FEMS Microbiology Letters, 364, 2017, fnx211

doi: 10.1093/femsle/fnx211
Advance Access Publication Date: 4 October 2017
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M I N I R E V I E W – Biotechnology & Synthetic Biology

Metagenomics: novel enzymes from non-culturable

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microbes
†
Francesca Berini1,2,∗, , Carmine Casciello1,2 , Giorgia Letizia Marcone1,2
and Flavia Marinelli1,2
1
Department of Biotechnology and Life Sciences, University of Insubria, 21100 Varese, Italy and 2 ‘The Protein
Factory Research Center’, Politecnico di Milano and University of Insubria, 20131 Milan, Italy
∗
Corresponding author: Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese (Italy).
Tel: +39 0332421547; Fax: +39 0332421500; E-mail: f.berini@uninsubria.it
One sentence summary: Metagenomics is a powerful tool for identifying novel microbial biocatalysts.
Editor: Michael Sauer
†
Francesca Berini, http://orcid.org/0000-0003-1913-9929

ABSTRACT
In the transition to the post-petroleum economy, there is a growing demand for novel enzymes with high process
performances to replace traditional chemistry with a more ‘green’ approach. To date, microorganisms encompass the
richest source of industrial biocatalysts, but the Earth-living microbiota remains largely untapped by using traditional
isolation and cultivation methods. Metagenomics, which is culture independent, represents a powerful tool for discovering
novel enzymes from unculturable microorganisms. Herein, we summarize the variety of approaches adopted for mining
environmental DNA and, based on a systematic literature review, we provide a comprehensive list of 332 industrially
relevant enzymes discovered from metagenomes within the last three years.

Keywords: metagenomics; environmental libraries; screening; heterologous expression; industrial enzymes

INTRODUCTION cells estimated to be 4–6 × 1030 (Bunge, Willis and Walsh 2014),
inhabiting a widest variety of ecosystems, including the less-
Microbial enzymes are employed in almost all industrial sectors,
hospitable habitats, such as hot springs, nearly saturated salt
from the chemical, pharmaceutical and food industries to the
brines, acid mine waters at pH near zero, deep-sea hydrother-
manufacturing of detergents, textiles, leather, pulp and paper
mal vents and Antarctic ices (Mirete, Morgante and Gonzàlez-
(Demain and Adrio 2008). In 2015, the application of industrial
Pastor 2016). Environmental DNA (eDNA) analysis of microbial
enzymes generated a turnover of about 5 billion USD, a value
communities currently estimates that only from 0.1% to 1% of
that is forecasted to rise to 6.9 billion USD in 2017 and to 10.7
these prokaryotes are culturable by using traditional microbio-
billion USD in 2024 (World Enzyme to 2017, https://www.gmin
logical methods (Culligan et al. 2014a). Also part of eukaryotic
sights.com/industry-analysis/enzymes-market), thanks to the
microbes is unculturable (Hirst, Kita and Dawson 2011), although
increasing demand for novel biocatalysts with high process per-
the extent of this phenomenon is less clear. Magnuson and
formances to replace traditional chemistry. Enzyme-based pro-
Lasure (2002) reported that 10% to 30% of fungi from different
cesses are favored by reduced costs, increased efficiency, im-
soils can be cultivated.
proved product recovery, and reduced use of toxic compounds
In the last two decades, the development of culture-
and harsh conditions. Microorganisms represent the largest pro-
independent methods based on collecting biological material
portion of biomass on Earth, with a total number of prokaryotic

