Generalised Photocatalysis by Enzymes (GENPENZ)

243e12cf-1787-47fb-8d2e-3081bd7350e3

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BBSRC
BBSRC
BBSRC
BBSRC
BBSRC

£15,890,255

Feb. 1, 2023

Jan. 31, 2028

Enzyme catalysis is being industrialised at a phenomenal rate, offering routes to chemical transformations that avoid expensive heavy metal catalysts, high temperatures and pressures, and providing impressive enantio-, regio- and chemo-selectivities. In short, biocatalysts are a cornerstone of the bioeconomy: they are required individually, or as cascades, in live cells or cell-free preparations to manufacture every day chemicals, materials, healthcare products, fuels and pharmaceuticals; and they are integral to many diagnostic and industrial sensing applications. They are central components of technologies underpinning the circular economy and offer engineering biology routes to realising global challenges, including net zero, clean growth and the bioeconomy. An ability to exploit and tailor biocatalyst activities both rapidly and predictably is essential to realising the contemporary global challenges and the UK Government's Innovation Strategy.

Despite their central importance, the vast majority of natural and engineered enzymes are thermally-activated. This dependence on thermally-activated catalysis: i) limits biocatalysis to those reaction types found naturally in biology; ii) places a high dependence on expensive and unstable cofactors / coenzymes; and iii) places a sizeable demand on the provision of energy source (biochemical / artificial reductants), 'bioreactor' designs (e.g. within cell-free formats, nanoscale devices or microbial cell factories); and iv) restricts approaches to regulating biocatalyst / bioprocess activity.

The use of light to drive enzyme catalysis would bypass many of these hurdles. However, with only three known exceptions, nature does not make use of enzymatic photocatalysis. Therefore, biology cannot access a broad range of 'difficult-to-achieve' reactions that would be transformational in catalysis science, and applications of these reactions in the modern world. Light is freely available and non-invasive, yet the photochemical versatility of natural cofactors such as flavin is seldom used by enzymes. Therefore, securing generalised routes to predictive photobiocatalysis design is a fundamental biological challenge. If successful, identifying generalised routes to the engineering and design of photobiocatalysts would be transformative for catalysis science in the emerging bioeconomy.

This project will address this urgent need by using the natural photochemistry of flavin to make possible photocatalysis by any flavin-containing protein. This programme (termed GENPENZ) is positioned at the frontier of biological photocatalysis and enzyme design and engineering. It will generalise the concept of photo-biocatalyst design and engineering using existing (top down) and man-made (bottom up) protein scaffolds to biologically encode new photo-biocatalysts with wide reaction scope, or to assemble de novo protein frameworks from synthetic peptides. It will unite time-resolved 1D / 2D spectroscopy in the visible / infra-red spectral regions, across 12 decades of time (fs - s), with emerging capabilities in photo mass spectrometry (ion mobility; hydrogen-deuterium exchange), EPR spectroscopy, and photo-biocatalyst design engineering. High-level computational chemistry will underpin all protein-design/engineering work, spectroscopy, and structure elucidation. GENPENZ is based on breakthroughs in discovery science relating to mechanisms of enzyme photocatalysis. Realisation of a generalised platform for photo-biocatalyst design will open up new high-energy reaction pathways, enrich catalysis outcomes, and sidestep many of the scientific / economic constraints of working with thermally-activated biocatalysis in the emerging bioeconomy.

Technical Abstract:
GENPENZ is at the frontier of biological photocatalysis and enzyme design & engineering. It will deliver transformative photocatalysis technology by generalising the concept of photo-biocatalyst design/engineering using existing (top-down, genetically encoded) and man-made (bottom-up, chemically synthesised) protein scaffolds to produce first-in-class photo-biocatalysts of wide chemical reaction scope. Light is freely available and non-invasive but the photochemical versatility of flavin is seldom used by enzymes. Establishing routes to generalised photo-biocatalysts would impact on the bioeconomy, opening up new high-energy reaction pathways, enriching catalysis outcomes, and side-stepping constraints (scientific/economic) of conventional biocatalysis.

GENPENZ will innovate in protein engineering and design, developing data-driven strategies and tools to deliver design principles for cofactor and substrate binding to protein scaffolds. It will establish high-throughput workflows to generate and test the new enzymes. It will combine these with state-of-the-art biophysics (e.g. laser spectroscopy, photo-mass spectrometry, and time-resolved EPR spectroscopy) to develop a detailed mechanistic understanding of photo-biocatalysis.

These activities will be underpinned by high-level computational biochemistry; and they will be done using innovative Engineering Biology Design-Build-Evaluate-Learn cycles. In these ways, GENPENZ will generalise photo-biocatalysis design and engineering, resulting in broad access to 'difficult-to-achieve' reactions, by de novo synthesis and genetic encoding of new flavin-based photo-enzymes.

Thus, GENPENZ is frontier bioscience: it will bring photo-biocatalysis into the mainstream and expand reaction space beyond that available with natural photo-enzymes. Outputs will impact widely on industrial biocatalysis, cell-factory engineering, nanoscale-device construction, diagnostics, chemical processing and related applications.