Biology subjects

Santos-Aberturas, J.

Publications and source records attributed to Santos-Aberturas, J..

5 recordsLinked to original sources

bioRxiv2026

Efficient in vitro refactoring and biosynthetic gene cluster amplification for the overproduction and accelerated discovery of anticancer thioamitides

Thioamitides, a class of highly modified bacterial ribosomally synthesised and post-translationally modified peptides (RiPPs), have potent activities against multiple cancer cell lines. Among these compounds, the structurally divergent thioalbamide combines promising in vivo antiproliferative activity with a superior chemical stability respect to its counterparts. However, thioalbamide is produced in low yields by its genetically intractable native producer and its biosynthetic pathway was initially not productive when transferred into the heterologous host Streptomyces coelicolor M1146. These circumstances substantially hamper to increase the production of this promising compound. Here, we show how in vitro Gibson-like assemblies can be employed for the quick and efficient refactoring of the thioalbamide biosynthetic gene cluster (BGC), leading to substantially increased levels of production in S. coelicolor M1146 through a prioritised selection of promoters. Via this work, PtsrA and PgroEL2 were identified as beneficial additions to the Streptomyces synthetic biology toolbox. We then assessed bacterial genomes for biosynthetic gene clusters (BGCs) predicted to produce thioalbamide-like compounds with improved hydrophilicity. This rational discovery campaign led to the identification a silent thioamitide BGC encoding a thioalbamide-like core peptide but clustered with additional tailoring enzymes, including a previously unknown cupin-fold protein. Applying the refactoring strategy together with the simultaneous expression of multiple BGC copies, we characterised the product of this pathway, thiocupinamide, a polyhydroxylated thioamitide closely related to thioalbamide. We show that thiocupinamide has potent anticancer and antibacterial activities.

synthetic biology↗

bioRxiv2024

Discovery of lydiamycin A biosynthetic gene cluster in the plant pathogen Rhodococcus fascians guides structural revision and identification of molecular target

The natural products actinonin and matlystatin feature an N-hydroxy-2-pentyl-succinamyl (HPS) chemophore that facilitates metal chelation and confers their metalloproteinase inhibitory activity. Actinonin is the most potent natural inhibitor of peptide deformylase (PDF) and exerts antimicrobial and herbicidal bioactivity by disrupting protein synthesis. Here, we used a genomics-led approach to identify candidate biosynthetic gene clusters (BGCs) hypothesised to produce novel HPS-containing natural products. We show that one of these BGCs is on the pathogenicity megaplasmid of the plant pathogen Rhodococcus fascians and produces lydiamycin A, a macrocyclic pentapeptide. The presence of genes predicted to make a HPS-like chemophore informed the structural recharacterisation of lydiamycin via NMR and crystallography to show it features a rare 2-pentyl-succinyl chemophore. We demonstrate that lydiamycin A inhibits bacterial PDF in vitro and show that a cluster-situated PDF gene confers resistance to lydiamycin A, representing a novel self-immunity mechanism associated with the production of a PDF inhibitor. In planta competition assays showed that lydiamycin enhances the fitness of R. fascians during plant colonisation. This study highlights how a BGC can inform the structure, biochemical target and ecological function of a natural product.

microbiology↗

bioRxiv2024

Understanding the biosynthesis, metabolic regulation, and anti-phytopathogen activity of 3,7-dihydroxytropolone in Pseudomonas spp.

The genus Pseudomonas is a prolific source of specialized metabolites with significant biological activities, including siderophores, antibiotics, and plant hormones. These molecules play pivotal roles in environmental interactions, influencing pathogenicity, inhibiting microorganisms, responding to nutrient limitation and abiotic challenges, and regulating plant growth. These properties mean that pseudomonads are candidates as biological control agents against plant pathogens. Multiple transposon-based screens have identified a Pseudomonas biosynthetic gene cluster (BGC) associated with potent antibacterial and antifungal activity that produces 7-hydroxytropolone (7-HT). In this study, we show that this BGC also makes 3,7-dihydroxytropolone (3,7-dHT), which has strong antimicrobial activity towards Streptomyces scabies, a potato pathogen. Both molecules exhibit broad biological activities, suggesting roles in competitive soil and plant microbial communities. Through metabolomics and reporter assays, we unveil the involvement of cluster-situated genes in generating phenylacetyl-coenzyme A, a key precursor for tropolone biosynthesis via the phenylacetic acid catabolon. The clustering of these phenylacetic acid genes within tropolone BGCs is unusual in other Gram-negative bacteria. Our findings support the interception of phenylacetic acid catabolism via an enoyl-CoA dehydratase encoded in the BGC, as well as highlighting an essential biosynthetic role for a conserved thioesterase. Biochemical assays were used to show that this thioesterase functions after a dehydrogenation-epoxidation step catalysed by a flavoprotein. We use this information to identify diverse uncharacterised BGCs that encode proteins with homology to flavoproteins and thioesterases involved in tropolone biosynthesis. This study provides insights into tropolone biosynthesis in Pseudomonas, laying the foundation for further investigations into the ecological role of tropolone production.

microbiology↗

bioRxiv2021

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation.

Stress memory is a phenomenon whereby exposure to initial stress event influences a response to subsequent stress exposures. Studying stress memory is important to understand the cellular behaviour in dynamic environment, especially nowadays, in times with growing environmental instability. Stress memory has been characterized in vascular plants but its occurrence in non-vascular plant species has been rarely investigated. We hypothesized that stress memory occurs in non-vascular plants in relation to metabolic stress. We sought to test it using accumulation of lipids (triacylglycerols) in model green alga Chlamydomonas reinhardtii subjected to nitrogen deprivation stress as a model system. Here, we established stress memory protocol on C. reinhardtii cells. Using a blend of microscopy and gas chromatography methods, we showed that the cells exposed to recurrent stress show differential accumulation of triacylglycerols on the quantitative level without qualitative changes in lipid composition, comparing to single stress controls. Overall, our results suggest that metabolic stress memory does occur in non-vascular plant C. reinhardtii and provides a starting point to characterize metabolic stress memory mechanism. Due to the commercial potential of algae, our findings are relevant for basic science, as well as industrial production of algae-derived compounds.

molecular biology↗

bioRxiv2018

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

The rational discovery of new specialized metabolites by genome mining represents a very promising strategy in the quest for new bioactive molecules. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of natural product that derive from genetically encoded precursor peptides. However, RiPP gene clusters are particularly refractory to reliable bioinformatic predictions due to the absence of a common biosynthetic feature across all pathways. Here, we describe RiPPER, a new tool for the family-independent identification of RiPP precursor peptides and apply this methodology to search for novel thioamidated RiPPs in Actinobacteria. Until now, thioamidation was believed to be a rare post-translational modification, which is catalyzed by a pair of proteins (YcaO and TfuA) in Archaea. In Actinobacteria, the thioviridamide-like molecules are a family of cytotoxic RiPPs that feature multiple thioamides, and it has been proposed that a YcaO-TfuA pair of proteins also catalyzes their formation. Potential biosynthetic gene clusters encoding YcaO and TfuA protein pairs are common in Actinobacteria but the chemical diversity generated by these pathways is almost completely unexplored. A RiPPER analysis reveals a highly diverse landscape of precursor peptides encoded in previously undescribed gene clusters that are predicted to make thioamidated RiPPs. To illustrate this strategy, we describe the first rational discovery of a new family of thioamidated natural products, the thiovarsolins from Streptomyces varsoviensis.

microbiology↗