Periodic Reporting for period 1 - DiPaC_MC (Direct Pathway Cloning of Neglected Bacteria in the Hunt for Novel (Bio-)Chemistry) Reporting period: 2017-09-01 to 2019-08-31 Summary of the context and overall objectives of the project Natural products are low molecular weight molecules that comprise a vast array of chemical diversity and bioactivity. Bioactivities include anti-bacterial, anti-fungal, or anti-cancer, amongst many others. Since the initial discovery of penicillin in 1928, there was a ‘golden age’ of natural product discovery, particularly from microbiological sources. Unfortunately, mainly due to the tedious isolation and production procedures and the increased problem of rediscovery, commercial interest in natural products sharply declined over time. However, there has been a recent acknowledgement of the urgent need for novel bioactive molecules due to the current rise in resistance to antibiotics, chemotherapy and pesticides.To address these issues, we recently developed techniques for the rapid discovery of novel natural products. Specifically, this includes sequencing the genomes of various underexplored bacteria, screening the genome data for the genetic elements responsible for producing natural products, using synthetic biology to capture and produce the desired compounds in a heterologous host. The synthetic biology techniques devised in this proposal is termed Direct Pathway Cloning (DiPaC). Using this approach, the degree of novel discovery is increased and the amount of product obtained is usually higher than in the native producer. This is important for society because it overcomes the previous issues with natural product discovery while providing a quicker and more successful method for identifying novel molecules. As more novel natural products are identified, the number available as potential medicines increases and with new structural diversity comes the ability to identify molecules with novel modes of action. The project objective within this research program is to investigate natural product biosynthesis within neglected bacteria of the ZIEL Culture Collection using a heterologous expression approach. This includes:1. Genome mine and prioritise natural product gene clusters from the ZIEL Institute Culture Collection2. Design and clone selected pathways by application and further development of DiPaC3. Express pathways within E. coli or Streptomyces expression hosts4. Alter expression vectors via DiPaC and Red/ET homologous recombination and characterise the effect on biosynthesisThe outcomes from this proposal are beneficial to the scientific community and to society by providing improved methods for drug discovery. Significant results were achieved for all four project objectives, including the discovery of over 700 natural product pathways, the production of 7 molecules using DiPaC and the characterisation of an enzyme performing a novel reaction, which is key in the biosynthesis of a family of bioactive natural products. The development of the DiPaC methodology was also immensely successful. The methodology was improved further than expected, with an increase in cloning speed and efficiency while decreasing cost. DiPaC has the potential to transform biomolecular natural product chemistry and set precedence for quick and efficient activation of pathways encoding novel natural products. DiPaC is generally applicable to a broad range of methods such as the metabolic engineering of entire pathways with industrial (e.g. biofuel) or medical (e.g. novel small molecules with bioactivity) significance, and thus, has far reaching benefits for society. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far The organisms of interest in the project were extremely underrepresented from a natural products perspective yet were found to harbour the genetic machinery required to produce many natural products. A total of 789 putative natural product pathways were identified in 13 genomes, of which 207 belong to well-known structural families. Approximately >95% of pathways could not be linked to any known pathway and therefore, are likely the produce a novel compound. A total of 10 biosynthetic gene clusters have been cloned using the DiPaC method within this project. Thus, the number of cloned pathways was significantly higher than planned in the original proposal. Over the course of the project, the DiPaC methodology has achieved significant advancements in cloning technology. Firstly, we significantly increased the size of PCR amplicons compatible with the DiPaC method. Secondly, we significantly lowered the price and cloning time of the DiPaC method by utilising T4 DNA polymerase, termed sequence- and ligation-independent cloning (SLIC). Of the 10 cloned biosynthetic gene clusters, 8 encode an unknown natural product and 7 have been successfully heterologously expressed. The DiPaC methodology is now being exploited via its application to an even broader array of organisms and natural product types.The proposal has led to the isolation and discovery of 2 novel natural products with another 5 undergoing optimisation for structure elucidation. Biosynthesis of the 2 novel structurally elucidated natural products was believed to occur via a novel enzymatic pathway requiring 4 enzymes. Molecules with similar structures are known to be bioactive, making the elucidation of their enzymatic pathway significant. We were able to characterise the function of each enzyme resulting in the identification of a protein involved in precursor cyclisation to form the core ring structure via a novel mechanism.The outputs of this proposal were disseminated via several national (3) and international (5) conferences including 1 invited lecture, as well as 3 first-author peer-reviewed scientific publications. It is expected that a minimum of two more very high-ranking and first-author publications will result directly from the project output. Selected major conferences and publications disseminated as part of the project are listed below.• Directing Biosynthesis V, United Kingdom, 2017. Poster.• 3rd European Conference on Natural Products, Germany, 2018. Oral Presentation• The Chemistry and Biology of Natural Products Symposium XIII, UK, 2019. Invited Oral Presentation• *Mojićević, M., *D’Agostino, PM., Pavic, A., Vojnović, S., Senthamaraikannan, R., Vasiljević, B., Gulder, TAM., Nikodinović-Runić, J. MicrobiologyOpen (In revision).• *Duell, E., *D’Agostino, PM., Shapiro, N., Woyke, T., Fuchs, TM., Gulder, TAM. Microbial Cell Factories, 2019, 18, 32.• D’Agostino, PM., Gulder, TAM. ACS Synthetic Biology, 2018, 7(7), 1702-1708. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) It is known that resistances to pharmaceuticals is a global crisis facing humanity. Thus, developing efficient mechanisms for discovering novel natural products as well as the ability to exploit these resources is key to tackle the resistance emergency. The major impacts of the project with societal and scientific relevance can be seen from two outputs. Firstly, we have shown that previously underrepresented microorganisms are capable of encoding a plethora of natural product biosynthetic gene clusters. Thus, these types of underrepresented microorganisms should be targeted in the future for genome sequencing to increase the availability and expand databases of gene clusters likely to encode novel products. Further, the successful application of DiPaC is expected to spur research in this field. The ability of DiPaC to significantly increase the efficiency of natural product discovery will directly lead to more novel molecules being identified. Thus, we believe this project has directly provided significant outcomes to benefit both the scientific community and society as a whole.