Screening Of New Coronatine-producing Strains And Construction Of Engineering Strains

Coronatine ?COR? is a secondary metabolite produced by several pathovars of Pseudomonas syringae. It is a structural and functional mimic of methyl jasmonate ?MeJA? and abscisic acid ?ABA?.It can improve plant stress resistance, induce the accumulation of secondary metabolites, suppress germination and promote abscission. COR has a board application prospects as a new plant growth regulator.Currently COR production relies mainly on microbial fermentation. The existing wild strains have a low COR yield and new microbial resource for COR production is desired. Our knowledgement about the regulatory network on COR biosynthesis is incomplete and indistinct. In this study, we attempted to exploit new COR-producing strain resources by isolation and purification of microorganism. Transposon Tn5 was introduced for obtaining mutant population. A preliminary study on the relationship between mutation genes and COR biosynthesis was conducted by insertion gene mapping of mutants with significant variation in COR yield, which provided the basis for the subsequent genetic engineering. We cloned the biosynthetic genes of CMA, constructed a heterologous expression engineered yeast capable of biosynthesizing CMA,and developed a new model for COR production. We also investigated the degradation characteristics of COR in aqueous solution, which aimed to provide scientific guidance and reference for the production, storage and field application of COR. The main results are as follows:1. According to the key genes corS and corR in COR biosynthesis gene clusters, two sets of PCR primers and PCR procedure were designed. The PCR method was applied to preliminary screening of 104 wild stains isolated from diseased plants in the field. The specific PCR assay displayed that a 850 bp ?corS? band and a 450 bp ?corR? band could be obtained from the strains numbered 7 and 18. Subsequent HPLC analysis showed that the strains of 7 and 18 could synthesis COR and the yields of COR were 6.5 mg/L and 2.1 mg/L,respectively.2. Tn5 transposon mutagenesis was applied to the wild COR-producing strain Pseudomonas syringae pv. tomato DC3000. Insertion loci in nineteen mutants with significant COR yield variation were successfully identified by random PCR or genome resequencing. Several genes that play important roles in regulating COR biosynthesis were found, including PSPTO₂603, PSPTO₂737, PSPTO₀367 and PSPTO₁081. Among the genes,pspto₂603 and pspto₂737 mutations resulted in the inability of COR synthesization.3. A yeast expression system containing complete CMA biosynthesis genes was constructed by synthesis biology methods. CMA was biosynthesized in the recombinant yeast and the yield of CMA reached 6.1 mg/L.4. COR had a good tolerance to temperature and pH tested. The stability of COR was significantly affected by UV light rather than visible light and the half-life was only 61.9 h under UV irradiation. A significant damage to COR was also observed in the presence of oxidant. Approximately 75% COR was degraded in the presence of H2O2 for 12h at room temperature.