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com
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Functional metagenomics of extreme environments
Salvador Mirete, Veronica Morgante and Jose Eduardo
Gonzalez-Pastor
The bioprospecting of enzymes that operate under extreme
conditions is of particular interest for many biotechnological
and industrial processes. Nevertheless, there is a considerable
limitation to retrieve novel enzymes as only a small fraction of
microorganisms derived from extreme environments can be
cultured under standard laboratory conditions. Functional
metagenomics has the advantage of not requiring the
cultivation of microorganisms or previous sequence
information to known genes, thus representing a valuable
approach for mining enzymes with new features. In this review,
we summarize studies showing how functional metagenomics
was employed to retrieve genes encoding for proteins involved
not only in molecular adaptation and resistance to extreme
environmental conditions but also in other enzymatic activities
of biotechnological interest.
Address
Laboratory of Molecular Adaptation, Department of Molecular Evolution,
Centro de Astrobiologa (CSIC-INTA), Torrejon de Ardoz, Madrid, Spain
Corresponding author: Gonzalez-Pastor, Jose Eduardo
(gonzalezpje@cab.inta-csic.es)
Current Opinion in Biotechnology 2016, 38:143149
This review comes from a themed issue on Environmental
biotechnology
Edited by Bernardo Gonzalez and Regina-Michaela Wittich
For a complete overview see the Issue and the Editorial
Available online 20th February 2016
http://dx.doi.org/10.1016/j.copbio.2016.01.017
0958-1669//# 2016 Elsevier Ltd. All rights reserved.
Introduction
Extreme environments on Earth are colonized by micro-
organisms called extremophiles, which can thrive under
diverse harsh conditions such as acid (pH <4) and
alkaline (pH >9) pH values, high salinity (>10%) and
high and low temperatures (>50 8C and <15 8C, respec-
tively), high metal concentrations, high radiation and
high pressures. The molecular strategies employed for
survival in such environments are still not completely
characterized. However it is known that extremophiles
have biomolecules called extremozymes that are cata-
lytically active under extreme conditions. Diverse in-
dustrial processes need to operate under extreme
conditions (e.g. biofuel production, degradation of car-
bon polymers, paper manufacturing, food and textile
www.sciencedirect.com
industries) and extremozymes could therefore be used
as biocatalysts. Nevertheless, the majority of the
enzymes used to date originate from mesophilic organ-
isms and, despite their many advantages, mesophilic
enzymes usually lack stability during severe industrial
processes. However, extremozymes represent only a
small fraction of the potential industrial market [1,2].
Consequently, the discovery of microorganisms that are
able to thrive in extreme environments, their extremo-
zymes, and their molecular mechanisms of adaptation
have the potential to have a great impact on the field of
biocatalysis. The efficiency of harsh industrial processes
will be improved by employing extremophiles or meso-
philes which have been genetically modified to be more
resistant to different extreme conditions and to express
extremozymes.
A considerable limitation for genetic and biotechnological
studies is that only a minor fraction of the microorganisms
on Earth are culturable on standard laboratory media [3
7]. To overcome this problem, diverse methodological
approaches have been developed to access the genetic
potential of microorganisms present in the environment.
These procedures allow the culture-independent analysis
of the totality of microbial genomes, called the metagen-
ome, in a particular environment. One of these
approaches is functional metagenomics, which consists
of the isolation of functional genes from the metagenome
cloned into libraries that are expressed in culturable
microorganisms. The construction of metagenomic librar-
ies involves the selection of a DNA extraction method
that ensures enough yield and an appropriate average
insert size (Figure 1). The isolated DNA is subsequently
fragmented with suitable restriction enzymes or by me-
chanical shearing and the resulting fragments are then
ligated into a linearized cloning vector such as plasmids,
fosmids, cosmids or bacterial artificial chromosomes
(BACs). The resulting metagenomic library is expressed
in a microbial host, mostly the well-known model bacte-
rium Escherichia coli, which is amenable to genetic ma-
nipulation and for which many genetic tools have been
developed. Through an appropriate screening assay, a
single gene or a set of genes expressing a particular
enzymatic function can be identified and their products
further analyzed (Figure 1). One of the main limitations
of this approach is the proper expression of heterologous
genes in E. coli [8]. In this bacterium, many genes from
extremophiles cannot be translated properly, and also
heterologous proteins that are expressed may not fold
correctly, and would not be functional. Thus, the use of
Current Opinion in Biotechnology 2016, 38:143149
144 Environmental biotechnology

Figure 1

DNA fragmentation

Microbial communities from

extreme environments Vector
Metagenomic DNA fragmented DNA (fosmid, BAC, plasmid)

