An Isoprenol Biosensor for Combinatorial High Throughput Strain Engineering in Pseudomonas putida

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Putida Kt2440

SUBMITTER: Christopher Petzold  

LAB HEAD: Christopher J. Petzold

PROVIDER: PXD061547 | Pride | 2025-09-12

REPOSITORIES: Pride