**4. Conclusions**

Marine invertebrates with associated microbiota form complex holobiont assemblages, which are attractive sources of biologically active organic compounds and (poly-)peptides, including enzymes. Marine enzymes have a high potential to be applied in green organic synthesis and in pharmaceutical and industrial biotechnology. The search for improved biocatalysts can be carried out using different strategies, such as screening a huge number of environmental samples, pursuing enzyme engineering, mining genomic and proteomic data, or a combination of more than one approach. The process of data mining transcriptomes has some advantages over genomic analysis; the most obvious advantage is that only enzymes that are expressed in a given environmental context are retrieved, including enzymes not completely characterized and with unknow enzyme-substrate specificity. This is particularly advantageous in the case of the marine assemblages of microbionts that form species-specific holobionts, from which the purification of enzymes with a high yield may be a concern. Thus, once identified, the cloning and the recombinant production of desirable marine biocatalysts can be structure-guided and based on the nature of expressed transcripts.

According to data reported in this work, the zoantharian *P. variabilis* expresses a variety of putative enzymes that could potentially be converted into biotechnologically useful biocatalysts and biopharmaceuticals. This holo-transcriptomic data demonstrates that a single holobiont assemblage comprises a unique repository of relevant biotechnological enzymes. Finally, the integrative analyses of this holo-transcriptome point to a valuable marine resource for the discovery of improved enzymes with applications in green chemistry, industrial and pharmaceutical biotechnology.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-3397/16/6/207/s1. Figure S1: Summary of the unigenes mapped to the public databases. Figure S2: Characteristics of the sequence homology search results. Figure S3: GO and KEGG pathways assignments. Figure S4: Maximum Likelihood (ML) phylogenetic tree of the predicted *P. variabilis* beta-*N*-acetylhexosaminidase (3.2.1.52) and their closest homologous sequences. Figure S5: Prediction of enzymes with two activities closely positioned in given metabolic pathway. Table S1: Species from the BLAST search of the barcode sequences from the *P. variabilis* holobiont transcriptome against the NCBI nr database. Table S2: KEGG pathways mapping summary. Table S3: List of predicted enzymes in *P. variabilis*. Table S4: List of enzymatic activities with relevance in treatment of rare diseases

and other pharmaceutical fine chemicals predicted in *Protopalythoa variabilis* holo-transcriptome. Table S5: List of enzymatic activities with relevance in colorant, aromas, flavor, fragrance, cosmetic and hygienic industries predicted in *Protopalythoa variabilis* holo-transcriptome. Table S6: List of enzymatic activities with relevance in agrochemical, and food and feed industries predicted in *Protopalythoa variabilis* holo-transcriptome. Table S7: List of enzymatic activities with relevance in bioconversion and biopolymer synthesis predicted in *Protopalythoa variabilis* holo-transcriptome. Table S8: List of enzymatic activities with relevance in other industries predicted in *Protopalythoa variabilis* holo-transcriptome. Table S9: List of enzymatic activities with relevance in molecular biology and analytical applications predicted in *Protopalythoa variabilis* holo-transcriptome. Table S10: RNA-sequencing and assembling statistics. File S1: KEGG pathways.

**Author Contributions:** Conceived and designed the experiments: J.-É.R.L.M. and G.R.-B. Performed the experiments: J.-É.R.L.M., C.H. and Q.L. Analyzed the data: J.-É.R.L.M., A.R.B.P.-S. and G.R.-B. Contributed reagents, materials and/or analysis tools: S.M.-Y.L. and G.R.-B. Field trip missions, species identification and deposit collection: P.B.G. and C.D.P. wrote the manuscript with the contribution from all other authors: J.-É.R.L.M. and G.R.-B. All authors have read and approved the final article.

**Funding:** Financial support for G.R.-B. and research at the Institute for Marine Sciences, Federal University of Ceará, was provided by the Brazilian National Council for Scientific and Technological Development, CNPq (Marine Biotechnology Network—Proc. 408835/2013-3), the Ministry of Science, Technology and Innovation (MCTI), Brasília, DF, Brazil. At the University Federal of Pernambuco, financial support was CNPq (Proc. 408934/2013-1) granted to C.D.P. Research at the University of Macau was supported by grants from the Science and Technology Development Fund (FDCT) of Macao SAR and Research Committee, University of Macau, Macau, China.

**Acknowledgments:** Our gratitude to the Program on Toxinology (Issue 2010), the Coordination for the Improvement of Higher Education Personnel (CAPES), the Ministry of Education of the Federal Government of Brazil, Brasília, DF, Brazil. J-ÉRM was a doctoral fellowship recipient from CAPES.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
