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

Halophiles and Xerophiles

Halophiles are microorganisms that require elevated salt concentrations to grow. There are four main classifications of halophiles based on their optimal NaCl concentration for growth: slight halophiles (0.2 M), moderate halophiles (0.5–2.5 M), borderline extreme halophiles (2.5–4.0 M), and extreme halophiles (4.0–5.9 M). Halophilic organisms use several adaptation mechanisms to live in these conditions. These include modified electrostatic charge of their proteins, balancing osmotic pressure either by compatible solutes, such as betaine and ectoines, or with chloride and potassium uptake into the cells by transporters (primary or secondary) and the coordinated action of bacteriorhodopsin and ATP synthase (Karan et al., 2012a).

Halophilic organisms and their enzymes are used in several different fields, including production of fermented foods, manufacturing of solar salt from seawater, leather industries, environmental bioremediation and textile, pharmaceutical (Karan and Khare, 2010; Oren, 2010; Karan et al., 2012a; de Lourdes Moreno et al., 2013; Akal et al., 2019). The biochemical properties of the molecules, enzymes, and compatible solutes synthesized by halophiles also present potential implications in fine chemicals, medicines, and bioimplants (Irwin, 2010; Karan et al., 2012a, b; Chen and Jiang, 2018; Jin et al., 2019). One of the main selling points of these enzymes is their stability in such solvents as benzene, toluene or chloroform, which are frequently used in different industrial environments (Oren, 2010). Halophiles are a potential source of novel extremozymes like amylases, proteases, nucleases, cellulases, chitinases, xylanases, esterases, alcohol dehydrogenases and lipases (Karan et al., 2012a; Coker, 2016). For example, nuclease from Micrococcus varians has long been used commercially for production of a flavoring agent 5′-guanylic acid (5′-GMP) due to its efficiency in degrading RNA at 60°C and 12% (w/v) salt (Kamekura et al., 1982). Several other enzymes have been heterologously expressed and characterized (Table 1). Interestingly, some of the halophilic enzymes display polyextremophilicity i.e., stability toward more than one extreme condition e.g., high salt, elevated or low temperature, alkaline or acidic pH and non-aqueous medium having great potential application for industrial and biotechnological processes (Grötzinger et al., 2017; Akal et al., 2019; Karan et al., 2020).

Along with the enzymes, halophiles possess a number of bioactive molecules that found its application in different areas. The biocompatible solute ectoine has potential use in respiratory medicine (Galinski et al., 1985) and can reduce cell stress effects in nanoparticle-induced lung inflammation by inhibiting the signals (Sydlik et al., 2009). Some halophiles, such as Halobacterium and Haloferax, were found to accumulate polyhydroxyalkanoates (PHAs), which are a family of biopolyesters with diverse uses in the medical, environmental, and industrial fields (Kirk and Ginzburg, 1972; Quillaguaman et al., 2010; Chen and Patel, 2012). Halophiles, such as Natronococcus occultus and Naloterrigena hispanica, have been found to generate antimicrobial peptides and diketopiperazines (Charlesworth and Burns, 2015; Coker, 2016). Some haloarchaea produce internal protein gas vesicle nanoparticles (GVNPs), which have been tested as a vaccine scaffold platform and ultrasonic contrast agents (Stuart et al., 2001; Shapiro et al., 2014; DasSarma and DasSarma, 2015). Notably, halophilic nature makes cell lysis, hence farming of the enzymes and other molecules, easier. Since halophiles require high salt to maintain their cell integrity, lowering the salt concentration in the buffer system disrupts the cell wall of most halophiles.

Xerophiles are microorganisms that can survive and grow in arid conditions with water activity a_w < 0.75 by forming spores that help them mitigate environmental stress. Adaptive mechanisms are connected to water loss prevention and increased water retention through the accumulation of compatible solutes, production of extracellular polymeric substances (EPSs), adaptations on the cell membrane to retain intracellular water, and synthesis of DNA repair proteins (Lebre et al., 2017). These unique adaptations allow xerophiles to be used in microbial electrochemical systems (Dopson et al., 2015) or in next-generation industrial biotechnology, where they can be used for treating long-chain fatty acids, cellulose, chitin, rubbers, or other compounds (Chen and Jiang, 2018).

