Targeted Mutations in d-ldh and budA Differentially Affect Adaptive Traits and Fermented Milk Quality in Companilactobacillus crustorum MN047

Abstract

This study examined how targeted mutations in two pyruvate-associated genes, d-ldh and budA, affected adaptive phenotypes and fermented milk quality in Companilactobacillus crustorum MN047. Premature stop codons were introduced into d-ldh and budA by CRISPR-assisted base editing and verified by Sanger sequencing. The resulting nonsense mutants, designated ΔbudA and Δd-ldh, retained growth comparable to the wild-type strain (wt) but showed contrasting phenotypes. At 24 h, auto-aggregation was 4.44-fold the wt value in Δd-ldh and 0.71-fold the wt value in ΔbudA, whereas ΔbudA showed lower aggregation and stress survival. Viable counts remained 7.2–7.7 log10 CFU/mL, although titratable acidity and textural properties differed among fermented milk groups. ΔbudA produced higher firmness and consistency, whereas Δd-ldh showed lower values for both parameters. Electronic nose PCA clearly separated the three groups, while GC-MS identified strain-dependent differences in selected relative volatile compounds. These findings show that the two pyruvate-associated mutations affected bacterial robustness and fermented milk quality in distinct ways.