Discovery of Latent Cannabichromene Cyclase Activity in Marine Bacterial Flavoenzymes.

Production of phytocannabinoids remains an area of active scientific interest due to the growing use of cannabis by the public and the underexplored therapeutic potential of the over 100 minor cannabinoids. While phytocannabinoids are biosynthesized by Cannabis sativa and other select plants and fungi, structural analogs and stereoisomers can only be accessed synthetically or through heterologous expression. To date, the bioproduction of cannabinoids has required eukaryotic hosts like yeast since key, native oxidative cyclization enzymes do not express well in bacterial hosts. Here, we report that two marine bacterial flavoenzymes, Clz9 and Tcz9, perform oxidative cyclization reactions on phytocannabinoid precursors to efficiently generate cannabichromene scaffolds. Furthermore, Clz9 and Tcz9 express robustly in bacteria and display significant tolerance to organic solvent and high substrate loading, thereby enabling fermentative production of cannabichromenic acid in Escherichia coli and indicating their potential for biocatalyst development.

Caged luciferins enable rapid multicomponent bioluminescence imaging.

Bioluminescence is a sensitive technique for imaging biological features over time. Historically, though, the modality has been challenging to employ for multiplexed tracking due to a lack of resolvable luciferase-luciferin pairs. Recent years have seen the development of numerous orthogonal probes for multi-parameter imaging. While successful, generating such tools often requires complex syntheses and lengthy enzyme evolution campaigns. This work showcases an alternative strategy for multiplexed bioluminescence that takes advantage of already-orthogonal caged luciferins and established uncaging enzymes. These probes generate unique bioluminescent signals that can be distinguished via a linear unmixing algorithm. Caged luciferins enabled two- and three-component imaging on the minutes time scale. We further showed that the tools can be used in conjunction with endogenous enzymes for multiplexed studies. Collectively, this approach lowers the barrier to multicomponent bioluminescence imaging and can be readily adopted by the broader community.

Expedient Synthesis and Characterization of π-Extended Luciferins.

Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging.

Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

Longitudinal monitoring of individual infection progression in Drosophila melanogaster.

The innate immune system is critical for infection survival. Drosophila melanogaster is a key model for understanding the evolution and dynamics of innate immunity. Current toolsets for fly infection studies are limited in throughput and, because of their destructive nature, cannot generate longitudinal measurements in individual animals. We report a bioluminescent imaging strategy enabling non-invasive characterization of pathogen load. By using Escherichia coli expressing the ilux operon, we demonstrate that photon flux from autobioluminescent bacteria can be used to monitor pathogen loads in individual, living flies. Because animal sacrifice is not necessary to estimate pathogen load, stochastic responses to infection can be characterized in individuals over time. The high temporal resolution of bioluminescence imaging enables visualization of the dynamics of microbial clearance on the hours time-scale. This non-invasive imaging strategy provides a simple and scalable platform to observe changes in pathogen load in vivo over time.

Fluorogenic Cyclopropenones for Multicomponent, Real-Time Imaging.

Fluorogenic bioorthogonal reactions enable biomolecule visualization in real time. These reactions comprise reporters that "light up" upon reaction with complementary partners. While the spectrum of fluorogenic chemistries is expanding, few transformations are compatible with live cells due to cross-reactivities or insufficient signal turn-on. To address the need for more suitable chemistries for cellular imaging, we developed a fluorogenic reaction featuring cyclopropenone reporters and phosphines. The transformation involves regioselective activation and cyclization of cyclopropenones to form coumarin products. With optimal probes, the reaction provides >1600-fold signal turn-on, one of the highest fluorescence enhancements reported to date. The bioorthogonal motifs were evaluated in vitro and in cells. The reaction was also found to be compatible with other common fluorogenic transformations, enabling multicomponent, real-time imaging. Collectively, these data suggest that the cyclopropenone-phosphine reaction will bolster efforts to track biomolecule targets in their native settings.

Coumarin luciferins and mutant luciferases for robust multi-component bioluminescence imaging.

Multi-component bioluminescence imaging requires an expanded collection of luciferase-luciferin pairs that emit far-red or near-infrared light. Toward this end, we prepared a new class of luciferins based on a red-shifted coumarin scaffold. These probes (CouLuc-1s) were accessed in a two-step sequence via direct modification of commercial dyes. The bioluminescent properties of the CouLuc-1 analogs were also characterized, and complementary luciferase enzymes were identified using a two-pronged screening strategy. The optimized enzyme-substrate pairs displayed robust photon outputs and emitted a significant portion of near-infrared light. The CouLuc-1 scaffolds are also structurally distinct from existing probes, enabling rapid multi-component imaging. Collectively, this work provides novel bioluminescent tools along with a blueprint for crafting additional fluorophore-derived probes for multiplexed imaging.

Caged Cumate Enables Proximity-Dependent Control Over Gene Expression.

Cell-cell interactions underlie diverse physiological processes yet remain challenging to examine with conventional imaging tools. Here we report a novel strategy to illuminate cell proximity using transcriptional activators. We repurposed cumate, a small molecule inducer of gene expression, by caging its key carboxylate group with a nitrile. Nitrilase-expressing activator cells released the cage, liberating cumate for consumption by reporter cells. Reporter cells comprising a cumate-responsive switch expressed a target gene when in close proximity to the activator cells. Overall, this strategy provides a versatile platform to image and potentially manipulate cellular interactions over time.

Seeing (and Using) the Light: Recent Developments in Bioluminescence Technology.

Bioluminescence has long been used to image biological processes in vivo. This technology features luciferase enzymes and luciferin small molecules that produce visible light. Bioluminescent photons can be detected in tissues and live organisms, enabling sensitive and noninvasive readouts on physiological function. Traditional applications have focused on tracking cells and gene expression patterns, but new probes are pushing the frontiers of what can be visualized. The past few years have also seen the merger of bioluminescence with optogenetic platforms. Luciferase-luciferin reactions can drive light-activatable proteins, ultimately triggering signal transduction and other downstream events. This review highlights these and other recent advances in bioluminescence technology, with an emphasis on tool development. We showcase how new luciferins and engineered luciferases are expanding the scope of optical imaging. We also highlight how bioluminescent systems are being leveraged not just for sensing-but also controlling-biological processes.