Received: 5 June 2017; Accepted: 2 October 2017


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 2 FEMS Microbiology Letters, 2017, Vol. 364, No. 21
from environmental samples (metagenomics, metatran-
scriptomics and metaproteomics) revealed the potential of
unculturable microorganisms as sources of novel enzymes.
Metagenomics is generally considered the most promising
methodology for identifying innovative biocatalysts from
prokaryotic eDNA (Lorenz and Eck 2005), although recent ad-
vances in metaproteomics have shown great potential (Wilmes,
Heintz-Buschart and Bond 2015) leading to the discovery of
novel lipases (Sukul et al. 2017) and cellulases (Speda et al.
2017). Differently from prokaryotic genes that can be directly
transcribed and translated in a suitable heterologous host, the
presence of large introns in eukaryotic genes limits metage-
nomic analyses. Consequently, starting from the pioneering
work of Grant et al. (2006), metatranscriptomics (based on
selective enrichment of eukaryotic 3 -polyadenylated mRNAs
and reverse transcription) was preferred in the search for novel
eukaryotic enzymes. By this approach, few industrially relevant
biocatalysts were characterized as a cellulase (Takasaki et al.
2013) and a phosphatase (Kellner et al. 2011).
The emphasis of this review is on metagenomics applied to
prokaryotic eDNA, underlining recent trends and discussing in-
novative examples in mining metagenomes for discovering po-
tential biocatalysts. It does so by looking at studies published
in the last three years (since 1 January 2014 to 31 March 2017)
on 332 metagenome-sourced enzymes (refer to Table 1 for crite-
ria adopted in literature search and enzyme selection). With the
exception of two β-glucosidases affiliated to archaea (Bergmann
et al. 2014; Schröder et al. 2014), all the listed enzymes are of bac-
terial origin.
HOW TO MINE eDNA IN THE SEARCH FOR
NOVEL ENZYMES
The term ‘metagenomics’ is defined as the analysis of the ge-
netic complement of an entire habitat by direct extraction and
subsequent cloning of DNA from an assemblage of microorgan-
isms (Handelsman et al. 1998). Figure 1 summarizes the general
workflow of metagenomics in discovering novel enzymes, out-
lining the multiplicity of possible screening paths from eDNA
extraction to heterologous expression of selected protein se-
quences. How eDNA is extracted, fragmented and cloned largely
dictates the success of metagenomic experiments, but readers
are referred to comprehensive reviews of these methods that, al-
though still posing some technical challenges, might be consid-
ered quite consolidated (Delmont et al. 2011; Lombard et al. 2011;
Felczykowska et al. 2015). Herein, looking at the metagenome
sources of the enzymes listed in Table 1, it appears clear that,
nowadays, metagenomic libraries could be successfully assem-
bled from widely diverse ecological sources (Fig. 2A). Since a low
hit rate of positive clones in screening still represents a per-
sisting bottleneck limiting the success of metagenomics (see
below), pre-selecting environmental samples is an increasingly
used practice to enrich libraries with target genes. For instance,
in 2014 we identified the first metagenome-sourced chitinase
from a soil known to be suppressive for chitin-containing phy-
topathogens (Hjort et al. 2014), whereas two esterases were dis-
covered in a soil contaminated with petroleum hydrocarbons
(Pereira et al. 2015; Maester et al. 2016). A promising extension
of this concept is the ecological enhancement (also termed tar-
geted metagenomics), by which microbial communities are ar-
tificially manipulated prior to DNA extraction in order to in-
crease the in situ prevalence of target functions (Ekkers et al.
2012). For example, the addition of chitin to agricultural soils
stimulates active chitinolytic microbial communities, increas-
ing the chance of finding novel chitinases (Cretoiu et al. 2015;
Stöveken et al. 2015). Similar approaches were used to identify
novel hemicellulases (Sun et al. 2015; Zhao et al. 2017) and ox-
idoreductases (Nagayama et al. 2015) from cellulose-amended
samples or artificially polluted soils. A hormone-sensitive li-
pase was recovered from an olive oil-fed permafrost-derived mi-
crobiome (Petrovskaya et al. 2016). Incorporating stable-isotope
probing is another tool for innovating target library construc-
tion: for example, soil sample incubation with stable-isotope-
labeled carbohydrates enriched the eDNA of those bacteria ac-
tively engaged in degrading plant-derived biomasses (Verastegui
et al. 2014).
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Analysis of Table 1 focusing on how metagenomic libraries
are preferentially built up indicates that the majority of the re-
cently isolated metagenome-sourced enzymes (194) were dis-
covered by screening fosmid libraries (Fig. 2B). Although small-
insert libraries (in plasmids or phagemids) make it possible
to isolate single genes, large-insert libraries (in cosmids, fos-
mids or bacterial artificial chromosomes) were recently pre-
ferred since they permit easily recovering of full-length genes,
gaining information on their genetic context in natural sources,
and unveiling gene clusters encoding cocktails of enzymes
synergistically acting on the same natural substrate. In ex-
ample, Cretoiu et al. (2015), Maruthamuthu et al. (2016) and
Matsuzawa and Yaoi (2017) reported gene annotation of fos-
mid inserts containing the targeted open reading frames (ORFs)
when searching for novel chitinases and hemicellulases (Ta-
ble 1). Using large-insert libraries also permitted the dis-
covery of complex gene clusters encoding for natural prod-
ucts, which include multiple enzymatic activities that might
be useful for expanding the chemical diversity of bioactive
molecules. Although potentially interesting as biocatalysts, our
review does not cover these biosynthetic enzymes and read-
ers are referred to recent extensive reviews on the subject
(Milshteyn, Schneider and Brady 2014; Katz, Hover and Brady
2016).
Nowadays, the choice of appropriate screening method
remains crucial to eDNA mining (Figs 1 and 2C). Screens
could be based on metabolic activity detection (hereby named
functional screening or functional metagenomics) or on nu-
cleotide sequence analysis (also indicated as genetic screening)
(Table 1, Figs 1 and 2C). Actually, functional screening is consid-
ered the best strategy for identifying genes encoding functional
products and for potentially discovering completely new classes
of enzymes that lack homologies to known sequences (Culli-
gan et al. 2014a; Coughlan et al. 2015; Batista-Garcı́a et al. 2016).
Ferrer et al. (2016) reported that out of ∼6100 total targets (clones
and/or enzymes and/or sequences encoding enzymes) identified
by metagenomic studies in the last two decades, 5800 were dis-
covered by functional metagenomics. Our analysis—focusing on
enzymes that had been (at least partially) characterized at bio-
chemical/functional level (Table 1 and Fig. 2C)—confirms that,
in the last three years, the majority of the metagenome-sourced
biocatalysts (273) were discovered by functional screening.
Despite the enormous potentialities of functional metage-
nomics, there are some constraints that still limit its success.
The first one is the low level of gene expression in the library
host, which might introduce a bias in library representativeness
and dramatically reduce the hit output from screening. Indeed,
Escherichia coli typically remains the first-choice host for con-
structing a metagenomic library (Table 1). However, it is limited
in heterologous protein expression because of its bias in codon
usage, improper heterologous promoter recognition, spurious
Table 1. Industrially relevant enzymes discovered from metagenomics since 1 January 2014 to 31 March 2017.

