A synthetic growth switch based on controlled expression of RNA polymerase

Abstract

The ability to control growth is essential for fundamental studies of bacterial physiology and biotechnological applications. We have engineered an Escherichia coli strain in which the transcription of a key component of the gene expression machinery, RNA polymerase, is under the control of an inducible promoter. By changing the inducer concentration in the medium, we can adjust the RNA polymerase concentration and thereby switch bacterial growth between zero and the maximal growth rate supported by the medium. We show that our synthetic growth switch functions in a medium‐independent and reversible way, and we provide evidence that the switching phenotype arises from the ultrasensitive response of the growth rate to the concentration of RNA polymerase. We present an application of the growth switch in which both the wild‐type E. coli strain and our modified strain are endowed with the capacity to produce glycerol when growing on glucose. Cells in which growth has been switched off continue to be metabolically active and harness the energy gain to produce glycerol at a twofold higher yield than in cells with natural control of RNA polymerase expression. Remarkably, without any further optimization, the improved yield is close to the theoretical maximum computed from a flux balance model of E. coli metabolism. The proposed synthetic growth switch is a promising tool for gaining a better understanding of bacterial physiology and for applications in synthetic biology and biotechnology.

Optimal feedback strategies for bacterial growth with degradation, recycling, and effect of temperature

“…Regulations of an external inducer (based on specific chemical or light effects) can modify cellular processes [3]. Then it seems promising to consider the problem of finding an inducing strategy that maximizes the biotechnological production.…”

“…For both fundamental biology and engineering applications, it is relevant to investigate how microorganisms adapt to changing environmental conditions [1][2][3]. In compliance with evolutionary principles, internal regulation mechanisms of bacteria are expected to maximize appropriate fitness criteria.…”

Dynamic control of endogenous metabolism with combinatorial logic circuits

“…As the circuits get larger, one can imagine implementing complex control over a metabolic pathway by turning on different portions of the pathway at different times, coordinating stress responses, and staging a process to including steps before (seeding and growth) and after (biomass recycling or disposal) the production phase. Examples of advanced approaches include the following: (i) only expressing oxygen‐sensitive enzymes under anaerobic conditions (Burgard et al , ; Immethun et al , ), (ii) transiently responding to localized stresses within larger bioreactors (Bylund et al , ; Enfors et al , ), (iii) lysis system for product recovery (Borrero‐de Acuna et al , ), (iv) flocculation for sedimentation for biomass removal and inhibition of cell growth (You et al , ; Izard et al , ), and (iv) elimination of synthetic DNA before disposal (Caliando & Voigt, ; Chan et al , ). Such approaches have been implemented individually, but one can envision linking many together into one large system.…”

Computational design of Small Transcription Activating RNAs (STARs) for versatile and dynamic gene regulation

“…Finally, the relatively small size, functionality on the genome, and gene-independent regulation could make STARs ideally suited as a tool for strain engineering and reprogramming of cellular phenotypes. For example, STARs could be used to create synthetic switches for decoupling biomass and pathway production 57,58 .…”