Periodic Reporting for period 1 - RiPPs from the Gut (Functional Exploration of Biosynthetic Dark Matter in the Human Gut Microbiome)
Reporting period: 2021-09-01 to 2023-08-31
The number of microbial cells in the human body is projected to be in the trillions, amounting to a 10-fold excess of microbial to human cells. Most of the microbial cells in the body are located in the gut, which harbors a great diversity of bacterial species. Functional studies of gut bacteria are generally challenging, since most are as-yet unculturable, fastidious growers or genetically intractable. Nonetheless, metagenomic sequencing of gut samples has generated an abundance of data, and “sequence-gazing” insights have provided interesting clues into microbial metabolism.
The interactions within bacterial communities and between bacteria and host are often mediated by the molecular language of secondary metabolites, also termed natural products (NPs). Understanding the secondary metabolism of microbiome bacteria can elucidate their ecological roles within the microbiome and their contribution to human health. The molecular machinery required to synthesize secondary metabolites is encoded in bacterial genomes by autonomous units called biosynthetic gene clusters (BGCs). Remarkably, very little is known about the small molecule products of these BGCs or about why bacteria are producing them
The great complexity, diversity and density of the gut microbiome results in a rich environment for the discovery and functional study of molecules that mediate interactions between members of the community and the host. To address the lack of structural and functional knowledge of the secondary metabolism of gut bacteria, our project used genome mining and synthetic biology to study the function of novel NPs from bacteria from human gut microbiome.
In Work Package 1 (WP1), we explored the biosynthetic dark matter of human gut bacteria through genome mining approaches for the discovery of spliceotide and origamin RiPP natural products. And in Work Package 2 (WP2), we explored the function of spliceotide and origamin natural products and possible applications therapeutics.
We uncovered new biosynthetic gene clusters encoding for spliceotides from gut bacteria. While we were unable to obtain functional data for these systems, we were uncovered the biological activity of 4 other spliceotide products. All products showed protease inhibition activity and, remarkably, two spliceotides reproducibly demonstrated low-nanomolar activity against Human Neutrophil Elastase.
We also discovered origamin BGCs from gut microbes. We had previously demonstrated the activities for two enzymes in origamin biosynthesis in E. coli, but were not able to reproduce those results in origamins from gut bacteria. Nonetheless, we expanded our search of origamin clusters with the goal of isolating a final origamin product by obtaining activity of all key biosynthetic enzymes. We identified a new type of origamin BGC and were able to obtain activity for all three tailoring enzymes which produced one of the most extensively modified RiPPs known to date.
The interactions within bacterial communities and between bacteria and host are often mediated by the molecular language of secondary metabolites, also termed natural products (NPs). Understanding the secondary metabolism of microbiome bacteria can elucidate their ecological roles within the microbiome and their contribution to human health. The molecular machinery required to synthesize secondary metabolites is encoded in bacterial genomes by autonomous units called biosynthetic gene clusters (BGCs). Remarkably, very little is known about the small molecule products of these BGCs or about why bacteria are producing them
The great complexity, diversity and density of the gut microbiome results in a rich environment for the discovery and functional study of molecules that mediate interactions between members of the community and the host. To address the lack of structural and functional knowledge of the secondary metabolism of gut bacteria, our project used genome mining and synthetic biology to study the function of novel NPs from bacteria from human gut microbiome.
In Work Package 1 (WP1), we explored the biosynthetic dark matter of human gut bacteria through genome mining approaches for the discovery of spliceotide and origamin RiPP natural products. And in Work Package 2 (WP2), we explored the function of spliceotide and origamin natural products and possible applications therapeutics.
We uncovered new biosynthetic gene clusters encoding for spliceotides from gut bacteria. While we were unable to obtain functional data for these systems, we were uncovered the biological activity of 4 other spliceotide products. All products showed protease inhibition activity and, remarkably, two spliceotides reproducibly demonstrated low-nanomolar activity against Human Neutrophil Elastase.
We also discovered origamin BGCs from gut microbes. We had previously demonstrated the activities for two enzymes in origamin biosynthesis in E. coli, but were not able to reproduce those results in origamins from gut bacteria. Nonetheless, we expanded our search of origamin clusters with the goal of isolating a final origamin product by obtaining activity of all key biosynthetic enzymes. We identified a new type of origamin BGC and were able to obtain activity for all three tailoring enzymes which produced one of the most extensively modified RiPPs known to date.
In the spliceotide, work, we uncovered new biosynthetic gene clusters encoding for spliceotides from Eubacterium rectale and a number of Bacteroidetes strains. A number of experimental designs were tested to obtain enzymatic activity for splicease enzymes: heterologous expression in E. coli and Microvirgula aerodenitrificans, and homologous and heterologouns expression in Bacteroides thetaiotaomicron. Most recently, we attempted to express the protein precursor and splicease individually to optimize expression and purification conditions. We hoped that by exploring the in vitro activity of the splicease, we could determine what are the critical parameters for catalysis. However, attempts to get in vitro activity failed.
