Study On The Selection Of High-yield Strains Producing HSAF Antibacterial Metabolites Of Enzyme-producing Lysobacterium

The genus of Lysobacter enzymogenes belongs to Xanthomodaceae family,it is a kind of new plant disease biocontrol bacteria widely existing in the environment.The secondary metabolite from OH11,HSAF(Heat-Stable Antifungal Factor),is a kind of small molecule antifungal substance,could inhibit a variety of fungi and oomycetes,and play an important role in the biological control,it is a new broad-spectrum antifungal oomycete active substances with a novel structure and mode of action.The production of agricultural products without pesticide residues and without pests and diseases has always been an important way to promote human health and protect the ecological environment,and it is also a major goal in the field of biological control drug research.HSAF,as an environmentally friendly fungicide,has great potential for the development of new biogenic pesticides.However,the production of HSAF at this stage is too low to achieve industrialized large-scale production,which restricts its widespread application and promotion in disease prevention to some extent.Therefore,obtaining high-yield strains of HSAF and raising HSAF production is a problem that needs to be solved urgently.In this study,the HSAF as the research object,the Lysobacter enzymogenes OH11 as the parent strain,three methods be used for HSAF high-yield strains breeding.Multiple mutants strains were constructed by UV mutagenesis,knockout of HSAF negative regulatory genes,and the promoter modification of HSAF biosynthetic gene and its positive regulatory genes,and the HSAF yields were evaluated.Three HSAF high-yield strains were screened by UV mutagenesis.Their HSAF yields were increased by 36%,33%and 25%,respectively,compared to the wild type.But genetic stability experiments showed that their genetic traits were not stable.The second messenger c-di-GMP is known to regulate the biosynthesis of HSAF in Lysobacter enzymogenes OH11,in this study,two negatively-regulated genes,the c-di-GMP metabolic protein encoding gene le3756 and le1974,were knocked out,double mutant strain ?3756?1974 was obtained,its HSAF yield was increased by 67%compared to wild type.And then the gene hfq encoding the RNA chaperone was knocked out in the double mutant strain to obtain the strain?3756?1974?hfq,and the HSAF yield was doubled compared to the wild type strain OH11.In addition,since the gene le1632 encoding c-di-GMP metabolic protein has positive regulation on the biosynthesis of HSAF,the promoter of le1632 was replaced by the strong promoter Ptac in the double mutant strain ?3756?1974,the strain?2-1632-Ptac was obtained and its HSAF production was increased 1.7-fold compared to the wild-type strain.By measuring the growth curves of the strains?3756?1974Ahfq and ?2-1632-Ptac in 10%TSB medium,it was found that their growth was not adversely affected,and the maximum cell density could basically reach the wild-type level,and a stable HSAF high-yield trend was always maintained during the fermentation process.Overall,this study used a variety of methods to screen two strains of stable HSAF high-yield strains,which provided a reference for the subsequent selection of high-yield strains of HSAF,and laying a foundation for realizing its industrial production.