Within the phosphatases, phytases are of special interest because in addition to their relatively poorly studied in vivo functions, commercialized phytases have a growing international market and are one of the most important type of biocatalyst for the global enzyme industry (15). With very few exceptions, which includes the five enzymes described in the chapters 3, 4 and 5 of this study most characterized phytate degrading enzymes are derived exclusively from cultured individual microorganisms. Consequently, the real diversity of phytate-degrading enzymes remains underestimated. This partially limits our understanding of the biological functions of phytate degrading enzymes and also keeps concealed interesting enzymes with putative biotechnological relevance. In
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contrast to other biocatalyst such as lipases or cellulases, metagenome-derived phytases have not been commercialized so far (16).
Several groups around the world have made efforts aiming to retrieve phytase homologous genes from the unculturable fraction of microorganisms. The initial attempts in this direction were performed based on sequence similarity, mostly by using PCR amplification with degenerated primers. The designed primers were based on sequences of previously reported phytases from culturable microorganisms. This method has two major disadvantages. First, it limits largely the recovered genes to close relatives of the preceding genes (17-19). Moreover, it is uncertain if the identified and subsequently cloned gene will encode a functional enzyme capable of using phytate as substrate. This is due to the fact that almost all types of phytases are part of larger phosphatases superfamilies, which are capable of processing phosphorylated substrates but not necessarily phytate (20).
The subsequent improvement was the combination of PCR-based methods with large-scale sequencing experiments. The sequence information is used to design primers and other probes which are suitable to recover full-length versions of specific target genes (21). This type of procedure is called gene-targeted-metagenomics and was first used for the recovery of genes encoding aromatic dioxygenases from contaminated soil samples.
Originally, gene-targeted-metagenomics utilized pyrosequencing and required that the targeted gene contained enough conserved regions of suitable distance for PCR. Without those conditions, the designed sets of primers did not had the essential coverage for the amplification of the genes of interest (22). Nowadays, by combining shotgun sequencing methods and bioinformatics, these limitations are ovoid. Under the existent methodologies, all DNA is extracted and subsequently sheared into smaller fragments for library preparation which allows independent sequencing of the fragments. The resulting DNA sequences (i.e., reads) can be mined for genes with specific biological functions (23).
In the specific case of phytases, few sequence-based metagenomic surveys have been employed. Most of them focused on mining metagenome-derived gene sequences from sources such as subsurface groundwater, acidic peat-soil microbiomes, rumen and insect-cultivated fungus-gardens. The resulting phytases were characterized and exhibited habitat-related characteristics such as thermostability and acid resilience(24-26). The major limitation of those approaches is the low performance identifying novel phytase-encoding genes with no or low similarity to known ones. Mootapally et al. (26) identified
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a HAPhy by mining the metagenomic data obtained from the rumen of buffalos. However, the identified phytase showed more than 95% sequence identity to a previously reported phytase encoded by species belonging to genus Prevotella (26). Although, other sequence-based attempts reported lower sequence identity with known phytases (≈50 %) the recovered phytases were again strictly limited to a typical type of known phytases i.e.
HAPhys (25). Thus, the scavenge of phytases by sequence-driven approaches can provide results but remains limited to recover only enzymes harboring known molecular signatures.
Consequently, function-based approaches are preferred over the sequence-based methods to analyze the diversity of protein families with specific functions and for the discovering genes with novel functions (21). In the case of the phytases, function-driven screenings are also required to encounter “real” novel genes encoding phytases.
Different function driven approaches are broadly used to recover novel biocatalysts e.g.
heterologous complementation, induced gene expression and phenotypical detection of the activities (21, 27). Heterologous complementation of host strains or mutants is a simple fast and highly selective method in which host strains or their mutants require the expression of the targeted genes for growth under selective conditions. Different types of genes and biocatalysts have been recovered by this method (e.g. DNA polymerases and RNAses) (28, 29). Conversely, induced gene expression does not limit the growth of the clones carrying the library, but instead this method involves the use of different strategies for the detection of genes of interest. The strategies are based on the coupled expression of those genes with reporter genes e.g. gfp. Compared with the screening by heterologous complementation, the induced gene expression might lead to a higher rate of false positives, because the transcriptional activation of the reporter genes could be occasionally induced by cellular effectors and not necessarily by specific substrates (21).
Finally, one of the most broadly used function-based screenings is the phenotypical detection of the target activities. This method utilizes chemical dyes or chromophores derivated of enzyme substrates for the activity detection (27). These substances are incorporated into the growth medium, allowing the recognition of individual clones bearing the specific targeted metabolic capabilities (21). Plenty of successful examples of the application of this approach are reported in the literature e.g. proteases, esterases, lipases, cellulases among many others (30-33).
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The register of the positive clones/activities derived from function-based screening strategies is achieved by different methods. Those methods have variable levels of technological requirements from common Petri dishes to coupled droplet-laser devices.
Fig. 2 shows a simplified overview of the library construction steps and different methods for the detection of the target activities in function-based metagenomics (27).
Fig. 2. Simplified overview of function-based metagenomic screening procedures. A. Steps involved in the construction of a small DNA fragment library from environmental metagenome or from culturable bacteria. B. Agar plate activity screening. C. Microtiter plate screening. D. Microfluidics coupled with fluorescence-activated cell sorting. Modified from (27).
Different from other relevant biocatalysts such as lipases or glycosidases, phytases derived from function-based screenings are barely reported. Although, some progress has been made in order to get phytases from metagenomes, a large gap of information still exists. One of the main hindrances is the lack of reliable screening strategies. To my knowledge besides this study only two other published reports have pursuit the obtention of phytate-degrading enzymes by using function-based approaches (34, 35).
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6.4. A Simple and Effective Function-Based Method for the Retrieval of