Study On Genome Sequencing And Antimicrobial Metabolites Produced By Endophytic Bacterium Bacillus Velezensis FZ06 Isolated From Leaves Of Camellia Assamica

The symbiotic endophytes in the leaves of Camellia assamica take part in many processes of the production of Pu-erh tea,which will inevitably produce many microbial metabolites in Pu-erh tea.Although researchers have done extensive research on the plant metabolites of C.assamica and their health function,there are few studies focused on the endophytes and their active microbial metabolites in the leaves of C.assamica.Previous studies have shown that the leaves of tea plants are rich in antagonistic endophyte germplasms.Therefore,exploring antagonistic endophytes from fresh leaves of C.assamica and applying their microbial bioactive substances to inhibit food spoilage bacteria and toxin-producing fungi,has been a solution to the problem of microbial food contamination.At the same time,the research on the endophytes and their active metabolites in the leaves of C.assamica can further expand our understanding of the microbial bioactive components in Pu-erh tea and have a certain reference value for finding the potential microbial molecular basis for the health function of Pu-erh tea.In this study,a fungal antagonistic endophytic bacterium FZ06 was isolated from fresh leaf samples of C.assamica.The synthetic ability of the secondary metabolites,the molecular structure of the antimicrobial substances and the inhibitory effect against food spoilage bacteria and toxin-producing fungi were studied.The results and conclusions are as follows:（1）Thirty-two strains of endophytic bacteria and 14 strains of endophytic fungi were isolated from fresh leaf samples of C.assamica by a culture-dependent method.By the methods of confrontation culture and Oxford cup,the endophytic bacterium FZ06 with the strongest antimicrobial activity was considered as the starting strain.The results of morphological,physiological and biochemical indexes,and 16S r DNA sequence analysis showed that the endophytic bacteria FZ06 was a member of Bacillus,named Bacillus velezensis FZ06.And the antimicrobial activity of the fermentation antimicrobial substance of the strain can be well maintained in the heat treatment condition of 100^oC for 30 minutes.It can be stored stably for more than 28 days at 4^oC.Moreover,it has good p H tolerance within the range of p H 2～10.The antimicrobial components in fermentation substance can be extracted by anhydrous methanol,and no antimicrobial activity was detected in the residue.（2）Draft whole genome sequence of endophytic bacterium B.velezensis FZ06 was obtained by using Illumina second-generation sequencing technology,and the genome function of draft whole genome sequence of the strain was analyzed by using a variety of genome annotation service systems.The results of functional classification of COG,GO,and KEGG showed that the strain had relatively perfect growth and metabolism-related abilities.VFDB annotation service did not find critical genes for toxin synthesis in the genome of the strain.The results of Pan genomic analysis showed that the number of specific genes of the strain was much higher than that of most of its related strains,which means that this strain is different from its related strains of B.velezensis in evolutionary level.Using anti SMASH software to analyze the synthetic gene clusters of the secondary metabolites of the strain,and finally integrate and recognize the synthetic gene clusters of three NRPS products and four PKS products.（3）The antimicrobial products of B.velezensis FZ06 were extracted and purified by anhydrous methanol extraction combined with Sephadex LH-20 column chromatography.And the purified components with antimicrobial activity were collected.Using UPLC-MS/MS technology,combined with the prediction results of NRPS products,the antimicrobial products were identified.A total of 15 antimicrobial lipopeptides were identified as C₁₂-Leu⁷-surfactin,C₁₃-Leu⁷-surfactin,C₁₄-Leu⁷-surfactin,C₁₅-Leu⁷-surfactin,C₁₆-Leu⁷-surfactin,C₁₄-iturin A,C₁₅-iturin A,C₁₆-iturin A,C₁₇-iturin A,C₁₅-Ala⁶-fengycin,C₁₆-Ala⁶-fengycin,C₁₇-Ala⁶-fengycin,C₁₅-Val⁶-fengycin,C₁₆-Val⁶-fengycin,and C₁₇-Val⁶-fengycin.The results of bacteriostatic spectrum test showed that the lipopeptide extract had an excellent inhibitory effect against typical food spoilage bacteria（Escherichia coli,Salmonella typhimurium,Staphylococcus aureus,Listeria monocytogenes,and Bacillus cereus）and toxigenic fungi（Aspergillus niger,Aspergillus flavus,Aspergillus parasiticus,and Fusarium moniliforme）.The results of MIC test showed that the lipopeptide extract has a positive effect against Gram-positive bacteria at concentration from 512 to 2048μg/m L,Gram-negative bacteria at concentration of about 1024μg/m L,and indicator fungi at concentration from 128 to 256μg/m L.