Thermophiles and Psychrophiles

Thermophiles are heat-loving microorganisms with an optimum growth temperature of 45°C or above, while hyperthermophiles grow at temperatures above 80°C (Sarmiento et al., 2015). Thermophiles and hyperthermophiles exist in various natural ecosystems, such as geothermal waters, hot springs, volcanoes, deep-sea hydrothermal vents, and other ecosystems with high-temperature parameters. Thermophiles have thermostable proteins and cell membranes that do not become denatured at high temperatures, and some may also resist proteolysis (Sarmiento et al., 2015). Notably, polymerases from thermophiles have laid the foundation for the discovery of polymerase chain reaction (PCR), a technique that has become crucial in medicine and research. Today we may find various enzymes from thermophiles (e.g., from Thermus aquaticus and Pyrococcus furiosus) finding their use in PCR due to their stability and reasonable cost (Brock, 1997; Bruins et al., 2001; Irwin and Baird, 2004). Along with polymerases, a lot of different thermophilic enzymes, such as lipases, laccases, and xylanases, are also on the market, thus making the industrial process much more environmentally friendly (Damiano et al., 2003; Atalah et al., 2019).

Psychrophiles are cold-loving microorganisms that can grow at temperatures between −20 and 20°C. Psychrophiles possess diverse adaptive molecular mechanisms to survive and thrive at such low temperatures. Psychrophilic bacteria have increased (i) unsaturated fatty acids, cyclopropane-containing fatty acids, and short-chain fatty acids in their membranes, which prevent the loss of membrane fluidity; (ii) cold-shock proteins (CSPs) and chaperones to protect the synthesis of RNA and proteins; (iii) antifreeze proteins (AFPs) that bind to ice crystals and create a state of thermal hysteresis; and (iv) mannitol and other compatible solutes that act as cryoprotectants to prevent cell damage by ultraviolet (UV) radiation and ice formation (Sarmiento et al., 2015; De Santi et al., 2016a).

Psychrophiles have a promising future in pharmaceuticals and medicine since their cell membranes hold surfactants capable of sustaining stability at low temperatures (Cavicchioli et al., 2011). Psychrophiles, such as Pandalus borealis, Euphausia superba, Moraxella species, and Flavobacterium species, have been found to produce anticancer and antitumor agents (Margesin and Feller, 2010). Psychrophilic enzymes display high catalytic activity, stability at low temperatures, and pronounced heat lability and may offer useful industrial and biotechnological applications in various domains, such as pharmaceutical science, molecular biology, textiles, paper, food, feed technologies, detergents, and cosmetics (Margesin and Feller, 2010; Cavicchioli et al., 2011; Sarmiento et al., 2015; Dhaulaniya et al., 2019; Al-Ghanayem and Joseph, 2020).

Acidophiles and Alkaliphiles

Acidophiles are microorganisms that grow at an optimum pH < 3 (Baker-Austin and Dopson, 2007). Acid-tolerant microbes have optimum growth at pH > 5 but are still active in lower pH environments. Acidophiles maintain their cytoplasmic pH close to neutrality to protect acid-labile cellular constituents by active pumping of protons (proton flux system); by decreased permeability of the cell membrane, which helps to suppress the entry of protons into the cytoplasm; and by improved protein and DNA repair systems compared to those of neutrophiles (Baker-Austin and Dopson, 2007). Acidophilic enzymes can block the activity of matrix metallopeptidases (MMPs), which are essential for tumor metastasis (Irwin, 2010). MMP inhibitors from an acidophilic Penicillium species isolated from Berkeley Pit Lake promise a therapeutic approach for cancer (Stierle et al., 2006). Proteolytic enzymes from acidophiles have also been reported as nonallergenic preservatives in medicines (Sharma et al., 2012). Amylolytic enzymes, such as trehalase isolated from acidophilic Sulfolobus solfataricus, are used in medicine as preservatives and stabilizers (Schiraldi et al., 2002).

