Periodic Reporting for period 1 - NAfrAM (Novel antibiotics from Antarctic microbes)
Période du rapport: 2022-10-01 au 2024-09-30
Microbial resistance to antimicrobial compounds has become one of the biggest threats to global health. Infections and diseases caused by resistant microbes require prolonged health care, are easy to spread, are harder to treat, and increase the risk of severe health conditions or death. Although antimicrobial resistance (AMR) is a long-term issue, the speed and extent of AMR have been underestimated over a long time. Nowadays, the cost of AMR has reached to large extent, not merely economically, but especially for human health and life. The antibiotic-resistant infections are nowadays among the biggest threats to global health with an estimated contribution to 4.95 million deaths in 2019 and a predicted increase to approximately 10 million deaths per year by 2050. Due to a long-lasting decline in antibiotic development since the 1970s, the lack of high-quality, effective antimicrobials remains one of the most severe issues; thus, novel antibiotics are urgently needed.
A promising way to discover novel antibiotics is the identification of novel natural products (NPs). Largely diverse and complex scaffolds of NPs continue to be a major source for drug discovery including antibiotics. During the last decades, detailed bioinformatic and experimental investigation of genome-encoded NPs clustered into BGCs provided fascinating insights into the tremendous chemical diversity of bacteria-produced compounds. Besides, since high proportions of microbes across biomes remain uncultured, our knowledge about the natural diversity of specialized microbial metabolites is limited. Moreover, some molecules are biosynthesized only by a specific group of microorganisms or are specific to particular ecological niches and/or grow solely under natural conditions. Therefore, bioprospecting of the rarely studied environments represents a promising approach to discovering novel bioactive metabolites. One of the largely underexplored and simultaneously very extreme environments is Antarctica, where extensive genetic diversity of genes responsible for biosynthesis of NPs have been reported.
The NAfrAM project investigated diverse microbial communities from different ecosystems in Antarctica with the major goal of discovering novel antibiotics and characterising the overall biosynthetic potential of these unique microbial communities to produce unknown bioactive compounds. The project stands on deep-sequencing data that allow precise exploration of genetic information of studied Antarctic microbial communities. The bioinformatic search for biosynthetic gene clusters (BGC), which represents a recipe for the biosynthesis of natural products available in genetic information, enables the identification of novel BGCs and prioritization of BGCs likely-encoding antimicrobial compounds. The BGCs prioritized in this way are further used for experimental work in the laboratory, which allows the development of methodology resulting in the production of novel antimicrobial compounds, the above-mentioned primary goal of the project NAfrAM.
A promising way to discover novel antibiotics is the identification of novel natural products (NPs). Largely diverse and complex scaffolds of NPs continue to be a major source for drug discovery including antibiotics. During the last decades, detailed bioinformatic and experimental investigation of genome-encoded NPs clustered into BGCs provided fascinating insights into the tremendous chemical diversity of bacteria-produced compounds. Besides, since high proportions of microbes across biomes remain uncultured, our knowledge about the natural diversity of specialized microbial metabolites is limited. Moreover, some molecules are biosynthesized only by a specific group of microorganisms or are specific to particular ecological niches and/or grow solely under natural conditions. Therefore, bioprospecting of the rarely studied environments represents a promising approach to discovering novel bioactive metabolites. One of the largely underexplored and simultaneously very extreme environments is Antarctica, where extensive genetic diversity of genes responsible for biosynthesis of NPs have been reported.
The NAfrAM project investigated diverse microbial communities from different ecosystems in Antarctica with the major goal of discovering novel antibiotics and characterising the overall biosynthetic potential of these unique microbial communities to produce unknown bioactive compounds. The project stands on deep-sequencing data that allow precise exploration of genetic information of studied Antarctic microbial communities. The bioinformatic search for biosynthetic gene clusters (BGC), which represents a recipe for the biosynthesis of natural products available in genetic information, enables the identification of novel BGCs and prioritization of BGCs likely-encoding antimicrobial compounds. The BGCs prioritized in this way are further used for experimental work in the laboratory, which allows the development of methodology resulting in the production of novel antimicrobial compounds, the above-mentioned primary goal of the project NAfrAM.