To tackle the function of spliceotide products, we turned to characterizing better behaved systems. We tested the activities of spliceotide products from Cystobacter fuscus, Pseudoalteromonas phenolica, Rheinheimera aquimaris, and Pseudoalteromonas piscicida against a number of bacterial strains and proteases. While no antibacterial activity was observed, all spliceotides had some protease inhibitory activity:
• C. fuscus spliceotide: IC50 of 2.6 mM against cathepsin B and of 2.6 nM against human neutrophil elastase (HNE)
• P. piscicida spliceotide: IC50 of 0.4 mM against chymotrypsin
• R. aquimaris spliceotide: IC50 of 0.5 mM against chymotrypsin, 1.3 against Cathepsin B and 36 nM against HNE
• P. phenolica spliceotide: IC50 of 20 mM against HNE
These results are detailed in the manuscript: “Widespread microbial utilization of ribosomal β-amino acid-containing peptides and proteins“, currently under review
In the origamin work, we discovered origamin BGCs from gut microbes such as E. coli, Bifidobacterium longum, and Collinsella aerofaciens. We had previously demonstrated the activities for two enzymes in origamin biosynthesis in E. coli, but were not able to reproduce those results in origamins from gut bacteria. In the E. coli system, we were also unable to characterize all biosynthetic enzymes and therefore were not able to obtain the final origamin product.
Nonetheless, we expanded our search of origamin clusters with the goal of isolating a final origamin product by obtaining activity of all key biosynthetic enzymes. In our bioinformatic search, we discovered a new type of origamin cluster which contains 3 key biosynthetic enzymes, in addition to the precursor peptide: an epimerase rSAM, a SAM-dependent N-methyltransferase, and a B12-dependent C-methyltransferease. We identified this cluster in organisms such as Polymorphum gilvum and Actinosynnema sp. We then expressed the precursor peptide and tailoring enzymes from P. gilvum. Since E. coli does not have the metabolic capability to produce the B12 cofactor, we turned to an alternative heterologous expression host, Microvirgula aerodenitrificans. M. aerodenitrificans is a Gram-negative denitrifier and was isolated from activated sludge. Our research group engineered M. aerodenitrificans for heterologous expression, through the development of a suitable expression plasmid and transformation procedures. We thus heterologously expressed the partial or whole P. gilvum cluster, and, upon co-expression of all tailoring enzymes, we observed 18 epimerizations, 13 N-methylations, and additional 13 C-methylations. Our group is currently working on the full structural elucidation of this final product and on measuring its biological activity.
A manuscript detailing these findings is in preparation and will be submitted to publication later this year.
To tackle the function of spliceotide products, we turned to characterizing better behaved systems. We tested the activities of spliceotide products from Cystobacter fuscus, Pseudoalteromonas phenolica, Rheinheimera aquimaris, and Pseudoalteromonas piscicida against a number of bacterial strains and proteases. While no antibacterial activity was observed, all spliceotides had some protease inhibitory activity:
• C. fuscus spliceotide: IC50 of 2.6 mM against cathepsin B and of 2.6 nM against human neutrophil elastase (HNE)
• P. piscicida spliceotide: IC50 of 0.4 mM against chymotrypsin
• R. aquimaris spliceotide: IC50 of 0.5 mM against chymotrypsin, 1.3 against Cathepsin B and 36 nM against HNE
• P. phenolica spliceotide: IC50 of 20 mM against HNE
These results are detailed in the manuscript: “Widespread microbial utilization of ribosomal β-amino acid-containing peptides and proteins“, currently under review
In the origamin work, we discovered origamin BGCs from gut microbes such as E. coli, Bifidobacterium longum, and Collinsella aerofaciens. We had previously demonstrated the activities for two enzymes in origamin biosynthesis in E. coli, but were not able to reproduce those results in origamins from gut bacteria. In the E. coli system, we were also unable to characterize all biosynthetic enzymes and therefore were not able to obtain the final origamin product.
Nonetheless, we expanded our search of origamin clusters with the goal of isolating a final origamin product by obtaining activity of all key biosynthetic enzymes. In our bioinformatic search, we discovered a new type of origamin cluster which contains 3 key biosynthetic enzymes, in addition to the precursor peptide: an epimerase rSAM, a SAM-dependent N-methyltransferase, and a B12-dependent C-methyltransferease. We identified this cluster in organisms such as Polymorphum gilvum and Actinosynnema sp. We then expressed the precursor peptide and tailoring enzymes from P. gilvum. Since E. coli does not have the metabolic capability to produce the B12 cofactor, we turned to an alternative heterologous expression host, Microvirgula aerodenitrificans. M. aerodenitrificans is a Gram-negative denitrifier and was isolated from activated sludge. Our research group engineered M. aerodenitrificans for heterologous expression, through the development of a suitable expression plasmid and transformation procedures. We thus heterologously expressed the partial or whole P. gilvum cluster, and, upon co-expression of all tailoring enzymes, we observed 18 epimerizations, 13 N-methylations, and additional 13 C-methylations. Our group is currently working on the full structural elucidation of this final product and on measuring its biological activity.
A manuscript detailing these findings is in preparation and will be submitted to publication later this year.
For both spliceotide and origamin projects, we hope that the discovered compounds can become therapeutically relevant or inspire new therapeutically relevant molecules. For the spliceotides, we already validated the activity of the molecules against a host of proteases that are important targets to treat disease. We are now currently working on further understanding the mode of action for protease inhibition by co-crystallizing the spliceotide and the target proteases.
Regarding the origamin project, we are working towards elucidating the structure and testing the biological activity of the putatively beta-helical final product. The polytheonamide precedent suggests that the origamin products will have similar pore-forming activity. Further, the presence of immunity proteins on the origamin BGCs is suggestive of anti-bacterial activity.
Regarding the origamin project, we are working towards elucidating the structure and testing the biological activity of the putatively beta-helical final product. The polytheonamide precedent suggests that the origamin products will have similar pore-forming activity. Further, the presence of immunity proteins on the origamin BGCs is suggestive of anti-bacterial activity.