Transient MutS-Based Hypermutation System for Adaptive Evolution of Lactobacillus casei to Low pH

Area of Science:

• Microbiology
• Genetics
• Biotechnology

Background:

• Adaptive evolution is crucial for enhancing microbial industrial traits like acid resistance.
• Lactobacillus casei is a key bacterium in food fermentation, but its acid tolerance can limit applications.
• The DNA mismatch repair system, including MutS, influences microbial adaptation and evolution.

Purpose of the Study:

• To investigate transient MutS inactivation as a strategy to boost adaptive evolution in Lactobacillus casei.
• To enhance lactic acid resistance in L. casei strains 12A and ATCC 334 at low pH.
• To identify genetic factors contributing to improved acid resistance.

Main Methods:

• Construction of MutS deletion derivatives (ΔmutS) in L. casei 12A and ATCC 334.
• Subjecting wild-type and ΔmutS strains to a 100-day adaptive evolution process at low pH with lactic acid.
• Phenotypic characterization (growth, lactic acid production, cell morphology, survival) and whole-genome sequencing of adapted strains.
• Functional analysis of candidate genes (ndh, pstB, hpk) through targeted inactivation.

Main Results:

• Adapted ΔmutS strains exhibited significantly enhanced growth, higher cell densities, and increased lactic acid production compared to adapted wild-type strains.
• The adapted L. casei 12A ΔmutS derivative showed smaller cell volume, rougher surface, and improved survival at pH 2.5.
• Genome sequencing revealed decreased DNA replication fidelity and identified specific genetic mutations in adapted strains.
• Inactivation of ndh, pstB, and hpk genes in L. casei 12A contributed synergistically to increased lactic acid resistance.

Conclusions:

• Transient MutS inactivation is an effective method for accelerating adaptive evolution and enhancing lactic acid resistance in L. casei.
• The study identified specific genes (ndh, pstB, hpk) that play a significant role in conferring acid resistance to L. casei.
• This approach provides valuable insights for improving microbial fitness and industrial applications through directed evolution and gene manipulation.