Public Funding for Hydregen Limited

Europe needs a sustainable chemical industry which will only be realized by new breakthrough technologies. Industrial biotechnology is established in chemical manufacturing, offering more efficient, more specific, safer and less energy demanding production, but is held back by the limited number of enzyme classes in industrial use. This project opens up an important new enzyme class of tungstencontaining enzymes (W-enzymes) which catalyse amazing chemical reactions involving challenging low redox potential reduction reactions, but are currently impossible to obtain economically and on scale to match industrial needs. We need to produce W-enzymes using an industrial workhorse micro-organism such as E. coli. Yet, we discovered that W-cofactor biosynthesisisthe bottleneck preventing successful production of W-enzymes in E. coli. We can solve this challenge by using cutting-edge computational enzyme design approaches we recently developed, to create a completely new W-cofactor biosynthesis pathway for E. coli. The W-BioCat strains developed in this project will enable expression of new W-enzymes from genetic databases, and facilitate production of new engineered W-enzymes. The catalytic potential of these new W-enzymes will be established and implemented in new processes. Exciting new reaction scope in biocatalytic CO2 reduction to valuable chemicals and Birch reduction of aromatic compounds will be explored, alongside the already-established and broadly applicable carboxylic acid reductions. W-BioCat will be the breakthrough to make W-enzymes accessible for industry. As a proof of concept, a hydrogen-driven processto convert plant-derived oleic acid to the emollient ester oleyl oleate will be created. Oleyl oleate is used in many cosmetic products used daily by millions of people. This process will be demonstrated in multi-gram yield in scalable, industrially-relevant hydrogenation reactors, together with market research to address a pathway to commercialisation.

**HydRegen vision:** The HydRegen technologies offer the potential for cleaner, safer, faster and cheaper chemical manufacture. Deep understanding of biology and chemistry allow the HydRegen team to select the best components from biology and use them to develop robust biotechnologies that solve real problems in the chemicals sectors. We expect our technologies to play a part in the UK chemicals sector meeting ambitious Net Zero emissions targets (by 2050) and increasing adoption of enabling technologies such as Industrial Biocatalysis and continuous flow process. Currently we are addressing challenges in the manufacture of pharmaceuticals and speciality chemicals (e.g. flavours and fragrances) and exploring the potential of our technologies for larger-scale, lower-value chemical production. **AMT:** The patented active mixing technology opens up continuous manufacturing for a wide range of processes, mitigating challenges associated with the presence of solid particles, such as in heterogenous catalytic hydrogenation reactions. The range of Coflore continuous flow reactors offer a scalable manufacturing solution from grams to kilotonnes production per annum, de-risking tech transfer from R&D to pilot to commercial scale. According to a 2021 report by PwC, continuous manufacturing can increase energy efficiency up to 50% and reduce waste by 33% compared to existing batch methods of production. Global pressures such as the energy crisis and covid, have led EU and USA based companies to look to "re-shore" production away from India and China to strengthen supply chains. Project outcomes: * Showcase de-risked, bio-manufacturing route for two generic active pharmaceutical ingredients and package for licensing to UK and global chemical producers. * Improved competitivity and commercial-reputation in delivering bio-manufacturing capabilities providing a disruptive sustainable bio-manufacturing strategy by 2050\. * Evaluation of bio-based route to 'nitration' chemistry and consequently bio-cascades for nitration-reduction in specialty chemical manufacturing.

