Isolation And Identification Of Antifungal Active Substances From Pseudomonas Aeruginosa And Construction Of High-yield Engineering Bacteria

Pseudomonas aeruginosa has unique genes and complex metabolites,which is an important opportunistic pathogen in hospital,and is also an important biocontrol bacterium in agricultural production.The secondary metabolite phenazine which is synthesized and secreted,plays a major role in biocontrol.Therefore,the efficiency of phenazine synthesis is an important index to evaluate the biocontrol value of Pseudomonas aeruginosa.The strain 2016NX1 used in this study was isolated from the root of Millettia speciosa Champ.Firstly,the supernatants of fermentation cultures of 2016NX1 were extracted with an equal volume of chloroform to obtain the blue crude extract.Secondly,the silica gel column chromatography of the blue crude extract was conducted in order to purify the crude extract.For silica gel column chromatography,the mobile phase is chloroform/methanol(v:v=15:1).Meantime,thin layer chromatography was used to test the separated components.The experiment was performed on a sheet of glass which was coated with a thin layer of silica gel.The mobile phase is also chloroform/methanol(v:v=15:1).Thirdly,the primary purified products were subjected to HPLC detection to determine the various kinds and further purify the products.Lastly,the nuclear magnetic resonance was used to identify the purified products(yellow component and blue component).The results showed that the yellow component was identified as1-hydroxyphenazine,and the blue component was identified as Cereusitin A.Because the yield of phenazine in the wild strain 2016NX1 is relatively low(about 200 mg/L),and therefore this study attempts to construct engineering strain with a high yield of phenazine.The phzR gene of Pseudomonas aeruginosa regulating phenazine biosynthesis was selected based on the former studies in this study.Based on the conserved nucleotide sequence of phzR from other species,the primers were synthesized to amplify the core fragment of phzR using genomic DNA of 2016NX1 as template.The 5? end of this gene was obtained using genome walking method.Based on the above results,the terminal primers were designed to clone the complete coding region(from initiation codon to termination codon).The full-length phzR gene was cloned into the expression vector pBBR1MCS-5 under the control of strong promoter lac.Then the recombinant vector was transformed into wild strain 2016NX1 to obtain the engineering strain 2019 NES.The test using UV-Vis spectrophotometer showed that the phenazine content of 2019 NES was about 1.4-fold compared to the wild strain 2016NX1.