Production of novel biologically active molecules based on the functional analysis of glycosylation enzymes related to secondary metabolism

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This research deals with the functional analysis of the biosynthetic enzymes of C-glycosylated antibiotics produced by Streptomyces spp. The target compounds are a class of aromatic antibiotics known as benzoisochromanequinones (BIQs). Representative are medermycin and granaticin, both carry the unique deoxysugar components, which are of great pharmaceutical interest. The achievements in this project are as follows.(1) The functional expression of the deoxysugar biosynthetic enzymes involved in medermycin biosynthesis.The Med-17 and 18 enzymes, both related to the formation of nucleotide-sugar intermediates in angolosamine component in medermycin, are heterologously expressed in Escherichia coli, and the recombinant enzymes were functionally characterized. Med-17 and 18 were revealed to TDP-glucose synthase and TDP-glucose 4,6-dehydratase, respectively.(2) The functional characterization of the reducing and oxidizing enzymes involved in the BIQ molecules.Actinorhodin(ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic. n the ACT biosynthesis, a stereospecific ketoreductase(RED1) was heterologously expressed in E. coli, and enzymatic studies on RED1 with synthetic analogues. The enzyme preferred to accept an free acid form as its substrate, strongly suggesting that RED1 recognizes a protein free keto acid as the first committed intermediate of later biosynthetic steps. Separately, a novel shunt product, actinoperylone, has been isolated front a deletion mutant of the actVA-5and 6 genes involved for ACT biosynthesis. Spectoroscopic analysis revealed its perylenquinone-type skeleton with the four chiral centers, obviously derived from the dimerization of an ACT intermediate. The structure of actinoperylone indicates the essential role of ActVA-ORF5 in the oxygen introduction at C-6.