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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Unravelling the secrets of oxidative biomass decomposition

Alternative title: Oksidasjonsprosesser i enzymatisk nedbryting av biomasse

Awarded: NOK 10.0 mill.

Enzymatic depolymerization of difficult-to-degrade polysaccharides such as chitin and cellulose is a crucial element of the global carbon cycle and of great scientific and commercial interest. A better understanding of Nature's tools for degrading cellulose- or chitin-rich co-polymeric structures, as in plant cell walls, insect cuticles and shrimp shells, will have a profound impact on important areas of biological research, for example microbial behaviour and virulence, and plant pathology. Furthermore, efficient degradation of non-edible plant material, such as straw and wood, is a crucial step in second generation "biorefining", i.e. the conversion of these materials to useful products. In 2010 we discovered Lytic Polysaccharide Monooxygenases (LPMOs), which has had a major impact on the field. In contrast to what was previously thought, it is now clear that oxidative processes catalyzed by LPMOs and other enzymes are important. This opens up completely new perspectives, both in fundamental science and in industrial applications. We need to know more about LPMOs, especially since some organisms may have as many as 30 LPMO-encoding genes, showing that these enzymes are truly important. In this project, we carry out fundamental studies of LPMOs aimed at answering the following questions: (1) What are the structural determinants of the substrate specificity and catalytic efficiency? (2) What are the rate-limiting factors determining LPMO activity in vitro and in vivo? (3) How does microbial biomass conversion depend on LPMO action? (4) What is the interplay between LPMOs and other redox enzymes involved in plant cell wall degradation? (5) How exactly do LPMOs do their job? This fundamental project, with the ambition and potential to deliver ground-breaking research, is aligned with a Marie Curie training network (ended in 2021) and with applied projects in biorefining. In 2020, the project leader received an ERC Synergy grant and the projects are aligned, while building on what we have achieved so far. The Synergy project focuses on the most basic questions, but also, importantly, on an ambitious applied goal, namely to get these unique enzymes to catalyze other types of useful reactions. Highlights from the final phase of the project include: - The PhD fellow defended his thesis on 23 June 2022. - An article in PNAS and an article in Nature Communications that show how sunlight can be used to drive LPMO reactions. The discovery described here can provide a partial explanation for why light affects what happens to plant biomass in nature (increased light -> increased decomposition) and also provides opportunities to use less energy (namely light instead of chemical energy) when breaking down biomass with enzymes. - We have continued to work on our previous discovery of LPMOs that act specifically on xylan (a so-called "hemicellulose"). This may provide new understanding of how LPMOs can do much more than just break down cellulose and chitin. It may be that the large arsenal of LPMOs exists for organisms to be able to break down different parts of the highly complex plant cell wall. - We have initiated an even broader approach to the question of which natural substrates LPMOs can act on, including bacterial and fungal cell walls. It is now clear that finding out more about this can lead to completely new biological insights and biotechnological applications. This work will be continued in an NMBU-funded new PhD project. - We have also carried out a unique "directed evolution" study where we have tried to introduce new, industrially relevant properties into an LPMO, while at the same provide insights into the structural determinants of substrate specificity. This work will be completed in the ERC project. Our findings are also used in other, more applied projects at NMBU, such as in the FMEen Bio4Fuels and in an international ERA-Net project where LPMOs are used to produce better cellulose fibres. In 2022, we got a new EU project on biorefining of chitin-rich biomass where LPMOs are used. Finally, it can be mentioned that this project has also helped form the basis for a completely new project at NMBU, Enzyclic, where we will, among other things, try to engineer LPMOs to break down plastic.

