(164ao) Understanding Strain Instability in Yarrowia Lipolytica

The model oleaginous yeast species, Yarrowia lipolytica, is a promising biomanufacturing chassis well suited for production of oleochemicals, terpenoids, and other valuable chemicals of the carotenoid family. Scaleup of these processes, however, has been marred by phenotypic changes, such as loss of titer (or titer instability). The titer instability problem is exemplified by an engineered β-carotene producing Y. lipolytica strain that was developed by enhancing flux from acetyl-CoA to terpene precursors and overexpressing carB and carRP enzymes. This engineered strain produced ~4 g/L β-carotene titer using benchtop bioreactors. However, when moving this strain to a larger bioreactor, cell performance significantly dropped during 60 generations of cell growth. We hypothesized that loss of productivity is due to accumulation of mutations in genes for β-carotene production during prolonged generations of culture growth. To test this hypothesis, we collected samples from shake flask cultures under optimal continuous fermentation conditions (50 mL working volume) and performed Single Molecule, Real-Time (SMRT) HiFi resequencing using PacBio’s Sequel II system. The mutation rate and locations of the β-carotene producing strain were determined at 2 different time points in culture growth, associated with 50% (generation 30) and 0% (generation 60) β-carotene titer, respectively. Similarly, RNAseq, targeted metabolomics, and targeted proteomics of the time course samples will be used to identify correlations between gene expression rate and mutation rate/locations, and key genes or enzymes of frequent mutations causing production loss or metabolic shifts. The goal of this work is to investigate the nature of titer instability in this β-carotene strain as a model to understand more broadly the factors that lead to cellular heterogeneity during cell line development and scaleup.