The oligosaccharide antibiotics avilamycin A and C are produced by Streptomyces viridochromogenes Tu57. Both consist of a heptasaccharide chain, which is attached to a polyketide-derived dichloroisoeverninic acid moiety. They show excellent antibiotic activity against Gram-positive bacteria. Both molecules are modified by O-methylation at different positions, which contributes to poor water solubility and difficulties in galenical drug development. In order to generate novel avilamycin derivatives with improved polarity and improved pharmacokinetic properties, we generated a series of mutants with one, two, or three mutated methyltransferase genes. Based on the structure of the novel avilamycin derivatives, the exact function of three methyltransferases, AviG2, AviG5, and AviG6, involved in avilamycin biosynthesis could be assigned.
The oligosaccharide antibiotic avilamycin A is composed of a polyketide-derived dichloroisoeverninic acid moiety attached to a heptasaccharide chain consisting of six hexoses and one unusual pentose moiety. We describe the generation of mutant strains of the avilamycin producer defective in different sugar biosynthetic genes. Inactivation of two genes (aviD and aviE2) resulted in the breakdown of the avilamycin biosynthesis. In contrast, avilamycin production was not influenced in an aviP mutant. Inactivation of aviGT4 resulted in a mutant that accumulated a novel avilamycin derivative lacking the terminal eurekanate residue. Finally, AviE2 was expressed in Escherichia coli and the gene product was characterized biochemically. AviE2 was shown to convert UDP-D-glucuronic acid to UDP-D-xylose, indicating that the pentose residue of avilamycin A is derived from D-glucose and not from D-ribose. Here we report a UDP-D-glucuronic acid decarboxylase in actinomycetes.
Streptomycetes are typical soil-dwelling bacteria with intricate morphological and biochemical differentiation of colonies, resulting in onset of secondary metabolite production. Compounds produced during these stages of differentiation comprise nearly two-thirds of bioactive molecules synthesized by microorganisms, including antibiotics, antitumor agents and immunosuppressants. 1,2 The biosynthesis of antibiotics and other secondary metabolites is controlled by interactions of both global and pathway-specific regulators. However, in all cases, the influence of the environment is reflected by the activity of pathway-specific regulatory genes, which are located within a biosynthetic gene cluster and which control the expression of biosynthetic genes. 3 On account of high structural and functional similarity, such regulators were grouped into two families, namely Streptomyces antibiotics regulatory proteins (SARP), mostly found within the aromatic polyketides clusters, and large ATP-binding regulators of LuxR family (LAL), controlling the production of macrolides and glycopeptides. [4][5][6] Recent efforts in cloning and characterization of biosynthetic gene clusters for new groups of secondary metabolites revealed novel classes of transcriptional factors that differ from typical SARPs or LAL representatives. 7,8 Avilamycins are secondary metabolites produced by Streptomyces viridochromogenes Tu57. They are active against Gram-positive bacteria. It is known that avilamycins bind to the 23S rRNA in the region proximal to the channel, where tRNA enters the A-site and blocks the protein synthesis. 9 Avilamycin resistance is mediated by 23S rRNA methylation and active avilamycin transport. 10 The avilamycin resistance genes together with the structural genes were identified and cloned as an entire biosynthetic gene cluster containing 54 open reading frames. 11 The biosynthetic steps leading to the formation of the avilamycin molecule were established by the analysis of secondary metabolites produced by mutants. 11,12 Within the cluster also, two putative regulatory genes named aviC1 and aviC2 were identified. Both genes are located in close proximity to each other and are transcribed in the same direction (Figure 1). Genes are separated by a 271-bp non-coding region. We succeeded in identifying the presence of putative bacterial promoter sequences upstream of aviC1 initiation codon by the use of BPROM bacterial promoter prediction server (Softberry Inc., Mount Kisco, NY, USA). It consists of a putative À10 box GGTTTTCAT (Score 34) and a À35 box ATGCGA (Score 12). Another putative promoter sequence is located upstream of the aviC2 translation start site consisting of a À10 box GCCCATGAT (Score 31) and a À35 box at TTTCTA (Score 34) similar to consensus Streptomycetes promoters. 13
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