Biology subjects

Hotel, L.

Publications and source records attributed to Hotel, L..

4 recordsLinked to original sources

bioRxiv2024

Role of a FAD-dependent monooxygenase in diazo group functionalisation of kinamycin in Streptomyces ambofaciens

Kinamycin biosynthesis is a complex process which has been extensively studied over the years, yet specific enzymatic steps continue to be unveiled. A diazo group present in the molecule is responsible for the promising antitumoral activity of kinamycins but its specific installation in Streptomyces ambofaciens is not fully understood. In this study, we explore the diazo functionalization of kinamycin in this strain. A new FAD-dependent monooxygenase is identified which is essential for kinamcyin biosynthesis. In its absence, stealthin C accumulates instead likely as a pathway shunt-product. Furthermore, as a result of the position of the gene encoding this monooxygenase, named alp2F, we also propose new boundaries of the kinamycin biosynthetic gene cluster resulting in a large cluster spanning over 72kb. This work paves the way for the continued understanding of the biosynthetic steps that are characteristic of diazo-containing natural products, and provides new biocatalysts for molecular engineering and accelerate bioactive compounds production.

microbiology↗

bioRxiv2024

Exploiting the inherent promiscuity of the acyl transferase of the stambomycin polyketide synthase for the mutasynthesis of analogues

The polyketide specialized metabolites of bacteria are attractive targets for generating analogues, with the goal of improving their pharmaceutical properties. Here, we aimed to produce C-26 derivatives of the giant anti-cancer stambomycin macrolides using a mutasynthesis approach, as this position has been shown previously to directly impact bioactivity. For this, we leveraged the intrinsically broad specificity of the acyl transferase domain (AT12) of the modular polyketide synthase (PKS), which is responsible for the alkyl branching functionality at this position. Feeding of a panel of synthetic and commercially available dicarboxylic acid mutasynthons to an engineered strain of Streptomyces ambofaciens (Sa) deficient in synthesis of the native -carboxyacyl-CoA extender units, resulted in six new series of stambomycin derivatives as judged by LC-HRMS and NMR. Notably, the highest product yields were observed for substrates which were only poorly accepted when AT12 was transplanted into a different PKS module, suggesting a critical role for domain context in the overall functioning of PKS proteins. We also demonstrate the superiority of this mutasynthesis approach - both in terms of absolute titers and yields relative to the parental compounds - in comparison to the alternative precursor-directed strategy in which monoacid building blocks are supplied to the wild type strain. We further identify a malonyl-CoA synthetase, MatB_Sa, with specificity distinct from previously identified promiscuous enzymes, making it a useful addition to a mutasynthesis toolbox for generating atypical, CoA activated extender units. Finally, we show that two of the obtained (deoxy)-butyl-stambomycins exhibit antibacterial and antiproliferative activities similar to the parental stambomycins, while an unexpected butyl-demethyl congener is less potent. Overall, this works confirms the interest of biosynthetic pathways which combine a dedicated route to extender unit synthesis and a broad specificity AT domain for producing bioactive derivatives of fully-elaborated complex polyketides.

synthetic biology↗

bioRxiv2024

SYNTERUPTOR: mining genomic islands for non-classical specialised metabolite gene clusters

Microbial specialised metabolite biosynthetic gene clusters (SMBGCs) are a formidable source of natural products of pharmaceutical interest. With the multiplication of genomic data available, very efficient bioinformatic tools for automatic SMBGC detection have been developed. Nevertheless, most of these tools identify SMBGCs based on sequence similarity with enzymes typically involved in specialised metabolism and thus may miss SMBGCs coding for under characterised enzymes. Here we present SYNTERUPTOR (https://bioi2.i2bc.paris-saclay.fr/synteruptor), a program that identifies genomic islands, known to be enriched in SMBGCs, in the genomes of closely related species. With this tool, we identified a SMBGC in the genome of Streptomyces ambofaciens ATCC23877, undetected by earlier versions of antiSMASH, and experimentally demonstrated that it directs the biosynthesis of two metabolites, one of which was identified as sphydrofuran. SYNTERUPTOR is also a valuable resource for the delineation of individual SMBGCs within antiSMASH regions that may encompass multiple clusters, and for refining the boundaries of these SMBGCs.

bioinformatics↗

bioRxiv2023

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways

Iron is essential to many biological processes, but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include unprecedented tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate NRPS-independent siderophore (NIS) pathways in the producing organism. The new derivatives, whose biological role(s) remain to be elucidated, not only illustrate the unanticipated biosynthetic potential of S. ambofaciens, but have interest in immuno-PET imaging applications. ImportanceIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload, as well as in advanced imaging applications. In this study we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens - the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance 89Zr based immuno-PET imaging) and other applications of metal chelators.

microbiology↗