A modular protein engineering toolkit

Natural proteins perform an amazing repertoire of functions. A key goal of synthetic biology technology development is to design new functional proteins that are not derived from natural sequences. Protein design is a young field, beginning with DNA and peptide synthesis technologies. The first designed proteins were four-helix bundles, surprisingly stable, but not functional. Protein design is not yet routine, and has several limitations. Most successful design methods use a potential energy optimisation approach in an all-atom forcefield, which is computationally expensive and therefore limited to smaller domains. In contrast, the highly regular and predictable structure of DNA allows large "origami" objects to be predictably designed and constructed.

Here, we propose a modular protein construction kit that uses regular and extendable backbone scaffolds, but can have smaller computationally designed functional domains inserted with atomic precision. Such a system would have some of the composability and designability of highly regular DNA origami, with all of the advantages of computational protein design. The project begins with synthetic beta-solenoid proteins in the RFR family. This family has a 5 residue repeat, consensus ADLSG. Four of these repeats form a near-square and these squares stack to form the solenoid. The A and L residues of the repeat form the hydrophobic core, and there is a great deal of freedom for the other residues which form the surface. The solenoid core is about the same width as DNA, but much more chemically versatile.

We have made synthetic solenoid proteins of variable lengths with repeat sequences drawn from a residue frequency table. Into these solenoids we have inserted computationally designed loops, using a novel hierarchical fragment-free technique, and entire protein domains (MacDonald et al. 2016). This work defined an extendable protein scaffold part, which we now wish to extend into a multi-part modular protein design toolkit. We will extend the scaffold with functional loops, and design interfaces to enable the modular building of higher order structures. The project will involve computational protein design, protein characterization and structural biology.

The 2016 UK Roadmap Bio-design for the Bio-economy highlighted the substantial impact that synthetic biology can bring to the UK and global economies by developing: frontier science and technology; establishing a healthy innovation pipeline; a highly skilled workforce and an environment in which innovative science and businesses can thrive. Synthetic biology promises to transform the UK Bio-economy landscape, bringing bio-sustainable and affordable manufacturing routes to all industrial sectors and will ensure society can tackle many contemporary global Grand Challenges including: Sustainable Manufacturing, Environmental Sustainability Energy, Global Healthcare, and Urban Development. Whilst synthetic biology is burgeoning in the UK, we now need to build on the investments made and take a further lead in training next generation scientists to ensure sustained growth of a capable workforce to underpin the science base development and growth in an advanced UK bio-economy.
This training provided by this CDT will give students from diverse backgrounds a unique synthesis of computational, biomolecular and cellular engineering skills, a peer-to-peer and industrial network, and unique entrepreneurial insight. In so doing, it will address key EPSRC priority areas and Bioeconomy strategic priorities including: Next-generation therapeutics; Engineered biomaterials; Renewable alternatives for fuels, chemicals and other small molecules; Reliable, predictable, and scalable bioprocesses; Sustainable future; Lifelong health & wellbeing.
Advances created by our BioDesign Engineering approach will address major societal challenges by delivering new routes for chemical/pharma/materials manufacture through to sustainable energy, whilst providing clean growth and reductions in energy use, greenhouse gas emissions and carbon footprints. Increased industry awareness of bio-options with better civic understanding will drive end-user demand to create market pull for products. The CDT benefits from unrivalled existing academic-industry frameworks at the host institutions, which will provide direct links to industrial partners and a direct pathway to early economic and industrial impact.

This CDT will develop 80-100 next-generation scientists and technologists (via the funded cohort and wider integration of aligned students at the three institutions) as adept scientists and engineers, instilled with technical leadership, who as broadly trained individuals will fill key skills gaps and could be expected to impact internationally through leadership roles in the medium term. Importantly the CDT addresses key skill-gaps identified with industry, which are urgently required to create and support high value jobs that will enable the UK to compete in global markets. Commercialisation and entrepreneurship training will equip the next generation of visionaries and leaders needed to accelerate and support the creation of new innovative companies to exploit these new technologies and opportunities.

The UK government identified Synthetic Biology as one of the "Eight Great Technologies" that could be a key enabler to economic and societal development. This CDT will be at the forefront of research that will accelerate the clean growth agenda and the development of a resilient circular bioeconomy, and will align with key EPSRC prosperity outcomes including a productive, healthy and resilient nation. To foster wider societal impact, the CDT will expect all students to contribute to public outreach and engagement activities including: open days, schools visits, and science festival events: students will participate in an outreach programme, with special focus on widening participation.

This CDT will contribute to the development of industrial strategy through the Synthetic Biology Leadership Council (SBLC), Industrial Biotechnology Leadership Forum (IBLF), and wider Networks in Industrial Biotechnology and Bioenergy and Professional Institutes.