Biosynthesis of the antibiotic actinorhodin. Analysis of blocked mutants of Streptomyces coelicolor.

Abstract

Fromtwo types of class V act mutants of Streptomyces coelicolor two monomericprecursors of actinorhodin have been isolated and their structures determined. One is the known antibiotic kalafungin (2) and the other a new compound(6). Their relationship to actinorhodin biosynthesis is discussed.Streptomyces coelicolor A3(2) is the genetically most characterized actinomycete, with a well developed circular chromosomelinkage map1}. A fragment of this chromosomebetween the markers gua A and his D contains a cluster of structural genes for the biosynthesis of the isochromanequinone antibiotic, actinorhodin (1) (Scheme 1)2). Rudd and Hopwoodisolated 75 mutants which are blocked in the biosynthesis of this antibiotic, and were able to group them into seven classes. The classification was based on the mutants' ability to secrete intermediates which other mutants, blocked earlier in the biosynthetic pathway, could convert into actinorhodin. Production of this antibiotic could easily be recognized by fuming the agar plates over ammonia, since actinorhodin is a red-blue acidbase indicating compound. If a donor strain secreted a precursor which a converter strain could use to produce actinorhodin, then a blue zone would appear on the edge of the converting strain colonies closest to the donor. The classes could thus be ordered as to their place in the actinorhodin biosynthetic pathway.Class V mutants secreted (a) diffusible intermediate(s) into the medium which all other classes (except a putative regulatory class, II) could convert into actinorhodin. Membersof this class could not convert compoundssecreted by any other mutants into actinorhodin, and therefore must be blocked at the latest stage in the biosynthesis.The steps of isochromanequinone antibiotic biosynthesis following polyketide chain formation have recently been of interest since the act genes from class V have been cloned and inserted into another species, Streptomyces sp. AM-71613). This latter species normally produces the yellow-brown acid-base indicating antibiotic, medermycin but after transfer of the class V act genes, a new, hybrid orange-purple acid-base indicating pigment named mederrhodin A was produced. Another new hybrid structure, dihydrogranatirhodin was produced by a transformant of the dihydrogranticin-producing S. coelicolor A3(2). This compoundhas the granaticin stereochemistry at one chiral center and the actinorhodin stereochemistry at the other. With this ability to clone antibiotic genes and transfer them between species, the design and syn-

An Aromatic Hydroxylation Reaction Catalyzed by a Two-component FMN-dependent Monooxygenase

“…The enzyme ActVB was originally proposed to be involved in the dimerization reaction by analysis of the products produced by strains containing lesions in the biosynthetic cluster (23). However, more recent work has characterized this enzyme and close homologs as members of the FMN:NADH oxidoreductase family (2,6,20) that act to supply reduced flavins to a partner enzyme potentially catalyzing a hydroxylation.…”

“…Here we report the results of our enzyme assays of the ActVB-ActVA system using the presumed natural substrate dihydrokalafungin (DHK) 3 (23). Unexpectedly, we demonstrate that the DHK substrate appears to be utilized in its hydroquinone form and the only product formed is hydroxylated DHK.…”

“…Examples are those involved in the synthesis of the antibiotic pristinamycin in Streptomyces pristinaespiralis (14,15), utilization of sulfur from aliphatic sulfonates in E. coli (18), or desulfurization of fossil fuels by Rhodococcus species (19). In our laboratory, we have been investigating the FMN-dependent two-component enzyme system, consisting of ActVB (6), the flavin reductase, and ActVA-Orf5 (termed ActVA herein) (20), the oxygenase, thought to participate in the last steps of the biosynthesis of the antibiotic actinorhodin in Streptomyces coelicolor (23)(24)(25) (Scheme 1). Our results have provided new insights into the mechanism of the reaction, especially regarding flavin reduction as well as flavin transfer from the oxidoreductase to the oxygenase (20).…”

Cloning, sequencing and heterologous expression of the medermycin biosynthetic gene cluster of Streptomyces sp. AM-7161: towards comparative analysis of the benzoisochromanequinone gene clusters

“…A homologue, med-ORF12, was indeed found. The product ('chiral alcohol') of the C-3 reduction would cyclize to the hemiketal, followed by dehydration to afford (S)-DNPA (Cole et al, 1987) (Fig. 3).…”

“…Sequence analysis of ORFs in these clusters revealed rather diverse genetic organizations, especially for genes encoding tailoring enzymes (Ichinose et al, 1998b). Some common 'unknown' genes are also present, whose deduced functions are unavailable from database searches, but are apparently important for BIQ biosynthesis (Cole et al, 1987;Taguchi et al, 2000a, b). The present paper describes the complete DNA sequence of the medermycin biosynthetic gene cluster of Streptomyces sp.…”