Dataset Information

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):

ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold

LAB HEAD: Christopher J.

PROVIDER: PXD054609 | Pride | 2025-05-28

REPOSITORIES: Pride

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Evolution-guided tolerance engineering of Pseudomonas putida KT2440 for production of the aviation fuel precursor isoprenol.

Lim Hyun Gyu HG Srinivasan Aparajitha A Menchavez Russel R Yunus Ian S IS Noh Myung Hyun MH White Megan M Chen Yan Y Gin Jennifer W JW Palsson Bernhard O BO Lee Taek Soon TS Petzold Christopher J CJ Eng Thomas T Mukhopadhyay Aindrila A Feist Adam M AM

Metabolic engineering 20250519

Isoprenol (3-methyl-3-buten-1-ol) is a precursor to aviation fuels and other commodity chemicals and can be microbially synthesized from renewable carbon streams. Its production has been demonstrated in Pseudomonas putida KT2440 but its titers, rates, and yields have yet to reach commercially viable levels, potentially due to toxicity to the bacterial chassis. We hypothesized that utilization of Tolerization Adaptive Laboratory Evolution (TALE) would generate P. putida hosts more tolerant to iso ...[more]

PMID: 40398593

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Project description:Our group has evaluated the productivity of the alcohol isoprenol (3-methyl-3-buten-1-ol), platform commodity chemical with attractive properties specifically as a diesel fuel blendstock and precursor for the bio-jetfuel DMCO, across a number of microbial hosts. While there are strengths and drawbacks to producing isoprenol in any one microbe, Pseudomonas putida KT2440 has a naturally versatile catabolic profile competent of consuming isoprenol. The pathway for this catabolism was revealed using functional genomics data (RB-TnSeq - Thompson et al) and we speculated that this data could also provide a path to developing an isoprenol biosensor. Refactoring knowledge of the isoprenol catabolism signaling system could provide a readout for intracellular isoprenol, in turn enabling an alternative approach to strain engineering that did not rely on a mechanistic understanding of how a heterologous pathway modulates native metabolism. We define a biosensor as the system where a ligand is recognized by a transcriptional activator, which in turn binds to a cognate DNA sequence driving transcription of a downstream reporter gene. The resulting gene expression linearly increases in response to the initial ligand concentration. There are undoubtedly other biosensor modalities (such as directly converting the ligand into a colored molecule) but the advantage of a biosensor that activates a genetic circuit is that the response can also be integrated back into cellular physiology for high throughput selection with growth based assay. For example, strains can be devised where cell growth is concomitant with the increased production of the ligand. Prokaryotic systems are also advantaged over eukaryotes for biosensor development as the determinants of ribosome translation efficiency are dictated by simple RBS sequences adjacent to the start codon as opposed to complex transcriptional regulators, enabling fine protein level expression tuning while maintaining inducibility.

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