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ENERGIX-Stort program energi

Development of a plant biotechnology platform for low cost production of industrial enzymes to boost biorefinery of lignocellulose biomass

Alternative title: Development of a plant biotechnology platform for low cost production of industrial enzymes to boost biorefinery of lignocellulose biomass

Awarded: NOK 9.2 mill.

Project Number:

243974

Application Type:

Project Period:

2015 - 2020

Funding received from:

Subject Fields:

Partner countries:

Second-generation biofuels are considered sustainable, overcoming the problems of first-generation biofuels by using non-food materials such as forest raw materials, and forest and agricultural residues. However, there are many challenges involved in the conversion of lignocellulosic biomass into biofuels. The Bioboost project has the main goal of producing cheaper enzymes by using tobacco plants in a CO2-neutral way, thereby reducing the carbon footprint as well as producing cheap cell wall degrading enzymes. With a consortium consisting of NIBIO (leader), NMBU, NTNU and the international partners Max Planck Institute in Germany and Michigan State University, USA, and with strong support from our industrial partner Borregaard, we completed the project before the end of the project in November 2019. Cooperation between the partners has gone very well, and the Bioboost consortium has produced results that are shown in the final project report and are summarized here. 1) The Bioboost project has investigated the use of green plants (the Green Factory) to produce cell wall degrading enzymes for future biorefining of lignocellulosic biomass. We selected four candidate enzymes that represent the minimal enzyme cocktails and play central roles in the breakdown of pretreated lignocellulosic biomass. These are TrCel5A, TrCel6A, TrCel7A and TrCel7B from the fungus Trichoderma. All enzymes were produced in Nicotiana benthamiana plants, a relative of tobacco plants, using NIBIOs plant genetic transformation platform. The genes that encode for several important enzymes involved in lignocellulosic biomass conversion, Cel6B, Cel9A, Xeg74, and a thermostable betaglucosidase, BGLI, were introduced into tobacco plants through chloroplast transformation. Enzymes were highly expressed with normal growth and phenotype. Moreover, we focused on analyses of these eight plant-produced enzymes. 2) Different enzymatic analyses were carried out on the N. Benthamiana-produced key enzymes TrCel5A, TrCel7B, TrCel7A and TrCel 6A, with both NMBU?s own substrate and Borregaard?s Bali-substrate. The result of enzyme analyses showed that plants and fungi have different glycosylation patterns (N-and O-glycosylation). Thus, we have analysed the glycosylation pattern of our plant produced TrCel7A compared to TrCel7A from fungi, in collaboration with the Glycosylation Research Group at the University of Oslo. The results were very interesting and were published in Plant Biotechnology Journal in 2019 (van Eerde et al. 2019, Doi: 10.1111/PBI. 13227). The same analyses (protein characterization and purification, enzyme analysis and protein glycosylation study) were performed on the other plant-produced enzymes TrCel5A, TrCel 6A and TrCel7B. Manuscripts based on these results are expected to be published next year. We have also performed molecular and enzyme analyses of the Cel6B, Cel9A, Xeg74, and BGLI enzymes, produced in tobacco chloroplasts and met challenges of protein stability and solubility. The results will be ready for scientific publication next year. 3) We have conducted various studies on the laccase LAC51 enzyme. As reported previously, lac51 was produced in N. Benthamiana plants. Enzyme activity analysis showed an interesting result with the help of a commercial substrate and lignin monomer. A manuscript based on the results of plant produced LAC51 is ready for submission. 4) In addition to producing the enzymes described above in plants, we carried out a genome study to identify and characterize new cell wall degrading enzymes in Thermoascus Aurantiacus, a fungus that has been thoroughly examined for its ability to secrete large amounts of thermostable enzymes for depolymerization of cellulose and hemicellulose of plant biomass. We found over 1500 genes that were significantly upregulated in spruce compared to a control. Of these, we have selected three laccase candidates for further work; these laccases have been produced in N. Benthamiana plants and protein analyses are in progress. 5) Bioboost is concerned with dissemination, communication, publication and project impact: all partners have presented and disseminated Bioboost and its results in international and national conferences, meetings, seminars and social media, in addition to two conferences with stakeholders. The consortium has published both peer-reviewed scientific papers and popular scientific articles, lectures/posters, as well as articles in newspapers and social media as shown in the publication list. Moreover, we have also spin-off a new EEA Norway-Romania project (EEA-RO-NO-2018-0078). The network we have established through Bioboost shall be continued in future collaboration and joint publications. We thank the Research Council of Norway's ENERGIX programme for supporting the project. Although the Bioboost project is finished we will continue to publish our results, as well as communicate and disseminate the knowledge we have obtained through the project.

