Bioprospecting of Novel Extremozymes From Prokaryotes-The Advent of Culture-Independent Methods

Abstract

Extremophiles are remarkable organisms that thrive in the harshest environments on Earth, such as hydrothermal vents, hypersaline lakes and pools, alkaline soda lakes, deserts, cold oceans, and volcanic areas. These organisms have developed several strategies to overcome environmental stress and nutrient limitations. Thus, they are among the best model organisms to study adaptive mechanisms that lead to stress tolerance. Genetic and structural information derived from extremophiles and extremozymes can be used for bioengineering other nontolerant enzymes. Furthermore, extremophiles can be a valuable resource for novel biotechnological and biomedical products due to their biosynthetic properties. However, understanding life under extreme conditions is challenging due to the difficulties of in vitro cultivation and observation since > 99% of organisms cannot be cultivated. Consequently, only a minor percentage of the potential extremophiles on Earth have been discovered and characterized. Herein, we present a review of culture-independent methods, sequence-based metagenomics (SBM), and single amplified genomes (SAGs) for studying enzymes from extremophiles, with a focus on prokaryotic (archaea and bacteria) microorganisms. Additionally, we provide a comprehensive list of extremozymes discovered via metagenomics and SAGs.

Keywords: SAG; culture-independent methods; extremophile; halophiles; metagenomics; psychrophile; thermophiles.

FIGURE 1

Selection of different extremophiles. The corresponding image displays the extreme condition(s) in which they thrive. Overlaps are examples of potential polyextremophiles and are not limited to the displayed configurations.

FIGURE 2

The timeline of computing power shows a dramatic increase over the last two decades (A). Sequencing cost per megabase (Mb; one million bases) of DNA in USD (B).

FIGURE 3

Schematic comparison of sequence-based metagenomics (SBM) and single amplified genomes (SAGs). In contrast to the multitude of sequences obtained in metagenomics, SAG allows the correct assignment of one genome to one sample. Different amounts of diverse species result in an uneven distribution of DNA fragments for metagenomics, drastically increasing complexity in the assignment of DNA fragments to potential genomes and their assembly.

FIGURE 4

Protein sequence entries in the UniProt database (2020_03 release, https://www.ebi.ac.uk/uniprot/TrEMBLstats). Number of proteins annotated using the listed evidence of their functional annotation prediction from 185 million sequence entries. Only 0.09% of all entries, corresponding to ∼169 thousand entries, show any functional annotation evidence at the protein level (A). Taxonomic origin of the proteins in percent, separated into Kingdoms (B) (data from https://www.ebi.ac.uk/uniprot/TrEMBLstats).