Cloning And Functional Analysis Of Genes For L-rhodinose Biosynthesis In Streptolydigin

Streptolydigin, inhibiting RNA synthesis, is a kind of acyltetramic acids produced by streptomyces lydicus. It is glycosylated at later steps of biosynthesis by adding L-rhodinose. L-rhodinose is a kind of 2, 3, 6-trideoxysugar which belongs to the 6-deoxyhexoses family. The first two biosynthetic steps of L-rhodinose involve the activation of D-glucose into NDP-D-glucose and a further dehydration step generating NDP-4-keto-6-deoxy-D-glucose, which are common to the biosynthesis of 6-deoxyhexoses family. These two reactions are catalyzed by NDP-hexose synthase and NDP-D- glucose -4, 6-dehydratase respectively.By comparing gene sequences of NDP-D-glucose-4, 6-dehydratase, primers were designed for cloning this gene from streptomyces lydicus. Finally, a 724 bp fragment Lid1 was obtained and sequenced, which showed high similarity to the gene encoding NDP-D-glucose-4, 6-dehydratase.Based on comparison of gene clusters structure for L-rhodinose biosynthesis in five antibiotics biosynthesis gene clusters which were sequenced by now, all genes encoding NDP-hexose synthase were found locating upstream of the genes encoding NDP-glucose-4, 6 dehydratase, and furthermore, they were next to each other in four antibiotics gene clusters. Accordingly, by using two primers from consensus regions of genes encoding NDP-hexose synthase and NDP-D-glucose-4, 6-dehydratase. Respectively, one 1234 bp section Lid2 has been obtained and sequenced. Database search revealed that the upstream 500 bp section showed high similarity to gene encoding NDP-hexose synthase and the downstream 724 bp section showed same sequences with previous results. The two genes are orderly designated lidY and lidH. They are confirmed to encoding NDP-D-glucose-4, 6 dehydratase and NDP-hexose synthase by bioinfermation analysis. This result showed that the gene encoding NDP-hexose synthase was located upstream the gene encoding NDP-glucose-4, 6 dehydratase and these two genes are next to each other in biosynthetic gene clusters of L-rhodinose for streptolydigin biosynthesis.This study was first time of clonging genes of L-rhodinose in streptolydigin from streptomyces lydicus with PCR amplification. It is make a good basis for unveiling the biosynthetic gene cluster of streptolydigin from streptomyces lydicus, and further study on molecular engineering of streptomyces lydicus and combinatorial biosynthesis for hybrid antibiotics.