Mechanism Of The Biosynthesis Of Validamycin In Streptomyces Hygrosopicus 5008

Streptomyces hygroscopicus var. Jinggangensis Yen, isolated from the soil of the Jinggang Mountain in Jiangxi province in 1973, was a new subspecie of Streptomyces hygroscopicus. Validamycin, an aminoglycoside antibiotic produced by S. hygroscopicus var. Jinggangensis 5008, is used widely especially in Asia to control sheath blight disease of rice plants and dumping-off of cucumber seedlings caused by the fungus Pellicularia sasakii (Rhizoctonia solani). The biosynthesis gene cluster of validamycin has been cloned and fully sequenced, and the results are helpful for further research on the mechanism of the biosynthesis of validamycin.The bioinformatics analysis of validamycin gene cluster indicated that the protein encoded by gene valG was highly homologous with E. coli K4 chondroitin polymerase KfoC catalyzing the polymerization of chondroitin skeleton. There are 422 amino acid in ValG, and 264 amino acid less than 686 amino acid of KfoC. The region containing 250 amino acid residues on the N-terminal of ValG is the binding domain of glycosyl ligand, it is very similar to the glycosyltransferase, and the conserved module DGS and two DXD modules are proposed to NDP-glycosyl and Mn2+ binding relevant. These indicated that ValG was responsible for the last glycosyltransfer step of validamycin biosynthesis. After the replacing mutation of valG gene, validamycin can not be detected by HPLC, however, validoxylamine as the substrate of validamycin biosynthesis was accumulated. Complementation of valG by high-copy vector with strong promoter PermE* could restore the validamycin synthesis. It indicated that valG encode a glycosyltransferase, and glycosyl transfer was the limited step in the process of validamycin biosynthesis. Then, ValG was expressed in E. coli heterologously, and its function of glycosyl transfer was confirmed in vitro. The results of TLC experiment showed that both UDP-glucose and GDP-glucose could be used by ValG, but UDP-glucose was optimum.ValL, encoded by gene valL, was high homology with trehalose-6-phosphate synthase Rxyl021033 in Rubrobacter xylanophilus, which has homology with protein OtsA in E. coli. The trehalose-6-phosphate synthase OstA and the trehalose phosphatase OstB in E. coli were responsible for biosynthesis of trehalose with 6-phosphate glucose and UDP-glucose as substrates. Validamycin could not be detected in valL replaced mutant LL2, and validamycin production could be restored by complemented with the integrative vector which has a strong promoter PermE*. These results indicated that valL was necessary for validamycin synthesis catalyzing the condensation reaction of validamine and valienone.ValH has 33% identities and 50% similarities with the transporter SAV6900 in S. avermitilis and the trehalose permease FucP in Mesorhizobium sp. BNC1, respectively. Their functions were cotransporting the fructose, glucose or galactose with the help of H+. The analysis of ValH by TMHMM Server v. 2.0 indicated that ValH has 11 transmembrane domains belonging to The Major Facilitator Superfamily. ValH maybe has two functions, one is transfering the extracellular UDP-glucose into the cell and increasing the validamycin yield by accumulating the substrate of glycosyltransferase. The other is transfering the intracellular validamycin out of the cell, which can decrease the product of glycosyltransfer reaction, and induce the reaction to produce validamycin. Probably decreasing the intracellular content of validamycin is a mechanism of self-protection. The yield of validamycin in mutant LL5 still could be detected, but it was reduced.The normal growth temperature of S. hygroscopicus 5008 is 30℃, but the fermentation temperature in the lab is 37℃,and it even up to 42℃in the industry production, the validamycin yield at fermentation temperature is decuple higher than that at 30℃. The phenomenon of thermoregulation of the validamycin production is very special, and there is no report on the thermoregulation of antibiotic yield now. A two-component system including valP and valQ was found in the gene cluster by bioinformatics analysis, three mutants LL3, LL4, LL8 were obtained in absenc of valP and valQ respectively and at the same time. HPLC analysis indicated that the yields of validamycin in these three mutants reduced about 50%, and they could be restored by complemented by the integrative vector with a strong promoter PermE*. The structure genes in val cluster were composed of three operons, valABC,valKLMN and valGH. The RT-PCR results show that transcription of valA, valK, valG were obviously decreased at 37℃. It indicated that valP and valQ were participated in the thermoregulated of validamycin. ValQ was predicted to be a histidine kinase self-activating by autophosphorylating the conserved histidine residue in response to the extracellular signals, then transferred the phosphate groups to the aspartic acid residue of the response regulator ValP in absence of ATP. The phosphated ValP binded to the RNA fragment of targetting gene and enhanced the transcription.