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

本研究对链霉菌产生的C-糖基化抗生素生物合成酶进行了功能分析。目标化合物是一类被称为苯并异色满醌（BIQ）的芳香族抗生素。代表性的是美得霉素和石榴苷，两者都携带独特的脱氧糖组分，这具有很大的药学意义。本项目取得的成果如下。(1)与美得霉素生物合成有关的脱氧糖生物合成酶的功能性表达：与美得霉素中安哥罗胺组分中核苷酸-糖中间体形成有关的Med-17和Med-18酶在大肠杆菌中异源表达，并对重组酶进行了功能性鉴定。Med-17和Med-18分别为TDP-葡萄糖合成酶和TDP-葡萄糖4，6-脱氢酶。(2)BIQ分子中还原酶和氧化酶的功能特性天蓝色链霉菌A3（2）产生的放线菌紫素（ACT）是一种芳香族聚酮抗生素。在ACT生物合成中，一种立体特异性的酮还原酶（RED 1）在E.大肠杆菌，并与合成类似物的RED 1的酶促研究。该酶更喜欢接受游离酸形式作为其底物，这强烈表明RED 1识别蛋白质游离酮酸作为随后生物合成步骤的第一个承诺中间体。另外，一个新的分流产品，actinoperylone，已被分离前的actVA-5和6基因参与ACT生物合成的缺失突变体。光谱分析显示其具有四个手性中心的苝醌型骨架，显然来自ACT中间体的二聚化。放线菌酮的结构表明ActVA-ORF 5在C-6处的氧引入中的重要作用。

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.