Study On The Dye-decolorizing Peroxidase And Its Translocation System Of Streptococcus Thermophilus

Streptococcus thermophilus is a major dairy starter with Lactobacillus delbrueckii subsp. bulgaricus or Lact. helveticus for the manufacture of yoghurt and cheese, contributing to flavor, texture and shelf life. While various stress conditions including temperature, oxygen and osmotic pressure would decrease the viability of the starters, resulting in quality losses of the final fermented product. Hence clarifying the resistance mechanism against stress conditions was crucial for the preparation of starters with high viability. Among the environmental stresses, the presence of toxic reactive oxygen species (ROS) is the most severe survival challenge to S. thermophilus that belongs to facultative anaerobic bacteria. S. thermophilus has the ability to grow and survive in aerobic environment. However, the mechanism of S. thermophilus to resist H2O2 remains unknown. In this study, a dye-decolorizing peroxidase was discovered in S. thermophilus CGMCC7.179 and found able to resist oxidative stress. Then we characterized this enzyme and clarified its function to eliminate H2O2. In addition, the translocation system of the dye-decolorizing peroxidase, a twin-arginine translocation (Tat) system was identified and transplanted into Lactococcus lactis MG1363. The details of the contents and results are listed as follows:1. Genetic structure and anti-oxidative capacity of efeB gene in S. thermophilus CGMCC7.179S. thermophilus possesses a superoxide dismutase which produces H2O2. While the eliminative mechanism of H2O2 remains unclear. In this study, an efeB gene coding a dye-decolorizing peroxidase was identified in the genome of S. thermophilus CGMCC 7.179. The putative amino acid sequence of EfeB contained a Tat signal peptide. Strikingly, two component genes (tatA and tatC) of Tat system were also found in the efeOBU-tatCA operon with the efeB gene. Knocking out efeB gene resulted in the decrease of the strain growth under aerobic condition, suggesting that the efeB gene was able to defense the strain against oxidative stress. Interestingly, deletion of tatC gene showed similar phenomenon to the deletion of efeB gene, and complementation of efeB gene in the efeB-deficient strains enhanced the biomass of the hosts only in the presence of the tatC gene, which suggested that the capability of EfeB to resist oxidative stress needed the assistance of Tat system. Moreover, it was proved both in S. thermophilus CGMCC 7.179 and Escherichia coli DE3 that EfeB could be translocated by the Tat system from S. thermophilus. In addition, the transcriptional level of efeB and tatC increased when the strain was cultured under aerobic condition. Overall, these results provided the evidences firstly that EfeB played a role in protecting cells from oxidative stress after translocated by the Tat system in S. thermophilus.2. Function of EfeB from S. thermophilus CGMCC7.179Dye-decolorizing peroxidases were classified as a novel peroxidase family because of their broad substrate specificity, low pH optima, lack of a conserved active site distal histidine, and structural divergence from classical plant and animal peroxidases. To clarify the function of EfeB, its amino acid sequence was firstly aligned with those of three bacterial and four fungal dye-decolorizing peroxidases. The heme-binding residue His313 and the highly conserved G-X-X-D-G box of dye-decolorizing peroxidases were found in EfeB. In particular, EfeB showed 48.13% similarity to FepB of Staphylococcus aureus N315, suggesting that EfeB possessed the structure of dye-decolorizing peroxidases. To clarify the function of EfeB, it was expressed in E. coli DE3. The purified EfeB could decolorize reactive blue 5 when H2O2 was added as electron acceptor, with the optimum temperature and pH of 30℃ and 4.0 respectively, indicating that EfeB possessed peroxidase activity. Moreover, deletion of the efeB gene resulted in higher concentration of H2O2 in the shaking cultures, which suggested that EfeB could eliminate the environmental H2O2. In addition, the S. thermophilus CGMCC7.179 efeB-deficient mutant lost the ability to utilize ferrous. Taken together, EfeB could help the strain relieve oxidative stress and assimilate ferrous.3. Function of the Tat components from S. thermophilus CGMCC7.179Twin-arginine translocation (Tat) system for folded protein