Target DNA source Library Heterologous Screening Number of Number of Hit Number of Reference
type host approach screened positive frequency characterized
Activity EC number clones clones enzymes

Cellulase/hemicellulase
Endoglucanase 3.2.1.4 Soil Plasmid E. coli Function-based (A) 2.4 × 104 na na 1 Pimentel et al. (2017)
Endoglucanase 3.2.1.4 Hot spring / / Sequence-based (G) / / / 1 Zhao et al. (2017)
sediment
Endoglucanase 3.2.1.4 Ovine rumen BAC E. coli Function-based (A) 1.3 × 104 6 1:2150 1 Cheng et al. (2016)
Endoglucanase 3.2.1.4 Soil Plasmid E. coli Function-based (A) na 1 na 1 Garg et al. (2016)
Endoglucanase 3.2.1.4 Compost Cosmid E. coli Function-based (A) 1.0 × 104 1 1:10 000 1 Meneses et al. (2016)
Endoglucanase 3.2.1.4 Beer lees / / Sequence-based (G) / 23 na 3 Yang et al. (2016)
Endoglucanase 3.2.1.4 Paddy soil Fosmid E. coli Function-based (A) 2.5 × 104 na na 1 Zhou et al. (2016)
Endoglucanase 3.2.1.4 Soil / / Sequence-based (E) / 1 na 1 Hua et al. (2015)
Endoglucanase 3.2.1.4 Compost Fosmid E. coli Function-based (A) 2.1 × 104 3 1:7000 2 Okano et al. (2015b)
Endoglucanase 3.2.1.4 Soil Fosmid E. coli Function-based (A) 1.0 × 104 1 1:10 000 1 Mai et al. (2014)
Endoglucanase 3.2.1.4 Algae Plasmid E. coli Function-based (A) 4.0 × 104 20 1:2000 1 Martin et al. (2014)
Endoglucanase 3.2.1.4 Insect gut Fosmid E. coli Function-based (A) 9.2 × 104 1 1:92 000 1 Lee et al. (2014)
Endoglucanase 3.2.1.4 Soil Plasmid E. coli Function-based (A) 2.4 × 104 1 1:24 000 1 Xiang et al. (2014)
β-Glucosidase 3.2.1.21 Soil Fosmid E. coli Function-based (A) 1.0 × 105 1 1:100 000 1 Matsuzawa and Yaoi (2017)
β-Glucosidase 3.2.1.21 Insect gut Plasmid E. coli Function-based (A) 8.0 × 105 13 1:61 500 1 Gao et al. (2016a)
β-Glucosidase 3.2.1.21 Soil Cosmid E. coli Sequence-based (F) na na na 1 Gomes-Pepe et al. (2016)
β-Glucosidase 3.2.1.21 Bovine rumen Fosmid E. coli Function-based (A) 1.4 × 104 1 1:14 000 1 Ramı́rez-Escudero et al.
(2016)
β-Glucosidase 3.2.1.21 Soil Plasmid E. coli Function-based (A) 5.0 × 104 5 1:10 000 1 Cao et al. (2015)
β-Glucosidase 3.2.1.21 Bovine rumen Fosmid E. coli Function-based (A) 3.0 × 104 45 1:650 1 Loaces et al. (2015)
β-Glucosidase 3.2.1.21 Kusaya gravy Plasmid E. coli Function-based (A 1.0 × 104 7 1:1450 1 Uchiyama, Yaoi and
and B) Miyazaki (2015)
β-Glucosidase 3.2.1.21 Soil Fosmid E. coli Function-based (A) 9.8 × 104 2 1:49 000 2 Bergmann et al. (2014)
β-Glucosidase 3.2.1.21 Soil Plasmid E. coli Function-based (A) 9.0 × 104 4 1:22 500 2 Biver et al. (2014)
β-Glucosidase 3.2.1.21 Bovine rumen Fosmid E. coli Function-based (A) 2.0 × 104 1 1:20 000 1 Li et al. (2014b)
β-Glucosidase 3.2.1.21 Hydrothermal Plasmid E. coli Function-based (A) na na na 1 Schröder et al. (2014)
spring
β-Glucosidase 3.2.1.21 Insect gut Fosmid E. coli Function-based (A) 1.0 × 104 12 1:850 3 Zhang et al. (2014a)
β-Xylanase 3.2.1.8 Insect gut Fosmid E. coli Function-based (A) 1.0 × 104 13 1:750 1 Qian et al. (2015)
β-Xylanase 3.2.1.8 Compost / / Sequence-based (E) / na / 1 Sun et al. (2015)
β-Xylanase 3.2.1.8 Ovine rumen Fosmid E. coli Function-based (A) 1.2 × 104 18 1:650 1 Wang et al. (2015b)
β-Xylosidase/ 3.2.1.37 Bovine rumen Phagemid E. coli Function-based (A) na na na 1 Jordan et al. (2016)
arabinofuranosidase
α-Xylosidase/ 3.2.1.177 Soil Fosmid E. coli Function-based (A) 5.0 × 104 1 1:50 000 1 Matsuzawa et al. (2016)
arabinofuranosidase
β-Xylosidase/ 3.2.1.21 Compost Plasmid E. coli Function-based (A) 3.0 × 104 40 1:750 1 Matsuzawa, Kaneko and
arabinofuranosidase Yaoi (2015)
α-Arabinofuranosidase 3.2.1.55 Insect gut Fosmid E. coli Function-based (A) 4.0 × 104 87 1:450 4 Arnal et al. (2015)
α-Fucosidase 3.2.1.51 Soil Fosmid E. coli Function-based (A) 1.0 × 105 7 1:14 300 7 Lezyk et al. (2016)
Berini et al.
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Table 1. (Continued)

