Identification of trans-AT polyketide clusters in two marine bacteria reveals cryptic similarities between distinct symbiosis factors.

Glutarimide-containing polyketides are known as potent antitumoral and antimetastatic agents. The associated gene clusters have only been identified in a few Streptomyces producers and Burkholderia gladioli symbiont. The new glutarimide-family polyketides, denominated sesbanimides D, E and F along with the previously known sesbanimide A and C, were isolated from two marine alphaproteobacteria Stappia indica PHM037 and Labrenzia aggregata PHM038. Structures of the isolated compounds were elucidated based on 1D and 2D homo and heteronuclear NMR analyses and ESI-MS spectrometry. All compounds exhibited strong antitumor activity in lung, breast and colorectal cancer cell lines. Subsequent whole genome sequencing and genome mining revealed the presence of the trans-AT PKS gene cluster responsible for the sesbanimide biosynthesis, described as sbn cluster. Strikingly, the modular architecture of downstream mixed type PKS/NRPS, SbnQ, revealed high similarity to PedH in pederin and Lab13 in labrenzin gene clusters, although those clusters are responsible for the production of structurally completely different molecules. The unexpected presence of SbnQ homologues in unrelated polyketide gene clusters across phylogenetically distant bacteria, raises intriguing questions about the evolutionary relationship between glutarimide-like and pederin-like pathways, as well as the functionality of their synthetic products.

Tailoring modifications in labrenzin synthesis: a-la-carte production of pathway intermediates.

Pederin-family polyketides today constitute a group of more than 30 molecules being produced as natural products by different microorganisms across multitude of ecological niches. They are mostly known for their extreme cytotoxic activity and the decades of long exploration as potential antitumor drugs. The difference in their potency and biological activity lies in the tailoring modifications of the core molecule. Despite the isolation of many pederin-like molecules until the date, only marine bacterium Labrenzia sp. PHM005 was reported as a cultivable producer and able to be genetically modified. Here, we study the role of tailoring enzymes from the lab gene cluster responsible for methylation and hydroxylation of labrenzin core molecule. We managed to produce a spectrum of differently tailored labrenzin analogs for the development of future drugs. This work constitutes one-step forward in understanding the biosynthesis of pederin-family polyketides and provides the tools to modify and overproduce these anticancer drugs in a-la-carte manner in Labrenzia sp. PHM005, but also in other producers in the future.

In vivo production of pederin by labrenzin pathway expansion.

Pederin is a potent polyketide toxin that causes severe skin lesions in humans after contact with insects of genus Paederus. Due to its potent anticancer activities, pederin family compounds have raised the interest of pharmaceutical industry. Despite the extensive studies on the cluster of biosynthetic genes responsible for the production of pederin, it has not yet been possible to isolate and cultivate its bacterial endosymbiont producer. However, the marine bacterium Labrenzia sp. PHM005 was recently reported to produce labrenzin, the closest pederin analog. By cloning a synthetic pedO gene encoding one of the three O-methyltraferase of the pederin cluster into Labrenzia sp. PHM005 we have been able to produce pederin for the first time by fermentation in the new recombinant strain.

Genome of Labrenzia sp. PHM005 Reveals a Complete and Active Trans-AT PKS Gene Cluster for the Biosynthesis of Labrenzin.

The complete genome of the strain Labrenzia sp. PHM005, a free-living producer of a pederin analog 18-O-demethyl pederin, hereinafter labrenzin, has been sequenced. This strain contains two replicons comprising a circular chromosome of 6,167,349 bp and a circular plasmid (named p1BIR) of 19,450 bp. A putative gene cluster responsible for the synthesis of labrenzin (lab cluster) has been identified showing that it encodes a trans-AT mixed type PKS/NRPS biosynthetic pathway that is responsible for the synthesis of pederin and possibly an onnamide analog. The putative boundaries of the lab gene cluster were determined by genetic comparisons with other related strains, suggesting that the cluster consists of a 79-kb region comprising 3 genes encoding multidomain hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) proteins (PKS4, PKS/NRPS13, and PKS/NRPS15), and 16 auxiliary enzymes. Transcriptomic analyses suggest that all the genes of the cluster are expressed in our culture conditions (i.e., in minimal medium in the absence of any specific inducer) at detectable levels. We have developed genetic tools to facilitate the manipulation of this strain and the functional characterization of the cluster genes. We have created a site-directed mutant unable to produce pederin, demonstrating experimentally for the first time the role of the cluster in the synthesis of pederin. This work paves the way to unravel the clues of the biosynthesis of pederin family compounds and opens the door to modify and overproduce these anticancer drugs for industrial and pharmaceutical purposes.