Optogenetics as a new paradigm for dynamic control in metabolic engineering

We recently developed optogenetic circuits to control engineered metabolic pathways for microbial chemical production with light1. Light offers unique capabilities for dynamic control of fermentation processes. It is highly tunable and can be applied or removed instantly, and in any desired schedule, to elicit reversible metabolic responses without chemical inducers or complex media changes. In this talk, I will present our recent progress in the development of this new technological platform. I will describe new optogenetic circuit designs that enhance the transcriptional activation kinetics of inverter (OptoINVRT) circuits upon exposure to darkness, which improve the robustness of light-controlled cell growth and chemical production. In addition, I will introduce a new class of optogenetic circuits for metabolic engineering (OptoAMP), which amplify the transcriptional response to light, enabling strong light-induced gene expression in high cell-density fermentations in lab-scale bioreactors of up to five liters. Furthermore, I will present new optogenetic post-translational controls based on light-dependent assembly of synthetic organelles, which we use to control flux through branched metabolic pathways. I will demonstrate how each of these technologies can be applied to dynamically control engineered metabolisms to boost yields, titers, productivities, and product specificities of fuels and chemicals in microbial fermentations. Finally, I will provide a perspective on how optogenetics may emerge as a new paradigm for dynamic control in metabolic engineering. 1. Zhao EM, Zhang Y, Mehl J, Park H, Toettcher JE*, Avalos JL*. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature 29; 555 (7698):683-87 (2018). * Co-corresponding

Relating topology and dynamics in cell signaling networks

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2009.Cataloged from PDF version of thesis.Includes bibliographical references (p. 153-163).Cells are constantly bombarded with stimuli that they must sense, process, and interpret to make decisions. This capability is provided by interconnected signaling pathways. Many of the components and interactions within pathways have been identified, and it is becoming clear that the precise dynamics they generate are necessary for proper system function. However, our understanding of how pathways are interconnected to drive decisions is limited. We must overcoming this limitation to develop interventions that can fine tune a cell decision by modulating specific features of its constituent pathway's dynamics. How can we quantatively map a whole cell decision process? Answering this question requires addressing challenges at three scales: the detailed biochemistry of protein-protein interactions, the complex, interlocked feedback loops of transcriptionally regulated signaling pathways, and the multiple mechanisms of connection that link distinct pathways together into a full cell decision process. In this thesis, we address challenges at each level. We develop new computational approaches for identifying the interactions driving dynamics in protein-protein networks. Applied to the cyanobacterial clock, these approaches identify two coupled motifs that together provide independent control over oscillation phase and period. Using the p53 pathway as a model transcriptional network, we experimentally isolate and characterize dynamics from a core feedback loop in individual cells. A quantitative model of this signaling network predicts and rationalizes the distinct effects on dynamics of additional feedback loops and small molecule inhibitors. Finally, we demonstrated the feasibility of combining individual pathway models to map a whole cell decision: cell cycle arrest elicited by the mammalian DNA damage response. By coupling modeling and experiments, we used this combined perspective to uncover some new biology. We found that multiple arrest mechanisms must work together in a proper cell cycle arrest, and identified a new role for p21 in preventing G2 arrest, paradoxically through its action on G1 cyclins. This thesis demonstrates that we can quantitatively map the logic of cellular decisions, affording new insight and revealing points of control.by Jared E. Toettcher.Ph.D

Supplemental Data: Stochastic Gene Expression in a Lentiviral Positive Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity

Supplemental data for "Stochastic Gene Expression in a Lentiviral Positive Feedback Loop: HIV-1 Tat Fluctuations Drive Phenotypic Diversity" [q-bio.MN/0608002, Cell. 2005 Jul 29;122(2):169-82].Comment: Supplemental data for q-bio.MN/060800

Optogenetic Control of Subcellular Protein Location and Signaling in Vertebrate Embryos.

This chapter describes the use of optogenetic heterodimerization in single cells within whole-vertebrate embryos. This method allows the use of light to reversibly bind together an "anchor" protein and a "bait" protein. Proteins can therefore be directed to specific subcellular compartments, altering biological processes such as cell polarity and signaling. I detail methods for achieving transient expression of fusion proteins encoding the phytochrome heterodimerization system in early zebrafish embryos (Buckley et al., Dev Cell 36(1):117-126, 2016) and describe the imaging parameters used to achieve subcellular light patterning

Photoswitchable diacylglycerols enable optical control of protein kinase C.

Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling

Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation

© Ankers et al. Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators. These interactions modulate the NF-κB response. In S-phase, the NF-κB response was delayed or repressed, while cell cycle progression was unimpeded. By contrast, activation of NF-κB at the G1/S boundary resulted in a longer cell cycle and more synchronous initial NF-κB responses between cells. These data identify new mechanisms by which the cellular response to stress is differentially controlled at different stages of the cell cycle

The DNA damage response—Repair or despair?

The term “the DNA damage response” (DDR) encompasses a sophisticated array of cellular initiatives set in motion as cells are exposed to DNA-damaging events. It has been known for over half a century that all organisms have the ability to restore genomic integrity through DNA repair. More recent discoveries of signal transduction pathways linking DNA damage to cell cycle arrest and apoptosis have greatly expanded our views of how cells and tissues limit mutagenesis and tumorigenesis. DNA repair not only plays a pivotal role in suppressing mutagenesis but also in the reversal of signals inducing the stress response. If repair is faulty or the cell is overwhelmed by damage, chances are that the cell will despair and be removed by apoptosis. This final fate is determined by intricate cellular dosimeters that are yet to be fully understood. Here, key findings leading to our current view of DDR are discussed as well as potential areas of importance for future studies. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78214/1/20597_ftp.pd

Functioning Nanomachines Seen in Real-Time in Living Bacteria Using Single-Molecule and Super-Resolution Fluorescence Imaging

Molecular machines are examples of “pre-established” nanotechnology, driving the basic biochemistry of living cells. They encompass an enormous range of function, including fuel generation for chemical processes, transport of molecular components within the cell, cellular mobility, signal transduction and the replication of the genetic code, amongst many others. Much of our understanding of such nanometer length scale machines has come from in vitro studies performed in isolated, artificial conditions. Researchers are now tackling the challenges of studying nanomachines in their native environments. In this review, we outline recent in vivo investigations on nanomachines in model bacterial systems using state-of-the-art genetics technology combined with cutting-edge single-molecule and super-resolution fluorescence microscopy. We conclude that single-molecule and super-resolution fluorescence imaging provide powerful tools for the biochemical, structural and functional characterization of biological nanomachines. The integrative spatial, temporal, and single-molecule data obtained simultaneously from fluorescence imaging open an avenue for systems-level single-molecule cellular biophysics and in vivo biochemistry

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions