Adaptive lab evolution and omics characterization enable tolerance engineering of Pseudomonas putida KT2440 to produce the jet-fuel precursor isoprenol

ABSTRACT: Pseudomonas putida KT2440 (KT2440) has been established as an industrially relevant chassis for the production of the jetfuel precursor isoprenol. However, this strain assimilated isoprenol as sole carbon source and also showed impaired growth at isoprenol concentrations > 2 g/L which is a significant impediment to high TRY production. In this study, we used tolerization adaptive laboratory evolution and functional genomics to demonstrate improved growth of KT2440 strains in the presence of up to 8 g/L isoprenol. DNA sequencing of the evolved strains revealed mutations – a combination of SNPs and large deletions acquired in response to both isoprenol and a trace formaldehyde impurity. Mutations associated to isoprenol tolerance in the evolved isolates were specific to SNPs in the transcriptional regulators gnuR, xxxR and xxxR and two large deletions in the regions that also encoded the multidrug efflux pump TtgABC and certain phage proteins. Proteomic analysis of selected evolved strains implied a concerted detoxification process for isoprenol and formaldehyde even in the absence of the offending trace contaminant. The evolved strains also activated different pathways to enhanced tolerance. These included either upregulation of a global stringent response regulator SpoT in one or by the action of multiple alcohol and other dehydrogenases

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold  

LAB HEAD: Christopher J.

PROVIDER: PXD054609 | Pride | 2025-05-28

REPOSITORIES: Pride