Biology subjects

d'Oelsnitz, S.

Publications and source records attributed to d'Oelsnitz, S..

4 recordsLinked to original sources

bioRxiv2022

Directed evolution of generalist biosensors for single ring aromatics

Biosensors can accelerate the engineering of new biosynthetic pathways. Phloroglucinol is a platform chemical of wide utility that can be produced at limited titers in Escherichia coli. Starting from the TetR family repressor RolR that is responsive to the related compound resorcinol, we were able to employ a combined selection and screen to identify variants that had greatly improved activities with phloroglucinol (EC50 for phloroglucinol of 131 uM, relative to an estimated 42 mM for wild-type RolR). The variants obtained were further screened with a panel of similar single ring aromatics, and several were found to be generalists, consistent with the hypothesis that both natural and directed evolution tend to first create semi-specific pockets prior to further optimization for new function

synthetic biology↗

bioRxiv2022

GroovDB: A database of ligand-inducible transcription factors

Genetic biosensors are integral to synthetic biology. In particular, ligand-inducible prokaryotic transcription factors are frequently used in high-throughput screening, for dynamic feedback regulation, as multi-layer logic gates, and in diagnostic applications. In order to provide a curated source that users can rely on for engineering applications, we have developed GroovDB (available at https://groov.bio), a Web-accessible database of ligand-inducible transcription factors that contains all information necessary to build chemically-responsive genetic circuits, including biosensor sequence, ligand, and operator data. Ligand and DNA interaction data has been verified against the literature, while an automated data curation pipeline is used to programmatically fetch metadata, structural information, and references for every entry. A custom tool to visualize the natural genetic context of biosensor entries provides additional information that provides potential insights into alternative ligands and systems biology.

synthetic biology↗

bioRxiv2021

Evolving a generalist biosensor for bicyclic monoterpenes

Prokaryotic transcription factors can be repurposed as analytical and synthetic tools for precise chemical measurement and regulation. Monoterpenes encompass a broad chemical family that are commercially valuable as flavors, cosmetics, and fragrances, but have proven difficult to measure, especially in cells. Herein, we develop genetically-encoded, generalist monoterpene biosensors by using directed evolution to expand the effector specificity of the camphor-responsive TetR-family regulator CamR from Pseudomonas putida. Using a novel negative selection coupled with a high-throughput positive screen (Seamless Enrichment of Ligand-Inducible Sensors, SELIS), we evolve CamR biosensors that can recognize four distinct monoterpenes: borneol, fenchol, eucalyptol, and camphene. Different evolutionary trajectories surprisingly yielded common mutations, emphasizing the utility of CamR as a platform for creating generalist biosensors. Systematic promoter optimization driving the reporter increased the systems signal-to-noise ratio to 150-fold. These sensors can serve as a starting point for the high-throughput screening and dynamic regulation of bicyclic monoterpene production strains.

synthetic biology↗

bioRxiv2021

Using structurally fungible biosensors to evolve improved alkaloid biosyntheses

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can greatly improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a unique combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors, and using RamR as a starting point we evolve highly specific (>100-fold preference) and sensitive (EC50 <30 M) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine, and noscapine. High resolution structures reveal multiple evolutionary avenues for the fungible effector binding site, and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, an immediate precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors now enable the rapid engineering of pathways for therapeutic alkaloids.

synthetic biology↗