Adaptive responses and molecular mechanisms of Lactiplantibacillus plantarum to citric acid stress

Abstract

Lactiplantibacillus plantarum, known for its environmental adaptability due to genetic flexibility, was subjected to 180 days of citric acid exposure (a prevalent stressor derived from plants) to understand its adaptive response to environmental stress. The evolved strain demonstrated a 2.4-fold increase in cell density under 10 g/L citric acid stress, indicating significantly improved citric acid tolerance compared to the ancestral strain. It also demonstrated cross-protection, particularly under HCl and cold temperature challenges. During early-stage adaptation, reversal mutations were observed, and most plastic changes indicated by gene expression alterations in the ancestral strain were reversed via genetic adaptation, indicating a compensatory effect of these mutations. Whole-genome sequencing identified a key mutation in the citrate transporter gene nacT (F178V). This mutation, validated through point mutation and homologous modeling, enhanced citrate transport and utilization. When applied to ferment citric acid-rich materials (green plums, lemons, passion fruits, pineapples, oranges, and white grapefruits), the evolved and point-mutated strains exhibited 3.7- and 3.4-fold higher citric acid consumption than the ancestral strain, respectively. Notably, the fruits fermented by evolved strain demonstrated significantly elevated viable cell counts, as well as a richer taste and more pleasant aroma profile based on sensory analysis. These findings advance our understanding of environmental stress adaptation and underscore the potential application of evolved strain in food fermentation.

Keywords: Adaptive laboratory evolution; Citric acid; Cross-protection; Genetic changes; Lactiplantibacillus plantarum; Plastic changes; Reverse mutations.