Short, auto-inducible promoters for well-controlled protein expression in Escherichia coli.

Expression of recombinant proteins in Escherichia coli often requires use of inducible promoters to shorten the lag phase and improve protein productivity and final protein titer. Synthetic molecules that cannot be metabolized by E. coli, such as isopropyl thiogalactopyranoside (IPTG), have been frequently used to trigger the protein expression during early exponential growth phase. This practice has many drawbacks, including high cost and toxicity of IPTG, complex operating procedure, and non-uniform protein expression pattern (some cells in the population do not express recombinant proteins). A few auto-inducible protein expression systems have been developed recently to overcome some of these limitations, but they required use of an additional plasmid or presence of large (a few kilobases) DNA part to be functional, making plasmid construction to be difficult, especially when multiple genes need to be expressed. In this study, by using RNA sequencing, we identified a short, endogenous promoter (PthrC) that can be auto-induced during early exponential growth phase, and improved its performance by use of native and mutated regulatory elements. We found that the developed mutants of PthrC drove uniform protein expression-close to 100% of cells were fluorescent when green fluorescence protein was used as target protein-and cells carrying them could achieve much higher cell density than those with T7 promoter (PT7), a commonly used inducible promoter. In terms of promoter strength (product protein quantity per cell), the developed promoter mutants can cover a range of strength, from 30 to 150% of maximal strength of PT7. One strong mutant (PthrC3_8) was found to work well at a large range of temperature (22, 30, 37 °C) and in various media, and was also confirmed to cause less stress to host cell than PT7 when they were used to express a toxic protein. We foresee that PthrC3 and its mutants will be useful genetic parts for various applications including metabolic engineering and biocatalysis.