Ligation
DNA isolation

Functional screening

EXTREMOZYMES
(cellulases, lipases,
esterases, etc) Expression in laboratory
RESISTANCE GENES microbial hosts
(metals, acid pH,etc)
Metagenomic library
Current Opinion in Biotechnology

Schematic diagram describing the steps involved in the construction of a metagenomic library. Environmental DNA can be isolated from an
extreme environment using a DNA extraction method that ensures sufficient DNA yield and an appropriate average insert size. The isolated DNA is
subsequently fragmented with suitable restriction enzymes or by mechanical shearing. The DNA fragments are then ligated into a linearized
cloning vector, and the resulting library of recombinant vectors are used to transform a laboratory microbial host. Amplification of the
metagenomic library will allow its use in different types of screenings.

alternative hosts and broad-host-range vectors may be Acidic environments

required to optimize gene expression and protein trans-
lation [9,10,11]. Advantages of the use of functional
metagenomics are: first, entire genes can be isolated;
second, genes identified from the screening are function-
al; and third, novel genes can be isolated, since this
approach does not depend on the information about
the sequences of known genes [12,13]. Therefore, func-
tional-based screening of metagenomic libraries can de-
tect both novel and previously known classes of genes
related to diverse cellular functions.
In this review, we summarize different studies employing
the functional screening of metagenomic libraries to re-
trieve genes involved in molecular adaptation to extreme
environmental conditions and also genes encoding enzy-
matic activities of biotechnological interest (Table 1).
Among the most acidic environments on Earth are acid
mine drainages, which are characterized by pH values as
low as 3.6 [14] and elevated concentrations of metals
(e.g. iron, nickel, aluminum, manganese, copper, cadmi-
um, etc.) and metalloids (e.g. arsenic) depending on the
mineralogy of the host rock. The microorganisms adapted
to these environments are referred to as acidophiles, and
their networked cellular adaptations maintain a neutral
pH internally, thus their intracellular enzymes do not
need to be adapted to low pH [15,16]. However, the
extracellular enzymes from acidophiles have to function
at low pH, and many extremozymes such as amylases,
proteases, cellulases, xylanases, a-glucosidases, endoglu-
canases, and esterases derived from them, are functionally
active under harsh industrial conditions [17]. Therefore
these enzymes have been used or have been proposed for

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 Metagenomics of extreme environments Mirete, Morgante and Gonzalez-Pastor 145

Table 1

Summary of the functional metagenomic studies included in this review

Environment Geographical origin Activity Library References

Acid Tinto river (SW Spain) Nickel resistance Plasmid [20]
Tinto river (SW Spain) Acid resistance Plasmid [22]
Tinto river (SW Spain) Arsenic resistance Plasmid [23]
Hypersaline Hypersaline Basins in the Eastern Mediterranean Sea Esterase Phagemid [28]
Brines Well Zigong, Sichuan (SW, China) Na+/H+ antiporter Plasmid [29]
Brines and rhizosphere (Mallorca, Spain) NaCl resistance Plasmid [31]
Cold temperatures Pointe Geologie archipelago, Terre Adelie (Antarctic soil) Cellulose biosynthesis BAC [37]
Pointe Geologie archipelago, Terre Adelie (Antarctic soil) Lipase/esterase, amylase, BAC [36]
protease and cellulase
Miers Dry Valley (Antarctic soil) Esterase Fosmid [38]
Miers Dry Valley (Antarctic soil) Esterase Fosmid [39]
Adelie penguin rookery, King George Island (Antarctic soil) Lipase Plasmid [40]
Dasan Station at Ny-Alesund (Arctic soil) Esterase Fosmid [41]
Kapp Wijk in the Svalbard archipelago (Arctic sediment) Esterase Fosmid [42]
Spitsbergen island, Svalbard Archipelago (Arctic sediment) Esterase Fosmid [43]
Ikaite columns (SW Greenland) b-Galactosidase, BAC [44]
a-amylase
and a phosphatase
Glacial ice of the Northern Schneeferner (Germany) DNA polymerase Plasmid, [45]
Fosmid
High temperatures Yellowstone hot spring (USA) DNA polymerase Plasmid [49]
Hot sulfur springs in northern Himalayan region (India) b-Galactosidase Plasmid [50]
Black smoker chimney at the Juan de Fuca Ridge Amylase Fosmid [51]
Hot springs from Sao Miguel island (Azores, Portugal) Esterase Fosmid [52]
Hot springs in the Sileri region (Indonesia) Esterase Fosmid [53]
Blacksmoker chimney in Guaymas Basin Patatin-like protein (PLP) Fosmid [54]
Jae Sawn hot spring (Thailand) Esterase and patatin-like Plasmid [55]
protein (PLP)

