Use of sigma factors from Bacillus subtilis in the development of an orthogonal expression system in Escherichia coli

Background: Technological advances in synthetic biology, systems biology, and metabolic engineering have boosted applications of 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 regulatory mechanisms. Hence, fine-tuning expression of synthesis pathways in specific conditions is mandatory. Objectives: Here we develop a new genetic circuit for regulated expression specifically in stationary phase due to clear advantages during this period (reduction of toxicity, competition). Methods: This circuit consists of a heterologous sigma factor (𝛔) recognizing specific promoter sequences, which are not recognised by the native 𝛔 factors of E. coli and is expressed upon entering the stationary phase. First, several 𝛔 factors of B. subtilis were tested for their orthogonality in E. coli on the level of promoter recognition, by using a red-fluorescent reporter system. Secondly, the potential of 𝛔 factors of B. subtilis to work together with the E. coli core RNA polymerase was tested, by expressing these proteins together with their promoters. Based on the results a specific factor will be chosen for further optimalisation and the corresponding gene can be cloned in the 𝛔S factor operon of E. coli, which is most abundantly expressed in stationary conditions. Conclusions: Combining all these elements should allow us to create an orthogonal genetic circuit that is able to transcribe specific genes under stationary phase with a limited influence on the host cell's metabolism.

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