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.

Identification of the Biosynthetic Gene Cluster of New Piperazic Acid-Containing Lipopeptides with Cytotoxic Activity in the Genome of Marine Streptomyces PHM034.

Three novel lipopeptides, PM130391 (1), PM130392 (2), and PM140293 (3) were obtained from cultures of Streptomyces tuirus PHM034 isolated from a marine sediment. Structural elucidation of the three compounds showed they belong to the nonribosomal peptides family, and they all contain an acylated alanine, three piperazic acids, a methylated glycine, and an N-hydroxylated alanine. The difference between the three compounds resides in the acyl chain bound to the alanine residue. All three compounds showed cytotoxic activity against human cancer cell lines. Genome sequence and bioinformatics analysis allowed the identification of the gene cluster responsible for the biosynthesis. Inactivation of a nonribosomal peptide synthase of this cluster abolished the biosynthesis of the three compounds, thus demonstrating the involvement of this cluster in the biosynthesis of these lipopeptides.

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.

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.

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.

Bacterial Production of a Pederin Analogue by a Free-Living Marine Alphaproteobacterium.

The polyketide pederin family are cytotoxic compounds isolated from insects, lichen, and marine sponges. During the past decade, different uncultivable bacteria symbionts have been proposed as the real producers of these compounds, such as those found in insects, lichen, and marine sponges, and their trans-AT polyketide synthase gene clusters have been identified. Herein we report the isolation and biological activities of a new analogue of the pederin family, compound 1, from the culture of a marine heterotrophic alphaproteobacterium, Labrenzia sp. PHM005. This is the first report of the production of a pederin-type compound by a free-living marine bacteria that could be cultured in the laboratory.

Streptenols F-I Isolated from the Marine-Derived Streptomyces misionensis BAT-10-03-023.

A marine-derived bacterium, Streptomyces misionensis BAT-10-03-123, has produced four new streptenol derivatives, F, G, H, and I (1-4), as well as the known streptenols A and C (5 and 6). Their planar structures were elucidated by detailed analysis of spectroscopic data. The absolute configurations of the new streptenol compounds were determined by chemical and spectroscopic methods, including Mosher's ester method. All of the compounds were tested for cytotoxicity against four selected cancer cell lines.

PM100117 and PM100118, new antitumor macrolides produced by a marine Streptomyces caniferus GUA-06-05-006A.

Two new bioactive polyhydroxyl macrolide lactones PM100117 (1) and PM100118 (2) were isolated from the culture broth of the marine-derived Streptomyces caniferus GUA-06-05-006A. Their structures were elucidated by a combination of spectroscopic methods, mainly one-dimensional and 2D NMR and HRESI-MS. They consist of 36-membered macrolides with a side chain containing three deoxy sugars and a 1,4-naphthoquinone chromophore. Compounds 1 and 2 displayed potent cytotoxicity against three human tumor cell lines with GI50 values in the micromolar range, as well as slight antifungal activity against Candida albicans ATCC10231. In addition, both compounds alter the plasma membrane of tumor cells, inducing loss of membrane integrity and subsequent cell permeabilization leading to a fast and dramatic necrotic cell death.

Antitumor actinopyranones produced by Streptomyces albus POR-04-15-053 isolated from a marine sediment.

Four new antitumor pyranones, PM050511 (1), PM050463 (2), PM060054 (3), and PM060431 (4), were isolated from the cell extract of the marine-derived Streptomyces albus POR-04-15-053. Their structures were elucidated by a combination of spectroscopic methods, mainly 1D and 2D NMR and HRESIMS. They consist of an α-methoxy-γ-pyrone ring containing a highly substituted tetraene side chain glycosylated at C-10 in the case of 1 and 4. Compounds 1 and 4 displayed strong cytotoxicity against three human tumor cell lines with GI50 values in the submicromolar range, whereas 2 showed subnanomolar activity as an inhibitor of EGFR-MAPK-AP1-mediated mitogenic signaling, causing inhibition of EGF-mediated AP1 trans-activation and EGF-mediated ERK activation and slight inhibition of EGF-mediated JNK activation. Taken together, these results suggest that members of the pyranone family of compounds could be developed as potential antitumor agents.

