Production of Propene from n-Butanol: A Three-Step Cascade Utilizing the Cytochrome P450 Fatty Acid Decarboxylase OleT_JE

Propene is one of the most important starting materials in the chemical industry. Herein, we report an enzymatic cascade reaction for the biocatalytic production of propene starting from n-butanol, thus offering a biobased production from glucose. In order to create an efficient system, we faced the issue of an optimal cofactor supply for the fatty acid decarboxylase OleT_JE, which is said to be driven by either NAD(P)H or H₂O₂. In the first system, we used an alcohol and aldehyde dehydrogenase coupled to OleT_JE by the electron-transfer complex putidaredoxin reductase/putidaredoxin, allowing regeneration of the NAD⁺ cofactor. With the second system, we intended full oxidation of n-butanol to butyric acid, generating one equivalent of H₂O₂ that can be used for the oxidative decarboxylation. As the optimal substrate is a long-chain fatty acid, we also tried to create an improved variant for the decarboxylation of butyric acid by using rational protein design. Within a mutational study with 57 designed mutants, we generated the mutant OleT_V292I, which showed a 2.4-fold improvement in propene production in our H₂O₂-driven cascade system and reached total turnover numbers >1000.