Enhanced acid-stress tolerance in Lactococcus lactis NZ9000 by overexpression of ABC transporters

Abstract

Background: Microbial cell factories are widely used in the production of acidic products such as organic acids and amino acids. However, the metabolic activity of microbial cells and their production efficiency are severely inhibited with the accumulation of intracellular acidic metabolites. Therefore, it remains a key issue to enhance the acid tolerance of microbial cells. In this study, we investigated the effects of four ATP-binding cassette (ABC) transporters on acid stress tolerance in Lactococcus lactis.

Results: Overexpressing the rbsA, rbsB, msmK, and dppA genes exhibited 5.8-, 12.2-, 213.7-, and 5.2-fold higher survival rates than the control strain, respectively, after acid shock for 3 h at pH 4.0. Subsequently, transcriptional profile alterations in recombinant strains were analyzed during acid stress. The differentially expressed genes associated with cold-shock proteins (csp), fatty acid biosynthesis (fabH), and coenzyme A biosynthesis (coaD) were up-regulated in the four recombinant strains during acid stress. Additionally, some genes were differentially expressed in specific recombinant strains. For example, in L. lactis (RbsB), genes involved in the pyrimidine biosynthetic pathway (pyrCBDEK) and glycine or betaine transport process (busAA and busAB) were up-regulated during acid stress, and the argG genes showed up-regulations in L. lactis (MsmK). Finally, we found that overexpression of the ABC transporters RbsB and MsmK increased intracellular ATP concentrations to protect cells against acidic damage in the initial stage of acid stress. Furthermore, L. lactis (MsmK) consistently maintained elevated ATP concentrations under acid stress.

Conclusions: This study elucidates the common and specific mechanisms underlying improved acid tolerance by manipulating ABC transporters and provides a further understanding of the role of ABC transporters in acid-stress tolerance.

Keywords: ABC transporters; Acid-stress tolerance; Anti-acid components; Lactococcus lactis NZ9000; Transcriptomics.

Fig. 1

The survival rates of the control and recombinant strains under acid-stress conditions. a L. lactis (RbsA); b L. lactis (RbsB); c L. lactis (MsmK); d L. lactis (DppA). Error bars represent the mean ± standard deviation of three replicates

Fig. 2

Heatmap of important differentially expressed genes in the recombinant strain [L. lactis (RbsA)] relative to the control strain [L. lactis (Vector)] under normal (0 h) and acid-stress (2.5 h) conditions. Each gene shows the expression ratio (log₂-fold change). NA represents the expression of gene was upregulated or downregulated with a less than twofold change. Genes with at least a twofold change are shown. Adjusted p < 0.05 for all data selected

Fig. 3

Important differentially expressed genes in the recombinant strain (L. lactis (RbsB)) relative to the control strain (L. lactis (Vector)) under normal (0 h) and acid-stress (2.5 h) conditions. a Differentially expressed genes involved in the galactose metabolism, starch and sucrose metabolism, pyrimidine metabolism, purine metabolism, histidine metabolism, and valine and isoleucine biosynthesis. b Heatmap of differentially expressed genes involved in another biological process. Each gene shows the expression ratio (log₂-fold change). NA represents the expression of gene was upregulated or downregulated with a less than twofold change. Genes with at least a twofold change are shown. Adjusted p < 0.05 for all data selected

Fig. 4

Heatmap of important differentially expressed genes in the recombinant strain (L. lactis (MsmK)) relative to the control strain (L. lactis (Vector)) under normal (0 h) and acid-stress (2.5 h) conditions. Each gene shows the expression ratio (log₂-fold change). NA represents the expression of gene was upregulated or downregulated with a less than twofold change. Genes with at least a twofold change are shown. Adjusted p < 0.05 for all data selected

Fig. 5

Heatmap of important differentially expressed genes in the recombinant strain (L. lactis (DppA)) relative to the control strain (L. lactis (Vector)) under normal (0 h) and acid-stress (2.5 h) conditions. Each gene shows the expression ratio (log₂-fold change). NA represents the expression of gene was upregulated or downregulated with a less than twofold change. Genes with at least a twofold change are shown. Adjusted p < 0.05 for all data selected

Fig. 6

Effects of over-expressed ABC transporters on the intracellular ATP concentrations during acid stress. All strains were exposed to acid stress at pH 4.0 for various times (0, 1 and 2.5 h). Error bars represent the mean ± standard deviation of three replicates