**HydRegen** is a 2021 spin-out from the University of Oxford's Department of Chemistry. **Our vision:** The HydRegen technologies offer the potential for cleaner, safer, faster and cheaper chemical manufacture. Deep understanding of biology and chemistry allow the HydRegen team to select the best components from biology and use them to develop robust biotechnologies that solve real problems in the chemicals sectors. We expect our technologies to play a part in the UK chemicals sector meeting ambitious Net Zero emissions targets (by 2050) and increasing adoption of enabling technologies such as Industrial Biocatalysis and continuous flow process. Currently we are addressing challenges in the manufacture of pharmaceuticals and speciality chemicals (e.g. flavours and fragrances) and exploring the potential of our technologies for larger-scale, lower-value chemical production. **Dr Morra's Group (University of Nottingham)** have expertise in novel hydrogenase enzymes that are able to cycle H2 and H+. Theses enzymes are of interest to HydRegen due to their unusual balance in 'ease of production' and 'ease of handling'. Dr Morra's team have know-how and facilities in enzyme production spanning early-stage academic research through to evaluation of scalable enzyme production in bioreactors (up to 100L). **Project focus:** here we tackle challenges in manufacturing readiness of the HydRegen technologies by intensifying and scaling enzyme production, and applying the enzyme generated to a demonstrator process for quinuclidinol production. Quinuclidinol is an important chemical building-block present in a number of active pharmaceuticals. Dr Morra and HydRegen started their collaboration in 2022 via BBSRC-NIBB funding. This project allows both partners to realise the potential of this collaboration. **Project outputs:** * Lower cost-of-production of three enzymes that are critical to HydRegen. * Use of these enzymes for production of chemical building-block quninuclidinol at \>10g scale. * Validation of the cost and sustainability metrics for quinuclidinol manufacture. * New IP (enzyme production, biocatalyst formulation, process chemistry). * 'License-ready' bio-based manufacturing route for quinuclidinol that meets the needs for UK-based production (cost, foot-print, productivity, safety, sustainability).

**HydRegen** is a 2021 spin-out from the University of Oxford's Department of Chemistry. **Our vision:** The HydRegen technologies offer the potential for cleaner, safer, faster and cheaper chemical manufacture. We expect our technologies to play a part in the UK chemicals sector meeting ambitious Net Zero emissions targets (by 2050) and increasing adoption of enabling technologies such as Industrial Biocatalysis and continuous flow process. Currently we are addressing challenges in the manufacture of pharmaceuticals and speciality chemicals (e.g. flavours and fragrances). However, these high-value chemicals are typically required only at a small scale. **Focus:** In this project, we tackle challenges with using our technologies in the manufacture of bulk chemicals -- that is, the chemicals that underpin all manufacturing: '_businesses who make chemical products and solutions are integral \[...to ca\]96% of all manufactured goods'_ (source: CIA). These low-value chemicals are required at a large scale, therefore the potential benefits of improving sustainability are maximised. To enter the low-value, high-volume bulk chemical market, we first need to lower the cost-of-production of our technology -- a biocatalyst system that allows companies to replace toxic heavy metals and decrease waste produced by typical bio-processes - to provide a resource and energy efficient 'slot-in' alternative to state-of-the-art processes. Next, we select a demonstrator process (case-study), the manufacture of a bulk chemical that is used for the production of drugs, polymers and consumer products. At present there is no UK-based manufacture of this chemical, but we hope to develop a process than enables cost-competitive manufacture in the UK.

We have developed sustainable technologies that address unmet needs in the chemicals sector through academic research at the University of Oxford. Chemicals companies are interested in our technologies because they remove the need for toxic reagents, minimise the production of carbon-based waste products and lower energy demands compared to existing manufacturing routes. Our biocatalyst systems can be operated in continuous flow, allowing sustainability at the same time as improving productivity, overall allowing cheaper, faster, cleaner and safer production of speciality chemicals (e.g. pharmaceutical, flavours and fragrances). During the project we will produce prototype catalysts, and distribute them to industry partners. Using their feedback we will carry out product development research towards launch of our first commercial product: a 'bio-flow column' that allows clean and safe reactions with exquisite precision. We will also demonstrate our catalyst systems at pilot scale for kg production of a fine chemical. This project will allow us to form a spinout company, and take our technologies from academic research to commercial product, and will ultimately play a part in cleaning up the chemicals sector.