Mining Of Nonribosomal Peptides From Burkholderia Gladioli ATCC 10248

Microbial natural products with diverse structures and biological activities are important sources of clinical,agricultural and animal husbandry drugs,such as antibiotics,anticancer drugs,immunosuppressants and anti-insect drugs.In the past few decades,actinomycetes have been the focus of microbial natural products.With the development of genome sequencing technology,researchers have discovered more and more active natural products from Burkholderia genus.The establishment of multiple microbial recombination enzyme systems and gene clusters heterologous expression platform based on 'Recombineering' have provided technical support for the mining of microbial natural products.This study focused on the mining of nonribosomal peptides from Burkholderia gladioli ATCC 10248.Firstly,six unknown NRPS gene clusters were modificated in situ by the established recombination system,two of them were activated successfully and several novel lipopeptides were obtained.Then NRPS gene cluster BGC7 were cloned using recombineering.The results are summarized as follows:Establishment of recombination system in B.gladioli ATCC 10248.Firstly,the minimal inhibitory concentration of antibiotics against strain ATCC 10248 was tested.The results showed that three antibiotics,kanamycin,apramycin and gentamicin,could be used for genetic screening of strain ATCC 10248.Then,the recombinant efficiency of three recombinant systems,gbaA from E.coli phage,gBAS from P.aeruginosa phage and Redy-redaP7029 from Burkholderia,were tested through gene knockout experiments.The results revealed that the recombination system gBAS from P.aeruginosa phage could mediate the well recombination efficiency with homologous arms of 50 bp in strain ATCC 10248.Activation of two cryptic gene clusters BGC2 and BGC5 in situ.Firstly,six unknown NRPS gene clusters were modificated in situ by the constitutive promoter replacement of gentamicin resistance gene(Pgenta)and insert inactivation of gentamicin resistance gene(genta)using recombination system gBAS.Then,the metabolic profiles among mutants and wild type were compared by HPLC-MS.The results revealed that BGC2 and BGC5 were activated successfully.Finally,five compounds were isolated from the activated strain Bgl10248-P2 and two compounds were isolated from the activated strain Bgl10248-P5 by silica gel column chromatography and HPLC.The structures of the above seven compounds were identified by NMR,the structures of 4a,4b and 8 were predicted by HRMS/MS,are all novel linear lipopeptides which condensed by fatty acids and amino acids.Cloning of cryptic gene cluster BGC7 using recombineering.Firstly,the original promoter of BGC7 were replaced by the constitutive promoter of gentamicin resistance gene(Pgenta)in situ,but the gene cluster was not activated.Then,we cloned BGC7 directly using recombineering,and replaced its promoter with the constitutive promoter of gentamicin resistance gene(Pgenta),followed by heterologous expressed in multiple hosts including E.coli GB05-MtaA?B.thailandensis E264 and Burkholderiales DSM 7029,but it was failed.The reason could be deduced that the biosynthetic gene clusters of secondary metabolites are generally complex,and a single activation method couldn't work well.