(325e) Investigation of Genomic Loci between Essential Genes As Stable Harbors for Heterologous Expression in Yarrowia Lipolytica

As the field of metabolic engineering progresses from laboratory demonstrations to industrial production, genetic instability in production hosts emerges as a significant obstacle. Certain genomic regions are prone to genetic anomalies such as DNA polymerase slippage, recombination, transpositions, and base substitutions. Regardless of scale or application, microbial cultures are consistently susceptible to fitness-based competition, where cells with low or no production capabilities outcompete high-producing cells, leading to dominance of the former in the culture. In our research, we focus on nonconventional microbes and their genomic landscapes, with particular emphasis on Yarrowia lipolytica. This host has garnered significant interest due to its potential for generating a diverse array of value-added lipid-derived or hydrophobic compounds with applications as lubricants, surfactants, and nutraceuticals.

To address genetic instability issues and improve the scalability of microbial cell factories, we examined essential gene pools identified in two independent functional genomics studies. These studies utilized Hermes transposon or Cas9 to disrupt gene functions and identified 1963 and 1548 essential genes, respectively. Through a rigorous comparison of the two datasets, we identified a smaller subset of 13 candidate loci located in the intergenic regions between two essential genes. These loci were tested using three different heterologous genes and pathways of distinct lengths and sequences, including green fluorescent protein (GFP), fatty alcohol biosynthetic pathway, and beta-carotene biosynthetic pathway. In a mock test simulating fermentation scale-up, the stability of GFP expression and the production of fatty alcohols and beta-carotene enabled by each of these 13 loci were characterized and compared with commonly used integration sites. Among the 13 candidates, five loci exhibited significantly better stability compared to others. Our study provides a robust methodology for identifying optimal safe harbors for pathway integration, specifically focusing on minimizing genetic drifts during scale-up processes. These findings contribute to the development of reliable strategies for enhancing industrial production of desired compounds in Yarrowia lipolytica and other nonconventional microbial hosts.