Alkaliphiles are microorganisms that grow in alkaline environments with a pH > 9, usually showing optimal growth at pH ∼10. Alkaliphiles may coexist with neutrophiles under mild basic pH conditions and live in specific extreme environments. Alkaliphilic bacteria possess molecular mechanisms that compromise the activation of both symporter and antiporter systems. Electrogenic antiporters produce an electrochemical gradient of Na⁺ and H⁺, and the symporter system enables the uptake of Na⁺ and other solutes into the cells (Chinnathambi, 2015). Alkaliphilic enzymes have found use in several applications, such as tannery water treatment (Li et al., 2017), food, cosmetics, and pharmaceutical production (Horikoshi, 2016).

Radiophiles

Radiophiles or radiation-resistant extremophiles thrive in high oxidative stress and radiation environments, including UV radiation, gamma radiation and X-ray radiation, and have potential applications in therapeutics pharmacology and biotechnology. Direct intense or prolonged exposure to different forms of radiation, such as UV radiation, can lead to mutagenic and cytotoxic DNA lesions, resulting in various types of human cancers (Gabani and Singh, 2013). Primary and secondary metabolic products from radiophiles can protect the organism’s DNA and can be used to manufacture anticancer drugs, antioxidants, and sunscreens (Raddadi et al., 2015). Extremolytes, mycosporin-like amino acids (MAAs) from the red alga Porphyra rosengurttii, are commercially utilized to enhance the UV-protective properties of sunscreens (de la Coba et al., 2009) and are therapeutic candidates as preventive agents in UV radiation-induced cancers, such as melanoma. Bacterioruberin isolated from Halobacterium and Rubrobacter and deinoxanthin isolated from Deinococcus radiodurans are other therapeutic candidates for cancer diseases (Singh and Gabani, 2011; Choi et al., 2014).

Polyextremophiles

Polyextremophiles i.e., microorganisms growing preferentially under multiple extremes, have developed features that allow them to thrive in harsh environments such as Deep Lake Antarctica, where the temperature reaches as low as −20°C and stays liquid only due to extreme salt concentrations (DasSarma et al., 2013; Karan et al., 2013, 2020). Deep-sea anoxic brine pools at the bottom of the Red Sea are another type of polyextremophilic environment (Antunes et al., 2011). These brine pools are extreme in different physicochemical parameters and vary drastically, with temperatures ranging from 22.6 to 68.2°C and NaCl concentrations varying from 2.6 to 5.6 M (Antunes et al., 2011). Additionally, they show a characteristic sharp brine-seawater interface, with steep gradients of dissolved oxygen, density, pH, salinity, and temperature (Emery et al., 1969; Ross, 1972; Anschutz and Blanc, 1995). Because of this variation, brine pools offer a multitude of habitats for different kinds of extremophiles.

