DiPaC_MC · Direct Pathway Cloning of Neglected Bacteria in the Hunt for Novel (Bio-)Chemistry

Direct Pathway Cloning of Neglected Bacteria in the Hunt for Novel (Bio-)Chemistry

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 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.

Data: CORDIS, © European Union