Exploring Biosynthetic Pathways for the Production of Five Methyl Ethyl Ketone Precursors

Miskovic, L., EPFL
Tokic, M., Swiss Federal Institute of Technology (EPFL)
Hadadi, N., Swiss Institute of Bioinformatics (SIB)
Ataman, M., EPFL
Neves, D., RWTH Aachen University
Ebert, B. E., RWTH Aachen University
Blank, L. M., RWTH Aachen University
Hatzimanikatis, V., Swiss Federal Institute of Technology (EPFL)
Limited reserves of oil and natural gas and the establishment of new environmental policies sparked off intensive research towards sustainable production of the 2nd generation of biofuels, with Methyl Ethyl Ketone (MEK) being one promising fuel candidate. MEK is a commercially valuable petrochemical with an extensive application as a solvent. However, as of today, a sustainable and economically viable production of MEK has not yet been achieved despite several attempts of introducing biosynthetic pathways in industrial microorganisms. We used BNICE.ch as a retrobiosynthesis tool to discover all novel pathways around MEK. Out of 1’325 identified compounds connecting to MEK with one reaction step, we selected 3-oxopentanoate, but-3-en-2-one, but-1-en-2-olate, butylamine, and 2-hydroxy-2-methyl-butanenitrile for further study. We reconstructed 3’679’610 novel biosynthetic pathways towards these 5 compounds. We then embedded these pathways into the genome-scale model of E. coli, and a set of 18’622 were found to be most biologically feasible ones based on thermodynamics and their yields. For each novel reaction in the viable pathways, we proposed the most similar KEGG reactions, with their gene and protein sequences, as candidates for either a direct experimental implementation or as a basis for enzyme engineering. Through pathway similarity analysis we classified the pathways and identified the enzymes and precursors that were vital for the production of the target molecules. These retrobiosynthesis studies demonstrate the potential of BNICE.ch for discovery, systematic evaluation, and analysis of novel pathways in synthetic biology and metabolic engineering studies.