ERA-NET: Advanced Marine Biotechnology toolbox for accessing the uncultivated marine microbial biodiversity and its novel biomolecules

Det europeiske ERA-net prosjektet MarBioTech siktet på å utvikle nye verktøy og teknologier til funksjonsbasert screening i kombinasjon med DNA-sekvensbaserte søk for raskere å levere nye, verdifulle biomolekyler fra det marine miljøet. Den innovative teknologien som skulle utvikles i prosjektet, skulle anvendes på et bredt spektrum av eksisterende marine mikrobielle konsortier fra marine alger, maneter, akvakulturer, m.m., og identifisere nye, aktive marine biomolekyler med høyt kommersielt potensiale. MarBioTech-konsortiet besto av universitetene i Hamburg og Kiel i Tyskland, universitetet i Vancouver, Canada, den norske forskningsinstitusjonen SINTEF og den norske bioteknologibedriften ArcticZymes AS, som er velkjent for sine produkter innen molekylærbiologisk forskning og diagnostikk. I prosjektet utviklet SINTEF, i tett samarbeid med de andre prosjektpartnerne, funksjonelle screening-metoder for å finne nye DNA-aktive enzymer (nukleaser) som kan brukes innen molekylære diagnostikk, samt nye enzymer som nedbryter plast, for eksempel polykaprolakton (PCL) og poly(ethylene terephthalat) (PET). I tillegg ble sekvens-baserte screening-metoder forbedret i prosjektet, som en komplementær tilnærming til funksjonell screening. Et nytt aktivt nuklease-enzym ble oppdaget i samarbeidet med prosjektpartner ArcticZymes gjennom kombinasjon av funksjonell og sekvens-basert screening, og enzymet blir nå karakterisert videre i oppfølgingsprosjekter. Sekvensen til den nye nukleasen brukes nå også til leting etter andre nye DNA-aktive enzymer til mulig bruk i molekylær diagnostikk av for eksempel kreft eller arvelige sykdommer, samt overvåkning av smittsomme sykdommer. På den andre siden tilpasset SINTEF assayer til screening av plast nedbrytende enzymer utviklet av prosjektpartner UniHH, til et høy-gjennomsats format som brukes i robotisert screening på SINTEF. En screening-kampanje av PCL-aktive enzymer ble gjennomført ved bruk av det tilpassede assayet og et bibliotek av 11.000 kloner fra et oljereservoar metagenom. Flere enn 50 aktive kandidater ble oppdaget i screeningen som nå beskrives videre i oppfølgingsprosjekter. En ny massespektrometri-basert metode for å måle nedbrytningsprodukter fra PET aktiv enzymer ble også utviklet av SINTEF. Den ble brukt for å bekrefte PETase kandidater fra sekvens-basert screening. Et nytt PETase-enzym ble oppdaget som viser aktivitet mot PET ved lav temperatur. Dette er interessant mht. bruk i mer energi-effektive industrielle enzymprosesser. I tillegg utvider disse nye plast-aktive enzymene porteføljen av biokatalyser til nye løsninger for plastforurensingsproblematikken.

The MarBioTech project has played an important role in developing new high throughput functional screening methods at SINTEF for enzymes in the applications of molecular diagnostics (nucleases) and degradation of plastic, namely polycaprolactone (PCL) and Polyethylene Terephthalate (PET) (PETases). Finding new active nucleases for molecular diagnostics will contribute to the development of industrial enzymes of high interest and values for project partner ArcticZymes besides the newly developed screening method that SINTEF can now offer in other projects. The close collaboration between SINTEF and ArcticZymes in the MarBioTech project has extended to other projects in which the developed technology, i.e., screening method, and obtained results, i.e., sequences of active enzymes, have been extensively used, for example in the following-up activity in the SFI-IB Centre for Industrial Biotechnology project (funded by the Norwegian Research Council, project no. 309558). Enzymes for molecular diagnostics are important in developing diagnosis methods in oncology or genetic diseases as well as in surveillance of infectious diseases. As plastic pollution has increasingly become a serious concern for environment and human health, finding new biocatalysts for plastic degradation is one of the approaches to tackle the problem as enzymatic processes are usually carried out in mild conditions compared to harsh conditions that are often employed in chemical or physical processes. In the project, a high throughput method for screening of enzymatic plastic degrading activity has been developed in close collaboration with project partner UniHH. The obtained results from the screening campaign contained more than 50 candidates having high PCL activity. These candidates will be characterized further in the BLUETOOLS project funded by Horizon Europe (grant agreement no. 101081957). In addition, a new PETase showing activity at ambient temperature was discovered using SINTEF's data mining pipeline and the protein profile provided by UniHH. The newly found candidates will contribute to the increasing portfolio of enzymes that can degrade plastics which can be incorporated into further processes to address the plastic pollution problem. The mentioned work has been enabled by the close international collaboration between SINTEF and UniHH. Moreover, the work of developing screening assays for biofilm inhibitors in the MarBioTech project was also in close interplay with the ERA BlueBio project AquaHealth (RCN project no. 312075) in which SINTEF and UniHH are also partners. SINTEF's data mining pipeline has also been improved using the codes provided by project partner University of Vancouver, Canada, to enable the creation of local Sequence Similarity Networks which was important for the process of choosing final candidates for further characterization. The improved pipeline has already been used by SINTEF in other enzyme mining projects.

Organisms in the marine environment represent a largely unexploited source of highly valuable biomolecules. Due to the development of sequencing technologies in the last few decades, we are now able access a vast amount of sequence information of metagenomes of cultivable and non-cultivable marine organisms. Unfortunately, our abilities to link such sequence information with function lags completely behind. The conventional system to annotate protein functions, e.g., annotation based on BLAST homology search, is very poor and often provides false predictions, in particular for classes of proteins for which biochemical characterization data has not been accumulated. Consequently, it is virtually impossible to identify novel proteins and enzymes based on sequence based screenings, only. Therefore, the goals of MarBioTech are to develop innovative tools and technologies to advance function-based searches in combination with sequence-based searches and to deliver valuable biomolecules of marine origin. Together with the innovative technology advancement, a wide range of existing marine resources including microbiomes of marine alga, jelly fish, and marine fish farms, among others, will be exploited by combining innovative function-, sequence-based, and in vitro screenings for the identification of novel active high-value marine biomolecules. The target molecules will include enzymes involved in marine plastic degradation (PET esterases), fluorescent proteins for molecular medicine, novel highly active RNA polymerases as well as DNA nucleases for metagenome mining and molecular biology, and quorum quenching (QQ) proteins to prevent biofilm formation. Within this consortium, the Universities of Hamburg, Kiel and Vancouver together with the research centre of SINTEF work closely together. In addition, ArcticZymes AS as a well-known company in the field of molecular research and diagnostics adds their complementary expertise to the consortium.