use in biotechnological applications such as biofuel pro-
duction, degradation of carbon polymers, in the pharma-
ceutical, food and textile industries, for laundry
detergents and paper manufacturing, and also for the
production of ultra-purified enzymes and substrates used
in molecular biology [17,18].
The Tinto River (Huelva, SW Spain) is considered a
well-known environment impacted by acid mine drain-
age, and its microbial communities have been a source of
novel genes conferring resistance to nickel, arsenic and
acid pH. Metagenomic libraries from the microorgan-
isms of the rhizosphere of the endemic heather Erica
andevalensis, which grows on the banks of the Tinto
River, were screened for nickel resistance, and thirteen
clones were detected and analyzed [19,20]. Two clones
encoded putative ABC transporters belonging to a fami-
ly not previously related to metal uptake, and possibly
involved in active efflux pumping outside the cells. One
clone encoded a bacterial serine O-acetyltransferase
(SAT), whose expression allows nickel accumulation
in the cell. In plants from the Thlaspi genus, expression
of the SAT enzyme increases the level of glutathione,
which protects against the oxidative damage caused by
nickel and allowing the hyperaccumulation of this metal
in plant cells [21]. Five clones encoded proteins similar
to well-characterized proteins but not previously
reported to be related to nickel resistance, and the
remaining six clones encoded unknown and conserved
hypothetical proteins [20]. The metagenomic libraries
from rhizosphere and from planktonic microorganisms at
the origin of the Tinto River (pH  1.8) were screened
to search for arsenic and acid resistant genes [22,23]. A
total of thirteen functionally active genes involved in
arsenic resistance were identified, and which are also
associated in different processes such as transport, stress
response, DNA damage repair, phospholipids biosyn-
thesis, amino acid biosynthesis and RNA-modifying
enzymes. The majority were not previously related to
arsenic resistance. This set of genes included three
which encoded a ClpB chaperone and two tRNA-modi-
fying enzymes capable of increasing the cell survival
under different stress conditions (heat shock and UV
radiation) [23]. As a result of the screening to search for
acid resistance genes, fifteen genes were identified
encoding a ClpXP protease, the transcriptional repressor
LexA, an RNA-binding protein, the DNA-binding pro-
teins HU and Dps, and also unknown and conserved
hypothetical proteins [22]. Nine of the acid resistance
genes identified in this study were cloned and expressed
in alternative expression hosts such as the model organ-
isms Pseudomonas putida and Bacillus subtilis. These

