ERA-NET: New tools for prospecting the marine bone-degrading microbiome for new enzymes

New value-chains based on protein-rich deboning residues from the meat and poultry industry are emerging. Herein, enzyme-based protein hydrolysis is an attractive refinement process to achieve fat- and protein-based products. However, industrial enzyme-based protein hydrolysis is still hampered by low yields due to limited degradation of recalcitrant material. Driven by industrial demands, ProBone aimed at discovery of novel hydrolytic enzymes to improve such processes. The marine bone-degrading microbiome harbours a highly specialized community of free-living and symbiotic microorganisms associated to diverse bone-thriving invertebrates. An in-depth metagenomic study of non-cultivable microbiomes associated with marine-deployed bones, has provided a better understanding of the microbial diversity as well as of the microbial pathways and enzymes involved in bone degradation in the marine environment. The bone microbiome displayed a high taxonomic diversity, whose interplay between the organisms is needed for the degradation of bone matrix components. This degree of specialisation is revealed with the discovery of a unique collagen-degradation gene cluster, which is a key function in this unique environment. These studies open the possibility of moving beyond isolated strains and single enzymes, towards microbial communities and/or enzyme cocktails in the attempt to degrade recalcitrant bone materials in protein hydrolysis. This understanding is useful not only to protein hydrolysis, but may extend to bioremediation of recalcitrant materials, such as plastic and other emerging pollutants. Moreover, the analysis of the comprehensive metagenomic data, exceeding 350 million reads and accounting for more than 52,5 Gbp, allowed identification of relevant enzyme-encoding genes that may have the capacity to act on bone materials. 15 000 candidate enzyme sequences were identified, including more than 500 full-length and highly diverse ProBone-relevant enzymes, representing peptidases, ester-hydrolases, oxidoreductases, etc.?The sequence identity and structural identity of their homologs reflect both functional novelty, and a high degree of specialisation towards bone-degradation. About a dozen selected collagenases and glycohydrolases enzymes were further described by functional and biochemical profiling. Enzymes were combined in a first proof-of-concept experiment, which indicate that the joint action of collagenase and glycohydrolases improves protein hydrolysis of bone. ProBone also aimed at developing new tools for the marine biotechnology. Enzyme-encoding genes, including those studied in ProBone, are often difficult to produce and express in a soluble form. The project has thus addressed production host optimization to handle low expression levels by adapting the host to low-temperature conditions and thereby improve expression. Enzyme development was also addressed by expanding the CRISPR-Cas9 toolbox to facilitate an easier integration of genes and clusters to chromosomal location in production hosts. Such tools allow early assessment of expression levels in industry-relevant hosts and conditions. The 3-year ProBone project responded to a call on metagenomic approaches for biodiscoveries in non-cultivable microbiomes announced by Marine Biotechnology ERA-NET (ERA-MBT) and funded under the European Commission?s Seventh Framework Programme, grant agreement no. 604814. The project was coordinated by NORCE Norwegian Research Centre, and partners were Instituto de Catálisis y Petroleoquímica in Spain (CSIC), GEOMAR Helmholtz Centre for Ocean Research Kiel in Germany and the Romanian Institute Science and Technology - University of Agricultural Sciences and Veterinary Medicine (UASVM).

ProBone has been the first comprehensive metagenomic study carried out to understand the fate of bone material in the marine environment. ProBone has contributed to define new refinement routes that are key for protein hydrolysis in general, and specifically towards recalcitrant material. These advancements have the potential to contribute not only to the profitability and waste-management, but it also aligns well with the large need for protein-rich food for an increasing global population and for innovative livestock and fish feed products. ProBone has brought new enzymes, but also new ground-breaking perspectives on how improvements can be made using novel enzyme combinations. Validation of enzyme performance on deboning residue was performed as a small-scale prototype experiment (TRL3). Further technological and commercial verifications are expected to follow, in order to complete a successful technology transfer between research and industry.

ProBone will develop enabling technologies for the discovery of enzymes involved in the hydrolysis of bone from genetic material of marine microorganisms associated to bone decay (herein defined as the marine bone-degrading microbiome) accessed by culture-independent methods. To do so, we will study the marine bone-degrading microbiome that harbors a highly specialized community of free-living and symbiotic microorganisms associated to diverse bone-thriving invertebrates. The hydrolysis of organic bone components is expected to occur in concerted action with different enzymatic activities, including peptidases, ester-hydrolases, oxidoreductases, etc. ProBone is expected to generate new knowledge and innovation by using state-of-the-art technologies and developing new methods that overcome key bottlenecks in the marine biotechnology toolbox. These developments will foster an improved discovery pipeline of new enzymes for use in bioprocessing applications. The project is ambitious but feasible within the proposed time period and budget, as the expertise needed to reach the goals have been gathered in an international consortium of trans-disciplinary, highly qualified and dedicated research partners.