Phenotyping transcription factors-related genotypes in Y. lipolytica across a range of industrially relevant process parameters and chemical stress conditions.

One of the factors contributing to “the death valley” in biotech process transfer is an assumption that once a synthetic trait is successfully introduced and optimized in a microbial host, the resulting strain attains “industrial potential”. In practice, most engineered strains lack the robustness required to perform sufficiently in the demanding conditions of industrial bioprocesses. Stress tolerance is a complex, non‑pathway trait that is tightly regulated and highly polygenic, making it challenging to engineer. Consequently, beyond constructing a desired phenotype, substantial effort must also be directed toward improving the overall “robustness” of engineered strains. In this work, we systematically phenotyped transcription factor (TF)–related genotypes of Yarrowia lipolytica under a broad spectrum of industrially relevant stress conditions. Eight TFs were chosen based on prior high‑throughput screening results (YaliFunTome database). For each TF, two additional genotypes—overexpression (OE) and knockout (KO)—were generated to enable a comprehensive assessment of TF function. All three genotypes per TF were evaluated for growth under diverse stress exposures, including process parameters (oxygen availability, pH, and temperature) and a range of toxic chemicals relevant to industrial processes: lignocellulosic biomass-derived inhibitors, short‑chain fatty acids, osmotic inducers, salt, mild acid, and oxidative stressors, antifungal agent, and alcohols. This study delivers new insights—or deepens existing understanding—of how selected TFs contribute to process parameters and chemical stress resistance in Y. lipolytica. Based on these findings, we propose global metabolic engineering strategies to enhance strain robustness under industrial stress conditions. These include Msn4‑, Hap1‑OE, and Yas1‑KO for improved thermotolerance; Yas1‑OE for enhanced osmotic stress resistance; Dal81‑OE and KO of TF011 or Mhy1 for increased tolerance to lactic acid; and Yas1‑OE or KO of TF011 and Mhy1 for improved growth in the presence of the valuable aroma and antifungal compound 2‑phenylethanol (2PE).