A sigma factor toolbox enables orthogonal gene expression for modular control of complex pathways in Escherichia coli

Indra Bervoets, Maarten Van Brempt, Jo Maertens, Marjan De Mey, Daniel Charlier

Research output: Unpublished contribution to conferenceUnpublished abstract

Abstract

Advances in synthetic biology and metabolic engineering have boosted applications in industrial biotechnology for an increasing number of complex and high added-value molecules. In general, the transfer of multi-gene or poorly understood heterologous pathways into the production host leads to imbalances due to lack of adequate gene expression and regulation, which results in high metabolic burden for the host organism and low production efficiencies/yields. Nowadays the size and complexity of genetic circuits is growing, but stay limited by a lack of regulatory parts that can be used without interference. Therefore there is need for orthogonal expression and regulation systems to minimise this undesired crosstalk.

In Bacteria the first step in gene expression is transcription initiation where a multi subunit core RNA polymerase binds to a sigma factor to form the holoenzyme. This complex is able to recognise a specific DNA sequence (called a promoter) and initiate transcription. While the bacterial core RNA polymerase is highly conserved, there are numerous sigma factors varying greatly in sequence, size, and promoter specificity. Most bacteria have several alternative sigma factors that all bind competitively to the core enzyme and target the holoenzyme to distinct classes of promoters in order to change the genetic expression program in response to stress conditions.

Here we develop a set of orthogonal expression systems in Escherichia coli based on a selection of heterologous sigma factors originating from the Gram-positive sporulating soil bacterium Bacillus subtilis. They all belong to the sig-70 family but have different domain make-ups and recognise specific promoter sequences. These expression systems proved to function orthogonal between each other and towards the host. In addition we expand the toolbox by creating promoter libraries for the different sigma factors without loss of their specificity and orthogonal status. As this set is highly orthogonal and covers a wide range of promoter strengths, it enables fine-tuning of multiple independent channels (modular systems) linking the outputs of a circuit to the control of different input signals. Furthermore, this parts toolbox has potential for the assembly of even more complex genetic circuits (/pathways) in the future.

Finally we tested several anti-sigma factors from B. subtilis as orthogonal on/off switches of their cognate sigma factors in E. coli. These could add an additional level of control in modular genetic circuits.
Original languageEnglish
Publication statusUnpublished - 30 Jul 2017
EventGordon Research Conference Synthetic Biology - Stoweflake Conference Center, Stowe VT, United States
Duration: 30 Jul 20174 Aug 2017

Conference

ConferenceGordon Research Conference Synthetic Biology
Abbreviated titleGRC Synthetic Biology
CountryUnited States
CityStowe VT
Period30/07/174/08/17

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