Spatial organization of enzymes for metabolic engineering

Lee, Hanson; DeLoache, William C.; Dueber, John E.

doi:10.1016/j.ymben.2011.09.003

Cited by 226 publications

(234 citation statements)

References 72 publications

Supporting

Mentioning

225

Contrasting

Unclassified

Order By: Relevance

“…Pathway encapsulation would not only increase flux through pathways with poor kinetic properties but also allow control of molecules that enter or exit, sequestration of intermediates, and optimization of the local chemical microenvironment. Several possible compartment platforms have been suggested for both in vivo and in vitro applications, including viral capsids, lumazine synthase capsids, polymeric cages, liposomes, and bacterial MCPs (184)(185)(186)(187). Bacterial MCPs seem particularly well suited for the development of intracellular bioreactors, since evolution has designed MCPs to isolate biochemical reactions with the purpose of regulating enzyme activity, enhancing pathway flux, and protecting cells from toxic intermediates.…”

Section: Mcps As Bionanoreactors For In Vivo Pathway Optimizationmentioning

confidence: 99%

Diverse Bacterial Microcompartment Organelles

Chowdhury

Sinha

Chun

et al. 2014

Microbiol Mol Biol Rev

207

278

View full text Add to dashboard Cite

SUMMARY Bacterial microcompartments (MCPs) are sophisticated protein-based organelles used to optimize metabolic pathways. They consist of metabolic enzymes encapsulated within a protein shell, which creates an ideal environment for catalysis and facilitates the channeling of toxic/volatile intermediates to downstream enzymes. The metabolic processes that require MCPs are diverse and widely distributed and play important roles in global carbon fixation and bacterial pathogenesis. The protein shells of MCPs are thought to selectively control the movement of enzyme cofactors, substrates, and products (including toxic or volatile intermediates) between the MCP interior and the cytoplasm of the cell using both passive electrostatic/steric and dynamic gated mechanisms. Evidence suggests that specialized shell proteins conduct electrons between the cytoplasm and the lumen of the MCP and/or help rebuild damaged iron-sulfur centers in the encapsulated enzymes. The MCP shell is elaborated through a family of small proteins whose structural core is known as a bacterial microcompartment (BMC) domain. BMC domain proteins oligomerize into flat, hexagonally shaped tiles, which assemble into extended protein sheets that form the facets of the shell. Shape complementarity along the edges allows different types of BMC domain proteins to form mixed sheets, while sequence variation provides functional diversification. Recent studies have also revealed targeting sequences that mediate protein encapsulation within MCPs, scaffolding proteins that organize lumen enzymes and the use of private cofactor pools (NAD/H and coenzyme A [HS-CoA]) to facilitate cofactor homeostasis. Although much remains to be learned, our growing understanding of MCPs is providing a basis for bioengineering of protein-based containers for the production of chemicals/pharmaceuticals and for use as molecular delivery vehicles.

show abstract

Section: Mcps As Bionanoreactors For In Vivo Pathway Optimizationmentioning

confidence: 99%

Diverse Bacterial Microcompartment Organelles

Chowdhury

Sinha

Chun

et al. 2014

Microbiol Mol Biol Rev

207

278

View full text Add to dashboard Cite

show abstract

“…3A). To ensure efficient signal relay between the TVMV-based signal sensor and the HCV-based signal transducer, the first SH3 domain of the Crk adaptor protein (29) was added to the N terminus of a TVMVbased ligand sensor, whereas its cognate peptide ligand PPPPLPPKRRR (30) was fused to the C terminus of the HCVbased signal transducer. In this way, the sensitivity of the assay could be improved between one and two orders of magnitude as equivalent responses could be recorded in a shorter period using less protein compared with the TVMV-based ligand sensor on its own (Fig.…”

Section: Resultsmentioning

confidence: 99%

Protease-based synthetic sensing and signal amplification

Stein

Alexandrov

2014

Proc. Natl. Acad. Sci. U.S.A.

104

View full text Add to dashboard Cite

The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.synthetic biology | protein engineering | protein switches | proteases A key objective in the emerging field of synthetic biology is to develop approaches for the systematic engineering of artificial signal transduction systems (1, 2), which is expected to advance our understanding of fundamental biological processes and create new biotechnological and medical applications (3). Engineering synthetic signaling systems have predominantly been realized with gene expression circuits that relay molecular queues either through transcription factors or functional nucleic acids (4-8). Here, rational engineering strategies are supported by the modular organization and function of transcription factors and regulatory DNA elements (4), as well as the predictability of base pairing interactions (8). However, transcription-based signaling circuits usually require hours to process and actuate a specific molecular queue (9). Further, the limited chemical diversity of nucleic acids compared with amino acids ultimately restricts their functionality. Thus, protein-based signaling networks that operate faster compared with gene-based signaling circuits are highly desirable.The systematic engineering of protein-based modules that sense, process, and amplify defined molecular queues has provided a formidable challenge to date. At the molecular level, many biological response functions depend on allosterically regulated protein activities that couple an input to an output function solely through conformational changes. Engineering these has proven difficult with only a few successful designs reported to ...

show abstract

“…Like other methods of pathway optimization, post-translational balancing does not benefit from reducing the production of surplus quantities of RNA or protein, but rather, by improving the efficiency of substrate transfer from enzyme to enzyme, minimizing diffusion, before the substrate reacts with the enzyme. Scaffold based optimization techniques benefit from the formation of microdomains with extremely increased metabolite concentrations in the cytosol [49]. If substrate trafficking and diffusion is not limiting, the application of scaffolding proteins for pathway optimization will show little to no improvement, or potentially worsen production due to the metabolic burden associated with scaffolding protein production or blocking the access of substrate to active site due to complicated 3-D structures of scaffold-enzyme complexes in overexpressing strains.…”

Section: Post-translational Optimizationmentioning

confidence: 99%

Metabolic pathway balancing and its role in the production of biofuels and chemicals

Jones

Toparlak

Koffas

2015

Current Opinion in Biotechnology

178

119

View full text Add to dashboard Cite

In the last decade, metabolic engineering benefited greatly from systems and synthetic biology due to substantial advancements in those fields. As a result, technologies and methods evolved to be more complex and controllable than ever. In this review, we highlight up-to-date case studies using these techniques, examine their potential, and stress their importance for production of compounds such as fatty acids, alcohols, and high value chemicals. Beginning with basic rational control techniques and continuing with advanced level modern approaches, we review the vast number of possibilities for controlling metabolic fluxes. Our aim is to give a brief and informative insight about commonly used tools and universalized methodologies for metabolic pathway balancing and optimization.

show abstract

Spatial organization of enzymes for metabolic engineering

Cited by 226 publications

References 72 publications

Diverse Bacterial Microcompartment Organelles

Diverse Bacterial Microcompartment Organelles

Protease-based synthetic sensing and signal amplification

Metabolic pathway balancing and its role in the production of biofuels and chemicals

Contact Info

Product

Resources

About