The project NAfrAM started with preliminary data of the four prioritized samples from four different Antarctic ecosystems (lake, stream, wetland, pond). The two samples were vertically stratified microbial mats and two samples were non-structured microbial mats. The preliminary data consists of 16S amplicon sequencing and shotgun metagenomic sequencing data prepared by Illumina short-read sequencing technology. During the first three months of the project, I extracted high molecular weight DNA from these samples and the samples were also sequenced by the Oxford Nanopore Technologies method to produce long-read sequencing data. Such a complex ultra-deep sequencing dataset was used to produce high-quality metagenomic assemblies where the long-read data were assembled first and then corrected by short-read Illumina data.
The resulting metagenomic assemblies were used for comprehensive bioinformatic analysis. The bioinformatics analysis consists of taxonomic classification of metagenomic contigs, BGCs detection, BGC taxonomic classification, BGCs clustering to gene cluster families (GCFs) to allow more accurate evaluation of biosynthetic potential and BGCs diversity, binning of metagenomic contigs into metagenome-assembled genomes (MAGs), linking the BGCs with MAGs, phylogenetic and taxonomic analysis of MAGs, analysis of the environmental distribution of BGCs, semi-manual identification of precursor peptides for RiPPs BGCs, prioritization of nearly fifty BGCs possibly producing antimicrobial compound. Further, the datasets of more than 6,000 BGCs detected across 4 Antarctic samples, more than 1,000 high-quality (HQ) and medium-quality (MQ) MAGs, and nearly 370 precursor peptides of diverse RiPPs pathways belong to the main achievements of the bioinformatic part. Such datasets constitute invaluable data for further data mining studies that can help in the investigation of Antarctic biodiversity and understanding of microbial evolution in Antarctica. Importantly, these data can directly serve the biosynthesis or chemical synthesis of specialized metabolites with pharmaceutical or biotechnological potential and help to understand their biological functions.
The bioinformatic part informed the experimental part which builds on prioritized BGCs possibly producing antimicrobial compounds. In this direction, ten novel BGCs from Antarctic microbes were further selected for the experimental part, namely 1x thiopeptide, 1x hybrid lanthipeptide-NRPS-PKS-Terpene, 1x hybrid NRPS-PKS, 3x lasso peptide, 3x lanthipeptide, and 1x hybrid homoserine lactone-lasso peptide. All BGCs were successfully PCR amplified from Antarctic microbial communities and assembled into expression vectors. Considering the project's timescale, only one lasso peptide and three lanthipeptide BGCs, containing unique features in precursor peptides (polyarginine regions) were selected for detailed experimental investigations. In the first part, the lasso peptide BGCs were further optimized to increase the chance for successful heterologous expression on the level of promoter, RBS and codon-optimization for Escherichia coli. After sequencing verification of expression plasmids with inserted BGCs, lasso peptide and lanthipeptide BGC were heterologously expressed in Escherichia coli BL21(DE3). The BGCs were expressed under several experimental setups (temperature 20°Cfor 72 hours/37°C for 24 hours, lasso peptide in M9 media, lanthipeptide in LB media. each in triplicate). However, neither the lasso peptide nor the lanthipeptide final product was detected using LC-ESI-MS. Thus, the next step was heterologous expression and purification of every single protein involved in biosynthesis. These experiments revealed soluble expression for all proteins from lanthipeptide BGCs; however, it revealed insoluble expression of peptidase from lasso peptide BGCs responsible for cleavage of the leader peptide from the core peptide in precursor peptide, a step necessary for cyclization resulting in a final matured product. Therefore, these experiments directed the effort to the lanthipeptide BGCs. All single proteins were purified and used for in vitro reaction. Although the final product of the in vitro reactions was not detected by LC-ESI-MS, the final product was detected by MALDI-TOF MS. It also suggested that original experiments with complete BGCs also resulted in the production of the final product, but the native product is hard to detect using LC-ESI-MS.