Target DNA source Library Heterologous Screening Number of Number of Hit Number of Reference
type host approach screened positive frequency characterized
Activity EC number clones clones enzymes

β-Galactosidase 3.2.1.23 Soil Cosmid E. coli and Function-based (B) 7.9 × 104 na na 3 Cheng et al. (2017)
Sinorhizobium
melitoti
α-Galactosidase 3.2.1.23 Hot spring water / / Sequence-based (G) / / / 1 Schröder et al. (2017)
and sediment
β-Galactosidase 3.2.1.23 Soil Plasmid E. coli Function-based (A) 1.3 × 106 6 1:216 700 1 Erich et al. (2015)
β-Galactosidase 3.2.1.23 Marine sediment Plasmid E. coli Function-based (A) na 28 na 1 Li et al. (2015)
β-Galactosidase 3.2.1.23 Hot spring water / / Sequence-based (E) / 1 / 1 Liu et al. (2015b)
β-Galactosidase 3.2.1.23 Soil Plasmid E. coli Function-based (A) 7.0 × 105 1 1:700 000 1 Wang et al. (2014)
β-N- 3.2.1.52 Soil Fosmid E. coli Function-based (A) 1.0 × 105 30 1:3300 2 Nyffenegger et al. (2015)
Acetylhexosaminidase
α-Rhamnosidase 3.2.1.40 Feces Fosmid E. coli Function-based (A) 2.0 × 104 na na 1 Rabausch, Ilmberger and
FEMS Microbiology Letters, 2017, Vol. 364, No. 21

