Studies On The Secondary Metabolites Of An Endophytic Bacteria YK-HLS-11 Isolated Form Salicornia Bigelovii Torr.

The study found that plant-derived endophytes are rich in species and they are a huge treasure of nature products.In order to obtain a variety of secondary metabolites which contained novel structures and unique activities,screening valuable plant-derived endophytes must be the first critical step.Because of the living environment of Salicornia bigelovii Torr.,which neared the beach,saline and others,it formed an unique metabolic mechanism,suggesting that it may contain a variety of plant-derived endophytes.In this study,endophytic bacterias were isolated and identified from the plants——Salicornia bigelovii Torr.,then the non-ribosomal peptide synthetases(NRPSs)were used as targets to select the active strains containing NRPSs.The target strain—Bacillus methylotrophicus was selected for further fermentation,meanwhile the monomeric compounds were isolated by separation and purification technology,and identified by modern spectroscopic methods,as well as evaluated the antibacterial activities of these compounds preliminaryly.There are ten endophytes were isolated from Salicornia bigelovii Torr.by plate scribing and slope purification.According to the morphological observation,molecular biology methods and physiological and biochemical experiments,the results showed that there were eight endophytic bacterias and two endophytic actinomycetes.The eight endophytic bacterias were identified as:YK-HLS-3 Bacillus atrophaeus?YK-HLS-4 Kocuria marina?YK-HLS-5-1 Bacillus sonorensis?YK-HLS-6 Bacillus sonorensis?YK-HLS-7 Bacillus amyloliquefaciens subsp.Plantarum?YK-HLS-8 Bacillus amyloliquefaciens subsp.Amyloliquefacien?YK-HLS-10 Bacillus aryabhattai and YK-HLS-11 B.methylotrophicus;two endophytic actinomycetes were identified as:YK-HLS-5-2 Streptomyces lateritius and YK-HLS-9-2 Gordonia terrae.By the PCR amplification of NRPSs functional genes and the antibacterial experiment of the selected strain,it was found that the strain YK-HLS-11 contained NRPSs gene fragments and had a strong inhibitory effect on pathogens,such as Bacillus anthracis.The whole genome sequences of the target strain were further analyzed to predict that the strain could produce cyclic lipopeptide compounds.Finally,the results were further confirmed by the detection of its fermentation products using an electrospray ionization mass spectrometry(ESI-MS)analysis.After extraction by organic reagents and purification by column chromatography,seven monomer compounds were obtained.The compounds were identified by mass spectrometry and NMR.Their structures were determined as follows:Macrolactin A(1)?C15-Surfactin-O-methyl(2)?C14-Surfactin-O-methyl(3)?1-palmitoyl-2-linoleoyl-3-?-galactopyranosyl-O-?-galactopyranosyl(4)?Glul-Phe2-Leu3-Glu4-Pro5-Glu6-Pro7 Peptides(5)?n-Butane-O-a-Glucose(6)??-Daucosterol(7).Among them,compound 1,compound 4,compound 5,compound 6 and compound 7 were founded for the first time from B.methylotrophicus and especially compound 4 was a new one,suggesting that the addition of metalions may be due to the activation of silencing genes.The antibacterial activities of the secondary metabolites of YK-HLS-11 were evaluated by the microplate reader method and pore method.The results showed that compound 1?compound 2 and compound 3 had broad-spectrum antimicrobial activity.Among the three compounds,Compound 1 had broad-spectrum antimicrobial activity against B.subtilis,E.aerogenes and C.albicans with MIC values 8?8?32 ?M respectively;Compound 2 and compound 3 had broad-spectrum antimicrobial activity against C.albicans,E.aerogenes and B.anthracis with MIC values 16?8?32 ?M and 32?8?32 ?M respectively.Unluckly,other compounds had no antimicrobial activity.The results of this study showed that using the NRPSs functional genes as a target can achieve to predict the compounds from gene type to chemical type,and screen and find valuable lipopeptide compounds.