The resulting metagenomic assemblies were used for comprehensive bioinformatic analysis. The bioinformatics analysis consists of taxonomic classification of metagenomic contigs, BGCs detection, BGC taxonomic classification, BGCs clustering to gene cluster families (GCFs) to allow more accurate evaluation of biosynthetic potential and BGCs diversity, binning of metagenomic contigs into metagenome-assembled genomes (MAGs), linking the BGCs with MAGs, phylogenetic and taxonomic analysis of MAGs, analysis of the environmental distribution of BGCs, semi-manual identification of precursor peptides for RiPPs BGCs, prioritization of nearly fifty BGCs possibly producing antimicrobial compound. Further, the datasets of more than 6,000 BGCs detected across 4 Antarctic samples, more than 1,000 high-quality (HQ) and medium-quality (MQ) MAGs, and nearly 370 precursor peptides of diverse RiPPs pathways belong to the main achievements of the bioinformatic part. Such datasets constitute invaluable data for further data mining studies that can help in the investigation of Antarctic biodiversity and understanding of microbial evolution in Antarctica. Importantly, these data can directly serve the biosynthesis or chemical synthesis of specialized metabolites with pharmaceutical or biotechnological potential and help to understand their biological functions.
The bioinformatic part informed the experimental part which builds on prioritized BGCs possibly producing antimicrobial compounds. In this direction, ten novel BGCs from Antarctic microbes were further selected for the experimental part, namely 1x thiopeptide, 1x hybrid lanthipeptide-NRPS-PKS-Terpene, 1x hybrid NRPS-PKS, 3x lasso peptide, 3x lanthipeptide, and 1x hybrid homoserine lactone-lasso peptide. All BGCs were successfully PCR amplified from Antarctic microbial communities and assembled into expression vectors. Considering the project's timescale, only one lasso peptide and three lanthipeptide BGCs, containing unique features in precursor peptides (polyarginine regions) were selected for detailed experimental investigations. In the first part, the lasso peptide BGCs were further optimized to increase the chance for successful heterologous expression on the level of promoter, RBS and codon-optimization for Escherichia coli. After sequencing verification of expression plasmids with inserted BGCs, lasso peptide and lanthipeptide BGC were heterologously expressed in Escherichia coli BL21(DE3). The BGCs were expressed under several experimental setups (temperature 20°Cfor 72 hours/37°C for 24 hours, lasso peptide in M9 media, lanthipeptide in LB media. each in triplicate). However, neither the lasso peptide nor the lanthipeptide final product was detected using LC-ESI-MS. Thus, the next step was heterologous expression and purification of every single protein involved in biosynthesis. These experiments revealed soluble expression for all proteins from lanthipeptide BGCs; however, it revealed insoluble expression of peptidase from lasso peptide BGCs responsible for cleavage of the leader peptide from the core peptide in precursor peptide, a step necessary for cyclization resulting in a final matured product. Therefore, these experiments directed the effort to the lanthipeptide BGCs. All single proteins were purified and used for in vitro reaction. Although the final product of the in vitro reactions was not detected by LC-ESI-MS, the final product was detected by MALDI-TOF MS. It also suggested that original experiments with complete BGCs also resulted in the production of the final product, but the native product is hard to detect using LC-ESI-MS.
The combination of short-read and long-read sequencing resulted in an outstanding dataset of deeply sequenced metagenomes that provided detailed insight into the genomic information of Antarctic microbial communities and allowed very accurate bioinformatic analysis that precisely informed the experimental part of this project.
