A tight cold-inducible switch built by coupling thermosensitive transcriptional and proteolytic regulatory parts

Abstract

Natural organisms have evolved intricate regulatory mechanisms that sense and respond to fluctuating environmental temperatures in a heat- or cold-inducible fashion. Unlike dominant heat-inducible switches, very few cold-inducible genetic switches are available in either natural or engineered systems. Moreover, the available cold-inducible switches still have many shortcomings, including high leaky gene expression, small dynamic range (<10-fold) or broad transition temperature (>10°C). To address these problems, a high-performance cold-inducible switch that can tightly control target gene expression is highly desired. Here, we introduce a tight and fast cold-inducible switch that couples two evolved thermosensitive variants, TFts and TEVts, as well as an additional Mycoplasma florum Lon protease (mf-Lon) to effectively turn-off target gene expression via transcriptional and proteolytic mechanisms. We validated the function of the switch in different culture media and various Escherichia coli strains and demonstrated its tightness by regulating two morphogenetic bacterial genes and expressing three heat-unstable recombinant proteins, respectively. Moreover, the additional protease module enabled the cold-inducible switch to actively remove the pre-existing proteins in slow-growing cells. This work establishes a high-performance cold-inducible system for tight and fast control of gene expression which has great potential for basic research, as well as industrial and biomedical applications.

Development of a growth coupled dynamic regulation network balancing malonyl-CoA node to enhance (2S)-naringenin synthesis inE. coli

“…6c). The mutant library sizes were 2.02×10 7 and 1.5×10 6 for module B and C, respectively. After fermentation, the high naringenin-producing strains were sorted ( Supplementary Fig.…”

“…Generally, biosensors that could respond to universal, environmental induction factors such as temperature (6), dissolved oxygen (5), and light (7) enable construction of environment-responding dynamic regulation networks (DRNs). For example, Zhao et al established a blue light inducible transcription factor (VP16-EL222) controlled OptoEXP system for isobutanol production and demonstrated a 4-fold improvement in the titer compared with the strain lacking dynamic regulatory circuits (8).…”

Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation

“…Although reasonable fold changes were observed in current systems, it can be further improved to suit specific applications potentially via the addition of positive feedbacks (Nistala et al, 2010) and/or introducing temperature-sensitive proteolytic modules (Yang Zheng et al, 2019).…”

“…There are several notable examples of transcriptional based heat-inducible systems as described (Fu et al, 2020;Hussaina et al, 2014;Pearce et al, 2017;Piraner et al, 2017). However, there are limited heat-repressible systems which rely on transcriptional regulation (Liang et al, 2007;Yang Zheng et al, 2019). A recent work has performed temperature-based screening on the modified bacteriophage λ Cl434 repressor that contained the tobacco etch virus (TEV) cleavage site (Yang Zheng et al, 2019).…”

Dynamic control in metabolic engineering: Theories, tools, and applications

“…2C ). Cold-inducible switches have also been developed via directed evolution ( Zheng et al, 2019 ). Temperature induction also has limitations: (1) heat can alter protein folded structures and folding dynamics; (2) large or rapid change in temperature can trigger heat- or cold-shock responses and alter the global regulatory and metabolic networks; and (3) implementing temperature change in large fermenters uniformly can be slow due to heat transfer limitations.…”