Laboratory evolution reveals general and specific tolerance mechanisms for commodity chemicals.

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• High product concentrations are a major limitation in economically viable biomanufacturing.
• Understanding strain tolerance mechanisms is crucial for overcoming these limitations.
• Current knowledge of chemical tolerance mechanisms in industrial microbes is limited.

Purpose of the Study:

• To reveal the genetic mechanisms underlying microbial tolerance to high concentrations of industrial chemicals.
• To develop an automated platform for evolving industrial chemical tolerance in Escherichia coli.
• To create a comprehensive genotype-phenotype map for chemical-tolerant isolates.

Main Methods:

• Automated evolution of Escherichia coli in the presence of 11 industrial chemicals.
• Genomic sequencing of 223 evolved isolates from 89 populations.
• Reverse engineering and cross-compound tolerance profiling to identify tolerance mechanisms.

Main Results:

• Evolved strains exhibited 60%-400% higher tolerance to industrial chemicals.
• Key mutations were identified in genes encoding membrane transporters, cell wall proteins, transcription/translation machinery, and stress signaling proteins.
• Osmotic stress was found to play a significant role at higher chemical concentrations.
• Cross-tolerance was observed for similar chemicals, with trade-offs for dissimilar ones.
• Pre-tolerized strains significantly enhanced subsequent chemical production in some cases.

Conclusions:

• The study provides a detailed genotype-phenotype map of chemical tolerance in E. coli.
• Identified mutations offer targets for improving microbial strains for biomanufacturing.
• Evolving multiple parallel populations is essential for robust strain development.
• This research advances the field of microbial industrial chemical production.