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.
