Genome Variations of Evolved Escherichia coli ET8 With a Rhodopsin-Based Phototrophic Metabolism

Area of Science:

• Microbiology
• Synthetic Biology
• Metabolic Engineering

Background:

• Previous work enhanced phototrophic metabolism in Escherichia coli ET5 via a dgcQ mutation.
• This study investigates genome variations in a new evolved descendant, ET8, harboring the pKJ606-GR plasmid.

Purpose of the Study:

• To identify and characterize genomic variations in the evolved ET8 strain.
• To understand the impact of these variations on phototrophic metabolism and proton pumping.
• To explore the physiological consequences of kdtA upregulation and galU disruption.

Main Methods:

• Whole genome sequencing to identify mutations.
• Analysis of gene transcription (kdtA).
• Growth assays on minimal media with specific carbon sources.
• Measurement of proton pumping activity.
• Genetic manipulation (kdtA-upregulation, galU-disruption) in the ancestor strain.

Main Results:

• ET8 strain identified with a kdtA upstream mutation and IS4 insertion at galU.
• Enhanced kdtA transcription observed in ET8.
• ET8 showed no growth on D-galactose or lactose minimal media.
• Phototrophic growth and proton pumping increased 1.5-fold and threefold in ET8+GR compared to the ancestor.
• Both kdtA-upregulation and galU-disruption significantly increased proton pumping in the ancestor strain.

Conclusions:

• Genomic variations, specifically kdtA upregulation and galU disruption, are key drivers of enhanced phototrophic metabolism in evolved E. coli.
• These mutations lead to significant improvements in proton pumping efficiency.
• The study provides insights into engineering microbial hosts for improved phototrophic capabilities.