The bone-degrading enzyme machinery: from multi-component understanding to the treatment of residues from the meat industry
15 pags, 5 figs, 3 tabs. -- Supplementary data to this article can be found online at https://doi.org/10.1016/j.csbj.2021.11.027. ; Many microorganisms feed on the tissue and recalcitrant bone materials from dead animals, however little is known about the collaborative effort and characteristics of their enzymes. In this study, microbial metagenomes from symbionts of the marine bone-dwelling worm Osedax mucofloris, and from microbial biofilms growing on experimentally deployed bone surfaces were screened for specialized bone-degrading enzymes. A total of 2,043 taxonomically (closest match within 40 phyla) and functionally (1 proteolytic and 9 glycohydrolytic activities) diverse and non-redundant sequences (median pairwise identity of 23.6%) encoding such enzymes were retrieved. The taxonomic assignation and the median identity of 72.2% to homologous proteins reflect microbial and functional novelty associated to a specialized bone-degrading marine community. Binning suggests that only one generalist hosting all ten targeted activities, working in synergy with multiple specialists hosting a few or individual activities. Collagenases were the most abundant enzyme class, representing 48% of the total hits. A total of 47 diverse enzymes, representing 8 hydrolytic activities, were produced in Escherichia coli, whereof 13 were soluble and active. The biochemical analyses revealed a wide range of optimal pH (4.0-7.0), optimal temperature (5-65 °C), and of accepted substrates, specific to each microbial enzyme. This versatility may contribute to a high environmental plasticity of bone-degrading marine consortia that can be confronted to diverse habitats and bone materials. Through bone-meal degradation tests, we further demonstrated that some of these enzymes, particularly those from Flavobacteriaceae and Marinifilaceae, may be an asset for development of new value chains in the biorefinery industry. ; We acknowledge financial support of ERA-NET Marine Biotechnology (GA no.: 604814) funded under the FP7 ERA-NET scheme and nationally managed from the German Federal Ministry of Education and Research and Norwegian Research Council, the grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET), BIO2017-85522-R, PID2020-112758RB-I00 and PDC2021-121534-I00 from the Ministerio de Economía, Industria y Competitividad, Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI) (Digital Object Identifier 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union ("NextGenerationEU/PRTR"), and the grant 2020AEP061 from the Agencia Estatal CSIC. F.J.P and J.S-A. acknowledges grants PID2019-105838RB-C31 and PID2019-105838RB-C33 from the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (EU). Additional funding was received from the Norwegian Biodiversity Information Centre (PA 809116 knr. 47-14) and NORCE Norwegian Research Centre. J.W. acknowledge the use of de.NBI cloud and the support by the High Performance and Cloud Computing Group at the Zentrum für Datenverarbeitung of the Eberhard Karls University of Tübingen and the Federal Ministry of Education and Research (BMBF) through grant no 031 A535A. K.S.M. is supported by intramural funds of the US Department of Health and Human Services (to the National Library of Medicine). ; Peer reviewed