Prosjektet har sterkt bidratt til at NMBUs verdensledende fagmiljø i feltet har behold og videreutviklet sin posisjon. Miljøet er respektert verden over og har blant annet produsert både originalpublikasjoner i toppjournaler og ledende oversiktsartikler, som siteres mye, noe som bidrar til å sette NMBU, og dermed Norge på kartet. NMBU er en ettertraktet samarbeidspartner for internasjonal akademia og industri. Prosjektet har ført til, eller muliggjort, flere typer (delvis ad hoc) internasjonalt samarbeid, som bidrar til (1) å få flere, bedre og mer relevante resultater, (2) å styrke forskningspartnernes og Norges renomme i feltet, og (3) å styrke grunnlaget for framtidig samarbeid. Når det gjelder publikasjoner har prosjektet vært enormt produktivt. Prosjektet har generert kunnskap som har blitt brukt av industrien (Borregaard, No og St1, Fi) i andre, prosjekter med et anvendt fokus. Borregaard har utviklet teknologi basert på NMBU kunnskap som har blitt verifisert i pilot (flere tusen liter) skala. Prosjektet har vært med på å danne grunnlaget for SFI Industriell Bioteknologi (2020-2028), hvor mange norske industrier er med. Grunnforskningsprosjekter som dette er av avgjørende betydning for at vår mer anvendte forskning har tilstrekkelig høyt nivå og kan bidra til å skape virkelige konkurransefordeler. Prosjektet har bidratt til at vi kan være en pålitelig og ikke minst uavhengig kunnskapsleverandør til norsk industri og andre interessenter. Prosjektet har gjort det mulig å bidra litt til forskning på LPMOenes rolle i patogene mikroorganismer. Dette er et svært viktig tema, siden LPMOer tilsynelatende spiller nøkkelroller i flere humane sykdommer og plantesykdommer, mens de til og med kan spille en rolle i overlevelse av lakselus i sjøvann. Det jobbes med å bygge videre på dette og NMBU har allerede valgt å bruke noen av sine egne ressurser til å utvikle dette feltet. Prosjektet har vært en vesentlig del av grunnlaget for tildeling av ERC-Synergy prosjektet «Cube» (Unravelling the secrets of Cu-based catalysts for C-H activation; 2020-2026), hvor Eijsink (NMBU) er av fire PI (de andre PI er fra UiO, Italia og Tyskland). Her er målet å bruke all den akkumulerte LPMO-kunnskapen til få LPMOer til å katalysere helt nye type «vanskelige» reaksjoner, med potensielt vesentlige bidrag til et mer bærekraftig samfunn. En annen follow-up er det nye Enzyclic prosjektet (ledet av Gustav Vaaje-Kolstad; NFR) hvor vi blant annet skal videreutvikle noen av de LPMOene som vi har studert i prosjektet for bruk i enzymatisk resirkulering av plast. En siste follow-up er et EU finansiert prosjekt på bioraffinering av kitin-rik biomasse, som heter Valuable (2022-2025).

Enzymatic depolymerization of recalcitrant polysaccharides such as chitin and cellulose is a crucial element of the global carbon cycle and of great scientific and commercial interest. A better understanding of Nature's tools for degrading cellulose- or chitin-rich co-polymeric structures, as in plant cell walls and insect cuticles, will have a profound impact on areas such as microbial physiology, plant pathology and biorefining. The 2010 discovery of Lytic Polysaccharide Monooxygenases (LPMOs), in our lab, has changed the classical paradigm of polysaccharide turnover being accomplished by hydrolytic enzymes only. It is now clear that LPMO-catalyzed oxidative processes play a major role. Accumulating experimental data, as well as the abundance of LPMO-encoding genes in biomass-degrading microorganisms, indicate that the action of these copper-enzymes is very important and, moreover, that novel functionalities are yet to be discovered. Today, we know that LPMO function is affected by reductants, molecular oxygen, bivalent metals, redox mediators and even light, but the implications of these features for LPMO function in natural biomass conversion remain largely unexplored. In this project, we will carry out fundamental studies of LPMO functionality, primarily aimed at answering the following outstanding questions: (1) What are the structural determinants of substrate specificity and catalytic efficiency? (2) What are the rate-limiting factors determining LPMO activity in vitro and in vivo? (3) How does microbial biomass conversion depend on LPMO action? (4) What is the interplay between LPMOs and other redox enzymes, e.g. enzymes acting on other polymers in natural substrates, such as lignin in plant cell walls? (5) How exactly do LPMOs act on their natural substrates? This fundamental project, with the ambition and potential to deliver ground-breaking research, will be aligned with a Marie Curie training network and with applied projects in biorefining.

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FRIMEDBIO-Fri prosj.st. med.,helse,biol