(326d) Developing Engineering Strategies to Enhance the Genetic Stability of Fatty Alcohol-Producing Strains for Production Scale-up

Industrial production requires stringent strain stability for successful scaling up, particularly when the target product is toxic to the host cells. This toxicity not only poses a metabolic burden, limiting productivity, but also promotes the growth of low-producing or non-producing subpopulations, leading to significant genetic heterogeneity and instability during large-scale fermentation. Yarrowia lipolytica has emerged as a promising host for the biosynthesis of fatty acid-derived oleochemicals due to its high lipid content. Among these compounds, fatty alcohols have garnered attention for their applications as surfactants and lubricants. However, the hydrophobic nature of fatty acyl chains causes their insertion into cellular and subcellular membranes, negatively impacting the production host. In this study, we aimed to develop systematic strategies to improve genetic stability of engineered Y. lipolitica strains producing fatty alcohols.

In our mock test simulating fermentation scale-up, it was observed that the host completely lost its ability to produce fatty alcohols after five consecutive passages. To address this issue, we fused fatty alcohol reductase (FAR) with a monomeric gene, phosphoglycerate kinase I (PGK1), which is essential to the growth of the host. Additionally, a control strain expressing the FAR-GFP fusion was constructed. To assess long-term stability, the fatty alcohol-producing strains were cultured for five days, with one percent of the culture passaged into fresh medium every 24 hours over a total span of nine passages. Our results indicated that the fusion strategy between the essential PGK1 and FAR elongated the stability of fatty alcohol production by one additional passage. Interesting, fusing FAR and GFP extended genetic stability by four additional passages.

In parallel, we constructed an inactive FAR mutant fused with BFP and co-cultured it with the high-producing variant (FAR’GFP) with different ratios to study the competition between a nonproducer and a high-producer. The results showed that when the non-producing mutant appeared as a frequency of 10^-5, it took approximately six passages for it to become the dominant genotype in the culture. If it appeared at a frequency of 10% , it only took two passages. To understand the mechanisms of genetic instability, genomic DNA from various strains demonstrating different levels of stability and fatty alcohol productivity were isolated for deep sequencing of the FAR expression cassette. By building an understanding of the patterns, dynamics and causes of mutagenesis in engineered yeast, we are able to develop effective strategies to mitigate undesirable mutations and optimize the evolutionary stability of the high-producing strain for large-scale fermentation.