Streit (2014)
Lichenase 3.2.1.73 Soil Plasmid E. coli Function-based (A) 2.0 × 104 1 1:20 000 1 Kim, Oh and Kwon (2014)
(endoglucanase)
Cellulase 3.2.1.- Compost Fosmid E. coli Function-based (A) 6.0 × 103 24 1:250 1 Okano et al. (2014)
Polygalacturonase 3.2.1.15 Soil Plasmid E. coli Function-based (A) 2.0 × 103 9 1:200 1 Sathya, Jacob and Khan
(2014)
Cellobiose epimerase 5.1.3.11 Ovine rumen / / Sequence-based (E) / 71 / 2 Wasaki et al. (2015)
Glycosyl hydrolase with 3.2.1.- Ovine rumen Fosmid E. coli Function-based (A) 1.2 × 105 155 1:750 1 Song et al. (2017)
multifunctional activity
Glycosyl hydrolase with 3.2.1.- Bovine rumen / / Sequence-based (G) / 2597 / 1 Patel et al. (2016)
multifunctional activity
Glycosyl hydrolase with 3.2.1.- Bovine rumen BAC E. coli Function-based (A) na na na 1 Gruninger et al. (2014)
multifunctional activity
Glycosyl hydrolase with 3.2.1.- Compost Fosmid E. coli Function-based (A) 2.5 × 102 5 1:50 1 Sae-Lee and Boonmee
multifunctional activity (2014)
4
β-Xylanase, cellulase, 3.2.1.8, Insect gut Fosmid E. coli Function-based (A) 4.0 × 10 31 1:1300 8 Rashamuse et al. (2017)
α-fucosidase 3.2.1.51
β-Galactosidase, 3.2.1.23, Wheat straw Fosmid E. coli Function-based (A) 4.4 × 104 71 1:600 7 Maruthamuthu et al. (2016)
β-xylosidase, 3.2.1.37,
α-glucosidase 3.2.1.20
Endoglucanase, 3.2.1.4, Sugarcane Fosmid E. coli Function-based (A) 1.0 × 105 7 1:14 300 2 Kanokratana et al. (2015)
endoxylanase 3.2.1.8 bagasse
β-Xylosidase, xylanase, 3.2.1.37, Digester Fosmid E. coli Function-based (A) 9.7 × 103 178 1:54 4 Wang et al. (2015a)
β-glucosidase, cellulase 3.2.1.8,
3.2.1.21

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Table 1. (Continued)

Target DNA source Library Heterologous Screening Number of Number of Hit Number of Reference
type host approach screened positive frequency characterized
Activity EC number clones clones enzymes

Glycosyl hydrolases 3.2.1.- Digester / / Sequence-based (G) / 163 / 6 Wei et al. (2015)
belonging to various
families
β-Glucosidase, 3.2.1.21, Subseafloor / / Sequence-based (G) / 60 / 10 Klippel et al. (2014)
endomannanase, 3.2.1.25, sediments
endoxylanase, 3.2.1.8,
β-xylosidase 3.2.1.37
Cellulase, xylanase 3.2.1.4, 3.2.1.8 Soil Plasmid E. coli Function-based (A) 1.5 × 105 6 1:25 000 3 Mori et al. (2014)
β-Glucosidase, 3.2.1.21 Soil Plasmid E. coli Function-based (A) 5.0 × 105 9 1:55 600 9 Stroobants, Portetelle,
glycosyltransferases Vandenbol (2014)
Amylase
Amylopullulanase 3.2.1.1 Insect gut Fosmid E. coli Function-based (A) 9.2 × 104 1 1:92 000 1 Lee et al. (2016b)
(α-amylase)
α-Amylase 3.2.1.1 Cow dung Plasmid E. coli Function-based (C) 1.0 × 105 200 1:500 1 Pooja et al. (2015)
α-Amylase 3.2.1.1 Submarine ikaite BAC E. coli Function-based (A) 2.8 × 103 3 1:900 3 Vester, Glaring and
column Stougaard (2014)
α-Amylase 3.2.1.1 Feces Fosmid E. coli Function-based (A) 5.0 × 104 8 1:6200 1 Xu et al. (2014)
Chitinase
Chitinase 3.2.1.14 Soil Fosmid E. coli Sequence- (D) and 7.8 × 103 1 1:7800 1 Hjort et al. (2014); Berini
function-based (A) et al. (2017)
Chitinase 3.2.1.14 Soil Fosmid E. coli Function-based (A) 5.0 × 104 15 1:3300 1 Thimoteo et al. (2017)
Chitinase 3.2.1.14, Soil Fosmid E. coli Sequence-based (D) 1.45 × 105 8 1:18 100 1 Cretoiu et al. (2015)
3.5.1.41
Chitinase 3.2.1.14 Pig feces / / Sequence-based (E) / 1 / 1 Liu et al. (2015a)
Chitinase 3.2.1.14, Soil / / Sequence-based(G) / 10 / 1 Stöveken et al. (2015)
3.5.1.41
Chitin deacetylase 3.5.1.41 Sediment Plasmid E. coli Sequence-based (F) 10 1 1:10 1 Liu et al. (2016)
Esterase/lipase
Carboxylesterase 3.1.1.1 Anaerobic Fosmid E. coli Function-based (A) 1.1 × 106 714 1:1500 77a Popovic et al. (2017)
digester, sunken
shipwreck’s tar,
water, soil etc.
Carboxylesterase 3.1.1.1 Marine mud Fosmid E. coli Function-based (A) 4.0 × 104 34 1:1200 1 Gao et al. (2016b)
Carboxylesterase 3.1.1.1 Soil Cosmid E. coli Function-based (A) 8.0 × 104 1 1:80 000 1 Jeon et al. (2016)
Carboxylesterase 3.1.1.1 Gill chamber Fosmid E. coli Function-based (A) 2.7 × 104 10 1:2700 3 Alcaide et al. (2015)
Carboxylesterase 3.1.1.1 Biogas digester Plasmid E. coli Function-based (A) 9.6 × 103 1 1:9600 1 Cheng et al. (2014)
Carboxylesterase 3.1.1.1 Hot vent Fosmid E. coli Function-based (A) 9.6 × 103 120 1:80 3 Placido et al. (2015)
sediment
Berini et al.
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Table 1. (Continued)

