Curated Optogenetic Publication Database

Abstract: Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae. Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.

Abstract: Diabetes mellitus and its associated secondary complications have become a pressing global healthcare issue. The current integrated theranostic plan involves a glucometer-tandem pump. However, external condition-responsive insulin delivery systems utilizing rigid glucose sensors pose challenges in on-demand, long-term insulin administration. To overcome these challenges, we present a novel model of antidiabetic management based on printable metallo-nucleotide hydrogels and optogenetic engineering. The conductive hydrogels were self-assembled by bioorthogonal chemistry using oligonucleotides, carbon nanotubes, and glucose oxidase, enabling continuous glucose monitoring in a broad range (0.5-40 mM). The optogenetically engineered cells were enabled glucose regulation in type I diabetic mice via a far-red light-induced transgenic expression of insulin with a month-long avidity. Combining with a microchip-integrated microneedle patch, a prototyped close-loop system was constructed. The glucose levels detected by the sensor were received and converted by a wireless controller to modulate far-infrared light, thereby achieving on-demand insulin expression for several weeks. This study sheds new light on developing next-generation diagnostic and therapy systems for personalized and digitalized precision medicine.

Abstract: Engineered light-dependent switches provide uniquely powerful opportunities to investigate and control cell regulatory mechanisms. Existing tools offer high spatiotemporal resolution, reversibility and repeatability. Cellular optogenetics applications remain limited with diffusible targets as the response of the actuator is difficult to independently validate. Blue light levels commonly needed for actuation can be cytotoxic, precluding long-term experiments. We describe a simple approach overcoming these obstacles. Resonance energy transfer can be used to constitutively or dynamically modulate actuation sensitivity. This simultaneously offers on-line monitoring of light-dependent switching and precise quantification of activation-relaxation properties in intact living cells. Applying this approach to different LOV2-based switches reveals that flanking sequences can lead to relaxation times up to 11-fold faster than anticipated. In situ-measured parameter values guide the design of target-inhibiting actuation trains with minimal blue-light exposure, and context-based optimisation can increase sensitivity and experimental throughput a further 10-fold without loss of temporal precision.

Abstract: Engineering light-sensitive protein regulators has been a tremendous multidisciplinary challenge. Optogenetic regulators of MAPKs, central nodes of cellular regulation, have not previously been described. Here we present OptoJNKi, a light-regulated JNK inhibitor based on the AsLOV2 light-sensor domain using the ubiquitous FMN chromophore. OptoJNKi gene-transfer allows optogenetic applications, whereas protein delivery allows optopharmacology. Development of OptoJNKi suggests a design principle for other optically regulated inhibitors. From this, we generate Optop38i, which inhibits p38MAPK in intact illuminated cells. Neurons are known for interpreting temporally-encoded inputs via interplay between ion channels, membrane potential and intracellular calcium. However, the consequences of temporal variation of JNK-regulating trophic inputs, potentially resulting from synaptic activity and reversible cellular protrusions, on downstream targets are unknown. Using OptoJNKi, we reveal maximal regulation of c-Jun transactivation can occur at unexpectedly slow periodicities of inhibition depending on the inhibitor's subcellular location. This provides evidence for resonance in metazoan JNK-signalling circuits.