Oenococcus oeni is a commercial wine-fermenting bacterial strain, owing to its high efficiency of malolactic fermentation and stress tolerance. The present study explored the function of key genes in O. oeni to enhance stress resistance by heterologous expression of these genes in another species. The orf00404 gene that encodes a two-component signal transduction response regulator in O. oeni was heterologously expressed in Lactiplantibacillus plantarum WCFS1. The expression of orf00404 significantly enhanced the growth rate of the recombinant strain under acid stress. At 60 h, 72 h, and 108 h of culture at pH 4.0, the recombinant strain had 1562, 641, and 748 differentially expressed genes compared to the control strain, respectively. At all three time points, 20 genes were upregulated in the recombinant strain, including the lamA-D operon-coding genes of the quorum-sensing two component signal transduction system and the spx5 RNA polymerase-binding protein coding gene, which may help adaptation to acid stress. In addition, 47 genes were downregulated in the recombinant strain at all three time points, including the hsp1 heat shock protein-coding gene, the trxA1 thioredoxin-coding gene, and the dinP, mutY, umuC, and uvrB DNA damage repair-related protein-coding genes, potentially indicating that the recombinant strain was less susceptible to stress and had less DNA damage than the control strain in acid stress conditions. The recombinant strain had higher membrane fluidity, permeability, and integrity at an early stage of logarithmic growth (72 h), suggesting that it had a more complete and active cell membrane state at this stage. The intracellular ATP content was significantly reduced in the recombinant strain at the beginning of logarithmic growth (60 h), implying that the recombinant strain consumed more energy at this stage to resist acid stress and growth. These results indicated that the recombinant strain enhances acid stress tolerance by regulating a gene expression pattern, increasing ATP consumption, and enhancing cell membrane fluidity, membrane permeability, and membrane integrity at specific growth stages. Thus, the recombinant strain may have potential application in the microbial biotechnology industry.

Oenococcus oeni is the main microorganism that undergoes malolactic fermentation (MLF) in the winemaking industry due to its excellent adaptability to harsh wine environments. The start of MLF is often delayed or even fails, and low pH appears to be a crucial parameter. To study the function of the trxA gene in acid stress, a plasmid containing the trxA gene of O. oeni SD-2a was heterologously expressed in Lactiplantibacillus plantarum WCFS1. The recombinant strain (WCFS1-trxA) grew better than the control strain (WCFS1-Vector) under acid stress. The expression of thioredoxin system genes was much higher in the recombinant strain compared with the control strain under acid stress. In addition, a series of physiological and biochemical assays were conducted. The ATP content was lower in the recombinant strain, while the cell membrane fluidity and integrity improved in the recombinant strain. Moreover, reactive oxygen species (ROS) accumulation, intracellular GSH level, and superoxide dismutase (SOD) activity assays showed that the recombinant strain decreased the intracellular reactive oxygen species (ROS) accumulation by improving the SOD activity. In conclusion, heterologous expression of trxA improves the SOD activity of L. plantarum WCFS1, reducing bacterial ROS and increasing cell membrane fluidity and integrity, enhancing the tolerance of Lactiplantibacillus plantarum WCFS1 under acid stress.

Oenococcus oeni (O. oeni) can initiate and complete the malolactic fermentation (MLF) process, which significantly improves wine quality. However, stress factors commonly encountered in wine, such as acid stress and ethanol stress, can hinder this process. Overexpression of certain key functional genes using genetic recombination technology can enhance the stress tolerance of O. oeni. In this study, the large-conductance mechanosensitive channel (mscl) gene was overexpressed in O. oeni SD-2a using genetic recombination technology. The results showed that overexpression of this gene enhanced the growth rate of O. oeni under 10% ethanol stress conditions. Physiological index measurements indicated that overexpression of this gene enhanced the control of cell membrane permeability in the recombinant strain at different time points under ethanol stress and altered cell membrane fluidity at these time points. Proteomic analysis after 12 h of treatment under 10% ethanol stress revealed that mscl overexpression significantly altered the protein expression pattern of O. oeni. The most significantly affected proteins included some cell membrane transporters (for sugars, lipids, amino acids, and nucleotides) and proteins involved in cell wall synthesis. These results suggest that mscl overexpression enhances the ethanol stress tolerance of O. oeni by altering its cell membrane properties and affecting the expression levels of proteins related to cell membrane transport and cell wall synthesis. This study provides a theoretical reference for obtaining O. oeni recombinant strains with enhanced stress tolerance through genetic recombination technology.