Establishment Of Recombinant System Of Xenorhabdus And Study On Gene Clusters Of Secondary Metabolites

Xenorhabdus sp.is a gram-negative bacterium of the enterobacteriaceae family that mutually symbiotic with the Steinernema sp.,which can produce a large number of secondary metabolites with biological activities such as insecticidal,bacteriostatic,anti-tumor.Based on the genome sequencing and bioinformatics analysis,a large number of recessive gene clusters was found in the Xenorhabdus stockiae HN?xs01 stain genome.However,the current research on secondary metabolites of X.stockiae lacks effective gene editing methods,which hinder the discovery of new natural products.Due to the Red/ET homologous recombination technology can effectively promote double-strand break repair and instant recombination merely by short homology arms and recombinases,it has gradually become a powerful means for exploring gene functions and constructing new strains.The research aims to build on the principle of Red/ET recombination engineering,based on the whole genome sequencing results of X.stockiae,and use the recombinant enzymes of X.stockiae's own phage to establish and optimize a new type of recombinant enzyme system in X.stockiae HN?xs01 strain in order to efficiently and quickly mining of secondary metabolites.The main results are as follows:?1?Research on the recombination system of Xenorhabdus sp.and Aeromonas sp..Based on blast alignment of Xenorhabdus bovienii and Aeromonas salmonicidal genome and phage gene and Red/ET homologous recombinant protein,homologous proteins of Red?and Red?were obtained in pathogenic bacillus.The homologous proteins of RecE,RecT and Red?were obtained in this study,and the expression plasmids of the recombinant system of Xenorhabdus sp.and Aeromonas sp.were constructed,and the recombination efficiency was verified and compared in Escherichia coli.?2?Establishment of electric conversion method.This study explored the influencing factors of electrotransformation by detecting the growth curve of X.stockiae HN?xs01 strain,investigating the optimal electrotransformation buffer,the voltage at the time of electric shock,and the culture medium.The best electroporation conditions for obtaining the X.stockiae are:When the LB medium was shake-cultured at 30°C for 4 h(OD₆₀₀00 was 0.8?1.0),competent cells were prepared by using solution A electroporation buffer?0.5 mol/L sorbitol,0.5 mol/L mannitol,10%glycerol?and adding proteinase K for 20 min at a voltage of 1200 V to obtain the best transformation efficiency.?3?Mining of secondary metabolites.HN?xs01 strain cluster1 was inactivated based on homologous recombination,and then a 16-minute difference peak?M16?and a 19-minute difference peak?M19?were obtained by comparing with secondary metabolites of wild-type strains.Combines biomolecule rapid purification system and high-performance liquid chromatography,as well as high resolution mass spectrometry and nuclear magnetic resonance spectroscopy,M16 was identified as a white amorphous powder with a characteristic absorption peak?max?MeOH?of230 nm and 260nm,a relative molecular mass of 843,and a chemical formula of C₄₅H₆₁N₇O₉,a cyclic lipopeptides formed by linking 7 amino acids?phe1-Thr2-phe3-Pro4-Pro5-Leu6-Val7?and 1 amino acid with a fatty chain.M19 was identified as a yellow-brown oil with characteristic absorption peaks?max?MeOH?of 240 nm and 274 nm,and a relative molecular mass of 148.Bioassay found that M16 and M19 have certain antibacterial effect on Candida albicans and Bacillus subtilis.Among them,M16 can cause cancer cell HeLa and insect cell CF-203 to shrink and round,with reduced adhesion and quantity reduced and inhibited proliferation of both cells in a dose-dependent manner.The above results not only offord effective methods for gene editing and genetic manipulation of Xenorhabdus,these also provide new ideas for the construction and transformation of other bacterial recombination systems.Meanwhile,this research method enriches the secondary metabolite pool,laying a foundation for digging more natural products with biological activity.