Elsevier

Current Opinion in Biotechnology

Volume 52, August 2018, Pages 145-152
Current Opinion in Biotechnology

Computational protein design  the next generation tool to expand synthetic biology applications

https://doi.org/10.1016/j.copbio.2018.04.001Get rights and content

Highlights

  • Recent computational protein design efforts show high potential for synthetic biology.

  • Examples include small molecule sensors and self-assembling extra-cellular vesicles.

  • We focus on computationally designed proteins validated in cells or animal models.

  • We predict that protein design will become an essential tool in synthetic biology.

One powerful approach to engineer synthetic biology pathways is the assembly of proteins sourced from one or more natural organisms. However, synthetic pathways often require custom functions or biophysical properties not displayed by natural proteins, limitations that could be overcome through modern protein engineering techniques. Structure-based computational protein design is a powerful tool to engineer new functional capabilities in proteins, and it is beginning to have a profound impact in synthetic biology. Here, we review efforts to increase the capabilities of synthetic biology using computational protein design. We focus primarily on computationally designed proteins not only validated in vitro, but also shown to modulate different activities in living cells. Efforts made to validate computational designs in cells can illustrate both the challenges and opportunities in the intersection of protein design and synthetic biology. We also highlight protein design approaches, which although not validated as conveyors of new cellular function in situ, may have rapid and innovative applications in synthetic biology. We foresee that in the near-future, computational protein design will vastly expand the functional capabilities of synthetic cells.

Introduction

Synthetic biologists manipulate and engineer cellular pathways to forge new cellular functions. Important achievements in the field to date include novel biosynthetic pathways [1], cellular sensing with complex logic [2], and even creation of cells with therapeutic applications [3]. A way to engineer these pathways is by introducing exogenous protein parts [4, 5] into an existing cellular pathway, knocking-out existing proteins in these pathways if necessary. Till now, most parts used in synthetic biology are sourced from natural organisms. Although Nature effectively constitutes a large catalog of protein-based parts, the catalog is intrinsically limited, and naturally sourced proteins with desired properties and activities may not be always readily available.

Protein engineering approaches hold the potential to create novel functional proteins, enabling the sourcing of custom, designer parts to build biological pathways. One fast growing protein engineering method is structure-based computational protein design (referred to as simply protein design), where structures and atomistic computational simulations guide the design of novel protein sequences, structures, and functions. Protein design has already delivered a number of hallmark achievements which illustrate the potential of this approach to design functional proteins with potential applications in synthetic biology, such as computationally designed enzymes [6, 7, 8], protein-based binders [9], vaccine-like immunogens [10], novel membrane transporters [11], and large macromolecular assemblies (e.g. [12]).

The use of protein design in synthetic biology, however, is only beginning. Although many computational protein designs are inspired by potential future applications in synthetic biology, the transition from in vitro assays to a synthetic in situ pathway may not always be straightforward. Studying efforts that have attempted to validate computational designs in cells can illustrate both the challenges and opportunities in the intersection of protein design and synthetic biology. Thus, in this review we cover primarily computationally designed proteins that have not only been validated in vitro, but have also been shown to control and manipulate different activities in living cells. These papers cover a broad range of synthetic biology applications, such as biofuel production, biosensors for toxic products, or chemical sensors for basic biology applications, among others. We have attempted to classify them into four broad categories, with corresponding subsections: enzyme engineering, protein specificity engineering, cellular pathway control, and high-order protein assemblies. Papers that meet our protein design and in vivo validation criterion are highlighted in Table 1, marked of special/outstanding interest, and the protein design methods used are described in the references’ summary at the end of this article. We also highlight protein design efforts which, though not validated as conveyors of new cellular function in situ, were inspired by and may have emerging applications in synthetic biology (Figure 1).

Section snippets

Engineering enzymes

One of the most promising applications of synthetic biology is the development of new biocatalysts, such as cells that produce biofuel, manufacture expensive chemicals, or biodegrade toxic waste. Protein design can be a powerful tool to engineer enzymes for reactions that may not occur in nature or where the natural counterparts may not have the desired characteristic. Three landmark papers have shown that the current computational methods are powerful enough for de novo design of enzymes [6, 7

Engineering protein specificity

Redesigning naturally occurring proteins provides ample opportunities to manipulate cellular pathways. Typically this involves repurposing and modulating existing protein–protein interactions (PPI) or protein–ligand interactions in terms of affinity or specificity. Kapp et al. [20] were the first to use computational design methods to fine-tune a protein–protein interface relevant for a signaling pathway, and tested its effect in situ. Their work repurposed an endogenous activator-effector pair

Controlling cellular pathways

The ability to control protein activity using external stimuli (e.g. light or small molecules) has many important applications in both fundamental and synthetic biology, due to the need to control the precise timing and/or localization of particular protein activities in cells. Designed proteins can act as controllers or switches by conjugating them to other proteins that carry out a specific activity.

Protein engineering techniques have been successfully used to create light-controlled protein

High-order protein assemblies

High-order protein assemblies are one of the most exciting areas in protein design with many potential applications in synthetic biology. This is particularly relevant in light of the many important roles that macromolecular machines play in central biological processes. Hsia et al. [38] designed a 60-subunit icosahedron particle. Their computational approach used known protein trimers as building blocks, which were then assembled into an icosahedron and the interfaces designed using Rosetta.

Outlook for synergies between protein design and synthetic biology

One of the major hurdles between protein engineers and synthetic biologists lies on the timespan that it takes to design an optimal functional protein to perform a specific purpose in the cell. Technical improvements in DNA synthesis and sequencing, high throughput screening, computational power, and computational protein design methodologies will expand our capability to quickly design and characterize novel protein sequences [45, 46]. These advances will in turn significantly diminish the

Conflict of interest statement

Nothing declared.

Acknowledgements

We thank Sabrina Vollers for critical reading. BE Correia is a grantee from the European Research Council (Starting grant  716058), the Swiss National Science Foundation, Novartis Foundation for Medical-Biological Research and the Biltema Foundation. P Gainza-Cirauqui is sponsored by an EPFL-Fellows grant funded by an H2020 Marie Sklodowska-Curie action.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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