-culture experiments, in which producing strains were cultured alongside non-producing mutants, revealed that when non-producers emerged with a frequency of 10^-5, they dominated the fermentation population within six passages; at 10%, they took only two passages. Furthermore, deep sequencing of strains that demonstrate different levels of stability and fatty alcohol productivity identified mutation patterns that contribute to strain instability. These findings provide insights into engineering Y. lipolytica with enhanced genetic stability for scale-up fatty alcohol production.