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146 Environmental biotechnology
experiments revealed that four of them, encoding HU,
the LexA regulator, an unknown protein and a hypo-
thetical protein showed a broad-host range as they
conferred acid resistance in all the different hosts
employed [22].
Hypersaline environments
Hypersaline habitats such as salt evaporation ponds,
lakes, and sediments associated to marine ecosystems
represent well-known examples of these extreme envir-
onments, which are inhabited by microorganisms called
halophiles that can thrive in the presence of more than
30% (w/v) total salts [24,25]. One of the strategies to
survive in this condition is the salt in strategy, which is
characterized by increasing the salt concentration inside
the cell, leading to significant changes in the enzymatic
machinery. Halophilic proteins typically have an excess of
acidic amino acids on their surface, preventing their
aggregation at high salt concentrations [26,27]. Therefore,
these salt-enriched habitats represent ideal systems to
search for halo-tolerant enzymes to be used in industrial
processes that require high salt concentrations (e.g. treat-
ment of saline wastewaters and production of fermented
foods) and also to address questions related to the molec-
ular mechanisms of adaptation to this condition. For
instance, the screening of a metagenomic expression
library, constructed with environmental DNA from a
deep-sea hypersaline anoxic basin using a phagemid
vector, led to the identification and subsequent purifica-
tion of five esterases [28]. Two of them exhibited no
significant sequence homology to previously known
esterases and, unlike other esterases, exceptional stability
and activity stimulation by polar solvents [28]. From the
metagenome of a man-made brine well in Sichuan (SW
China), a novel Na+/H+ antiporter was identified, denoted
as M-Nha [29] based on the functional complementation
of the E. coli strain KNabc, which is deficient in the major
Na+/H+ antiporters [30]. This antiporter showed a long
carboxyl terminal hydrophilic tail (140 amino acid resi-
dues) and a hydropathy profile with 10 putative trans-
membrane domains in contrast to the 12 transmembrane
domains found in similar antiporters [29]. A more recent
study revealed diverse genes involved in salt resistance
from the screening of metagenomic libraries from brines
and rhizosphere of a halophyte within a hypersaline
environment, a saltern in Mallorca (Spain) [31]. The
osmosensitive strain E. coli MKH13 was employed in
this study to favour the selection of genes conferring salt
resistance, because it carries mutations in the ProP and
ProU transport systems involved in the efficient uptake of
the osmoprotectant proline betaine (N,N-dimethyl-L-pro-
line) [32]. Among the eleven salt resistance genes found
some encoded for well-known proteins previously related
to osmoadaptation such as a glycerol transporter and a
proton pump, whereas others encoded for proteins not
previously related to this function in microorganisms such
as DNA/RNA helicases, an endonuclease III (Nth) and
Current Opinion in Biotechnology 2016, 38:143149
hypothetical proteins of unknown function [31]. In ad-
dition, four of these genes were cloned and expressed in
B. subtilis and interestingly they also increased salt resis-
tance in this bacterium [31].
Cold and hot environments
Other extreme environments where bioprospecting of
microbial communities based on functional metage-
nomics have been successfully applied include cold envir-
onments such as polar regions, glaciers and deep ocean
waters, and hot environments, such as hydrothermal vents
and hot springs [33,34]. Microorganisms adapted to cold
environments (<15 8C) are called psychrophiles, and
their enzymes can be more active than their mesophilic
homologues at cold temperatures. For example, the
esterases reviewed here and retrieved from cold habitats
showed highest activity at 2040 8C. The high activity at
cold temperatures of these extremozymes can represent a
reduction in the cost of industrial processes because less
concentration of the enzyme is necessary to reach a
particular activity and also they can provide lower energy
consumption because heating is not necessary during a
given process [35]. The potential applications of these
cold-active enzymes include their use in detergents at low
washing temperatures, food processing, molecular biolo-
gy applications and bioremediation at cold sites [35].
Among the most commonly studied extremozymes from
these environments through functional metagenomics,
are cellulases, lipases and esterases owing to their poten-
tial application in biocatalysis, detergents and industrial
processes [34,36]. For instance, the screening of metage-
nomic libraries from Antarctic soil samples revealed four-
teen lipase/esterase-, fourteen amylase-, three protease-,
