Adaptive evolution of genomically recoded Escherichia coli

Area of Science:

• Synthetic Biology
• Genomics
• Microbial Physiology

Background:

• Genomically recoded organisms offer multivirus resistance and enhanced nonstandard amino acid (nsAA) incorporation.
• The initial genomically recoded Escherichia coli strain C321.∆A exhibits significantly reduced fitness in defined media, limiting its applications.

Purpose of the Study:

• To improve the fitness of the genomically recoded Escherichia coli C321.∆A strain.
• To identify genetic adaptations that restore fitness in recoded organisms.

Main Methods:

• Adaptive laboratory evolution of C321.∆A for over 1,000 generations in glucose minimal media.
• Next-generation sequencing to identify mutations in evolved populations.
• Multiplex Automatable Genome Engineering (MAGE) to reconstruct and validate mutations.

Main Results:

• Evolved recoded populations demonstrated significantly higher growth rates than ancestral and nonrecoded strains.
• Identified selective mutations unique to evolved recoded populations, some affecting the translation apparatus.
• Discovered mutations correcting off-target mutations from initial genome engineering.

Conclusions:

• Laboratory evolution is an effective strategy to restore fitness in engineered recoded genomes.
• This approach streamlines recovery compared to further targeted engineering.
• Provides comprehensive physiological insights into the C321.∆A strain.