Cyclopropanes-functionalized hydrocarbons are excellent fuels. however, their synthesis is challenging and harmful for the environment. In this work we produced polycyclopropanated fatty acids in bacteria. These molecules can be easily converted into renewable fuels for high energy applications such as shipping, long-haul transport, aviation, and rocketry. We explored the chemical diversity encoded in the genome of thousands of bacteria to identify and repurpose naturally occurring cyclopropanated molecules. We identified a set of candidate iterative Polyketide Synthases (iPKSs) predicted to produce polycyclopropanated fatty acids (POP-FAs), expressed these PKSs in Streptomyces coelicolor and produced the POP-FAs. We determined the structure of the molecules and increased their production 22-fold. Polycyclopropanated fatty acid methyl esters (POP-FAMEs) were obtained by methyl esterifying the POP-FAs. Finally, we calculated the enthalpy of combustion of several POP fuel candidates to assess their potential as replacement for fossil fuels in energy demanding applications. Our research shows that POP-FAMEs and other polyketide derived POPs have the energetic properties for energy demanding applications for which sustainable alternatives are scarce.

Proteins from E. coli were extracted using a previously described chloroform/methanol precipitation method. The cell pellets (~10 OD/mL) were resuspended in 400 μl of methanol and briefly vortexed, followed by sequential additions of 100 μl of chloroform and 300 μl of water with short intervals of vortexing in between. For Streptomyces samples, cell pellets were resuspended in 1% octyl glucopyranoside solution and incubated at 100°C for 10 minutes, followed by centrifugation at 10,817 x g for 1 minute. The supernatant was decanted and a standard chloroform/methanol precipitation method was applied to the treated cell pellet to extract proteins. For both cases, extracted proteins were resuspended in 100 mM ammonium bicarbonate buffer supplemented with 20% methanol, and protein concentration was determined by the DC assay (BioRad). Protein reduction was accomplished using 5 mM tris 2-(carboxyethyl)phosphine (TCEP) for 30 min at room temperature, and alkylation was performed with 10 mM iodoacetamide (IAM; final concentration) for 30 min at room temperature in the dark. Overnight digestion with trypsin was accomplished with a 1:50 trypsin:total protein ratio. The resulting peptide samples were analyzed on an Agilent 1290 UHPLC system coupled to a Thermo scientific Obitrap Exploris 480 mass spectrometer for discovery proteomics and on an Agilent 1290 UHPLC system coupled to an Agilent 6460QqQ mass spectrometer (Agilent Technologies) for targeted proteomics analyses. Briefly, peptide samples were loaded onto an Ascentis® ES-C18 Column (Sigma–Aldrich) and introduced to the mass spectrometer operating in positive-ion mode. Multiple reaction monitoring (MRM) transitions for the targeted proteins were generated by Skyline software (MacCoss Lab Software) and selection criteria excluded peptides with Met/Cys residues, tryptic peptides followed by additional cut sites (KK/RR), and peptides with proline adjacent to K/R cut sites.