and eleven cellulase-producing clones [36] and also
allowed the isolation of a new enzyme involved in cellu-
lose biosynthesis [37]. Lipolytic enzymes displaying
optimal activity at an extremely high pH are of great
interest in biotechnology but unfortunately they are rare
in nature. Nevertheless, the identification of alkaliphilic
and cold-active esterases/lipases from psychrophilic
microorganisms were successfully identified by screen-
ing metagenomic libraries derived from Antarctic soil
samples using either fosmid [38,39] or plasmid [40]
vectors. From Arctic soil samples, two cold-active
esterases belonging to family VIII, which shared a rela-
tively low degree of sequence similarity (30%) with each
other [41], and novel cold-active esterases that displayed
low similarity with other lipolytic enzymes have been
retrieved from the screening of metagenomic fosmid
libraries [42,43]. The Ikaite columns of Greenland can
also be considered an extreme environment as these
particular sites are characterized by being permanently
cold (46 8C) and alkaline (above pH 10). A metage-
nomic BAC library was constructed with DNA isolated
from this environment and screened for different activi-
ties of industrial interest which allowed the identifica-
tion of novel cold-active enzymes (b-galactosidases,
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 Metagenomics of extreme environments Mirete, Morgante and Gonzalez-Pastor 147
a-amylases and a phosphatase) with low homology to
known sequences [44]. Interestingly, the a-amylase
clones and one of the b-galactosidase clones were found
active at 15 8C but not at 37 8C [44]. Metagenomic
libraries with different insert sizes were constructed
from glacial ice of the Northern Schneeferner in the
Bavarian Alps (Germany) and used for a function-driven
screening for the identification of DNA polymerases
[45]. The screening employed in this study was based
on complementation of a cold-sensitive lethal mutation
in the polA gene of E. coli [46]. As a result, sequence
analyses of eight recombinant plasmids and one fosmid
revealed the presence of genes encoding complete DNA
polymerase I proteins or domains that are typical of these
proteins [45].
Microorganisms adapted to hot environments are called
thermophiles (6080 8C) and hyperthermophiles
(>80 8C), which use thermo-active enzymes to tackle
these conditions. Biotechnological processes carried out
at high temperatures can have several advantages such as
ensuring proper availability and solubility of organic
compounds, and reducing the presence of microbial con-
taminants [47,48]. As a result of these advantages, high
temperature-active enzymes can be potentially used in
diverse industrial and biotechnological applications in-
cluding food and textile processing, chemical synthesis
and the production of pharmaceuticals [48]. Functional
screening of metagenomic libraries derived from high
temperature habitats have provided recent examples of
thermostable enzymes of biotechnological interest such
as esterases with highest activity at 7085 8C. Screening
of a viral metagenomic library from a hot spring (93 8C) in
Yellowstone National Park revealed the presence of a
thermostable DNA polymerase with reverse transcriptase
(RT) activity [49]. This DNA polymerase showed maxi-
mal activity at 77 8C and can be used in a single-enzyme
reverse transcription PCR (RT-PCR) reaction with high
sensitivity and specificity [49]. Also a novel and thermo-
stable alkalophilic b-D-galactosidase with an optimum
temperature at 65 8C and with high transglycosylation
activity was identified through functional screening of a
metagenomic library derived from a hot spring [50]. A
novel and thermostable amylase with highest activity at
90 8C was retrieved from a black smoker chimney by
combining fosmid library construction with pyrosequen-
cing [51]. An alternative expression host may be required
to overcome the heterologous expression of some genes
derived from extreme environments and thus identify a
broader range of enzymes. In a recent work, a strain of
Thermus thermophilus was employed as a screening host for
functional screening of metagenomic libraries and
allowed the identification of novel esterases from hot
springs in the Azores [52]. To date, this is the only report
of an extremophile used for functional screening of
metagenomic libraries. Other novel lipolytic enzymes
retrieved from metagenomic libraries of extremely hot
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environments include an esterase from hot springs and
mud holes in solfataric fields in Indonesia [53], a patatin-
like proteins (PLPs) from a blacksmoker chimney in
Guaymas Basin [54] and both a PLP and an esterase from
a hot spring in Thailand [55].
Conclusion/future perspectives
Overall, diverse studies in extreme environments using
functional metagenomics have revealed not only the
presence of genes encoding proteins of biotechnological
