Ligify: Automated Genome Mining for Ligand-Inducible Transcription Factors.

Prokaryotic transcription factors can be repurposed into biosensors for the ligand-inducible control of gene expression, but the landscape of chemical ligands for which biosensors exist is extremely limited. To expand this landscape, we developed Ligify, a web application that leverages information in enzyme reaction databases to predict transcription factors that may be responsive to user-defined chemicals. Candidate transcription factors are then incorporated into automatically generated plasmid sequences that are designed to express GFP in response to the target chemical.

A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules.

Halogen-containing molecules are ubiquitous in modern society and present unique chemical possibilities. As a whole, de novo fermentation and synthetic pathway construction for these molecules remain relatively underexplored and could unlock molecules with exciting new applications in industries ranging from textiles to agrochemicals to pharmaceuticals. Here, we report a mix-and-match co-culture platform to de novo generate a large array of halogenated tryptophan derivatives in Escherichia coli from glucose.

Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan.

Directed evolution is often limited by the throughput of accurate screening methods. Here we demonstrate the feasibility of utilizing a singular transcription factor (TF)-system that can be refactored in two ways (both as an activator and repressor). Specifically, we showcase the use of previously evolved 5-halo- or 6-halo-tryptophan-specific TF biosensors suitable for the detection of a halogenated tryptophan molecule .

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus.

Snowprint: a predictive tool for genetic biosensor discovery.

Bioengineers increasingly rely on ligand-inducible transcription regulators for chemical-responsive control of gene expression, yet the number of regulators available is limited. Novel regulators can be mined from genomes, but an inadequate understanding of their DNA specificity complicates genetic design. Here we present Snowprint, a simple yet powerful bioinformatic tool for predicting regulator:operator interactions.

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 multilayer 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.

Using fungible biosensors to evolve improved alkaloid biosyntheses.

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can 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 combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point, we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC) < 30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine and noscapine.

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 .

Sorting for secreted molecule production using a biosensor-in-microdroplet approach.

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection.

Directed Evolution of an Improved Aminoacyl-tRNA Synthetase for Incorporation of L-3,4-Dihydroxyphenylalanine (L-DOPA).

The catechol group of 3,4-dihydroxyphenylalanine (L-DOPA) derived from L-tyrosine oxidation is a key post-translational modification (PTM) in many protein biomaterials and has potential as a bioorthogonal handle for precision protein conjugation applications such as antibody-drug conjugates. Despite this potential, indiscriminate enzymatic modification of exposed tyrosine residues or complete replacement of tyrosine using auxotrophic hosts remains the preferred method of introducing the catechol moiety into proteins, which precludes many protein engineering applications. We have developed new orthogonal translation machinery to site-specifically incorporate L-DOPA into recombinant proteins and a new fluorescent biosensor to selectively monitor L-DOPA incorporation in vivo.

FABP5 coordinates lipid signaling that promotes prostate cancer metastasis.

Prostate cancer (PCa) is defined by dysregulated lipid signaling and is characterized by upregulation of lipid metabolism-related genes including fatty acid binding protein 5 (FABP5), fatty acid synthase (FASN), and monoacylglycerol lipase (MAGL). FASN and MAGL are enzymes that generate cellular fatty acid pools while FABP5 is an intracellular chaperone that delivers fatty acids to nuclear receptors to enhance PCa metastasis. Since FABP5, FASN, and MAGL have been independently implicated in PCa progression, we hypothesized that FABP5 represents a central mechanism linking cytosolic lipid metabolism to pro-metastatic nuclear receptor signaling.

Improving ionic liquid tolerance in Saccharomyces cerevisiae through heterologous expression and directed evolution of an ILT1 homolog from Yarrowia lipolytica.

Ionic liquids show promise for deconstruction of lignocellulosic biomass prior to fermentation. Yet, imidazolium ionic liquids (IILs) can be toxic to microbes even at concentrations present after recovery. Here, we show that dominant overexpression of an Ilt1p homolog (encoded by YlILT1/YALI0C04884) from the IIL-tolerant yeast Yarrowia lipolytica confers an improvement in 1-ethyl-3-methylimidazolium acetate tolerance in Saccharomyces cerevisiae compared to the endogenous Ilt1p (ScILT1/YDR090C).

Synthetic evolution.

The combination of modern biotechnologies such as DNA synthesis, λ red recombineering, CRISPR-based editing and next-generation high-throughput sequencing increasingly enables precise manipulation of genes and genomes. Beyond rational design, these technologies also enable the targeted, and potentially continuous, introduction of multiple mutations. While this might seem to be merely a return to natural selection, the ability to target evolution greatly reduces fitness burdens and focuses mutation and selection on those genes and traits that best contribute to a desired phenotype, ultimately throwing evolution into fast forward.

Custom selenoprotein production enabled by laboratory evolution of recoded bacterial strains.

Incorporation of the rare amino acid selenocysteine to form diselenide bonds can improve stability and function of synthetic peptide therapeutics. However, application of this approach to recombinant proteins has been hampered by heterogeneous incorporation, low selenoprotein yields, and poor fitness of bacterial producer strains. We report the evolution of recoded Escherichia coli strains with improved fitness that are superior hosts for recombinant selenoprotein production.

Lipocalin-Type Prostaglandin D Synthase Is a Novel Phytocannabinoid-Binding Protein.

Lipocalin-type prostaglandin D synthase (L-PGDS; EC:5.3.99.

Continuous directed evolution for strain and protein engineering.

Continuous directed evolution is the 'while loop' of synthetic biology, autonomous cycles of mutation, selection, and self-replication that can lead to the rapid development of industrially relevant organisms, pathways, or molecules. Although this engineering strategy requires particular mutagenesis methods and well-defined selections, recent advances have facilitated its implementation. Control over selection pressure has been augmented by novel cost-effective continuous culturing devices with open source designs.