| Validamycins are aminocyclitol antibiotics, consisting of a valienamine moiety, widely used in Asia as rice protectant. Biosynthetic gene cluster of validamycin has been cloned and sequenced in our previous work, including 45 kb and 27 ORFs defined from BLAST search. To confirm the involvement of this gene cluster in validamycin biosynthesis, we rearranged two operons, one contains valABC and the other contains valKLMN, on a vector named pSET152 and then it was transformed into heterologous host Streptomyces lividans 1326. Validoxylamine A was detected from the fermentation broth of the mutant with seven above genes integrated into the chromosome. Further introduction of valG which codes for the glycosyltransferase led to the production of validamycin A. This result suggested that validoxylamine A could be synthesized by valABCKLMN and ValG was the glycosyltransferase converting validoxylamine A to validamycin A as the last step.In the biosynthetic pathway to validamycin A, ValA has been characterized to be a cyclase which catalyzed the convertion from sedoheptulose 7-phosphate in phosphate pentose pathway to 2-epi-5-epi-valiolone. In this study, ValD was characterized to be an epimerase which converted 2-epi-5-epi-valiolone to 5-epi-valiolone. From the result of enzyme assay in vitro, sedoheptulose 7-phosphate was converted into 2-epi-5-epi-valiolone with ValA only and further converted into 5-epi-valiolone with both ValA and ValD. ValD belongs to the VOC family according to the sequence alignments. Like the other members of this family, each active site of ValD was formed by twoβαβββmotifs. ValD exsits as dimers and monomers of ValD had two active sites, which suggested that ValD is the largest member of VOC family to date.And in the following steps, valienone and validone have been proved to be intermediates and could be phosphorylated by kinase ValC. But intermediates further downstream haven't been purified or identified by enzyme assay in vitro.Mutant with the deletion of valN from validamycin biosynthetic gene cluster produced 1,1'-bis-valienamine and validienamycin, which has one more double bond than validoxylamine A and validamycin A, respectively. This indicated that ValN was responsible for the reduction of the double bond between C-5 and C-6 of validamycin A. It is proposed to use valienone or valienone 7-phosphate as substrate to produce validone or validone 7-phosphate.Mutant with the deletion of valM, the only gene that codes aminotransferase, couldn't produced validamycin A or validoxylamine A, which suggested that ValM was also involved in the biosynthesis of validamycins and the putative function is to introduce the nitrogen of validamycin A. And it is proposed to catalyzed the reaction from validone 7-phosphate to validamine 7-phosphate.ValB was suggested to be an ADP transferase from BLAST research and the activity was checked with different candidate substrates chemically synthesized in vitro. And the result showed that ValB used ATP as nucleotidyl donor, which catalyzed the conversion from valienol 1-phosphate to ADP-valienol. This also suggested that valienol 1-phosphate and ADP-valienol are both intermediates in the pathway. Valieno 1-phosphate was also the substrate for AcbR, a homologous protein of ValB with 60% indentities, from acarbose biosynthetic pathway. AcbR used ATP or GTP as nucleotidyl donor and produced ADP-valienol or GDP-valienol.Glycosyltransferase ValG could also use UDP-galactose as sugar donor in addition to UDP-glucose, and produce the new compound 4"-epi-validamycin A, which had antifungal activity.
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