Use of sigma factor M 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 () recognizing specific promoter sequences, which are not recognised by the native factors of E. coli and is expressed upon entering the stationary phase. First, several 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 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 S 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

A sigma factor toolbox for orthogonal gene expression in Escherichia coli

Synthetic genetic sensors and circuits enable programmable control over timing and conditions of gene expression and, as a result, are increasingly incorporated into the control of complex and multi-gene pathways. Size and complexity of genetic circuits are growing, but stay limited by a shortage of regulatory parts that can be used without interference. Therefore, orthogonal expression and regulation systems are needed to minimize undesired crosstalk and allow for dynamic control of separate modules. This work presents a set of orthogonal expression systems for use in Escherichia coli based on heterologous sigma factors from Bacillus subtilis that recognize specific promoter sequences. Up to four of the analyzed sigma factors can be combined to function orthogonally between each other and toward the host. Additionally, the toolbox is expanded by creating promoter libraries for three sigma factors without loss of their orthogonal nature. As this set covers a wide range of transcription initiation frequencies, it enables tuning of multiple outputs of the circuit in response to different sensory signals in an orthogonal manner. This sigma factor toolbox constitutes an interesting expansion of the synthetic biology toolbox and may contribute to the assembly of more complex synthetic genetic systems in the future

The protein–DNA contacts in RutR·carAB operator complexes

Pyrimidine-specific regulation of the upstream carP1 promoter of the carbamoylphosphate synthase operon of Escherichia coli requires numerous trans-acting factors: the allosteric transcription regulator RutR, the nucleoid-associated protein integration host factor, and the trigger enzymes aminopeptidase A and PyrH (UMP-kinase). RutR, a TetR family member, binds far upstream of carP1. Here, we establish a high-resolution contact map of RutR•carP1 complexes for backbone and base-specific contacts, analyze DNA bending, determine the DNA sequence specificity of RutR binding by saturation mutagenesis, demonstrate that uracil but not thymine is the physiologically relevant ligand that inhibits the DNA binding capacity of RutR and build a model of the RutR·operator DNA complex based on the crystal structures of RutR and of the DNA-bound family member QacR. Finally, we test the validity of this model with site-directed mutagenesis of the helix–turn–helix DNA binding motif and in vitro binding studies with the cognate purified mutant RutR proteins

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 () recognizing specific promoter sequences, which are not recognised by the native factors of E. coli and is expressed upon entering the stationary phase. First, several 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 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 S 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

Unlocking the bacterial domain for industrial biotechnology applications using universal parts and tools

Synthetic biology can play a major role in the development of sustainable industrial biotechnology processes. However, the development of economically viable production processes is currently hampered by the limited availability of host organisms that can be engineered for a specific production process. To date, standard hosts such as Escherichia coli and Saccharomyces cerevisiae are often used as starting points for process development since parts and tools allowing their engineering are readily available. However, their suboptimal metabolic background or impaired performance at industrial scale for a desired production process, can result in increased costs associated with process development and/or disappointing production titres. Building a universal and portable gene expression system allowing genetic engineering of hosts across the bacterial domain would unlock the bacterial domain for industrial biotechnology applications in a highly standardized manner and, doing so, render industrial biotechnology processes more competitive compared to the current polluting chemical pro-cesses. This review gives an overview of a selection of bacterial hosts highly interesting for industrial biotech-nology based on both their metabolic and process optimization properties. Moreover, the requirements and progress made so far to enable universal, standardized, and portable gene expression across the bacterial domain is discussed