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ACS Synth Biol. 2018 Nov 16;7(11):2686-2697. doi: 10.1021/acssynbio.8b00345. Epub 2018 Oct 22.

A Post-translational Metabolic Switch Enables Complete Decoupling of Bacterial Growth from Biopolymer Production in Engineered Escherichia coli.

Author information

1
Environmental Microbiology Group , Centro de Investigaciones Biológicas (CIB-CSIC) , 28040 Madrid , Spain.
2
Systems and Synthetic Biology Program , Centro Nacional de Biotecnología (CNB-CSIC) , 28049 Madrid , Spain.
3
Systems Environmental Microbiology Group, The Novo Nordisk Foundation Center for Biosustainability , Technical University of Denmark , 2800 Kgs Lyngby , Denmark.

Abstract

Most of the current methods for controlling the formation rate of a key protein or enzyme in cell factories rely on the manipulation of target genes within the pathway. In this article, we present a novel synthetic system for post-translational regulation of protein levels, FENIX, which provides both independent control of the steady-state protein level and inducible accumulation of target proteins. The FENIX device is based on the constitutive, proteasome-dependent degradation of the target polypeptide by tagging with a short synthetic, hybrid NIa/SsrA amino acid sequence in the C-terminal domain. Protein production is triggered via addition of an orthogonal inducer ( i.e., 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry), and further exploited to completely uncouple poly(3-hydroxybutyrate) (PHB) accumulation from bacterial growth. By tagging PhaA (3-ketoacyl-CoA thiolase, first step of the route), a dynamic metabolic switch at the acetyl-coenzyme A node was established in such a way that this metabolic precursor could be effectively redirected into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation (0.4 h-1) with a polymer content of ca. 72% (w/w) in glucose cultures in a growth-independent mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.

KEYWORDS:

Escherichia coli; PHB; metabolic engineering; pathway engineering; proteolysis; synthetic biology

PMID:
30346720
DOI:
10.1021/acssynbio.8b00345
[Indexed for MEDLINE]

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