To create a sustainable chemical industry in Europe, we need to harness innovative breakthrough technologies. Industrial biotechnology is already making strides in chemical manufacturing by offering processes that are more efficient, specific, safer, and less energy-intensive. However, the industry faces a limitation: there are not enough types of enzymes being used. The W-BioCat project aims to expand the range of enzymes available, specifically by focusing on tungsten-containing enzymes (W-enzymes). W-enzymes have the ability to facilitate complex chemical reactions, particularly those that involve low redox potential reductions. Unfortunately, producing these enzymes on a large scale and at a reasonable cost has been a challenge. To overcome this, we plan to produce W-enzymes using an industrially friendly microorganism, E. coli. One big hurdle is the difficulty of synthesizing the tungsten cofactor for these enzymes. We're addressing this issue through advanced computational enzyme design techniques. Our goal is to create a completely new pathway for producing the tungsten cofactor in E. coli. With Hop-on W-BioCat, we will introduce a new W-enzyme expression system using the microorganism Aromatoleum evansii. This system has already proven effective in producing W-enzyme AaAOR from Aromatoleum aromaticum. Notably, AaAOR can oxidize H2, enabling it to catalyze the reduction of carboxylic acids to aldehydes or converting NAD+ to NADH. This innovation allows us to streamline the reaction, using just one enzyme instead of two to drive the process with H2. By incorporating AaAOR into our project, we gain new ways to achieve the objectives of the W-BioCat project, particularly demonstrating a hydrogen-driven conversion of oleic acid—derived from plants—into the emollient ester oleyl oleate. The expansion of the W-BioCat project with Hop-on W-BioCat offers significant synergies that will enhance our prospects to provide W-enzymes for a sustainable chemical industry.

Lytic polysaccharide monooxygenases (LPMO) and cytochrome P450 (CYP) are copper- and iron-dependent, respectively, enzymatic systems that perform regio- and stereospecific oxidation of non-activated hydrocarbons in Nature. To control such reactions in modern industry and biotechnology is of utmost importance in creating products of value such as secondgeneration bioethanol and products of value for i.e. the pharmaceutical industry. Due to the major drawbacks of using CYPs, including their partially membrane bound nature and the requirement of a reductase in combination with reducing agents such as NAD(P)H to transfer electrons to the active site for oxygen activation, it is highly desirable to develop new type of catalyst that can perform the same type of reactions. An attractive alternative strategy is to engineer LPMOs to perform CYP catalysis. LPMOs are small, robust, easy to produce in large scale, and rigid water-soluble proteins with a plethora of electron donors. The extended, flat LPMO surface, with huge natural sequence variation and thus, likely, mutability, provides a fantastic scaffold for engineering access to the active site as well as substrate affinity. We propose to use LPMOs engineered to accommodate typical CYP substrates and immobilize this on solid supports to provide confinement necessary in bringing the oxygen species together with the C-H bond to be oxidized in a tailored, "closed" environment. Moreover, the rate of LPMO catalysis can be greatly enhanced compared to traditional CYP catalysis by the addition H2O2 in the presence of low, priming concentrations of an external reductant to achieve efficiency constants (kcat/Km) in the order of 106 M-1s-1, which is typical for peroxygenases. The proposed ground-breaking research fits excellently well with the work program "Future and Emerging Technologies" where the goal is to challenge current thinking.

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 tungsten-containing 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 biosynthesis is the 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 process to 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.

Heritage conservation preserves the tangible remains of society, but relies on toxic, unsustainable materials and on energy-consuming air conditioning of collections. GoGreen promotes preventive and remedial conservation practices based on green principles to spearhead the green revolution within conservation. Specifically, GoGreen will: (1) develop new damage functions that allow more flexible environmental control in collections, thus improving energy efficiency; (2) generate innovative nature-inspired, bio-based and historical conservation treatment-inspired methods for remedial conservation, including new cleaning solutions for paintings and metals, all using green solvents, bio-inspired reagents, green delivery systems; and stabilization methods for metal and glass that employ innovative techniques like biopassivation to stabilize metal surfaces and nanomaterials that mimic the growth of silica to stabilize glass; (3) assess our new materials and methods using cutting-edge analytical techniques and benchmark methods in collaboration with expert practitioners and museums, to determine their efficacy in the cleaning of paintings and metals, or the stabilisation of glass and metal objects; (4) develop a digital web-app to aid conservators in the design of green preventive and remedial conservation treatments, and a decision model integrating green thinking in complex conservation decision making. Crucially, GoGreen encompasses all relevant and necessary academic and socio-economic actors spanning the full stakeholder value chain to ensure future impact. Our uniquely advantageous composition coupled with the embedding of bottom-up education of professionals through modules and courses for conservation training programmes, emerging conservators and mid-career professionals, guarantees the next generation of conservators are fully prepared to embrace the GreenDeal.