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

The European ERA-net project MarBioTech project set out to develop new tools and technologies to advance function-based discovery in combination with DNA sequence-based approaches and to deliver valuable new biomolecules of marine origin. The innovative technology developed in the project should be applied to screen a wide range of existing marine microbial consortia of marine alga, jelly fish, and marine fish farms, among others, and to identify novel active marine biomolecules with high commercial potential. The MarBioTech consortium consisted of the Universities of Hamburg and Kiel, Germany, the University of Vancouver, Canada, the Norwegian research organization SINTEF, and Norwegian biotech company ArcticZymes AS, well known for their products in the field of molecular research and diagnostics. In the project SINTEF, in close collaboration with the other project partners, developed methods for functional screening for DNA-active enzymes, which can be used in molecular diagnostics applications and enzymes that can degrade plastic materials, namely polycaprolactone (PCL) and poly(ethylene terephthalat) (PET). In addition, sequence-based screening methods at SINTEF were improved in the project, as a complementary approach for functional screening. A new active nuclease was discovered by SINTEF in collaboration with project partner ArcticZymes through combining function- and sequence-based screening. The active enzyme is now being characterized further in a follow-up project. In addition, the sequence of the new enzyme is now being used as the template to look for new DNA active enzymes for molecular diagnostics of e.g., cancer or genetic diseases, as well as to monitor infectious diseases. On the other hand, SINTEF adapted assays for screening of plastic degradation enzymes first developed by project partner UniHH to the high throughput format used on the robotic screening platform at SINTEF. A screening campaign of PCL active enzymes was carried out with the new assay and a library of 11,000 clones from an oil reservoirs metagenome. More than 50 active enzyme candidates were identified in the screening, which can now be further characterized in follow-up projects. A new highly sensitive mass spectrometry-based method for detecting products from PET degradation by PET active enzymes was also developed and used to identify active enzymes candidates from sequence-based screening. That way, a new PETase showing activity at low temperature was discovered, with prospects for potential use in more energy efficient industrial enzyme processes. In addition, the newly discovered PCL and PET active enzymes contribute to an increasing portfolio of biocatalysts that can lead to new solutions for the plastic pollution problem.

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.