interest such as cellulases and esterases but also genes
involved in resistance mechanisms to tackle these con-
ditions. Functional metagenomic screening can be con-
sidered challenging, but it is still the only metagenomics
approach that allows the identification of novel full-length
genes involved in a variety of microbial functions. Fur-
thermore, previous knowledge of gene sequence infor-
mation is not a prerequisite and represents an advantage
over sequence-based metagenomics. The identification
of novel genes retrieved from the environment through
functional metagenomics, combined with further bio-
chemical studies, may pave the way for the molecular
elucidation of diverse microbial processes that take place
under extreme conditions. In addition, the possibility of
improving extremozymes by genetic engineering and
directed evolution will further enhance their industrial
applications. Since the cultivation of extremophiles is
associated with many potential difficulties, the genetic
engineering of the desired extremozymes into mesophilic
hosts may allow large-scale production. On the other
hand, genes isolated from extremophiles conferring resis-
tance to extreme conditions could be used to genetically
modify other organisms to expand their capabilities to
thrive under different physicochemical conditions. Ge-
netically modified microorganisms and plants could be
used in industrial processes, biomining, bioremediation
and phytoremediation. For instance, chemolithotrophic
microorganisms used in biomining could be modified to
resist more saline conditions, which could allow the use of
seawater if fresh water is not available in the mining areas.
Acknowledgement
This work was funded by the Spanish Ministry of Science and Innovation
(CGL2012-39627-C03-02).
References and recommended reading
Papers of particular interest, published within the period of review,
have been highlighted as:
 of special interest
 of outstanding interest
1. Elleuche S, Schroder C, Sahm K, Antranikian G: Extremozymes
biocatalysts with unique properties from extremophilic
microorganisms. Curr Opin Biotechnol 2014, 29:116-123.
2. Raddadi N, Cherif A, Daffonchio D, Neifar M, Fava F:
Biotechnological applications of extremophiles,
extremozymes and extremolytes. Appl Microbiol Biotechnol
2015, 99:7907-7913.
Current Opinion in Biotechnology 2016, 38:143149
148 Environmental biotechnology
3. Amann RI, Ludwig W, Schleifer KH: Phylogenetic identification
and in situ detection of individual microbial cells without
cultivation. Microbiol Rev 1995, 59:143-169.
4. Giovannoni SJ, Britschgi TB, Moyer CL, Field KG: Genetic
diversity in Sargasso Sea bacterioplankton. Nature 1990,
345:60-63.
5. Hugenholtz P, Pitulle C, Hershberger KL, Pace NR: Novel division
level bacterial diversity in a Yellowstone hot spring. J Bacteriol
1998, 180:366-376.
6. Torsvik V, Goksoyr J, Daae FL: High diversity in DNA of soil
bacteria. Appl Environ Microbiol 1990, 56:782-787.
7. Ward DM, Weller R, Bateson MM: 16S rRNA sequences reveal
numerous uncultured microorganisms in a natural
community. Nature 1990, 345:63-65.
8. Gabor EM, Alkema WB, Janssen DB: Quantifying the
accessibility of the metagenome by random expression
cloning techniques. Environ Microbiol 2004, 6:879-886.
9. Cheng J, Pinnell L, Engel K, Neufeld JD, Charles TC: Versatile
broad-host-range cosmids for construction of high quality
metagenomic libraries. J Microbiol Methods 2014, 99:27-34.
10. Craig JW, Chang F-Y, Kim JH, Obiajulu SC, Brady SF: Expanding
small-molecule functional metagenomics through parallel
screening of broad-host-range cosmid environmental DNA
libraries in diverse proteobacteria. Appl Environ Microbiol 2010,
76:1633-1641.
11. Liebl W, Angelov A, Juergensen J, Chow J, Loeschcke A,
 Drepper T, Classen T, Pietruzska J, Ehrenreich A, Streit W et al.:
Alternative hosts for functional (meta)genome analysis. Appl
Microbiol Biotechnol 2014, 98:8099-8109.
This review focuses on the use of diverse microbial species and genera as
well as broad-host range vectors to improve the heterologous expression
of genes cloned in metagenomic libraries.
12. Lopez-Perez M, Mirete S: Discovery of novel antibiotic
resistance genes through metagenomics. Recent Adv DNA
Gene Seq 2014, 8:15-19.
13. Simon C, Daniel R: Achievements and new knowledge
 unraveled by metagenomic approaches. Appl Microbiol
Biotechnol 2009, 85:265-276.
This comprehensive review covers different metagenomic approaches
including function-based screenings.
14. Nordstrom DK, Alpers CN, Ptacek CJ, Blowes DW: Negative pH
and extremely acidic mine waters from Iron Mountain,
California. Environ Sci Technol 2000, 34:254-258.
15. Baker-Austin C, Dopson M: Life in acid: pH homeostasis in
acidophiles. Trends Microbiol 2007, 15:165-171.
16. van de Vossenberg MJLC, Driessen MAJ, Konings NW: The
essence of being extremophilic: the role of the unique
