Metabolic Checkpoint Aldehyde Dehydrogenases Are Important for Diverting β-Oxidation into 1-Butanol Biosynthesis from Kitchen Waste Oil in Pseudomonas aeruginosa.

Abstract

1-Butanol (1-BD) is a promising fuel additive which can be biosynthesized via reversed β-oxidation pathway in bacteria. However, heterologous reversed β-oxidation pathway is a carbon chain prolongation process with several genes overexpressed in most of bacterial hosts, leading to low titer of 1-BD and high cost for production. Here we displayed a forward β-oxidation pathway for 1-BD production in a kitchen waste oil (KWO) degrading Pseudomonas aeruginosa PA-3, and we proved that aldehyde dehydrogenase (ALDH) is a checkpoint for diverting metabolic flux into 1-BD biosynthesis. With nitrogen source supplied, titer of 1-BD was increased accompanied with 12 ALDH coding genes transcriptionally promoted to different degrees. At the same time, binding energies of these ALDHs with different length of acyl-CoAs in β-oxidation were calculated to identify their specificities. Based on the above information, ALDH deletions were conducted. We certified that deletion of ALDH8 and ALDH9 led to significant decreased titers of 1-BD. Finally, these two ALDHs were separately overexpressed in PA-3, and titer of 1-BD was promoted to 1.36g/L at 72h in shake flask. Totally in this work, we provided a forward β-oxidation pathway for 1-BD production from KWO, and the roles of ALDHs were confirmed.