Investigation Of Bacterial Natural Product And Study Of Functional CDI Based On Homologous Recombination Technology

Homologous recombination technology is a technology that can modify genes rapidly.With the extensive application and development of recombination technologies,more and more recombination systems have been excavated.In addition,with the development and maturation of genome sequencing technology,the genmome information showed that there are many unexpressed biosynthetic gene clusters in the microbial genome.Activation and inactivation of gene clusters in situ based on homologous recombineering in the microbial genome investigate unknown compounds synthesized by gene clusters,which not only provides a rapid and effective novel way for studying the diversity and function of natural products,but also provides the research foundation for the potential application value of new natural products.Burkholderia HKI454(Burkholderia rhizoxinica HKI454)is an intracellular symbiosis of the plant pathogenic fungus Rhizopus microsporus.Burkholderia HKI454 not only conveys macrolide rhizoxin with anti-mitotic activity to plant pathogenic fungus,but also is important to form spores for fungus.The size of its genome containing a chromosome,and two plasmids is 3.75 Mb.The chromosome includes 8 biosynthetic gene clusters and pBRH01 includes 9 biosynthetic gene clusters from antismash prediction,and in addition,these gene clusters are mostly non-ribosomal peptide synthase(NRPS).Currently,only a polyketide rhizoxin synthesized by a heterozygous NRPS/PKS biosynthetic gene cluster of the chromosome is known,and other compounds synthesized by other 16 gene clusters are unknown structurally and functionally.Therefore,Burkholderia HKI454 has a potential for producing neotype compounds.In this study,gene and the other biosynthetic gene clusters were modified in situ using the recombinase BAS from the phage of Pseudomonas aeruginosa,respectively.Finally,two kinds of compounds expressed by clusterl and cluster7 of pBRH01 were obtained respectively.The three compounds rhizomide A,rhizomide B and rhizomide C,expressed by clusterl of pBRH01,were isolated and purified by the positive-phase silica gel column and semi-preparative HPLC.Finally,rhizomide A,rhizomide B and rhizomide C were macrolide compounds including seven amino acids,which were elucidated by NMR and marfey's hydrolysis.At the same time,the biological activities of rhizomide A demonstrated that rhizomide A not only had weak antitumor activities,but also had weak inhibitory activities against fungi and gram-positive bacteria.Burkholderia DSM7029 is gram-negative bacterium and produces glidobactins which are toxic to cells.The genome of Burkholderia DSM7029 contains 17 biosynthetic gene clusters predicted by antismash.At present,only glidobactins synthesized by a heterozygous NRPS/PKS biosynthetic gene cluster are known,and and other compounds synthesized by other 16 gene clusters are unknown structurally and functionally.Therefore,Burkholderia DSM7029 has a potential for producing neotype compounds.In this study,modifications of different genes and gene clusters were performed by recombinase(Reda7029-Red?7029)from the phage of strain DSM7029.And,the compounds expressed by cluster7 and cluster11 are acquired in Burkholderia DSM7029.A series of compounds expressed by cluster11 are firstly extracted from the XAD16 resin by methanol,separated by positive-phase silica gel column and reverse-phase silica gel column,and then purified by semi-preparative HPLC to give three compounds eventually.Escherichia coli Nissle 1917(EcN)is an enteric probiotic that produces a lot of antimicrobial compounds.Contact-dependent growth inhibition(CDI)is neo-type mechanism of inhibition,and canonical CDI included three genes,cdiA,cdiB and cdil.In this study,two kinds of CDI gene cluster were found in the EcN genome.One of both is the complete gene cluster CDI and another is orphan CdiA-CT/CdiI module.In order to study whether the completed gene cluster produced biologically active toxin protein CdiA in wild type EcN,the four mutants of EcN which were EcN ? cdiBAI,EcN ? cdiB,EcN ? cdiA,and EcN ? cdiI were constructed based on Red/ET homologous recombination.Meanwhile,CdiA-CT,CdiA-CTOl,and 11 kinds of different truncated structures of CdiA-CT were constructed using linear and linear homologous recombination,respectively,in order to seek the optimal biological activity of the toxin CdiA.In addition,coexpression of toxin proteins and hypothetical protein were constructed.The biomass assay demonstrated that cdiBAI gene cluster could produce toxin CdiA in wild type EcN,protein CdiA-K08 and protein CdiA-K09 had strong biological activity and the hypothetical protein was immune protein CdiI,and could neutralize the activity of toxin CdiA in the heterologous host.Therefore,we deduced that CDI might provide a novel competition for EcN to survive in the complex intestinal environment.Based on the bacterial genome sequencing,various genetic modifications of Burkholderia KI454 and Burkholderia DSM7029 were completed based on homologous recombination and four cryptic biosynthetic gene clusters were successfully excavated.One was silent gene cluster and three were cyptic gene clusters which expressed compounds with low yield.In addition,contact-dependent growth inhibiton(CDI)is a novel kind of competitive growth mechanism for probiotics EcN,and relative proteins of CDI gene cluster were studied preliminarily.Moreover,the results show that genome sequencing technology,direct cloning technology,and homologous recombination technology play a vital role in the mining of natural products and lay the foundation for further development of new natural products in the aspect of structure and function.