High-yield Modification Of Non-ribosomal Peptide Antibiotic Edeines In Tobacco Bacterial Wilt Biocontrol Bacteria X23

Bacillus was globally used in biological control of plant pathogens,enhancing plant growth,and increasing the capability of stress resistance.Edeines,as a nonribosomal peptide antibiotic with broad-spectrum,were produced by Brevibacillus brevis X23 which is a bio-control agent for bacterial wilt in solanaceae crops.Many factors restrain the possibility of extensive application of edeines in agriculture such as the low yield,unknown biosynthesis pathway,complicated chemical synthesis pathway,and high expense.This paper worked on the effect of edeines biosynthesis in Br.brevis X23 with different promoter and modification of regulator by recombination engineering、transcriptome and bioinformatics analysis.The main results of this research are listed below:（1）A highly expressed edeines producing strain was constructed by promoter replacement.The edeines operon area was identified by bioinformatics analysis,and the natural promoter was replaced by different constitutive promoters through Red/ET homologous recombination technology for 6 engineering strains named X23(P_ede::P_mwp)、X23（Pede::Pgrac）、X23（Pede::Pxyl A）、X23（Pede::Pspc）、X23（Pede::Pshuttle-09）and X23(P_ede::P₄₃)with high edeines production.The yields of edeines produced by engineering strains were evaluated by various tests such as plate antagonism,liquid chromatography-mass spectrometry（HPLC-MS）and real-time fluorescence quantitative PCR（q RT-PCR）analysis.All the results demonstrated that the expression level of engineering strain X23(P_ede::P_mwp)was 15.40 times more than wild type,and the yield of engineering strain was increased to 83.4μg/m L which is 16.70 times compared to wild type.The yields of edeines of other five engineering strains were increased from 3.98 to 8.00 times and the order from highest to lowest is X23（Pede::Pgrac）、X23（Pede::Pxyl A）、X23（Pede::Pspc）、X23（Pede::Pshuttle-09）and X23（Pede::P43）.（2）abr B was identified as a negative regulation gene for edeines biosynthesis which based on the results of whole-genome sequencing and bioinformatics analysis.The abr B deletion strain was obtained by gene knock out and then the fermentations of mutation strains collected from different phases were analyzed by plate antibacterial assay,HPLC-MS and q RT-PCR elucidating the variety in antibacterial activity,edeines production and transcription level of Biosynthetic Gene Cluster（BGC）.The result shows that after knocked out abr B,the transcription level of the ede operon was increased in the early stage and after 48 hours fermentation,the total production of edeine A and edeine B from mutation strains were increased to 1.10 times than wild type strain.