Periodic Reporting for period 1 - COMMUNITY (Unraveling the regulatory networks in Streptomyces that switch on antibiotic production on demand)
Periodo di rendicontazione: 2022-09-01 al 2025-02-28
As Nature's medicine makers, Streptomyces bacteria produce a plethora of natural products, which we harness for clinical, biotechnological and agricultural applications, including 70% of the antibiotics. Streptomycetes still have a vast reservoir of unexplored biosynthetic potential, but many biosynthetic gene clusters (BGCs) are not expressed in the laboratory. To bring the chemical dark matter to the light, we need to discover the keys to unlock the expression of cryptic BGCs. Aim is to add a new dimension to genome mining, via understanding and exploitation of the regulatory networks that control natural product biosynthesis in Actinobacteria. My team discovered the concept of antibiotic production on demand, showing that plant hormones activate antimicrobials. Predicting when instead of what BGCs produce will allow clustering of BGCs based on their response to ecological signals. This can serve as a beacon for prioritising BGCs, and aid in the discovery of new biosynthetic pathways. I will tackle three major challenges:
1. The systems biology challenge is to elucidate the regulatory circuitry of streptomycetes and to reliably predict how BGCs are controlled.
2. The metabolic challenge is to unwire the networks that tie carbon metabolism to antibiotic production, to bridge the gap from the complex polysaccharides in nature to the defined carbon sources of the laboratory.
3. The ecological challenge is to unravel the mechanisms and molecules via which plants invoke the power of Streptomyces' bioactive molecules to obtain protection against infections and pests, aimed at biological disease-suppression.
COMMUNITY is an open science project that will help to elucidate whether the yet unexplored BGCs will deliver a paradigm shift in drug discovery, for application in agriculture and human health. Deliverables are innovative systems biology tools and detailed transcription factor networks, elicitors for drug screening and disease-suppressive microbes
1. The systems biology challenge is to elucidate the regulatory circuitry of streptomycetes and to reliably predict how BGCs are controlled.
2. The metabolic challenge is to unwire the networks that tie carbon metabolism to antibiotic production, to bridge the gap from the complex polysaccharides in nature to the defined carbon sources of the laboratory.
3. The ecological challenge is to unravel the mechanisms and molecules via which plants invoke the power of Streptomyces' bioactive molecules to obtain protection against infections and pests, aimed at biological disease-suppression.
COMMUNITY is an open science project that will help to elucidate whether the yet unexplored BGCs will deliver a paradigm shift in drug discovery, for application in agriculture and human health. Deliverables are innovative systems biology tools and detailed transcription factor networks, elicitors for drug screening and disease-suppressive microbes
We obtained proof of concept for a core idea that was proposed in COMMUNITY, namely that we can functionally predict biosynthetic gene clusters (BGCs) entirely based on the way they are controlled, instead of by analysing the genes in the BGC or the natural products they specify. By analysing genomes for binding sites for the iron master regulator DmdR1, we discovered a new BGC involved in iron homeostasis. Detailed study of the BGC showed that it is a previously undetected BGC that plays a key role in the biosynthesis of the well-known siderophore desferrioxamine B.
This example indicates how important it is to understand how BGCs are controlled. However, so far we only knew some 3% of the transcription factor regulatory networks (TFRNs). To understand how BGCs for bioactive molecules are controlled, which is also key for their activation and thus the elucidation of the compounds they specify, we need to greatly expand our knowledge of the TFRNs. In close collaboration with the JGI in Berkeley, California, we are analysing DAPseq data, which are in vitro DNA binding studies for all TFs in the cell (some 800 TFs). This massively expands the state of the art relating to the known TFRNs in Streptomyces, and the data obtained provide unprecedented insights into the networks and to predict how BGCs are controlled. We will verify the in vitro data and then implement them into the BGC prediction software antiSMASH and LogoMotif.
We also developed a new Crispri system called CUBIC, which allows knocking down specific genes and monitoring the response of the cell, making use of a cumate-inducible promoter. These data and technologies are of great importance for the broad fields of Streptomyces biology and natural product discovery. Am ordered library targeting each regulator individually (900 genes, two probes per gene, so 1800 clones) was created and using the robotics set up from the COMMUNITY project, we are extensively screening the library to identify genes that belong to complex signal transduction pathways, such as regulators involved in the control of growth, development and antibiotic production.
CUBIC is available via the open access repository Addgene, and the TFBS have been integrated into the open access BGC prediction tool antiSMASH 7, so that scientists can for the first time obtain information on how their BGC of interest is controlled.
