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

Since their discovery, antibiotics have been medicinal products with greatest impact on human society. Meanwhile, they are absolutely essential in modern medicine. Unfortunately, the easy access to antibiotics and inappropriate antibiotic treatments on a global scale have led to an increasingly rapid development of antibiotic resistance in threatening human pathogens. Coupled to a severe reduction of antimicrobial research by pharmaceutical companies over the past 20 years, this has led to antibiotic resistance becoming a major threat to global public health. Traditional approaches of fighting resistance - total chemical synthesis of derivatives or semisynthesis - are no longer sufficient. As a consequence, we face an urgent unmet medical need for new antibiotics with significantly novel chemical scaffolds and new targets/mode-of-action (MOA). Bottromycins were originally discovered as antibacterial peptides of unknown biosynthetic origin with promising activity against Gram-positive bacteria. The naturally occurring variant bottromycin A2 is highly effective against major Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococci (VRE). More importantly, they provide a completely new scaffold with a molecular target at the A-site of the prokaryotic 50S ribosome, not tackled by any other antibiotic used in the clinic. In 2012, the biosynthetic origin of bottromycins was finally elucidated and the responsible gene cluster published – they belong to the growing family of ribosomally produced and post-translationally modified peptides (RiPPs). Like all RiPPs, the biosynthesis of bottromycins starts with the expression of a precursor peptide, which contains a core peptide (the eventual natural product), and, uniquely amongst bacterial RiPPs, a follower peptide (important for enzymatic recognition of the precursor peptide). We have established the order of biosynthetic reactions leading to bottromycins through a combined approach, using biochemistry and structural biology. In the process, we have identified a key regulator of bottromycin biosynthesis and an unusual resistance protein. Building on these data, we will determine the structure of the bottromycin macrocyclase, an unusual YcaO enzyme responsible for macroamidine formation. Four-amino-acid macrocycles are extremely challenging to obtain by organic synthesis. Using the structural insights will allow us to expand the catalytic scope of this enzyme to produce bespoke, four-amino-acid macrocycles and explore their potential. In addition, we will investigate the formation of the D-amino acid found in bottromycins, which is epimerized from an L-amino acid during biosynthesis by an as of yet unknown mechanism.

• DFG Programme: Independent Junior Research Groups