High-titer production of astaxanthin by the semi-industrial fermentation of Xanthophyllomyces dendrorhous.

An improved semi-industrial process for astaxanthin production by fermentation of Xanthophyllomyces dendrorhous has been developed. The culture medium was designed at the flask scale, reaching an astaxanthin cellular content of 3.0 mgg(-1) cell weight and a volumetric yield of 119 mgL(-1) broth. Astaxanthin production in flask was significantly improved by white light (4.0 mgg(-1) and 221 mgL(-1)), and by ultraviolet light (4.4 mgg(-1) and 235 mgL(-1)). The scale-up to 10- and 800-L fermentors was developed by feeding with glucose. Representative data for illuminated fermentation processes are presented and discussed at the 10-L scale, where 420 mgL(-1) (4.7 mgg(-1)) astaxanthin were produced, and the 800-L scale, with productivities of 350 mgL(-1) (4.1 mgg(-1)) astaxanthin. The purity of the astaxanthin in the broth was about 84%, with accumulation of the following carotenoids other than astaxanthin: 4% beta-carotene, 4% canthaxanthin, 5% HDCO, 1% zeaxanthin and 2% phoenicoxanthin. This technology can be easily scaled-up to an industrial application for the production of this xanthophyll widely demanded nowadays.

Tartrolon D, a cytotoxic macrodiolide from the marine-derived actinomycete Streptomyces sp. MDG-04-17-069.

Exploration of marine-derived actinomycetes as a source of antitumor compounds has led to the isolation of a new member of the tartrolon series, tartrolon D (4). This new compound was obtained from Streptomyces sp. MDG-04-17-069 fermentation broths and displayed strong cytotoxic activity against three human tumor cell lines. Additionally, the known compound ikarugamycin (5) was also found in the culture broths of the same microorganism. The structure of this new tartrolon was established by a combination of spectroscopic techniques (1D and 2D NMR, HRMS, and UV) as well as by comparison with published data for similar compounds.

Molecular characterization of the safracin biosynthetic pathway from Pseudomonas fluorescens A2-2: designing new cytotoxic compounds.

Safracin is an antibiotic with anti-tumour activity produced by Pseudomonas fluorescens A2-2. The entire safracin synthetic gene cluster spanning 17.5 kb has been identified, cloned and sequenced. The safracin cluster comprises 10 open reading frames (ORFs) encoding proteins for three non-ribosomal peptide synthetases (NRPS), three safracin precursor biosynthetic enzymes, two safracin tailoring enzymes, a safracin resistance protein and a small hypothetical protein of unknown function. These genes are organized in two divergent operons of eight and two genes respectively. This pathway exhibits unusual features when compared with other NRPS systems. We have demonstrated by heterologous expression of the cluster that it is able to direct the synthesis of safracin in other strains. Cross-feeding experiments have confirmed that 3-hydroxy-5-methyl-O-methyltyrosine is the precursor of two amino acids of the molecule. Genetic analyses have allowed us to demonstrate that the bicistronic operon encodes the hydroxylation and N-methylation activities of the pathway. The cloning and expression of the safracin cluster has settled the basis for the in vivo and in vitro production of a wide variety of compounds, such as the promising ecteinascidins anti-cancer compounds.

Catabolism of D-glucose by Pseudomonas putida U occurs via extracellular transformation into D-gluconic acid and induction of a specific gluconate transport system.

Pseudomonas putida U does not degrade D-glucose through the glycolytic pathway but requires (i) its oxidation to D-gluconic acid by a peripherally located constitutive glucose dehydrogenase (insensitive to osmotic shock), (ii) accumulation of D-gluconic acid in the extracellular medium, and (iii) the induction of a specific energy-dependent transport system responsible for the uptake of D-gluconic acid. This uptake system showed maximal rates of transport at 30 degrees C in 50 mM potassium phosphate buffer, pH 7.0. Under these conditions the K(m) calculated for D-gluconic acid was 6.7 microM. Furthermore, a different transport system, specific for the uptake of glucose, was also identified. It is active and shows maximal uptake rates at 35 degrees C in 50 mM potassium phosphate buffer, pH 6.0, with a K(m) value of 8.3 microM.