Aurantinins (ARTs) are antibacterial polyketides featuring a unique 6/7/8/5-fused tetracyclic ring system and a triene side chain with a carboxyl terminus. Here we identify the art gene cluster and dissect ART’s C-methyl incorporation patterns to study its biosynthesis. During this process, an apparently redundant methyltransferase Art28 was characterized as a malonyl-acyl carrier protein O-methyltransferase, which represents an unusual on-line methyl esterification initiation strategy for polyketide biosynthesis. The methyl ester bond introduced by Art28 is kept until the last step of ART biosynthesis, in which it is hydrolyzed by Art9 to convert inactive ART 9B to active ART B. The cryptic reactions catalyzed by Art28 and Art9 represent a protecting group biosynthetic logic to render the ART carboxyl terminus inert to unwanted side reactions and to protect producing organisms from toxic ART intermediates. Further analyses revealed a wide distribution of this initiation strategy for polyketide biosynthesis in various bacteria.
Along with the fast developing of DNA sequencing technology, a great number of natural product biosynthetic gene clusters have been discovered by bioinformatic analysis, which demands novel high-throughput genome mining methods to obtain the diverse compounds dictated by those gene clusters. In this work, a method based on the reporter gene xylE was established to screen for the activation conditions of thirteen different gene clusters from Streptomyces lavendulae CGMCC 4.1386. In this reporter-guided method, the key structure gene was replaced by a xylE-kanaR cassette with the xylE gene being controlled by the transcription and translation machinery of the key structure gene. It not only facilitated the screening of activation conditions, but also provided the null mutants of specific natural product gene clusters as controls to link those clusters with their products conveniently. The potential activation conditions of eleven gene clusters from S. lavendulae CGMCC 4.1386 were obtained. In addition, activation of three of the eleven gene clusters was confirmed and their products were identified.
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