secretion was rare in lactic acid bacteria (LAB). Interestingly, we previously identified a Tat system with TatA and TatC subunits in Streptococcus thermophilus CGMCC 7.179. However, the function of this system was unknown. Here, the tatA and tatC genes were cloned and transformed into Escherichia coli DE3 tat-deficiency mutants, generating complementation strains ArA, ArAB, CrC and ACrAD. Analysis of cellular morphology and sodium dodecyl sulphate (SDS) resistance showed that strains ArAB and CrC displayed relatively normal cellular morphology and SDS resistance. Strain ACrAD exhibited shortened cellular chains, but its ability to resist SDS was not restored. Oppositely, strain ArA that could tolerate SDS but exhibited long cellular chains. These results suggested that TatA and TatC could serve as active protein translocases to relieve cell chaining or reestablish resistance against SDS in E. coli DE3 tat-deficiency mutants. Moreover, TatA as a bifunctional subunit fulfilled the roles of both TatAe and TatBE (The subscript E represents the Tat component of E. coli), and the activity of TatA could be influenced by TatBE. Hence both TatA and TatC from S. thermophilus CGMCC 7.179 possessed the typical function of Tat components, and this minimal Tat system was transplantable between different bacteria.The function of the S. thermophilus CGMCC7.179 efeOBU-tatCA operon was as follows:(1) EfeB crosses the membrane via the polymerized TatC-TatA complex; (2) EfeB catalyzes ferrous iron oxidation; (3) Proteins EfeO and EfeU form a minimal complex for ferric iron uptake; (4) EfeB is also able to eliminate H2O2.4. Expression of S. thermophilus CGMCC 7.179 TatC A in Lactococcus lactis MG1363As GRAS strains, LAB were demonstrated to be an ideal cell factory for the production of food-grade recombinant proteins. Steady progress has since been made in research on the areas of recombinant enzyme production and secretion in LAB. Recombinant proteins were majorly secreted through Sec pathway in LAB, in which Tat system was rare. In this study, the secretion efficiency of the Tat system from S. thermophilus CGMCC 7.179 was tested in Lc. lactis MG1363. Unlike S. thermophilus CGMCC 7.179, the growth of Lc. lactis MG1363 was not affected by shaking condition. When EfeB and TatCA had a low expression level, it did not work in H2O2 resistance of Lc. lactis MG1363. However, high expression level of EfeB and TatCA in Lc. lactis MG1363 could remove the growth decrease resulted from the sole expression of EfeB and enhance the H2O2 resistance, indicating that TatCA could transport EfeB to resist oxidative stress in Lc. lactis MG1363. Moreover, subcellular localization showed that TatCA could translocate spNuc (Nuc with the EfeB signal peptide), but not spGFP. All results suggested that the Tat system of S. thermophilus CGMCC 7.179 functioned well in Lc. lactis MG1363, but its efficiency was distinct towards different substrate proteins.5. Carbon catabolite derepression in Lactobacillus fermentum 1001The broad ecological distribution and diverse habitats of Lact. fermentum reflects its metabolic flexibility. To explore the molecular mechanism of the carbon catabolite derepression in Lact. fermentum 1001 when this strain consumed xylose and glucose simultaneously, the transcriptional level of ccpAf was measured by real-time qPCR, revealing that ccpAf transcribed mRNA normally in Lact. fermentum 1001. The ccpAf gene could complement the ccpA-deficiency of a Lactococcus lactis mutant, indicating that CcpAf was functional. Moreover, when the phosphofructokinase from Lact. delbrueckii subsp. bulgaricus ATCC 11842 was expressed in Lact. fermentum 1001, the recombinant preferred glucose to fructose rather than to xylose. All data suggested that CcpAf was functional in Lact. fermentum 1001. In addition, the promoter(Plx) activity of the xyl operon from Lact. fermentum 1001 was further tested in Lact. casei BL23, and it could drive the expression of green fluorescent protein in the presence of glucose. Hence the ability of Lact. fermentum 1001 to co-utilize xylose and glucose resulted from the deficiency of catabolite responsive element in Plx rather than the null mutation of the ccpAf gene. Lact. fermentum 1001 is a potential candidate as a CCR-absent cell factory to transform biomass to high-value-added products. Plx was provided for engineering LAB to enhance fermentation efficiency by avoiding CCR.