Sensory photoreceptors underpin light-dependent adaptations of organismal physiology, development, and behavior in nature. Adapted for optogenetics, sensory photoreceptors become genetically encoded actuators and reporters to enable the noninvasive, spatiotemporally accurate and reversible control by light of cellular processes. Rooted in a mechanistic understanding of natural photoreceptors, artificial photoreceptors with customized light-gated function have been engineered that greatly expand the scope of optogenetics beyond the original application of light-controlled ion flow. As we survey presently, UV/blue-light-sensitive photoreceptors have particularly allowed optogenetics to transcend its initial neuroscience applications by unlocking numerous additional cellular processes and parameters for optogenetic intervention, including gene expression, DNA recombination, subcellular localization, cytoskeleton dynamics, intracellular protein stability, signal transduction cascades, apoptosis, and enzyme ac...

Blue-Light Receptors for Optogenetics.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

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Light-based control of metabolic flux through assembly of synthetic organelles.

Advanced Strategies for Production of Natural Products in Yeast.

Many natural transcription factors are regulated in a pulsatile fashion, but it remains unknown whether synthetic gene expression systems can benefit from such dynamic regulation. Here we find, using a fast-acting, optogenetic transcription factor in Saccharomyces cerevisiae, that dynamic pulsatile signals reduce cell-to-cell variability in gene expression. We then show that by encoding such signals into a single input, expression mean and variability can be independently tuned. Further, we construct a light-responsive promoter library and demonstrate how pulsatile signaling also enables graded multi-gene regulation at fixed expression ratios, despite differences in promoter dose-response characteristics. Pulsatile regulation can thus lead to beneficial functional behaviors in synthetic biological systems, which previously required laborious optimization of genetic parts or the construction of synthetic gene networks.

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Pulsatile inputs achieve tunable attenuation of gene expression variability and graded multi-gene regulation

Dynamic control in metabolic engineering: Theories, tools, and applications.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l-1 of isobutanol and 2.38 ± 0.06 g l-1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.

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Justin Mehl

Papers

Optogenetic regulation of engineered cellular metabolism for microbial chemical production