Adaptive Laboratory Evolution of Probiotics toward Oxidative Stress Using a Microfluidic-Based Platform

Area of Science:

• Microbiology
• Biotechnology
• Synthetic Biology

Background:

• Adaptive laboratory evolution (ALE) enhances bacterial resistance to oxidative stress.
• Microfluidic platforms offer efficient and economical alternatives to traditional batch cultures for ALE.
• Existing microfluidic ALE systems face challenges with cell passaging and spatial control.

Purpose of the Study:

• To develop and evaluate a novel microfluidic Evolution on a Chip (EVoc) system for ALE.
• To enhance the oxidative stress tolerance of Lacticaseibacillus rhamnosus GG using the EVoc platform.
• To investigate the adaptive mechanisms and genetic changes in L. rhamnosus GG under prolonged H2O2 exposure.

Main Methods:

• Utilized a microfluidic EVoc design with a progressive H2O2 gradient.
• Performed prolonged ALE (up to 72 hours) on L. rhamnosus GG.
• Analyzed adaptive strain morphology, gene expression, and performed whole-genome sequencing.

Main Results:

• Successfully evolved adaptive strains of L. rhamnosus GG exhibiting increased H2O2 tolerance.
• Demonstrated progressive growth enhancement in increasing H2O2 concentrations (1-3 mM) after ALE.
• Observed distinct morphological and gene expression profiles in adapted strains compared to wild-type.
• Identified a single nucleotide mutation in the omega-amidase gene in adapted strains.

Conclusions:

• The microfluidic EVoc platform is effective for ALE, enhancing bacterial oxidative stress resistance.
• ALE significantly improves L. rhamnosus GG's ability to tolerate and grow in the presence of hydrogen peroxide.
• Genetic adaptation, including a mutation in omega-amidase, underlies the enhanced stress tolerance.