Polyextremophiles have a diverse range of uses and applications. Halothermophiles and halopsychrophiles are promising sources of useful enzymes (Elleuche et al., 2014). Enzymes derived from these polyextremophilic microbes possess particularly attractive properties for biotechnology, namely, their function at high salt concentrations and high or low temperatures (Sarmiento et al., 2015). Elevated temperatures are used to shift equilibria, distill products, increase reaction’s speed, liquefy compounds, and eliminate microbial contamination (Grötzinger et al., 2017). Low temperatures can save energy (e.g., in washing processes, bioremediation, or food processing), avoid labile or volatile compound production (e.g., in biotransformations or food processing) or prevent bacterial growth. Therefore, cold-active enzymes have great potential for various biotechnological processes. Increasing the salt concentration in solution decreases the water activity, thus mimicking aqueous-organic solvent mixtures. Therefore, halophilic enzymes generally retain high activity and stability in high salt and organic or nonaqueous media (Karan and Khare, 2010, 2011; Karan et al., 2011, 2012a). In turn, organic solvents increase the solubility of hydrophobic substrates and alter the hydrolytic and kinetic equilibria (Sellek and Chaudhuri, 1999). Traditional mesophilic enzymes lose their native structure and thus catalytic activity in organic solvents, limiting their use. However, the high stability of halophilic enzymes toward salt is associated with tolerance to low water activity, such as in mixtures of aqueous and organic or nonaqueous media (Sellek and Chaudhuri, 1999), which emphasizes their high potential for biocatalysis. Cold-active or heat-stable enzymes operating in high-salt or organic solvents are of interest for the sustainable production of value-added chemicals. The world is currently looking for microorganisms with new enzymes, such as hydrolases, amylases, cellulases, peptidases, and lipases (Dumorne et al., 2017). Some examples include alkalithermophilic serine proteases from Alkalibacillus sp. NM-Da2, which can potentially be applied in different biotechnological and pharmaceutical industries (Abdel-Hamed et al., 2016), and alkalipsychrophilic esterase from the marine bacterium Rhodococcus sp., which can be used in the food industry since the process catalyzed by an esterase can be stopped by increasing the temperature, thus saving food properties (De Santi et al., 2014). The polyextremophilic characteristics of halophilic amylases potentially make them efficient catalysts under alkaline pH and high salinity in processes such as starch hydrolysis and applications such as detergent production, the food industry, and bioremediation (Ali et al., 2014; Kumar et al., 2016; Rekadwad and Khobragade, 2017).

Microbial Dark Matter—The Big Unknown

According to recent estimations, Earth is home to an upward range of 10¹² (1 trillion) microbial species, with microorganisms being the most abundant, widespread, and taxonomically, metabolically, and functionally diverse organisms (Locey and Lennon, 2016). During the past decade, high-throughput sequencing in combination with advanced bioinformatics algorithms has allowed for enhanced insight into microbial taxa and expanded the estimation of the global microbial load by orders of magnitude via projects such as the “Earth Microbiome project” (EMP), which analyzed more than 200,000 environmental samples (Gilbert et al., 2014). Out of the 10¹² microbial species, only approximately 10⁵ have been sequenced (0.00001%), 10⁴ have been cultured, and even the EMP, which also uses rRNA sequences for identification, has cataloged fewer than 10⁷ species (0.001%), of which 29% were detected only twice (Locey and Lennon, 2016). It is estimated that approximately 99.999% of microbial taxa remain undiscovered (Locey and Lennon, 2016). Focusing on the ∼10⁴ cultivated species and looking at the highest order, among the 60 significant lines of descent (phyla or divisions) that are known within the archaeal and bacterial domain (Hugenholtz and Kyrpides, 2009), 50% are uncultured and make up the “microbial dark matter” (Marcy et al., 2007). Furthermore, 88% of all isolated microbes are members of only four bacterial phyla, Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (Rinke et al., 2013).

The Challenge of Cultivation

Traditional cultural methods employ the ability of microorganisms to grow in a specific laboratory environment. Historically, these methods have been used in microbiology, including selective or differential media, microscopy, Gram-staining, and biochemical tests. These methods are sensitive, reliable, inexpensive, and provide qualitative and quantitative results on the bacterial populations (Nowrotek et al., 2019). However, only a small fraction of all microbes can be grown in a laboratory setting. Thus, although cultural methods are a well-established, simple, and inexpensive way of isolating, detecting, and quantifying microorganisms, the methods lack speed and are labor-intensive with the risk of contamination. Based on this legacy, microbial culturomics has emerged as a tool that complements metagenomics data and gives another way to determine composition of microbial populations. Several studies have shown that culture-dependent and culture-independent methods often deliver different results (Steven et al., 2007; Carraro et al., 2011; Stefani et al., 2015; Nowrotek et al., 2019; Rego et al., 2019). For example, using 212 different culture conditions, such as temperature, various oxygen levels, and selective media, many researchers have isolated 340 different bacterial species (Lagier et al., 2012). Interestingly, the metagenomics approach could identify only 51 species out of 340 cultured species in this study. Therefore, it is crucial to combine both culture-dependent and culture-independent ways to study a given microbiome (Steven et al., 2007; Lagier et al., 2016; Nowrotek et al., 2019; Sarhan et al., 2019).

