Genetic Determinants forn-Butanol Tolerance in Evolved Escherichia coli Mutants: Cross Adaptation and Antagonistic Pleiotropy betweenn-Butanol and Other Stressors

Abstract

bCross-tolerance and antagonistic pleiotropy have been observed between different complex phenotypes in microbial systems. These relationships between adaptive landscapes are important for the design of industrially relevant strains, which are generally subjected to multiple stressors. In our previous work, we evolved Escherichia coli for enhanced tolerance to the biofuel n-butanol and discovered a molecular mechanism of n-butanol tolerance that also conferred tolerance to the cationic antimicrobial peptide polymyxin B in one specific lineage (green fluorescent protein [GFP] labeled) in the evolved population. In this work, we aim to identify additional mechanisms of n-butanol tolerance in an independent lineage (yellow fluorescent protein [YFP] labeled) from the same evolved population and to further explore potential cross-tolerance and antagonistic pleiotropy between n-butanol tolerance and other industrially relevant stressors. Analysis of the transcriptome data of the YFP-labeled mutants allowed us to discover additional membrane-related and osmotic stress-related genes that confer n-butanol tolerance in E. coli. Interestingly, the n-butanol resistance mechanisms conferred by the membrane-related genes appear to be specific to n-butanol and are in many cases antagonistic with isobutanol and ethanol. Furthermore, the YFP-labeled mutants showed cross-tolerance between n-butanol and osmotic stress, while the GFP-labeled mutants showed antagonistic pleiotropy between n-butanol and osmotic stress tolerance.

Evolved Osmotolerant Escherichia coli Mutants Frequently Exhibit Defective N -Acetylglucosamine Catabolism and Point Mutations in Cell Shape-Regulating Protein MreB

“…Stronger NaCl selection could disfavor mutations that also improve n-butanol resistance, as it is reasonable to expect that different levels of osmotic stress select for distinct tolerance mechanisms. n-Butanol tolerance is also not always associated with improved osmotic stress resistance in evolved mutants (28), so there are at least some evolutionary paths on both the NaCl and n-butanol landscapes that lead to divergent tolerance phenotypes. The overall lack of significant cross-adaptation for the isolates in this case does indicate that specialist mutants with adaptations specific only to NaCl tolerance are favored under these evolutionary conditions.…”

“…Resistance to osmotic stress is known to affect other phenotypes of industrial interest, such as n-butanol or low pH tolerance (17,28) and growth at elevated temperatures (29). Growth assays of the mutants in the presence of inhibitory levels of glucose, 0.8% n-butanol, mild acidic pH, and thermal stress (Fig.…”

A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli

“…The percentage of tolerance (T) was calculated using equation 1 (61). This parameter was calculated using the measured growth parameters (OD 600 ) after 6 and 24 h. In addition, the percentage of inhibition (I) and the relative fitness coefficient (s) were calculated using equations 2 and 3, respectively (62). These parameters were calculated using the measured maximum specific growth rate ( max ) of each strain, which was determined during early exponential growth according to equation 4.…”

Characterizing the Phenotypic Responses of Escherichia coli to Multiple 4-Carbon Alcohols with Raman Spectroscopy

“…To maintain homeostasis and optimize the use of resources, host microbes activate several genetic programs in response to changing environmental conditions [5][6][7][8][9][10]. Previous studies have shown that the microbial response to alcohol toxicity is not universal but varies according to the host microbe and the type of alcohol [8][9][10][11][12][13][14]. Understanding the nature and dynamics of the host response to specific alcohols is essential for designing effective metabolic and process engineering solutions.…”

“…In addition to isobutanol, 1-butanol (or n-butanol) remains a promising biofuel (and chemical commodity). Yet, it is one of the most toxic alcohols due to its elevated hydrophobicity (relative to other alcohols) [15], and its toxicity is strain-dependent [8][9][10][11][12][13]. Despite significant advances, many details regarding the mode of action of different alcohols as well as the nature and adaptation mechanism to alcohol exposure are still being resolved.…”