The consequent bioinformatic analysis revealed more than 97% of GCFs detected across Antarctic microbial communities are significantly distant in sequence similarity to those in reference databases pointing to the extensive novelty of genome-encoded natural products originating from Antarctic microbes. The analysis of the environmental distribution of BGCs using the tool BGC Atlas indicated that more than 85% of GCFs are endemic to the Antarctic, based on the currently available metagenomes in the MGnify database. Moreover, the dataset of >1000 HQ and MQ MAGs represents the biggest MAGs dataset from Antarctica and allowed the identification of phyla with the highest density of BGCs per genome, Cyanobacteria and Myxococcota together with the phylogenetic origin of specific BGCs. The final part of the bioinformatic analysis prioritized BGCs putatively producing antimicrobial compounds. The outcome of this part was the dataset of 47 BGCs highly suitable for heterologous expression experiments. Although it is not feasible in the project's timescale to explore all these BGCs, the data will be further used for the collaboration of the supervisor and researcher and constitute excellent preliminary data for future research activities of the researcher.
The experimental part of the project NAfrAM achieved several outstanding results. The recovery of ten BGCs in size range from 5,000 bp to 37,000 bp from Antarctic metagenomes represents a unique dataset exploitable by the researcher for his future scientific activities and the establishment of collaborations with other researchers in the field of NPs. Within the time frame of the project NAfrAM, one lasso peptide and three lanthipeptide BGCs were successfully produced with detectable core peptide products. Since these peptides represent very promising molecules thanks to their predicted antimicrobial and neuroprotective properties, the researcher will continue with the comprehensive characterization of these peptides to fully exploit the potential of the discovered peptides.
In summary, (i) an outstanding dataset of deeply sequenced metagenomes, (ii) described extensive novelty of genome-encoded natural products originating from Antarctic microbial communities dominated by cyanobacteria, (iii) the largest dataset of HQ and MQ MAGs to date (11/2024) from Antarctica, (iv) the set of ten recovered (novel and uncharacterized) BGCs for Antarctic microorganisms, and (v) discovery of 4 novel unique RiPPs peptide together with a methodology for their biosynthesis represent results and knowledge beyond the state of the art, produced thanks to MSCA funded project NAfrAM.
The consequent bioinformatic analysis revealed more than 97% of GCFs detected across Antarctic microbial communities are significantly distant in sequence similarity to those in reference databases pointing to the extensive novelty of genome-encoded natural products originating from Antarctic microbes. The analysis of the environmental distribution of BGCs using the tool BGC Atlas indicated that more than 85% of GCFs are endemic to the Antarctic, based on the currently available metagenomes in the MGnify database. Moreover, the dataset of >1000 HQ and MQ MAGs represents the biggest MAGs dataset from Antarctica and allowed the identification of phyla with the highest density of BGCs per genome, Cyanobacteria and Myxococcota together with the phylogenetic origin of specific BGCs. The final part of the bioinformatic analysis prioritized BGCs putatively producing antimicrobial compounds. The outcome of this part was the dataset of 47 BGCs highly suitable for heterologous expression experiments. Although it is not feasible in the project's timescale to explore all these BGCs, the data will be further used for the collaboration of the supervisor and researcher and constitute excellent preliminary data for future research activities of the researcher.
The experimental part of the project NAfrAM achieved several outstanding results. The recovery of ten BGCs in size range from 5,000 bp to 37,000 bp from Antarctic metagenomes represents a unique dataset exploitable by the researcher for his future scientific activities and the establishment of collaborations with other researchers in the field of NPs. Within the time frame of the project NAfrAM, one lasso peptide and three lanthipeptide BGCs were successfully produced with detectable core peptide products. Since these peptides represent very promising molecules thanks to their predicted antimicrobial and neuroprotective properties, the researcher will continue with the comprehensive characterization of these peptides to fully exploit the potential of the discovered peptides.
In summary, (i) an outstanding dataset of deeply sequenced metagenomes, (ii) described extensive novelty of genome-encoded natural products originating from Antarctic microbial communities dominated by cyanobacteria, (iii) the largest dataset of HQ and MQ MAGs to date (11/2024) from Antarctica, (iv) the set of ten recovered (novel and uncharacterized) BGCs for Antarctic microorganisms, and (v) discovery of 4 novel unique RiPPs peptide together with a methodology for their biosynthesis represent results and knowledge beyond the state of the art, produced thanks to MSCA funded project NAfrAM.