The enormous lack of isolates is based on the fact that very few microbes are cultivable, e.g., only as few as 0.001–0.1% of the microbes found in seawater can be cultivated under laboratory conditions (Amann et al., 1995). Therefore, archaea enzymes are typically studied after expression in a heterologous host, mostly Escherichia coli (E. coli). However, this approach is impossible for some archaeal proteins due to low expression rates, inaccurate protein folding, or the lack of functionally necessary posttranslational protein modifications in eubacterial hosts. Besides, it is challenging to identify archaeal enzymes directly from genome samples in bacterial host-based functional screening assays, as archaeal promoter structures differ from bacterial ones (Zweerink et al., 2017). A direct in vivo investigation of archaeal enzymes in an archaeal strain or an archaeal host with the right expression machinery of the respective proteins represents a valuable alternative. For this reason, some archaeal expression models have been used, such as Halobacterium salinarum/NRC-1 or Haloferax volcanii. Using these expression systems, researchers have studied archaeal TATA-binding proteins, discovered 16S gene transfer between bacteria and archaea (Baliga et al., 2000; Fuchsman et al., 2017), and several different archaeal enzymes have been studied using these archaeal expression systems (Table 1). However, this approach requires technically sensitive and straightforward technologies to detect active enzymes and a methodology to express proteins in an archaeal host heterologously. Ultimately, as extremophiles preferentially live in harsh conditions, it is difficult to satisfy all of these microbes’ extreme requirements. However, with the progress in computational methods and improving bioinformatics algorithms, it is now possible to study the mysterious “dark matter” of the microbial world using culture-independent approaches, including bulk metagenomics and SAGs.

Function-Based Metagenomics (FBM), Sequence-Based Metagenomics (SBM), and Single Amplified Genomes (SAGs) as a Gateway to Novel Species

Culture-independent methods developed and utilized over the last few years, such as metagenomics, have recently gained momentum. Metagenomics analyses rely on the direct isolation of genomic DNA from the environment. These analyses can be either sequence-based (i.e., putative enzymes are discovered based on their conserved sequences) or function-based (i.e., functional enzymes are found based on the expressed features, such as a specific enzyme activity).

The Challenge of Gene Function Annotation

Although the accessibility of uncultured species’ genomes has increased, computational challenges hinder discovering novel extremozymes using culture-independent methods. These hindrances are not unique to extremophiles and affects all organisms, albeit every group has its own unique hurdles contributing to the major cause. The major challenge in mining uncultured organisms’ genomic data is the reliable large-scale annotation of DNA sequences from non-mesophilic organisms with low sequence homology to experimentally described organisms. Annotation by homology faces a significant dilemma: annotation reliability is reciprocally coupled to protein diversity. Therefore, the difference between gene sequences negatively correlates with the overall homology to any related gene. Moreover, studying extremozymes amplifies not only in culture-based approaches but also culture-independent methods. First reason is linked to the fact that extremophiles tend to alter the overall amino acid composition of enzymes to adapt to their environment, which can be observed in halophiles and thermophiles (Madern et al., 2000; Sterner and Liebl, 2001; Berezovsky and Shakhnovich, 2005; Radestock and Gohlke, 2011; Siglioccolo et al., 2011; Reed et al., 2013; Karan et al., 2020). The second reason is mainly ought to the very limited amount of characterized extremozymes. For example, alcohol dehydrogenases and γ-carbonic anhydrase (CA_D) discovered from uncharacterized archaea collected from brine pool at the bottom of the Red Sea showed sequence homology of about 30–37% to the nearest mesophilic homologs (Grötzinger et al., 2017; Akal et al., 2019; Vogler et al., 2020).

