Investigating bottromycin biosynthesis by X-ray crystallography and in vitro engineering of the pathway

• Applicant: Professor Dr. Jesko-Alexander Koehnke
• Subject Area: Biochemistry ; Biophysics
• Term: from 2014 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 261144906

• Final Report Year: 2021

Final Report Abstract

Since their discovery almost a century ago, antibiotics have become indispensable for modern medicine and are a main contributor to the long and healthy lives we enjoy today. The major problem we are faced with in antibiotic therapy today is the increasingly rapid development of antimicrobial resistance. This challenge can only be overcome by a confluence of factors, including better and more restricted use of current antibiotics, but also the discovery of new antibiotics with a novel mode of action. Bottromycins fit this bill: they are cyclic peptide antibiotics, which address a thus far unexploited cellular target. As a result, they are effective against problematic human pathogens, such as vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. The major hurdle in the development of bottromycins is the exceptionally difficult total synthesis and the poor yields when attempts have been made to produce the compounds via bioengineering. We set out to attain a detailed understanding of bottromycin biosynthesis, using structural biology and biochemistry. We intended to translate these insights into a system for the production of novel bottromycin variants in sufficient quantities for small-scale biological testing. We have determined the crystal structures of five key biosynthetic enzymes and elucidated the mechanisms of six enzymes. This includes mechanistic work on hitherto unprecedented biochemical transformations. As a result of our work we now have a facile, two-pot biosynthesis of the bottromycin core scaffold with excellent yields. In the coming months we are going to further optimize the system to arrive at a true one-pot system compatible with high-throughput production of bottromycin derivatives for biological testing.

Publications

• (2016): Structure and substrate recognition of the Bottromycin maturation enzyme BotP. Chembiochem, 17(23), pp. 2286-92
  Mann G, Huo L, Adam S, Nardone B, Vendome J, Westwood NJ, Müller R, Köhnke J
  
• (2017): Macroamidine formation in bottromycins is catalyzed by a divergent YcaO enzyme. J Am Chem Soc. 139(50), pp. 18158-61
  Franz L, Adam S, Santos-Aberturas J, Truman AW, Köhnke J
  
• (2019): The role of protein–protein interactions in the biosynthesis of ribosomally synthesized and post-translationally modified peptides. Nat Prod Rep 36(11), pp. 1576-88
  Sikandar A, Koehnke J
  
• (2019): Thiazoline-Specific Amidohydrolase PurAH Is the Gatekeeper of Bottromycin Biosynthesis. J Am Chem Soc. 141(25), pp. 9748-52
  Sikandar A, Franz L, Melse O, Antes I, Köhnke J
  
• (2020): Characterization of the Stereoselective P450 Enzyme BotCYP Enables the In Vitro Biosynthesis of the Bottromycin Core. J Am Chem Soc. 142, 49, pp. 20560-5
  Sikandar A, Franz L, Milhim M, Bernhardt R, Kalinina OV, Köhnke J
  
• (2020): New developments in RiPP discovery, enzymology and engineering. Nat Prod Rep
  Montalbán-López M, Scott TA, Ramesh S, Rahman IR, van Heel AJ, Viel JH, Bandarian V, Dittmann E, Genilloud O, Goto Y, Grande Burgos MJ, Hill C, Kim S, Köhnke J, Latham JA, Link AJ, Martínez B, Nair SK, Nicolet Y, Rebuffat S, Sahl HG, Sareen D, Schmidt EW, Schmitt L, Severinov K, Süssmuth RD, Truman AW, Wang H, Weng JK, van Wezel GP, Zhang Q, Zhong J, Piel J, Mitchell DA, Kuipers OP, van der Donk WA
  
• (2020): The bottromycin epimerase BotH defines a group of atypical α/β-hydrolase-fold enzymes. Nat Chem Biol 16(9), pp. 1013-8
  Sikandar A, Franz L, Adam S, Santos-Aberturas J, Horbal L, Luzhetskyy A, Truman AW, Kalinina OV, Köhnke J
  
• (2021): Bottromycins – Biosynthesis, Synthesis and Activity. Nat Prod Rep
  Franz L, Kazmaier U, Truman AW, Koehnke J