There is growing industrial, consumer and regulatory demand for sustainable alternative ingredients for a range of everyday products, with a particular focus on surfactant-based formulations. The necessity of sustainable, bio-based materials for the future economy is emphasised under "Materials for the future economy", Innovate UK Materials and manufacturing vision 2050\. Bio-based replacements for fossil derived synthetic surfactants have existed since the 1990s, though current production processes are severely limited by: low productivity (<2 g/L/h), high energy demand and complex downstream processing, leading to a high price point in excesses of the cost of synthetic surfactants, hindering market adoption. Holiferm's technology and fermentation expertise enable a step change in biosurfactant production cost, allowing manufacturers of formulated products to overcome current adoption barriers, ultimately benefiting consumers. NetZeroSoph will provide a significant change in trajectory and accelerated progress towards Holiferm's strategic goal of achieving net zero sophorolipid biosurfactant production, enhancing their world leading sophorolipid production process and diversifying the portfolio of sophorolipid molecules through application of advanced fermentation technology, holistic process development and engineering biology approaches. This collaboration with Evolutor and The University of Manchester will yield circular, novel, sophorolipids with significantly reduced life cycle impacts and enhanced technoeconomics. Market need has been validated through discussion with end-users and distributors such as Sasol, Star Brands and Ingredients Plus, with this project expanding on Holiferm's success in constructing a flexible pilot plant and the recent commissioning of a 1000 tpa commercial demonstration sophorolipid production facility, providing in house capabilities for accelerated development and rapid scaleup/translation (building on/exploiting outputs of prior Innovate UK funding). NetZeroSoph will accelerate CR&D aligned with long-term strategy to drive down cost whilst achieving net zero manufacture, providing bio-based replacements for traditional synthetic surfactants in applications including household, laundry and personal care, establishing Holiferm as a world leading manufacturer of biosurfactants.

Biomanufacturing using yeast/bacteria and precision fermentation processes is a powerful approach that can displace manufacturing processes that are currently extremely polluting and destructive such as fossil fuel extraction, non-degradable plastic production and intensive animal agriculture. But these harmful industries have decades of infrastructure, economies-of-scale and subsidisation behind them, making the commercial optimisation of biomanufacturing processes to achieve price parity on existing markets a huge challenge for biomanufacturers across all sectors. One way that commercial viability can be achieved is through using cheaper raw materials to feed into precision fermentation processes. Currently, the majority of biomanufacturers use virgin sugars as their feedstocks. These can make up almost 50% of overall biomanufacturing cost, and they also create competition with other markets where these sugars could arguably be better used, for example going directly into existing food manufacturing chains. In this project, Evolutor will use its proprietary microbe optimisation technology platform to develop entirely new microbial systems that can effectively utilise industrial side streams as fermentation feedstocks better than any existing microbial solutions. Side streams are challenging to capture as biomanufacturing feedstocks as they are generally complex, impure and inconsistent, making reliable fermentation difficult. But over 500M tonnes of waste goes to landfill or is incinerated every year (an estimated economic loss of $720B). With Evolutor's new microbe developments, we will unlock side stream valorisation for biomanufacturing on a global scale, pushing our economies closer to a much needed Bio-Industrial Revolution. To demonstrate the power of our new microbes in the first instance, we are partnering with Faber Futures. Faber are developing their "Project Coelicolor" to produce bacterial textile dyes and decentralise the production of high-quality, sustainable garments. By allowing them to use currently wasted or under-valorised side streams as raw materials for this dye production, this project will push Faber Futures' fabrics to market in late 2023 and fulfil their mission of building textile biomanufacturing supply chains that are entirely **ethical, equitable and sustainable** from end-to-end.

