Optimizing the Bioprocesses of Bacteriocin Production in Lacticaseibacillus paracasei HD1.7 by the "Acetate Switch": Novel Insights into the Labor Division Between Energy Metabolism, Quorum Sensing, and Acetate

Abstract

Acetate may act as a signaling molecule, regulating Paracin 1.7 production via quorum sensing (QS) in Lacticaseibacillus paracasei HD1.7. The "acetate switch" phenomenon requires mechanistic exploration to optimize Paracin 1.7 production. The "acetate switch" phenomenon delays with higher glucose levels (30 h, 36 h, and 96 h). Before the occurrence of the "acetate switch", the ATP content increases and peaks at the "acetate switch" point and the NAD⁺/NADH ratio decreases, indicating energy changes. Moreover, the QS genes used for the pre-regulation of bacteriocin, such as prcKR, comCDE, were highly expressed. After the "acetate switch", the ATP content decreased and the QS genes for the post-regulation of bacteriocin were highly expressed, such as rggs234 and sigma70-1/70-2. The "acetate switch" could act as an energy switch, regulating bacterial growth and QS genes. Before and after the "acetate switch", some metabolic pathways were significantly altered according to the transcriptomic analysis by HD1.7 and HD1.7-Δpta. In this study, acetate was used as an input signal to regulate the two-component system, significantly influencing the bacteriocin expression system. And this study clarifies the roles of acetate, energy, and quorum sensing in promoting Paracin 1.7 production, providing a theoretical basis for optimizing the bacteriocin fermentation process of HD1.7.

Keywords: Lacticaseibacillus paracasei HD1.7; acetate switch; bioprocess; quorum sensing; transcriptomic analysis.

Figure 1

Determination of the “acetate switch” points under different concentration of glucose. (A) Changes in intracellular acetate content as well as intracellular pH in the three treatments (Glu2, Glu5, Glu20) as time progressed; (B) Glucose consumption as well as bacterial growth in the three treatments as time progressed; (C) Heat map of the expression of AMRGs in the three treatments as time progressed; (D) Metabolic pathway map utilizing glucose and acetate.

Figure 2

Variation in energy, AMRGs, and QSRGs abundance before and after “acetate switch” points. (A) Changes in pyruvate in the three treatments over time; (B) Changes in NAD⁺/NADH in the three treatments over time; (C) Changes in ATP in the three treatments over time; (D–F) Heat map of the expression of QSRGs in the three treatments over time; (G) Heat map of correlation analysis of QSRGs with acetate and energy before and after the “acetate switch”; (H) Bacteriocin titer of HD1.7 in Glu 20. “*” indicates “p < 0.05”; Different lowercase letters indicate significant differences among groups (p < 0.05).

Figure 3

SEM analysis of the effects of intracellular acetate on energy as well as quorum sensing. (A,D) SEM analysis of Glu2, direct and indirect effects of Lacticaseibacillus paracasei HD1.7 intracellular acetate on ATP, reducing power, AMRGs, and QSRGs. (B,E) SEM analysis of Glu5, direct and indirect effects of Lacticaseibacillus paracasei HD1.7 intracellular acetate on ATP, reducing power, AMRGs, and QSRGs. (C,F) SEM analysis of Glu20, direct and indirect effects of Lacticaseibacillus paracasei HD1.7 intracellular acetate on ATP, reducing power, AMRGs, and QSRGs. Differently colored bars represent different groups; gray bars indicate statistically tested p > 0.05. Red symbolizes a positive correlation, and blue symbolizes a negative correlation; solid lines indicate a significant effect between variables, while dashed lines indicate a non-significant effect.

Figure 4

Changes of genes in HD1.7 with different acetate-producing capacity before and after the “acetate switch”. (A) Intracellular acetate content of HD1.7 and strain HD1.7-Δpta under 5 g/L glucose conditions; (B) Volcano gram of DEGs in the AOF group; (C) Volcano gram of DEGs in the LCA group; (D) Venn diagram of DEGs in AOF vs. LCA; (E) Variance analysis of DEG in LCA and LCA, the color labeling involved in this figure is consistent with that in 4D.; (F) WGCNA of DEGs in the AOF group; (G) WGCNA of DEGs in the LCA group; (H) Ringed heat map of modular trait in WGCNA associations of AOF, where each ring represents a module, the letters in the figure indicate the treatment groups; (I) Ring heatmap of module trait associations in WGCNA of LCA, where each ring represents a module, the letters in the figure indicate the treatment groups. “*” indicates “p < 0.05”; “***” indicates “p < 0.001”.

Figure 5

Changes in metabolic pathways of HD1.7 with different acetate-producing capacity before and after the “acetate switch”. (A) KEGG enrichment in AOF and LCA to analyze differential metabolic pathways and their enriched genes; (B) histogram of the number of up- and downregulated genes in the differential metabolic pathways of AOF and LCA; (C) dynamic Venn diagram of enriched genes in the differential metabolic pathways of AOF; (D) dynamic Venn diagram of enriched genes in the differential metabolic pathways of LCA.

Figure 6

Analysis of differential metabolic pathways in AOF and LCA. (A) QS and two-component system; (B) The other metabolic pathway. Solid arrows represent a direct reaction from the previous substance to the next, dotted arrows indicate that the reaction requires a complex process. Created with BioRender.com.

Figure 7

Conceptual model of production of bacteriocin by L. paracasei before and after the “acetate switch”. Created with BioRender.com.