Complete decoupling of bacterial growth from biopolymer production through proteolytic control of enzyme levels

Most current methods for controlling the rate of formation 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 targeted proteins. The 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. The protein degradation process can be reversed by activating the system via addition of an orthogonal inducer (e.g. 3-methylbenzoate) to the culture medium. The system was benchmarked in Escherichia coli by tagging two fluorescent proteins (GFP and mCherry) and further exploited for engineering poly(3-hydroxybutyrate) (PHB) accumulation completely uncoupled 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 directed into PHB formation upon activation of the system. The engineered E. coli strain reached a very high specific rate of PHB accumulation with a polymer content of ca. 72% (w/w) in glucose cultures set in the growth-decoupled mode. Thus, FENIX enables dynamic control of metabolic fluxes in bacterial cell factories by establishing post-translational synthetic switches in the pathway of interest.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC=\"FIGDIR/small/389809_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (34K):\norg.highwire.dtl.DTLVardef@1d9ec08org.highwire.dtl.DTLVardef@1722feorg.highwire.dtl.DTLVardef@fa77b2org.highwire.dtl.DTLVardef@1c8624a_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT C_FIG