Charactering Recombinants with Multiple Complex Phenotypes Developed Using the Genderless Recombination System

Strain robustness and productivity are key traits for bio-based production. Stressors, such as inhibitory compounds present in the feedstock, osmotic, temperature, and toxic products, can inhibit cell growth and productivity. The production host is often exposed to multiple stressors during fermentation. Strain tolerance to environmental stressors is a complex phenotype involving unknown genes and uncharacterized mechanisms that leads to the difficulty of phenotypic improvement by rational engineering. To solve the problem that rational engineering cannot, a new strategy that can facilitate the generation of strains with multiple complex phenotypes rapidly was developed. “Genderless strain” - a sexual Escherichia coli strain, which is capable of in situ DNA exchange, was evolved under stress of 1-ethyl-3-methylimidazolium chloride (EMIC)), which is an ionic liquid used for the pretreatment of lignocellulosic biomass, to generate populations with enhanced tolerance to EMIC (0.15 M). Populations with improved growth under 0.15 M EMIC, a concentration that inhibits growth of the unevolved parental strain, were mixed with populations separately evolved for tolerance to sodium chloride (NaCl) stress, for genome shuffling. Recombinant populations with tolerance to both ionic liquid and osmotic stressors were identified under high concentrations of both inhibitors: 140 mM EMIC and 495 mM NaCl. Omics analyses integrating genomic, proteomic and lipidomic analyses were applied to help identify mechanisms underlying the dual tolerance phenotype. A large fragment deletion involving rcdA, rybB and ybjL genes detected by whole genome resequencing was found to be partially responsible for the enhanced tolerance to both inhibitors. Proteomic analysis revealed increased expression levels of several proteins in recombinant isolates. Additionally, changes in fatty acid compositions in the cell, such as saturated and potentially trans-unsaturated fatty acids, was observed by Raman spectroscopy. The increased ratio of saturated-to-unsaturated fatty acids in dual tolerant recombinants likely helped to reduce membrane fluidity, which helps the cell to overcome the stressors that fluidizes the cell membrane. The results uncovered by integrated omics analyses could expand current knowledge on the tolerance to a combination of hyperosmotic and ionic liquid stress, and facilitate future rational design of strains. Furthermore, the method developed here can be broadly applied to develop strains with desired multiple tolerance phenotypes