archaeal membrane lipids. Extremophiles 1998, 2:163-170.
17. Sharma A, Kawarabayasi Y, Satyanarayana T: Acidophilic
bacteria and archaea: acid stable biocatalysts and their
potential applications. Extremophiles 2012, 16:1-19.
18. Dopson M: Physiological adaptations and biotechnological
applications of acidophiles. In Extremophiles: Microbiology and
Biotechnology. Edited by Anitori RP. Norwich: Horizon Scientific
Press; 2012:265-294.
19. Gonzalez-Pastor JE, Mirete S: In Novel Metal Resistance Genes
from Microorganisms: A Functional Metagenomic Approach, vol
668. Edited by Streit WR, Daniel R. Springer; 2010.
20. Mirete S, de Figueras CG, Gonzalez-Pastor JE: Novel nickel
 resistance genes from the rhizosphere metagenome of plants
adapted to acid mine drainage. Appl Environ Microbiol 2007,
73:6001-6011.
In this study, the screening of metagenomics libraries from microorgan-
isms of the rhizosphere of a naturally metal-enriched and extremely acidic
environment lead to the identification of novel genes involved in nickel
resistance.
21. Freeman JL, Persans MW, Nieman K, Albrecht C, Peer W,
Pickering IJ, Salt DE: Increased glutathione biosynthesis plays
Current Opinion in Biotechnology 2016, 38:143149
a role in nickel tolerance in Thlaspi nickel hyperaccumulators.
Plant Cell 2004, 16:2176-2191.
22. Guazzaroni M-E, Morgante V, Mirete S, Gonzalez-Pastor JE:
 Novel acid resistance genes from the metagenome of the
Tinto River, an extremely acidic environment. Environ Microbiol
2013, 15:1088-1102.
In this work, acid resistance genes were isolated from a metagenomic
library constructed in Escherichia coli, and some of them were success-
fully expressed in both Pseudomonas putida and Bacillus subtilis.
23. Morgante V, Mirete S, de Figueras CG, Postigo Cacho M,
 Gonzalez-Pastor JE: Exploring the diversity of arsenic
resistance genes from acid mine drainage microorganisms.
Environ Microbiol 2015, 17:1910-1925.
This study shows how the metagenome of an acid mine drainage
environment can be considered an appropriate source to retrieve genes
or activities not previously related to arsenic resistance.
24. Rodriguez-Valera F, Ventosa A, Juez G, Imhoff JF: Variation of
environmental features and microbial populations with salt
concentrations in a multi-pond saltern. Microbial Ecol 1985,
11:107-115.
25. Anton J, Rossello-Mora R, Rodrguez-Valera F, Amann R:
Extremely halophilic bacteria in crystallizer ponds from solar
salterns. Appl Environ Microbiol 2000, 66:3052-3057.
26. Paul S, Bag SK, Das S, Harvill ET, Dutta C: Molecular signature of
hypersaline adaptation: insights from genome and proteome
composition of halophilic prokaryotes. Genome Biol 2008,
9:R70.
27. Rhodes ME, Fitz-Gibbon ST, Oren A, House CH: Amino acid
signatures of salinity on an environmental scale with a
focus on the Dead Sea. Environ Microbiol 2010, 12:2613-
2623.
28. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, dos
 Santos VAM, Yakimov MM, Timmis KN, Golyshin PN: Microbial
enzymes mined from the Urania deep-sea hypersaline anoxic
basin. Chem Biol 2005, 12:895-904.
In this work, the authors point out the novelty of the esterases obtained
from extreme environments.
29. Xiang W, Zhang J, Li L, Liang H, Luo H, Zhao J, Yang Z, Sun Q:
Screening a novel Na+/H+ antiporter gene from a
metagenomic library of halophiles colonizing in the Dagong
Ancient Brine Well in China. FEMS Microbiol Lett 2010, 306:22-
29.
30. Nozaki K, Inaba K, Kuroda T, Tsuda M, Tsuchiya T: Cloning and
sequencing of the gene for Na+/H+ antiporter of Vibrio
parahaemolyticus. Biochem Biophys Res Commun 1996,
222:774-779.
31. Mirete S, Mora-Ruiz MR, Lamprecht-Grando M, de Figueras CG,
 Rossello-Mora R, Gonzalez-Pastor JE: Salt resistance genes
revealed by functional metagenomics from brines and
moderate-salinity rhizosphere within a hypersaline
environment. Front Microbiol 2015, 6:1121.
This study describes the identification of novel genes not previously
related to osmoadaptation from different microorganisms of a hypersaline
environment.
32. Haardt M, Kempf B, Faatz E, Bremer E: The osmoprotectant
proline betaine is a major substrate for the binding-protein-
dependent transport system ProU of Escherichia coli K-12.
Mol Gen Genet 1995, 246:783-796.
33. Lewin A, Wentzel A, Valla S: Metagenomics of microbial life in
extreme temperature environments. Curr Opin Biotechnol 2013,
24:516-525.
34. Lopez-Lopez O, Cerdan ME, Siso MIG: New extremophilic
lipases and esterases from metagenomics. Curr Protein Pept
Sci 2014, 15:445-455.
35. Margesin R, Feller G: Biotechnological applications of
psychrophiles. Environ Technol 2010, 31:835-844.
36. Berlemont R, Pipers D, Delsaute M, Angiono F, Feller G, Galleni M,
Power P: Exploring the Antarctic soil metagenome as a source
of novel cold-adapted enzymes and genetic mobile elements.
Rev Argent Microbiol 2011, 43:94-103.
www.sciencedirect.com
 Metagenomics of extreme environments Mirete, Morgante and Gonzalez-Pastor 149
