Abrogating the adenine methylation ability of Lacticaseibacillus paracasei improves its freeze-drying and storage resistance

Abstract

Freeze-drying is a widely adopted method for the long-term storage of starter cultures in the food industry but can cause cell instability and a decline in post-storage viability. We used an unmethylated Lacticaseibacillus paracasei Zhang mutant lacking adenine-specific DNA-methyltransferase. This mutant was subjected to freeze-drying and stored at 30 °C for two distinct durations (30 and 60 days), Our analysis revealed the unmethylated mutant outperformed the wild-type in cell viability and survival following freeze-drying and post-freeze-drying storage. And significant metabolic pathway differences between the stored mutant and wild-type bacteria. These differences were evident in the phosphotransferase system, carbohydrate, and amino acid metabolism, and fatty acid biosynthesis, and were consistent across transcriptomic, proteomic, and metabolomic analyses. This is achieved by modulating key metabolic pathways within the bacteria. This study contributes to the limited literature on the role of bacterial adenine methylation in industrial strain application and starter culture storage.

Fig. 1. Viable counts and survival rates of L. paracasei Zhang (wild-type) and L. paracasei Zhang ΔpglX (mutant) during storage at 30 °C.

Viable counts (A) and survival rates (B) of L. paracasei Zhang (wild-type) and L. paracasei Zhang ΔpglX (mutant) during storage at 30 °C. Error bars represent standard deviations. Significant differences between the two strains at each time point were evaluated using Student’s t-tests (* p < 0.05; ** p < 0.01; *** p < 0.001).

Fig. 2. The number of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) between L. paracasei Zhang ΔpglX (mutant) and wild-type freeze-dried cells stored for 30 and 60 days.

Venn diagram and histogram (A) show the number of DEGs in freeze-dried cells stored for 30 and 60 days. Gene sets of up- and down-regulated DEGs after 30 or 60 days of storage are represented as “30 up”, “30 down”, “60 up”, and “60 down”, respectively. Volcano plots of DEPs between the mutant and wild-type after (B) 30 days and (C) 60 days of storage are presented. The x- and y-axes show the fold change and p-value of the comparison between the two cell lines, respectively. Each dot in the figure represents a specific protein; grey, blue, and red dots represent non-significant, significantly upregulated, and down-regulated DEPs, respectively (cut-off level: |FC | ≥ 1.5; p ≤ 0.05). The number of non-significant, significantly up- regulated, and down-regulated DEPs is indicated in the suffix of the label.

Fig. 3. Transcriptomic and proteomic analysis results obtained at two storage time points (30 and 60 days).

A Kyoto Encyclopedia of Gene and Genomes (KEGG) enrichment analysis of common differentially expressed proteins (DEPs) identified at the two storage time points. The color scale represents the false discovery rate (FDR). Each circle represents a KEGG pathway, with the size of the circle indicating the number of enriched proteins in that specific KEGG pathway. B, C Scatter plots of Pearson correlation between fold changes identified by tandem mass tag (TMT) labeling and parallel reaction monitoring (PRM) after storing the freeze-dried cells for (B) 30 days and (C) 60 days. The correlation strength and statistical significance between fold changes found by TMT and PRM are represented by R and p values, respectively. The grey area shows the confidence interval. Gene ontology (GO) enrichment analysis of DEPs and differentially expressed genes (DEGs) after (D) 30 days and (E) 60 days of storage of freeze-dried cells. The x-axis represents the GO term, and the y-axis represents the enrichment ratio. The blue and green bars in the histograms represent DEPs and DEGs, respectively. * FDR < 0.05; ** FDR < 0.01; *** FDR < 0.001. KEGG enrichment analysis of identified differential DEPs and DEGs at the two storage time points: (F) 30 days and (G) 60 days. Each circle represents a specific KEGG pathway, with the size and color of the circle indicating the number of significant DEGs/DEPs in that KEGG pathway and FDR, respectively.

Fig. 4. Metabolomics analysis results obtained at two storage time points (30 days and 60 days).

Principal component analysis of metabolomics data generated in positive and negative ion modes at the two storage time points: (A, C) 30 days and (B, D) 60 days, respectively. Plots of OPLS-DA and permutation test (200 random permutations) of metabolomics data generated in positive ion mode at the two storage time points: (E, G) 30 days and (F, H) 60 days, respectively. Plots of OPLS-DA and permutation test (200 random permutations) of metabolomics data generated in negative ion mode at the two storage time points: (I, K) 30 days and (J, L) 60 days, respectively.

Fig. 5

Membrane fatty acid contents in freeze-dried cells of L. paracasei Zhang (wild-type) and L. paracasei Zhang ΔpglX (mutant) after storage for 60 days. Error bars represent standard deviations. Significant differences between groups are indicated by: *p < 0.05 and **p < 0.01, respectively.

Fig. 6. Changes in protein and gene expression of phosphotransferase system (PTS) components at the two storage time points (30 and 60 days).

Glc = glucose; Fru = fructose; Lac = lactose; Man = mannose; Gat = galactitol; L-Asc = L-ascorbate; P = phosphate; EI = enzyme I; Hpr = histidine phosphate carrier protein; EIIABCD = enzyme II complex. The superscript number after the protein or gene represents the locus tag (1: LCAZH_RS13340, 2: LCAZH_RS03235, 3: LCAZH_RS13630, 4: LCAZH_RS13635, 5: LCAZH_RS02965, 6: LCAZH_RS14555, 7: LCAZH_RS14220, 8: LCAZH_RS14235, 9: LCAZH_RS02390, 10: LCAZH_RS02395, 11: LCAZH_RS02405, 12: LCAZH_RS14305, 13: LCAZH_RS13395, 14: LCAZH_RS02245, 15: LCAZH_RS13400, 16: LCAZH_RS02230, 17: LCAZH_RS13335, 18: LCAZH_RS13210).