Target DNA source Library Heterologous Screening Number of Number of Hit Number of Reference
type host approach screened positive frequency characterized
Activity EC number clones clones enzymes

Carboxylesterase 3.1.1.1 Seawater Phagemid E. coli Function-based (A) 3.0 × 105 23 1:13 050 5 Tchigvintsev et al. (2015)
Esterase 3.1.1.1 Sediment Fosmid E. coli Function-based (A) 7.2 × 103 10 1:720 1 Zhang et al. (2017)
Esterase 3.1.1.1 Marine sediment Fosmid E. coli Function-based (A) 3.9 × 103 19 1:200 1 De Santi et al. (2016)
Esterase 3.1.1.1 Compost Fosmid E. coli Function-based (A) 2.3 × 104 18 1:1300 1 Lee et al. (2016a)
Esterase 3.1.1.1 Moss Fosmid E. coli Function-based (A) 9.0 × 104 83 1:1100 6 Müller et al. (2017)
Esterase 3.1.1.- Hot spring mud / / Sequence-based (G) / 1 / 1 Zarafeta et al. (2016)
Esterase 3.1.1.1 Glacier soil Fosmid E. coli Function-based (A) 1.0 × 104 5 1:2000 1 De Santi et al. (2015)
Esterase 3.1.1.1 Sediment Fosmid E. coli Function-based (A) 2.0 × 105 1 1.200 000 1 Hu et al. (2015)
FEMS Microbiology Letters, 2017, Vol. 364, No. 21

Esterase 3.1.1.1 Hot spring water, Fosmid E. coli and Function-based (A 8.0 × 103 8 1:2650 2b Leis et al. (2015)
sediment and Thermus for E. coli, B for T.
compost thermophilus thermophilus)
Esterase 3.1.1.1 Hot spring water Fosmid E. coli Function-based (A) 1.2 × 104 6 1:2000 1 López-López et al. (2015)
Esterase 3.1.1.1 Compost Fosmid E. coli Function-based (A) 6.0 × 103 19 1:300 1 Okano et al. (2015a)
Esterase 3.1.1.1 Bovine rumen Fosmid E. coli Function-based (A) 2.8 × 104 3 1:9200 1 Rodrı́guez et al. (2015)
Esterase 3.1.1.1 Soil Plasmid E. coli Function-based (A) 1.0 × 104 3 1:3300 1 Sudan and Vakhlu (2015)
Esterase 3.1.1.1 Sediment Fosmid E. coli Function-based (A) 3.2 × 104 1 1:32 000 1 Seo et al. (2014)
Hormone-sensitive lipase 3.1.1.79 Soil Plasmid E. coli Function-based (A) 1.7 × 105 1 1:170 000 1 Dukunde et al. (2017)
Hormone-sensitive lipase 3.1.1.79 Sediment Fosmid E. coli Function-based (A) 2.0 × 104 12 1:1700 1 Huang et al. (2016)
Hormone-sensitive lipase 3.1.1.79 Permafrost Fosmid E. coli Function-based (A) 5.0 × 103 7 1:700 1 Petrovskaya et al. (2016)
Hormone-sensitive lipase 3.1.1.79 Sediment Fosmid E. coli Function-based (A) 1.1 × 104 7 1:1600 1 Li et al. (2014a)
Hormone-sensitive lipase 3.1.1.79 Sediment Plasmid E. coli Function-based (A) 1.2 × 105 15 1:8000 1 Peng et al. (2014)
Lipase 3.1.1.3 Hot spring Plasmid E. coli Function-based (A) 1.3 × 104 7 1:1900 1 Sahoo et al. (2017)
sediment
Lipase 3.1.1.3 Hot spring water BAC E. coli Function-based (A) 6.8 × 104 4 1:17 000 1 Yan et al. (2017)
Lipase 3.1.1.3 Soil / / Sequence-based (E) / na / 1 Kumar et al. (2017)
Lipase 3.1.1.3 Soil Plasmid E. coli Function-based (A) 2.0 × 103 15 1:130 1 Khan and Kumar (2016)
Lipase 3.1.1.3 Soil Fosmid E. coli Function-based (A) 5.0 × 105 32 1:15 600 2 Martini et al. (2014); Alnoch
et al. (2015)
Lipase 3.1.1.3 Water / / Sequence-based (F) / 777 / 1 Masuch et al. (2015)
Lipase 3.1.1.3 Marine sponge Plasmid E. coli Function-based (A) 6.5 × 103 1 1:6500 1 Su et al. (2015)

