Synthetic biology approaches for targeted protein degradation

Chen, Rebecca P.; Gaynor, Andrew S.; Chen, Wilfred

doi:10.1016/j.biotechadv.2019.107446

Cited by 15 publications

(16 citation statements)

References 114 publications

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“…For example, compared to SPP/PDT-CRISPRi that integrated both transcriptional level and protein level regulations, higher concentrations of byproducts could be detected when using a single-layered transcriptional regulation (Dual-SPP-CRISPRi system) (Figure S9). To achieve protein level regulation, numerous endogenous proteolytic machines, such as degron-based proteolytic machines, proteasome-based proteolytic machines, and AAA+ proteolytic machines, could be used for metabolic applications. , These mechanisms, together with suitable protein circuit design, would provide new possibilities to dynamically program biological systems for controlling cellular behaviors as well as boosting microbial production.…”

Section: Discussionmentioning

confidence: 99%

Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli

Gao

Guo

et al. 2021

ACS Synth. Biol.

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Building autonomous switches is an effective approach for rewiring metabolic flux during microbial synthesis of chemicals. However, current autonomous switches largely rely on metabolite-responsive biosensors or quorum-sensing circuits. In this study, a stationary phase promoter (SPP) and a protein degradation tag (PDT) were combined with the CRISPR interference (CRISPRi) system to construct an autonomous repression system that could shut down multiple-gene expression depending on the cellular physiological state. With this autonomous CRISPRi system to regulate one target gene, a fermenter-scale titer of shikimic acid reached 21 g/L, which was the highest titer ever reported by Escherichia coli in a minimal medium without any chemical inducers. With three target genes repressed, 26 g/L glutaric acid could be achieved with decreased byproduct accumulation. These results highlight the applicability of the autonomous CRISPRi system for microbial production of value-added chemicals.

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Section: Discussionmentioning

confidence: 99%

Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli

Gao

Guo

et al. 2021

ACS Synth. Biol.

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“…Proteome editing technology represents a powerful strategy for posttranslational control of protein function based on the principle of "inhibition-by-degradation" whereby an inhibitor/degrader hijacks the cellular quality control machinery to selectively eliminate target proteins [1][2][3] . A common feature of proteome editing approaches is the ability to promote catalytic turnover of otherwise stable intracellular proteins, requiring only transient binding to virtually any site on the protein of interest (POI).…”

Section: Introductionmentioning

confidence: 99%

“…Proteome editing technology has emerged as a powerful approach to control protein function at the post-translational level. Proteome editing involves a molecular degrader that hijacks the cellular quality control machinery to selectively eliminate target proteins, thereby executing an “inhibition-by-degradation” mechanism. − A common feature of proteome editing approaches is the ability to promote catalytic turnover of otherwise stable intracellular proteins, requiring only transient binding to virtually any site on the protein of interest (POI). This is in stark contrast to traditional occupancy-type inhibitors, which depend on a distinct binding site that affects function ( e.g.…”

Section: Introductionmentioning

confidence: 99%

Engineering Single Pan-Specific Ubiquibodies for Targeted Degradation of All Forms of Endogenous ERK Protein Kinase

et al. 2021

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Ubiquibodies (uAbs) are a customizable proteome editing technology that utilizes E3 ubiquitin ligases genetically fused to synthetic binding proteins to steer otherwise stable proteins of interest (POIs) to the 26S proteasome for degradation. The ability of engineered uAbs to accelerate the turnover of exogenous or endogenous POIs in a post-translational manner offers a simple yet robust tool for dissecting diverse functional properties of cellular proteins as well as for expanding the druggable proteome to include tumorigenic protein families that have yet-to-be successfully drugged by conventional inhibitors. Here, we describe the engineering of uAbs composed of human carboxyl-terminus of Hsc70-interacting protein (CHIP), a highly modular human E3 ubiquitin ligase, tethered to differently designed ankyrin repeat proteins (DARPins) that bind to nonphosphorylated (inactive) and/or doubly phosphorylated (active) forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Two of the resulting uAbs were found to be global ERK degraders, pan-specifically capturing all endogenous ERK1/2 protein forms and redirecting them to the proteasome for degradation in different cell lines, including MCF7 breast cancer cells. Taken together, these results demonstrate how the substrate specificity of an E3 ubiquitin ligase can be reprogrammed to generate designer uAbs against difficult-to-drug targets, enabling a modular platform for remodeling the mammalian proteome.

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“…Another common method is the fusion of a degradation domain (DD) to a protein of interest (POI) (Li et al, 1998), which drastically reduces its half-life and allows faster fluctuations in the intracellular level (Mei et al, 2018; Sjaastad et al, 2018). As we recently reviewed (Chen et al, 2019), while several approaches can modulate protein degradation in response to a small molecule (Chung et al, 2015; Iwamoto et al, 2010; Lau et al, 2010), they do not allow protein concentration control in response to native cellular environments. Ideally, a modular platform that combines rapid protein turnover by DDs with temporal and autonomous responsiveness to cellular environments will greatly expand our ability to generalize the strategy for conditional protein rescue (CPR) in a wide range of biological contexts.…”

Section: Introductionmentioning

confidence: 99%

Conditional Protein Rescue (CPR) by Binding-Induced Protective Shielding

Gaynor

Chen

2020

Preprint

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1An effective method to modulate the stability of proteins is essential to biological 2 research. Herein, we describe a new technology that allows conditional stabilization of 3 proteins based on masking of a degron tag by a specific intracellular protein cue. A 4 target protein is fused to a degron tag and an affinity sensor domain. When the sensor 5 detects its target protein, the degron is effectively concealed and the target protein is 6 rescued. By introducing nanobodies as the sensor, we allow for virtually any endogenous 7 protein to be targeted. In a model system using yeast cytosine deaminase, we 8 demonstrate low cell death background yet maintain the ability to elicit strong activation 9 and prodrug-mediated cell killing using GFP as the rescue protein. The flexibility in 1 0 choosing different masking targets provides a straightforward method to generalize the 1 1 strategy for conditional protein rescue in a wide range of biological contexts, including 1 2 oncoprotein detection.

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Synthetic biology approaches for targeted protein degradation

Cited by 15 publications

References 114 publications

Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli

Engineering a CRISPRi Circuit for Autonomous Control of Metabolic Flux in Escherichia coli

Engineering Single Pan-Specific Ubiquibodies for Targeted Degradation of All Forms of Endogenous ERK Protein Kinase

Conditional Protein Rescue (CPR) by Binding-Induced Protective Shielding

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