Biosynthesis of the chinoline alkaloid aurachin in Stigmatella aurantiaca: The first characterized type II polyketide synthase from gram negative bacteria
ROLF
MÜLLER
Pharmaceutical Biotechnology
Saarland University
66041 Saarbrücken
rom@mx.uni-saarland.de
www.myxo.uni-saarland.de
In this project, we aimed at the isolation and characterisation of the aurachin biosynthetic genes in the myxobacterium Stigmatella aurantiaca Sg a15. Aurachines represent bacterial chinoline alkaloids revealing some striking biosynthetic and structural similarity to plant acridones. Because of the similarity to acridones we expected a type III polyketide synthase (PKS) system to be involved in aurachin biosynthesis. To identify the biosynthetic genes we used a mariner based transposon mutagenesis system and generated 4200 mutants of S. aurantiaca. Culture extracts of all mutants were analyzed by HPLC-MS in comparison to the S. aurantiaca Sg a15 wild type to identify a number of mutants affected in aurachin production. Surprisingly, a type II PKS gene cluster encodes the aurachin biosynthetic proteins. This is the first description of a type II PKS system from a myxobacterium and to our knowledge also the first characterized type II PKS from a gram negative bacterium. All known natural products derived from PKS II systems have been isolated from actinobacteria like those involved in actinorhodin (Streptomyces coelicolor), tetracenomycin (Streptomyces glaucescens) and oxytetracycline production (Streptomyces rimosus). By phylogenetic comparison of the DNA sequences of different type II PKS systems from actinobacteria with that of aurachin biosynthesis (and plant type III PKS) we could show that the aurachin PKS represents a new distinct class of type II PKS.The exceptional aurachin type II PKS is required for the biosynthesis of the methyl-chinoline core starting from an anthraniloyl-CoA starter moiety and two malonyl-CoA extender units. The previously unknown methyl-chinoline intermediate was synthesized, identified from the wild type and used to complement mutants of some biosynthetic genes. A CoA ligase required for activation of anthranilate was also identified and the loss of aurachin production in the respective mutant could be complemented by the addition of anthraniloyl-SNAC to the growth medium. The intermediate methyl-chinoline is subsequently prenylated by AuaA during aurachin biosynthesis to form aurachin D and then further modified by enzymes not encoded in the core biosynthetic gene cluster. Two of the latter genes were also identified in the mutant library as cytochrome P450 dependent monooxigenases inactivation of which leads to the loss of aurachin A production.
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