37. Berlemont R, Delsaute M, Pipers D, DAmico S, Feller G, Galleni M,
Power P: Insights into bacterial cellulose biosynthesis by
functional metagenomics on Antarctic soil samples. ISME J
2009, 3:1070-1081.
38. Heath C, Hu XP, Cary SC, Cowan D: Identification of a novel
alkaliphilic esterase active at low temperatures by screening a
metagenomic library from Antarctic desert soil. Appl Environ
Microbiol 2009, 75:4657-4659.
39. Hu XP, Heath C, Taylor MP, Tuffin M, Cowan D: A novel,
extremely alkaliphilic and cold-active esterase from Antarctic
desert soil. Extremophiles 2012, 16:79-86.
40. Cieslinski H, Bialkowskaa A, Tkaczuk K, Dlugolecka A, Kur J,
Turkiewicz M: Identification and molecular modeling of a novel
lipase from an Antarctic soil metagenomic library. Pol J
Microbiol 2009, 58:199-204.
41. Yu EY, Kwon M-A, Lee M, Oh JY, Choi J-E, Lee JY, Song B-K,
Hahm D-H, Song JK: Isolation and characterization of cold-
active family VIII esterases from an arctic soil metagenome.
Appl Microbiol Biotechnol 2011, 90:573-581.
42. Fu J, Leiros H-KS, de Pascale D, Johnson KA, Blencke H-M,
Landfald B: Functional and structural studies of a novel cold-
adapted esterase from an Arctic intertidal metagenomic
library. Appl Microbiol Biotechnol 2013, 97:3965-3978.
43. Jeon JH, Kim J-T, Kang SG, Lee J-H, Kim S-J: Characterization
and its potential application of two esterases derived from the
arctic sediment metagenome. Mar Biotechnol 2009, 11:307-316.
44. Vester JK, Glaring MA, Stougaard P: Discovery of novel enzymes
 with industrial potential from a cold and alkaline environment
by a combination of functional metagenomics and culturing.
Microb Cell Fact 2014, 13:72.
In this work a comparison between a culture-based approach and the
function-based metagenomics approach is described and it highlights
the convenience of combining both strategies.
45. Simon C, Herath J, Rockstroh S, Daniel R: Rapid identification of
 genes encoding DNA polymerases by function-based
screening of metagenomic libraries derived from glacial ice.
Appl Environ Microbiol 2009, 75:2964-2968.
In this study, the screening for genes encoding DNA polymerases from
glacial ice microorganisms was based on the complementation of an E.
coli mutant with a temperature-sensitive lethal mutation in the 50 30
exonuclease domain of DNA polymerase I.
46. Nagano K, Wachi M, Takada A, Takaku F, Hirasawa T, Nagai K:
fcsA29 mutation is an allele of polA gene of Escherichia coli.
Biosci Biotechnol Biochem 1999, 63:427-429.
www.sciencedirect.com
47. Niehaus F, Bertoldo C, Kahler M, Antranikian G: Extremophiles as
a source of novel enzymes for industrial application. Appl
Microbiol Biotechnol 1999, 51:711-729.
48. Unsworth LD, van der Oost J, Koutsopoulos S:
Hyperthermophilic enzymes stability, activity and
implementation strategies for high temperature applications.
FEBS J 2007, 274:4044-4056.
49. Moser MJ, DiFrancesco RA, Gowda K, Klingele AJ, Sugar DR,
 Stocki S, Mead DA, Schoenfeld TW: Thermostable DNA
polymerase from a viral metagenome is a potent rt-PCR
enzyme. PLoS ONE 2012, 7:e38371.
This work illustrates the interesting biochemical properties of a thermo-
stable DNA polymerase retrieved from a viral metagenome.
50. Gupta R, Govil T, Capalash N, Sharma P: Characterization of a
glycoside hydrolase family 1 b-galactosidase from hot spring
metagenome with transglycosylation activity. Appl Biochem
Biotechnol 2012, 168:1681-1693.
51. Wang H, Gong Y, Xie W, Xiao W, Wang J, Zheng Y, Hu J, Liu Z:
 Identification and characterization of a novel thermostable
gh-57 gene from metagenomic fosmid library of the Juan de
Fuca Ridge hydrothermal vent. Appl Biochem Biotechnol 2011,
164:1323-1338.
This study shows how the construction of a metagenomic library com-
bined with pyrosequencing was used to identify a novel thermostable
amylase.
52. Leis B, Angelov A, Mientus M, Li H, Pham VTT, Lauinger B,
 Bongen P, Pietruszka J, Goncalves LG, Santos H et al.:
Identification of novel esterase-active enzymes from hot
environments by use of the host bacterium Thermus
thermophilus. Front Microbiol 2015, 6:275.
To date, this is the only report of an extremophile used for functional
screening of metagenomic libraries.
53. Rhee J-K, Ahn D-G, Kim Y-G, Oh J-W: New thermophilic and
thermostable esterase with sequence similarity to the
hormone-sensitive lipase family, cloned from a metagenomic
library. Appl Environ Microbiol 2005, 71:817-825.
54. Fu L, He Y, Xu F, Ma Q, Wang F, Xu J: Characterization of a novel
thermostable patatin-like protein from a Guaymas basin
metagenomic library. Extremophiles 2015:1-12.
55. Tirawongsaroj P, Sriprang R, Harnpicharnchai P, Thongaram T,
Champreda V, Tanapongpipat S, Pootanakit K, Eurwilaichitr L:
Novel thermophilic and thermostable lipolytic enzymes from a
Thailand hot spring metagenomic library. J Biotechnol 2008,
133:42-49.
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