The biomanufacturing industry needs fit-for-purpose, market-ready microbial chassis that are pre-optimized for large-scale biomanufacturing of commodity products that are competitively viable against existing markets and embrace circularity in their supply chains. As biomanufacturing sectors expand rapidly towards 2030, this need cannot be satisfied by existing microbe development technologies that are restricted by the relatively minimal genetic understanding of non-model microbial systems. At Evolutor, we use our Accelerated Evolution Platform (AEP) to digitalise, amplify and rapidly accelerate adaptive laboratory evolution (ALE) to address this unmet need. Evolutor’s AEP is an integrated suite of platform technologies made up of: • Novel miniature bioreactor hardware, including advanced sensing tools and evolutionary stress controllers. • Software for evolutionary control, forecast and modelling. • Proprietary genetic tools to unlock the hidden biological potential of non-model microbes and massively amplify their evolutionary potential. Evolutor’s long-term ambition is to build Galapagos Towers: a world-leading and paradigm-shifting bio-foundry in the North of England which unlocks 24/7 massively parallel and fully automated ALE at a globally unprecedented scale. This will be achieved through 1000s of AEP units all managed through proprietary software and generative AI. Through Galapagos Towers, we will develop a diverse library of precision-evolved microbial chassis pre-optimized for large-scale circular biomanufacturing use across all sectors. Additionally, the vast amounts of evolutionary data generated will unlock development of generative AI models for predictive evolution to take ALE in silico and perpetually widen our global competitive moats. Overall, the Galapagos Towers vision will drive a Bio-Industrial Revolution of sustainable and circular manufacturing across all industries. To reach this ambition, Evolutor is beginning with targeted deployment of the unscaled AEP technology to solve a specific biomanufacturing problem: the inability of model microbial chassis systems to effectively utilise wastestream-derived feedstocks. It’s estimated that to realise the full potential of Bio-Industrial Revolution using virgin sugar feedstock would require 3 x current global commodity crop production. Alongside this, we are producing up to 10 billion tonnes of solid waste every year (UN Environment Programme). Using the AEP, Evolutor are developing waste-hungry microbes to address these 2 commercial and environmental problems. The first targeted waste is end-of-life tyres (ELTs). Through this project, Evolutor is iterating the hardware of its AEP for increased functionality and scalability before demonstrating it with proof-of-concept microbe evolution by evolving non-model microbes for rapid degradation of ELTs. These precision-evolved microbes will unlock a significant wastestream for use as fermentation feedstock in biomaterial production.

For novel low-emission foods to reach customers in a way that is affordable and widely accessible (and therefore maximise the positive impact they can have on reducing the carbon emissions and climate impact of our agriculture systems) one of the major challenges that needs to be overcome is **price parity.** After taste, cost is the greatest blocker to public acceptance and adoption of alternative proteins. It is therefore vital that we develop production methods and technologies that decrease the manufacturing cost of novel low-emission proteins to allow them to compete on the market with incumbent products. These incumbent products (e.g. traditional cow's milk, beef, hen's eggs, etc) have decades or centuries of infrastructure and economies-of-scale behind them. Competing with these industries is no small challenge, but a challenge that is vitally necessary to overcome as we push towards Net Zero Ambitions. At Evolutor, our focus is on the production of food proteins using microbes (yeast and bacteria) in precision fermentation systems. Much like how we use yeast to convert sugar into alcohol for beer and wine production, we can engineer these microbial strains to produce meat, dairy and egg proteins. There are many companies around the world already focussed on doing just this (e.g. Better Dairy, Motif Foodworks, Imagindairy, The Every Company and Onego Bio), but they all face a major challenge: how to maximise the protein production capabilities of their precision fermentation manufacturing to drive down cost and hit price parity _v.s._ traditional proteins. Through the innovations of this project, Evolutor and The University of Sheffield are developing **Protein-Infinity:** a microbe optimisation toolkit that will solve this problem in an entirely new way that is **protein-agnostic** and can be applied across diverse precision fermentation sectors to **maximise the project's commercial value and climate impact.** We are doing this by linking protein production to evolutionary selection pressures, creating environments where _"survival-of-the-fittest"_ means _"survival-of-the-highest-protein-producer"_. This strategy and the ground-breaking way in which Protein-Infinity will execute it will rapidly accelerate the commercial viability of precision fermentation UK-wide and beyond. We aim to double protein production, driving novel proteins towards price parity and moving our economy closer to a Bio-Industrial Revolution: producing food proteins that are **affordable, delicious, nutritious, sustainable** and **accessible by all**.

