Discovery and Functional Characterization of Novel Soil-metagenome Derived Phosphatases
von Genis Andrés Castillo Villamizar
Datum der mündl. Prüfung:2019-03-28
Erschienen:2019-07-22
Betreuer:Prof. Dr. Rolf Daniel
Gutachter:Prof. Dr. Rolf Daniel
Gutachter:PD Dr. Michael Hoppert
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-7565
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Name:2019_07_2015_Genis_Castillo_final.pdf
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Description:Genis Andrés Castillo dissertation
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Zusammenfassung
Englisch
Phosphatases, including phytases, play a major role in phosphorous cycle, cell metabolism, biotechnology, and pathogenic processes. Despite their relevance as biocatalysts, the study of phosphatases/phytases has been almost exclusively based on genes and enzymes derived from individual organisms, leaving out the ones associated to microbial communities and so far, uncultured microorganisms. One of the main limitations for the application of function-based metagenomic analysis on phosphatases and particularly phytases, is the lack of reliable and efficient screening methods. In this study we developed and standardized a function-based method to retrieve novel phosphatases/phytases from soil samples. The developed method is based on the induction of phosphatase/phytase activity in metagenomic library-bearing E. coli strains by the absence of inorganic phosphorus sources and the presence of phytate as sole phosphorus source in minimal media. A total of 612,910 clones from five different metagenomic soil libraries were analyzed. Twenty-three positive E. coli clones were recovered and their respective inserts sequenced and analyzed. The phylogenetic analysis of the inserts indicated that all cloned environmental DNA fragments were of bacterial origin. Sequence similarity searches of the of the 23 inserts resulted in the identification of 33 candidate genes. The deduced gene products of 31 of these genes showed at least some similarity to known phosphatases whereas the remaining two were similar to metallo-beta-lactamases. The protein sequence analysis revealed the presence of 11 different domains in the 33 deduced proteins. We detected the alkaline phosphatase and sulfatase superfamily domain (ALP-like cl23718) as the most frequent domain with eight representatives. In addition, two phosphatase-related genes of plasmids pLP04 and pLP15 did not encode known catalytic domains or signatures directly or indirectly associated with phosphatases. The produced proteins Pho4 and Pho15, respectively, showed phosphatase activity and low sequence identity to alkaline phosphatases, but the only catalytic domain detected in both proteins was the SNARE-associated superfamily domain (cl00429). In total, five genes out of the 33 candidates were selected on the basis of their biological significance for the individual subcloning, expression and characterization of the respective products. Pho07 was selected as representative of the most abundant superfamily detected in our survey from which to our knowledge no enzyme has been reported or comprehensively characterized regarding phytase activity. Pho18 was selected as a member of the classic purple acid phytases, which before this study were almost exclusively represented by plant-derived phytases. Pho16B was analyzed in more detail due to its affiliation to the protein tyrosine phytases. In addition, Pho16B possesses a molecular signature similar to members of a tyrosine phosphatases subtype, which are closely related to microbial pathogenesis. Finally, Mblp01 and Mblp02 were selected, as they are representatives of the metallo-β-lactamases. This superfamily is one of the most promiscuous types of enzymes. Moreover, the metallo-β-lactamases share an evolutionary relationship with phosphatases but did not comprise representatives with reported phytase activity prior to this study. The characterization of the selected proteins revealed different features among all analyzed enzymes. The lowest optimal pH of enzyme activity was 4.0 (Pho07) and the highest 7.0 (Mblp02). The highest optimal temperature was recorded at 50 °C (Pho18 and Mblp02) and the lowest at 30 °C (Pho07). The substrate specificity, was also variable among the analyzed enzymes. All 5 purified enzymes were able to degrade phytate but phytate was the preferred substrate only for Pho07. The metallo-β-lactamases (Mblp01 and Mblp02) showed their highest activity with ATP. Naphthyl phosphate and pyrophosphate were the preferred substrates for Pho16B and Pho18, respectively. Interestingly, both metallo-β-lactamases show potential mechanistic promiscuity as the ability to act on β-lactam antibiotics is indicated by reduced sensitivity of recombinant strains harboring mblp01 or mblp02 towards β-lactam antibiotics. This study provided a reliable function-based screening method to recover novel phosphatases/phytases and insights into the diversity of enzymes capable of phytate degradation. This study also demonstrates the potential of functional metagenomics to exploit phosphatase/phytase pools hidden in environmental samples by recovering novel phytate-degrading enzymes unrelated to previously known types of phytases.
Keywords: Metagenomics; Phytases; Functional metagenomics; Biocatalysis; Metagenomes; soil derived biocatalyst; Phosphatases; Phytate-degrading enzymes
