Exploration of Key Proteins for Plantaricin Q7 Biosynthesis and Metabolism Regulation

Author:Zhang Jian Ming

Supervisor:han xue


Degree Year:2019





Bacteriocins of lactic acid bacteria are secondary metabolites as ribosomally synthesised peptides or proteins and can be widely used in the field of food preservative due to their security features.At present,only Nisin has been permitted to be used as food additives in several countries for controlling some pathogenic bacteria.But Nisin has a narrow antimicrobial spectrum.Thus,screening a strain with broad spectrum and high antibacterial activity is very important.In this thesis,the key proteins for bacteriocin biosynthesis and feedback inhibition were mined by proteomics,bioinformatics and gene knockout firstly.Then,to enhance bacteriocin production by regulation of the key protein were researched.The objectives of this study were to lead to more efficient bacteriocin production and application in industry.The characterization of bacteriocin from three lactic acid bacteria showed that plantaricin Q7 produced by Lactobacillus plantarum Q7 had the best antimicrobial activity and processing stability,compared to Lactobacillus plantarum J23 and Enterococcus faecium T1.After that,synthetic timing and separation for plantaricin Q7 were studied.The results indicated that plantaricin Q7 biosynthesis and bacteria growth was partial coupling.The production of bacteriocin reached a maximum at end of logarithmic growth.In addition,two-step purification for SP Sepherose Fast Flow and Superdex Peptide 10/300 GL could obtain high purity bacteriocin.The effect of carbon source on plantaricin Q7 biosynthesis in Lactobacillus plantarum Q7 was investigated.It was determined that fructose induced and accelerated plantaricin Q7 production in the early stages of biosynthesis.The proteomes of plantaricin Q7 biosynthesis in MRS-fru and MRS broth were compared.A total of 14 protein spots were highly expressed(2.5-fold increase)in the MRS-fru broth.MALDI-TOF/TOF MS,bioinformatics and transcriptional level analysis showed that most proteins associated with protein transcription,translation and processing.So,these protein points in MRS-fru could prompt bacteria rapid protein expression.In addition,shock proteins were in the center of the protein interaction networks.They played an important role in the process of bacteriocin synthesis.A pre-heat-shock strategy was used to overexpress shock-related protein to enhance plantaricin Q7 production.The results showed that plantaricin Q7 production was significantly increased at 45 °C for 5 min The final plantaricin Q7 output reached 2873.08 IU/mL following pre-heat-shock,which was 23.36% higher than that of the control strategy without pre-heat-shock treatment.These results demonstrated that a pre-heat-shock was an efficient approach for improving plantaricin Q7 production.The quantitative real-time reverse transcriptase PCR analyses indicated that only GroEL was induced,by 10-fold,following pre-heat-shock treatment.Meantime,groEL knockout strains-Lactobacillus plantarum Q7ΔGroEL were successfully constructed.The results showed that bacteriocin production from Lactobacillus plantarum Q7ΔGroEL was significantly lower than the bactriocin production from wild strain.It was proved that GroEL was critical and essential protein for plantaricin Q7 biosynthesis.In addition,plantaricin Q7 production was inhibited by feedback regulation with its high concentrations.Adsorption assays for plantaricin Q7 were carried out with three clays Plantaricin Q7 was able to be adsorbed onto montmorillonite.Electrostatic,hydrogen bonding and hydrophobic interactions were the main adsorption force.The adsorption capacity of montmorillonite at room temperature could reach up to 1276.19 IU/mg.Thus,avoiding potential feedback inhibition,a strategy for the adsorption of plantaricin Q7 onto montmorillonite during fermentation was used to increase plantaricin Q7 production.Finally,the plantaricin Q7 level reached 3713.40 IU/mL in fermentation with adsorption,which was 41.61% higher than that of fermentation without adsorption.On the basis of the bioinformatics analysis,the results showed that Lactobacillus plantarum Q7 had six gene of histidine protein kinase(HPK)and response regulator protein(RR).Among them,feedback inhibition of plantaricin Q7 was regulated by chr_orf00356(HPK)and chr_orf00355(RR)using quantitative PCR analysis.HPK knockout strainsLactobacillus plantarum Q7ΔHPK were successfully constructed.The results showed that the feedback inhibition of plantaricin Q7 from Lactobacillus plantarum Q7ΔHPK was removed.It could contiue to bacteriocin.It was proved that HPK(chr_orf00356)was critical in feedback inhibition of plantaricin Q7,and a key protein in plantaricin Q7 biosynthesis.