Periodic Reporting for period 1 - DiPaC_MC (Direct Pathway Cloning of Neglected Bacteria in the Hunt for Novel (Bio-)Chemistry)
Berichtszeitraum: 2017-09-01 bis 2019-08-31
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 Collection
2. Design and clone selected pathways by application and further development of DiPaC
3. Express pathways within E. coli or Streptomyces expression hosts
4. Alter expression vectors via DiPaC and Red/ET homologous recombination and characterise the effect on biosynthesis
The 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.
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.