Error propagation further complicates the situation, especially in enzymes with few or highly diverged homologs. The function of the encoded protein can be proven experimentally only for a small and continuously decreasing fraction of gene sequences available from databases, which is currently 0.09% of the UniProt database (Figure 4A). Furthermore, more than 94% of all sequences originate from eukaryotes or bacteria (Figure 4B). Therefore, the challenge of a reliable annotation is even more pronounced for genes that originate from other organisms, such as archaea, including most extremophiles (Rampelotto, 2013). Hence, the more distinct a protein is, smaller the probability of being related to an experimentally described enzyme. At the same time, the more distinct an enzyme is, the higher is its impact on future annotations. For example, a gene from the newly discovered mesophilic microorganism can be compared to many homologs. The function of the majority of these homologs will be used as annotation of the new gene. In contrast, a gene from a newly discovered extremophilic microorganism might have far fewer homologs and therefore, the annotation of these homologs plays a significant role for future studies. Recent developments in annotation algorithms allow a higher degree of freedom between DNA sequences that will be annotated compared to the closest homologs. These methods include algorithms that focus on specific and short conserved sequences only, such as essential amino acids of the active center along with conserved regions such as catalytic sites and cofactor binding sites (Grötzinger et al., 2014). More elegant but substantially more complicated and computational resource-demanding are structure-based approaches. Here, the expression product of potential genes is modeled, and its function is assessed by its structure, not by the sequence. The biennial Critical Assessment of protein Structure Prediction (CASP) is a competition where their efficiency compares novel modeling algorithms to predict unusual and novel tertiary structures of proteins whose crystal and/or NMR structure was measured but not yet published. The overall in silico modeling success was limited until the rise of AI-based protein folding prediction. Last year, in 2020, a new version of a deep machine learning-based algorithm called AlphaFold 2 was released (Senior et al., 2020). This algorithm has substantially increased computational protein folding prediction potential and showed impressive results in the CASP assessment. AlphaFold 2 reached 92.4 out of 100 Global Distance Test (GDT), whereas the average crystal structure only gets 90. Thus, at least for the test samples, it was as good as a crystal structure. Such an impressive result gives hope for the future computational study of unknown proteins. In theory, it could mean that any given protein’s structure and function could be predicted solely based on its amino acid sequence in the future. Still, today only a limited amount of algorithms have experimentally been shown to be efficient in the annotation of far distant related genes (Grötzinger et al., 2014; Akal et al., 2019; Vogler et al., 2020). Reliable annotation of the entire genome of an organism that is very distantly related to described organisms is not yet available.

SAGs reduce data complexity significantly compared to that of the metagenomics approach, but the analysis and management of next-generation whole-genome sequencing (NGS) data include an impressive number of various software applications. These applications are used for sequence read assembly, mapping to the reference genome, variant/SNP calling and annotation, transcript assembly and quantification, and small RNA identification (Horner et al., 2010; Garber et al., 2011; Pabinger et al., 2013). Compared to classical genomic sequencing, experimental characterization of SAG gene products requires gene synthesis, expression, purification, and functional characterization and, therefore, is several orders of magnitude more time-consuming and cost-intensive. Hence, false-positive results from flawed annotation are much more problematic than false-negative results (due to incomplete annotation) when genomic data are searched for the desired function, which is particularly true for genes from extremophilic organisms that require slow-growing expression systems (Grötzinger et al., 2014).

Therefore, the initial challenge of cultivating organisms can be addressed to a considerable degree by using novel culture-independent methods. However, these advance shifts the problem toward bioinformatics-based handling and interpretation of the data, where developments are rapidly progressing but still need time and data, to allow reliable, functional annotation of whole genomes. In the Table 1, a comprehensive list of extremozymes discovered with metagenomics (Berini et al., 2017) and SAGs are shown.