We worked out large networks for the (control of) metabolism of polysaccharides such as cellulose, hemicellulose, pectin, chitin and chitosan. This is an important step towards a major aim in COMMUNITY, namely understanding how streptomycetes grow on polysaccharides in the natural habitat. Many new enzymes and regulators have been identified and await full functional analysis. We also identified new genes involved in carbon catabolite control and central metabolism. This includes NagS, an N-acetylglucosamine dehydratase, which is a new enzyme in N-acetylglucosamine metabolism. This enzyme forms the basis for a toxicity pathway that links growth, nutrient utilization and development in Streptomyces. Finally, we have identified new regulators involved in the perception of - and response - to plant hormones, such as jasmonic acid. This provides new insights into the concept of plant protection on demand, in other words, how plants stimulate the production of [protective molecules by streptomycetes.
This example indicates how important it is to understand how BGCs are controlled. However, so far we only knew some 3% of the transcription factor regulatory networks (TFRNs). To understand how BGCs for bioactive molecules are controlled, which is also key for their activation and thus the elucidation of the compounds they specify, we need to greatly expand our knowledge of the TFRNs. In close collaboration with the JGI in Berkeley, California, we are analysing DAPseq data, which are in vitro DNA binding studies for all TFs in the cell (some 800 TFs). This massively expands the state of the art relating to the known TFRNs in Streptomyces, and the data obtained provide unprecedented insights into the networks and to predict how BGCs are controlled. We will verify the in vitro data and then implement them into the BGC prediction software antiSMASH and LogoMotif.
We also developed a new Crispri system called CUBIC, which allows knocking down specific genes and monitoring the response of the cell, making use of a cumate-inducible promoter. These data and technologies are of great importance for the broad fields of Streptomyces biology and natural product discovery. Am ordered library targeting each regulator individually (900 genes, two probes per gene, so 1800 clones) was created and using the robotics set up from the COMMUNITY project, we are extensively screening the library to identify genes that belong to complex signal transduction pathways, such as regulators involved in the control of growth, development and antibiotic production.
CUBIC is available via the open access repository Addgene, and the TFBS have been integrated into the open access BGC prediction tool antiSMASH 7, so that scientists can for the first time obtain information on how their BGC of interest is controlled.
We worked out large networks for the (control of) metabolism of polysaccharides such as cellulose, hemicellulose, pectin, chitin and chitosan. This is an important step towards a major aim in COMMUNITY, namely understanding how streptomycetes grow on polysaccharides in the natural habitat. Many new enzymes and regulators have been identified and await full functional analysis. We also identified new genes involved in carbon catabolite control and central metabolism. This includes NagS, an N-acetylglucosamine dehydratase, which is a new enzyme in N-acetylglucosamine metabolism. This enzyme forms the basis for a toxicity pathway that links growth, nutrient utilization and development in Streptomyces. Finally, we have identified new regulators involved in the perception of - and response - to plant hormones, such as jasmonic acid. This provides new insights into the concept of plant protection on demand, in other words, how plants stimulate the production of [protective molecules by streptomycetes.
Our results highlight that we can indeed harness the understanding of the regulatory networks to functionally annotate biosynthetic gene clusters (BGCs) for natural products. In this way, we started to prioritize a small subset of BGCs in the enormous genomic space of millions of BGCs that are now available. We have developed LogoMotif and integrated the tool into antiSMASH, adding regulatory network functions to the world leading software antiSMASH. As a proof of concept, we have applied regulatory networks for the functional prediction of a novel biosynthetic gene cluster involved in siderophore biosynthesis.
The DAPseq data that are now available massively expand our knowledge of the TFRNs in Actinobacteria, and once we have done the full analysis and verification, this will allow scientists worldwide to predict how genes of interest are controlled, including BGCs for new bioactive compounds.
We have developed a new Crispri system called “CUBIC” that allows to knock down genes of interest and screen for new functions. The system is available to all scientists and of particular relevance for researchers in the broad field of actinomycete biology.
Finally, we have Identified toxicity pathways in Streptomyces that revolve around amino sugar metabolism, controlled by a novel enzyme that was not previously described in the textbooks.
The DAPseq data that are now available massively expand our knowledge of the TFRNs in Actinobacteria, and once we have done the full analysis and verification, this will allow scientists worldwide to predict how genes of interest are controlled, including BGCs for new bioactive compounds.
We have developed a new Crispri system called “CUBIC” that allows to knock down genes of interest and screen for new functions. The system is available to all scientists and of particular relevance for researchers in the broad field of actinomycete biology.
Finally, we have Identified toxicity pathways in Streptomyces that revolve around amino sugar metabolism, controlled by a novel enzyme that was not previously described in the textbooks.