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Table 1. (Continued)

Target DNA source Library Heterologous Screening Number of Number of Hit Number of Reference
type host approach screened positive frequency characterized
Activity EC number clones clones enzymes

Lipase 3.1.1.3 Fed-batch reactor Cosmid E. coli Function-based (A) 1.0 × 104 10 1:1000 1 Brault et al. (2014)
Lipase 3.1.1.3 Soil BAC E. coli Function-based (A) 3.0 × 103 6 1:500 1 Pindi, Srinath and
Pavankumar (2014)
Lipase 3.1.1.3 Human oral Plasmid E. coli Function-based (A) 4.0 × 104 20 1:2000 1 Preeti et al. (2014)
microbiome
Esterase/lipase 3.1.1.- Soil Fosmid E. coli Function-based (A) 4.2 × 103 30 1:140 2 Pereira et al. (2015); Maester
et al. (2016)
3
Esterase/lipase 3.1.1.- Mud flat Plasmid E. coli Function-based (A) 3.0 × 10 9 1:300 2 Kim et al. (2015)
Esterase/lipase 3.1.1.- Soil Fosmid E. coli Function-based (A) 1.4 × 104 1 1:14 000 1 O’Mahony et al. (2015)
Esterase/lipase/ 3.1.1.- Bovine rumen Fosmid E. coli Function-based (A) 2.4 × 104 14 1:1700 5 Privé et al. (2015)
phospholipase
Esterase/lipase 3.1.1.- Water BAC E. coli Sequence-based (F) / / / 1 Fang et al. (2014)
Thioesterase 3.1.2.- Activated sludge Plasmid E. coli Function-based (A) 1.0 × 104 1 1:10 000 1 Sánchez-Reyez et al. (2017)
Glucuronoyl esterase 3.1.1.- Marine sediment Fosmid E. coli Function-based (A) 1.7 × 102 1 1:170 1 De Santi, Willassen and
Williamson (2016)
Sulfatase, 3.1.6.-, 2.8.2.1 Soil and rumen Plasmid E. coli Function-based (A) 1.3 × 106 14 1:89 000 13c Colin et al. (2015)
sulfotransferase
Feruloyl esterase 3.1.1.73 Hindgut Fosmid E. coli Function-based (A) 4.0 × 104 7 1:5700 6 Rashamuse et al. (2014)
symbiont
Phospholipase 3.1.1.- Hydrothermal Fosmid E. coli Function-based (A) 1.8 × 104 7 1:2600 1 Fu et al. (2015)
(patatin-like) vent
Phosphodiesterase 3.1.1.- Coalfield Fosmid E. coli Function-based (A) na 1 na 1 Singh et al. (2015a)
Wax ester synthase 2.3.1.75 Soil Fosmid E. coli Function-based (A) 3.3 × 105 155 1:2100 1 Kim et al. (2016a)
Oxidoreductase
Dioxygenase 1.13.11.- Soil Fosmid E. coli Function-based (B) 1.5 × 105 62 1:2400 1 dos Santos et al. (2015)
Dioxygenase 1.13.11.- Soil Plasmid E. coli Function-based (A) na 1 na 1 Wang et al. (2015c)
Dioxygenase 1.13.11.- Soil / / Sequence-based (G) / 510 / 4 Chemerys et al. (2014)
Alcohol dehydrogenase 1.1.1.1 Soil Plasmid E. coli Sequence- (D) and 1.2 × 102 23 1:5 2 Itoh et al. (2014)
function-based (A)
Alcohol dehydrogenase 1.1.1.1 Soil and compost Plasmid E. coli Sequence- (D) and 2.0 × 103 40 1:50 2d Itoh, Kariya and Kurokawa
function-based (A) (2014)
d-Amino acid oxidase 1.4.3.3 Soil Plasmid E. coli Sequence-based (F) 3.2 × 104 1 1:32 000 1 Ou et al. (2015)
Monooxygenase, 1.14.-, Soil Cosmid E. coli and P. Function-based (B) 2.2 × 105 29 1:7600 2 Nagayama et al. (2015)
dioxygenase 1.13.11.- putida
β-Carotene 1.14.99.36 Human gut Fosmid E. coli Function-based (B) 2.3 × 104 53 1:450 1 Culligan et al. (2014b)
monooxygenase microbiome
4
Glucose dehydrogenase 1.1.99.10 Hay infusion Phagemid E. coli Function-based (A) 2.0 × 10 13 1:1550 1 Basner and Antranikian
(2014)
Berini et al.
7

