Identification Of Endophytic Bacteria With Antifungal Activity In Rice And Study On The Glycosylation In The Biosynthetic Pathway Of Aromatic Polyketide Medermycin

Many microorganisms with biological activity against pathogenic organisms could accumulate a vast number of bioactive compounds with structural and functional diversity. Isolation of more new microbes with potential application in crop protection and clinical therapy and elucidation of biosynthetic mechanism of bioactive natural products have been of great interest.The present study focused on two aspects:firstly, in order to get more microbes possessing the powerful characteristics such as stability, high effect and safety to hosts when used to control fungal diseases on crops, an endophytic bacteria from the rice with strong antifungal activity was screened and identified, and its colonized action and effect on the hosts were also studied; Secondly, in order to elucidate the mechanism of C-glycosylation of an aromatic antibiotic medermycin with antibacterial and antitumor activity, the function of several medermycin biosynthetic genes related to C-glycosylation was investigated:1.Here, several endophytic bacterial strains were isolated from the field rotated by rice and cole for many years.Among the isolated strains, CHM-1 was found to have the highest antifungal activity. The inhibitory effect of CHM-1 and its action mechanism on several kinds of plant pathogenic fungi were investigated by in vivo and in vitro tests, and the control efficacy was also determined against Bipolaris maydis and Rhizoctonia solani in vitro. On the basis of the comprehensive taxonomic data from morphological, physiological and biochemical characteristics, and 16S rDNA sequence analysis, CHM-1 was identified to be Bacillus licheniformis. In order to develop CHM-1 as a candidate for biocontrol in agriculture, the colonized action of this isolate was further probed in rice and cole by double-resistance tag. The results showed that CHM-1 has no any harm to the investigated plant hosts, and promotes strongly the growth of hosts.2.In the biosynthetic gene cluster of medermycin, there are six genes (med-ORF14,15,16,17, 19,20) proposed to encode glycosylsynthases and one C-glycosyltransferase-homologous gene med-ORF8,which are proposed for the glycosyl-synthesis and-transferring of a novel angolasamine during medermycin biosynthesis. A multigene coexpression system containing 7 genes as above was introduced into a streptomyces host which could offer a sugar acceptor. The LC/MS analysis on the resultant metabolites revealed that this coexpression system could covert the sugar acceptor into a glycosylated structure.Knock out and complementation experiments of med-ORF8 using PCR-Targeting RedirectTM technology indicated that med-ORF8 is an indispensable gene in the process of C-glycosylation of medermycin. But substrate specificity and enzymatic properties of Med-ORF8 remains obscured. Therefore, in order to obtain Med-ORF8 protein enough for further enzymatic characterization in vitro, several prokaryotic expression systems concerned with med-ORF8 gene were established, then an optimized system was screened to express efficiently soluble Med-ORF8 protein.