Characterization of new beta-amino acid-containing secondary métabolites in bacteria – BetaAAmetabolites

The methods and approaches used are based on those already successfully employed by the project partners in a previous project funded by the ANR and the DFG for the isolation and structural characterization and biosynthesis pathway of albicidin, an antibiotic and phytotoxin produced by the bacterium responsible for leaf scald disease in sugarcane.

The biosynthesis gene clusters of the two secondary metabolites studied in the current project are known. Techniques for overexpressing these genes, leading to the overproduction of the secondary metabolites of interest, are being implemented by the French partner. The isolation of the target molecules by chromatography is being carried out jointly by the French and German partners. The structural characterization of the secondary metabolites is carried out by the German partner using various mass spectrometry and nuclear magnetic resonance techniques. Subsequently, the respective biosynthesis pathways of these secondary metabolites are refined in light of the structures of the molecules. Finally, new biosynthesis gene clusters enabling the incorporation of ß-amino acids into secondary metabolites will be sought through genome mining among the bacterial genomes available on GenBank.

Two bacterial secondary metabolites were the focus of this project.

The first bacterial secondary metabolite is a lipopeptide produced by a phytopathogenic and opportunistic pathogen of animals and humans. This lipopeptide has been shown to be involved in the virulence of the bacterium in patients suffering from cystic fibrosis, but also to have antifungal properties against phytopathogenic fungi. Although an isolation protocol for this lipopeptide was known at the start of the project, the various project partners elucidated its structure, confirming the main hypothesis of the project regarding the presence of beta-amino acids in the structures of the metabolites studied, as well as its biosynthesis pathway involving enzymes involved in the non-ribosomal peptide biosynthesis pathway. The antifungal activity of this lipopeptide was also confirmed against four phytopathogenic fungi responsible for diseases in coffee, wheat, and rice.

The second bacterial secondary metabolite studied in this project is the signaling molecule involved in an original quorum sensing system implicated in the virulence of strains belonging to different species of a phytopathogenic bacterial genus. Isolating this signaling molecule has been difficult due to the very low concentrations produced naturally and the molecule's high instability. Numerous analyses of the genomes of this bacterial genus, and more specifically of the genes involved in the biosynthesis of this signaling molecule, have enabled us to identify the existence of several specificity groups within the bacterial genus studied. These specificity groups thus implement structural analogues of the quorum sensing signaling molecule. Thus, while quorum sensing is considered a specific language within certain bacterial species, the specificity groups identified in this project correspond to dialects understood by only certain strains within the bacterial genus. Experiments were also conducted to determine the stability of the various structural analogues of the quorum sensing signal molecule. Only part of the structure of these structural analogues could be determined.

Experiments have also been conducted to try to identify a biosynthesis pathway partially shared between different secondary metabolites whose biosynthesis gene clusters are close to each other on bacterial genomes, but without success.

This project aims to elucidate the chemical structure of bacterial secondary metabolites encoded by two groups of biosynthesis genes, decipher their biosynthesis pathways, and refine the function of their biosynthesis genes. This project contributes to a better understanding of the incorporation of ß amino acids by autonomous A domains in bacteria.

Since the targeted microorganisms are important pathogenic bacteria, this project will contribute significantly to a better understanding of their pathogenesis. Consequently, the fight against these pathogenic bacteria should be facilitated by the new data generated by the project. Characterizing the structure of a lipopeptide involved in the virulence of a human opportunistic pathogen will enable the development of methods to combat these bacteria.

Since genome exploration is an important approach for discovering new loci encoding natural products with potentially novel biological activities, the exploration of bacterial genomic sequences available in Genbank for the presence of new loci encoding an autonomous A domain specific to a ß amino acid will prove useful in identifying other bacterial secondary metabolites of interest.

No scientific production so far.

In recent years, the number of identified non-proteinogenic amino acid-containing secondary metabolites and their biosynthetic gene clusters has greatly expanded in bacteria. A new family of stand-alone adenylation (A) domains involved in the incorporation of ß-amino acids has been previously described. Based on the protein structural analyses of three members of this new family, new ß-amino acid specificity-conferring codes have been proposed. A common specific feature of these stand-alone A domains is that they are co-encoded with a stand-alone acyl carrier protein domain.
Based on these specific features, the French partner (BGPI) has identified two new stand-alone A domains expected to be involved in the incorporation of a ß-amino acid. These stand-alone A domains are present in two loci belonging to important pathogenic bacteria. According to their annotation, these loci encode two different new unknown molecules, respectively. Interestingly, by a genetic approach, these loci have both been shown to be required for the bacterial virulence. However, the chemical structure of the secondary metabolites synthesized by these loci remains unknown and the presence of a ß-amino acid has never been yet suspected and explored.
This project aims at elucidating the chemical structure of the molecules encoded by these two loci, deciphering their biosynthesis pathways, and refining the functional assignment of their biosynthesis genes. This project will contribute to a better understanding of the incorporation of ß-amino acids by stand-alone A domains in bacteria. Since the targeted microorganisms are important pathogens, this project will essentially contribute to a deeper understanding of their pathogenicity. Consequently, the fight against these pathogens should therefore be facilitated by new data arising from the project.
Since genome mining is an important approach to discover new loci encoding new natural products with potentially novel biological activities, this project proposes to mine bacterial genomic sequences available in Genbank for the presence of new loci encoding a stand-alone A domain specific of a ß amino acid.
Depending on the biological activity of the characterized ß-amino acid-containing secondary metabolites, this project potentially could lead to industrial applications as antibiotics or plant protection agents. Patents obtained would potentially also contribute to the visibility of the project. The French and German partners BGPI and TU Berlin have developed a long-standing collaboration since 2005, working together on several projects including the structural characterization of the potent antibiotic albicidin.