Both the climate crisis and public health crises such as the COVID-19 pandemic are presenting substantial challenges to our communities and economies. To address the climate collapse, we need to rapidly reduce our manufacturing industries' reliance on fossil fuels and build new decarbonised supply chains that are robust and resilient in an ever-changing world. In 2016/2017 healthcare associated infections cost the NHS £2.1B, and were responsible for approximately 22,800 inpatient deaths. To protect public health and reduce the burden on health care providers, we need new technologies and products that can reduce the spread of microbial pathogens carrying healthcare associated infections such as scarlet fever, strep A and bacterial co-infections that accompany conditions like COVID-19\. As well as fulfilling these functions, new products must also be manufacturable without any reliance on fossil fuels. In this project, Evolutor is addressing these challenges by developing production processes for the manufacture of advanced and highly-functionalized biomaterials with antimicrobial properties. We will use our specialist Accelerated Evolution Platform to develop microbial production hosts that, through precision fermentation, can turn waste products into biomaterials that are fully biodegradable and biocompatible. Additionally, we will test new strategies and manufacturing processes to impart natural antimicrobial properties to these materials by supplementing fermentation feedstocks with a range of natural compounds. Through these project outputs, we will produce and analyse the performance of several small batches of novel biomaterial that: * is 100% natural; * has customizable antimicrobial functionality; * is produced from waste; * requires no direct fossil fuel input; * is completely biodegradable and non-toxic; * produces significantly less chemical pollution than conventional materials; and * can be tailored to a wide range of applications. Overall, the success of this project will drive the expansion of the UK's bioeconomy, bring us closer to a Net Zero world, and help to reduce the spread of microbial pathogens and the burden of associated healthcare costs. It will also accelerate Evolutor's ambition to build its world-leading bio-foundry Galapagos Towers in the North of England. Beyond the completion of this project, the innovations achieved will allow Evolutor to explore scaled-up manufacture of its antimicrobial biomaterials as well as conduct onward R&D of feedstock supplementation to customise material properties beyond antimicrobial functionality. Example properties include colour (for advanced clothing and construction materials), fragrance (for cosmetics and detergents), tensile strength (for industrial and construction use) and permeability (for wastewater treatment and pollution control).

Palm oil is everywhere. It's in food, cosmetics and biofuels. It's in 50% of our supermarket products. Palm oil is unique in the vegetable oil market; it melts in your mouth, is great for cooking and is a high-yielding crop. However, palm oil is one of the biggest carbon emitters due to loss of tropical rainforest and peatland to make way for plantations - it contributes over 500 million tonnes of CO2e, over 1% of global emissions, from 19 million hectares of tropical land. It has caused significant habitat loss, including for the Orangutan and Sun Bear, and all too often the smallholders do not receive a fair price for their produce. The UK Government has committed to reaching Net Zero by 2050; 20% of UK emissions stem from agriculture and the food supply chain and these emissions must be tackled if we are to reach these targets. Sun Bear Bioworks is using precision fermentation to make an alternative to palm oil. We are optimising a yeast strain that naturally produces oil, in order to create our oil from food waste quickly and efficiently. Our process saves 5.7kg CO2 and 2000m2 land for every kg of palm oil - a saving in both cases of 80%. We are working with Evolutor on this project to optimise our strain's ability to process low-grade, sustainable feedstocks. Feedstock accounts for most of our process cost at scale, and finding a scalable sugar alternative is vital if we are to create maximum environmental impact. This builds on the work we carried out as part of our Fast Start award, project 10045430 "Alternative feedstock for precision fermentation of oil", which has allowed us to prove expression of key enzymes in our yeast strain for the metabolism of starch.