Bacterial two-component systems as sensors for synthetic biology applications.

Two-component systems (TCSs) are a ubiquitous family of signal transduction pathways that enable bacteria to sense and respond to diverse physical, chemical, and biological stimuli outside and inside the cell. Synthetic biologists have begun to repurpose TCSs for applications in optogenetics, materials science, gut microbiome engineering, and soil nutrient biosensing, among others. New engineering methods including genetic refactoring, DNA-binding domain swapping, detection threshold tuning, and phosphorylation cross-talk insulation are being used to increase the reliability of TCS sensor performance and tailor TCS signaling properties to the requirements of specific applications. There is now potential to combine these methods with large-scale gene synthesis and laboratory screening to discover the inputs sensed by many uncharacterized TCSs and develop a large new family of genetically-encoded sensors that respond to an unrivaled breadth of stimuli.

Dynamic Control of Gene Expression with Riboregulated Switchable Feedback Promoters.

One major challenge in synthetic biology is the deleterious impacts of cellular stress caused by expression of heterologous pathways, sensors, and circuits. Feedback control and dynamic regulation are broadly proposed strategies to mitigate this cellular stress by optimizing gene expression levels temporally and in response to biological cues. While a variety of approaches for feedback implementation exist, they are often complex and cannot be easily manipulated. Here, we report a strategy that uses RNA transcriptional regulators to integrate additional layers of control over the output of natural and engineered feedback responsive circuits. Called riboregulated switchable feedback promoters (rSFPs), these gene expression cassettes can be modularly activated using multiple mechanisms, from manual induction to autonomous quorum sensing, allowing control over the timing, magnitude, and autonomy of expression. We develop rSFPs in Escherichia coli to regulate multiple feedback networks and apply them to control the output of two metabolic pathways. We envision that rSFPs will become a valuable tool for flexible and dynamic control of gene expression in metabolic engineering, biological therapeutic production, and many other applications.

Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases.

The crystal structure is reported of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD). The structure was refined at 2.2 Šresolution with four polypeptide chains in the asymmetric unit. Based on the results of pairwise structure alignments, PobA from P. putida is structurally very similar to PobA from P. fluorescens and from P. aeruginosa. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Additionally, the structure was compared with two enzymes from the broader class of oxygenases: 2-hydroxybiphenyl 3-monooxygenase (HbpA) from P. nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from P. putida with HbpA from P. nitroreducens and MHPCO from M. japonicum revealed local similarities between these structures. Key secondary-structure elements important for catalysis, such as the ßαß fold, ß-sheet wall and α12 helix, are conserved across this expanded class of oxygenases.