Dette prosjektet har gitt virkninger på ulike områder både kortsiktig og langsiktig: 1) kompetansehevingen blant prosjektpartnerne. 2) Et langsiktig godt samarbeid mellom forskningsmiljø og næringsliv ble etablert, særlig med Borregård som våre viktige industripartner. Et spinn-off som viser virkningen av prosjektet er et nytt samarbeid gjennom NIBIOs Sinograin II Norge-Kinaprosjekt. 3) Bioboost-prosjektet har utnyttet plantebioteknologisktilnærminger for å produsere enzymer (ikke-mat og ikke-fôr produkter) i N. benthamiana planter. Bioboost-prosjektet har generert flere effekter gjennom prosjektperioden. Nasjonal og internasjonal samarbeid har blitt styrket. Tverrfaglig samarbeid mellom NIBIO, NMBU og NTNU er styrket. Samarbeidet med Max Planck Institutt i Tyskland og Michigan State universitet i Amerika har vært lærerikt og godt. Med Max Planck Institutt har NIBIO fått et nytt prosjekt som er et spin-off av Bioboost (EØS Norge-Romania prosjekt: EEA-RO-NO-2018-0078 SmartVac).

Summary: Sustainable and cost effective biorefinery of lignocellulose biomass for the production of biofuels and other higher value chemicals requires a large amount of enzymes. However, the cost of enzyme is a well-known bottleneck in present lignocellulose biorefinery. We propose here Bioboost, a research project building upon a previous "Greenzyme" concept, one of the 12 finalists during 2013 "new concept" competition but with some fresh input from our pilot experiments. Bioboost aims to develop a plant biotechnology platform for low cost production of key cell wall degrading enzymes (CWDEs) to boost biorefinery of lignocellulose biomass. We use tobacco, a non-food and non-feed crop (a primary CO2 binder), instead of energy demanding fermenter-based systems. Tobacco is an ideal choice for the production of key CWDEs because of its feasibility for genetic manipulation, excellent biomass (ca. 40 tons of leaf fresh weight/acre) and prolific seed production (ca. one million seeds per plant), thus hastening the time in which a product can be scaled up and brought to market. For proof-of-principle, we will express enzymes comprising minimal cocktails (including endocellulases, cellobiohydrolases, beta-glucosidase, and lytic polysaccharide monooxygenases) screened and identified as optimal for converting pretreated lignocellulose substrates in our consortium partner Eijsink's laboratory. Bioboost is organized in 6 workpackages (WPs) covering (i) effective management and successful dissemination of project results to the relevant stakeholders (WP1 and WP6); (ii) low cost production of key CWDEs in tobacco (WPs 2-3); (iii) Functionality and impacts of tobacco produced recombinant CWDEs (WP4-5). The WPs will be fulfilled by a consortium consisting of national and international partners with outstanding scientific expertise in plant genetic engineering, cell wall degrading enzymes and cell wall architecture, and an experienced industrial partner, Borregaard.

Publications from Cristin

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Funding scheme:

ENERGIX-Stort program energi

Thematic Areas and Topics

InternasjonaliseringInternasjonalt prosjektsamarbeidPolitikk- og forvaltningsområderOlje og gass - Politikk og forvaltningBransjer og næringerTransport og samferdselBransjer og næringerPolitikk- og forvaltningsområderMiljø, klima og naturforvaltningPolitikk- og forvaltningsområderNæring og handelDelportefølje InternasjonaliseringBransjer og næringerLandbrukBioteknologiIndustriell bioteknologiBransjer og næringerEnergi - NæringsområdeInternasjonaliseringBransjer og næringerSkog og trebrukDelportefølje Et velfungerende forskningssystemPolitikk- og forvaltningsområderSamferdsel og kommunikasjonBioteknologiGrunnforskningEnergiEnergibruk i transport, biodrivstoffBioøkonomiEnergiLTP3 Bioøkonomi og forvaltningLTP3 Et kunnskapsintensivt næringsliv i hele landetBransjer og næringerProsess- og foredlingsindustriMiljøteknologiLTP3 Fagmiljøer og talenterNaturmangfold og miljøDelportefølje KvalitetMiljøteknologiAnnen miljøteknologiLTP3 Miljøvennlig energi og lavutslippsløsningerNaturmangfold og miljøMiljøteknologiKlimarelevant forskningPolitikk- og forvaltningsområderEnergi - Politikk og forvaltningLTP3 Muliggjørende og industrielle teknologierPolitikk- og forvaltningsområderLTP3 Høy kvalitet og tilgjengelighetLTP3 Nano-, bioteknologi og teknologikonvergensPolitikk- og forvaltningsområderSkog, landbruk og matLTP3 Klima, miljø og energiLTP3 Styrket konkurransekraft og innovasjonsevnePortefølje Energi og transportPortefølje Banebrytende forskningPortefølje ForskningssystemetPortefølje Mat og bioressurserPortefølje InnovasjonPortefølje Muliggjørende teknologierMiljøvennlig energiEnergibruk i transport, biodrivstoff