Scalable Production of Precisely Engineered Proteins Using an Expanded Genetic Code

Proteins are biopolymers that perform a vast array of functions in nature, including speeding up the biochemical reactions needed for life, transporting molecules across membranes, providing structural support and controlling signalling processes. Beyond their natural functions, proteins are also used widely across chemistry, biotechnology and medicine - for example as therapeutics such as insulin or as biocatalysts used in laundry detergents or to break down plastics. Despite their remarkable structural and functional diversity, the vast majority of proteins are made from only 20 standard building blocks, the canonical amino acids. These amino acids only contain a narrow set of functional motifs, which ultimately restricts our ability to develop proteins with new and improved functions. To address this fundamental limitation, a powerful protein engineering technique called genetic code expansion (GCE) has been developed to allow proteins to be produced from >20 amino acid building blocks. Using this technique, hundreds of non-canonical amino acids (ncAAs) with new functional side chains can now be selectively introduced into proteins, leading to the development of new generations of biocatalysts, advanced materials and new biotherapeutics. However, despite great progress and its enormous commercial potential, the translation of GCE from academic labs into commercial protein products has been hindered by existing limitations of the technology. Current barriers to translation include the low yields of ncAA-containing proteins and the requirement for large excesses of ncAAs, which ultimately result in prohibitively high production costs. In this proposal we will develop a fully integrated engineering biology platform to translate GCE into commercial applications. Bringing together multidisciplinary researchers from across academia and industry, we will develop engineered strains capable of biosynthesizing new functional ncAAs and efficiently introduce them into proteins to deliver precisely functionalized proteins at substantially lower costs than existing platforms. To exemplify our technology, we will work in collaboration with our industrial partners AstraZeneca, GSK and Prozomix, to apply our engineered strains to the large-scale production of next generation biocatalysts and protein therapeutics. Moving forward, the versatile GCE platform and engineering biology tools developed within this proposal will enable the scalable production of diverse functionalized proteins in response to emerging societal needs.

Nature uses twenty canonical amino acids to produce proteins with diverse structures and functions. However, these amino acids contain limited chemical diversity, restricting our ability to engineer proteins with new functions and desirable properties. The recent emergence of powerful genetic code expansion (GCE) methods has allowed targeted introduction of non-canonical amino acids (ncAAs) (e.g. catalytic motifs, bio-orthogonal handles, photo-responsive elements) into proteins, providing engineered biopolymers with applications as biocatalysts, advanced biomaterials or protein therapeutics. Despite its great commercial potential, GCE remains largely an academic endeavour, and its translation into commercial products is hindered by existing limitations of the technology. Current barriers to translation include the limited efficiency of orthogonal translation components and the requirement for high excesses of ncAAs (typically >1000 equivalents) that lead to high costs. In this proposal we will develop a fully integrated platform to transition GCE from academic labs into industrial applications. Employing a combination of high-throughput experimentation and deep-learning based protein design methods, we will engineer orthogonal translation components with efficiencies approaching those of natural systems. We will develop biosynthetic pathways to produce key ncAAs from renewable feedstocks and applying recent advances in synthetic genomics, we will develop customized yeast strains that efficiently biosynthesize new functional ncAAs and produce modified proteins with high titres. In collaboration with our industrial partners AstraZeneca, GSK and Prozomix, we will apply our engineered strains and optimized translation components to the large-scale production of biocatalysts and protein therapeutics using bacterial, yeast and mammalian hosts. This application addresses engineering biology for clean growth and biomedicine.