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 8

Table 1. (Continued)

Target DNA source Library Heterologous Screening Number of Number of Hit Number of Reference
type host approach screened positive frequency characterized
Activity EC number clones clones enzymes

Aldehyde dehydrogenase 1.2.1.3 Hot spring water / / Sequence-based (E) / na / 1 Chen et al. (2014)
and soil
Mercuric reductase 1.16.1.1 Brine pool / / Sequence-based (G) / 1 / 1 Sayed et al. (2014)
Multicopper oxidase 1.10.- Coalbed Fosmid E. coli Function-based (C) 4.6 × 104 24 1.1900 1 Strachan et al. (2014)
Bilirubin oxidase (laccase) 1.3.3.5 Activated sludge Fosmid E. coli Function-based (A) 1.0 × 105 1 1:100 000 1 Kimura and Kamagata
(2016)
Protease
Serine protease 3.4.21.- Tannery sludge Plasmid E. coli Function-based (A) 1.0 × 104 1 1:10 000 1 Devi et al. (2016)
Serine protease 3.4.21.- Hot spring Plasmid E. coli Function-based (A) 9.0 × 103 1 1:9000 1 Singh et al. (2015b)
sediment
Subtilisin-like serine 3.4.21.62 Underground Fosmid E. coli Function-based (A) 2.1 × 104 23 1:900 2 Apolinar–Hernández et al.
protease water (2016)
Cysteine protease 3.4.22.- Ovine rumen Plasmid E. coli Sequence-based (G) na na na 1 Faheem et al. (2016)
Metalloprotease 3.4.- Sludge Fosmid E. coli Function-based (A) 2.8 × 104 2 1:14 000 1 Morris and Marchesi (2015)
FEMS Microbiology Letters, 2017, Vol. 364, No. 21

Phosphatase/phytase
Phytase 3.1.3.- Bovine rumen / / Sequence-based (G) / 1 / 1 Mootapally et al. (2016)
Phytase 3.1.3.- Peat soil na na Sequence-based (F) na 4 na 1 Tan et al. (2016a)
Phytase 3.1.3.- Fungus garden na na Sequence-based (F) na 11 na 1 Tan et al. (2016b)
Phytase 3.1.3.- Groundwater na na Sequence-based (F) na 1 na 1 Tan et al. (2015)
Phytase 3.1.3.- Soil Fosmid E. coli Function-based (B) 1.4 × 104 28 1:500 1 Tan et al. (2014)
Alkaline phosphatase 3.1.3.1 Sediment Fosmid E. coli Function-based (C) 8.0 × 104 6 1:13 350 1 Lee et al. (2015)
Other
Amine transferase 3.6.1.- Hot spring / / Sequence-based (G) / 3 / 3 Ferrandi et al. (2017)
β-Agarase 3.2.1.81 Soil Fosmid E. coli Function-based (A) 1.0 × 105 3 1:33 350 1 Mai, Su and Zhang (2016)
Penicillin G acylase 3.5.1.11 Sediment Cosmid E. coli Function-based (B) 7.0 × 103 1 1:7000 1 Zhang et al. (2014b)
Epoxide hydrolase 3.3.2.8 Hot spring / / Sequence-based (G) / 2 / 2 Ferrandi et al. (2015)
Nitrilase 3.5.5.1 Water / / Sequence-based (G) / 1 / 1 Sonbol, Ferreira and Siam
(2016)
Exonuclease 3.1.11.1 Soil Plasmid E. coli Function-based (B) 2.7 × 103 1 1:2700 1 Silva-Portela et al. (2016)
Rhodanese (sulfur 2.8.1.1 Soil Plasmid E. coli Function-based (A) 8.5 × 103 5 1:1700 1 Bhat et al. (2015)
transferase)

The list was created by searching Pubmed database (accession on 17 July 2017) with the following query: (metagenom∗[Title/Abstract]) AND (enzyme[Title/Abstract]) AND (‘2014/01/01’[Date—Publication]: ‘2017/03/31’[Date—
Publication]) (where ‘enzyme’ was substituted with the name of each enzymatic class considered). The results were manually checked in order to select only those publications dealing with enzymes fulfilling three criteria:
(i) identification by metagenomics, (ii) heterologous expression and at least partial purification (thus confirming the activity of the putative hit), (iii) biochemical and/or functional and/or structural characterization.
For functional screening: A = phenotypical detection; B = heterologous complementation; C = induced gene expression (see also Fig. 1). For genetic screening: D = PCR amplification of metagenomic library DNA; E = PCR
amplification of eDNA; F = in silico analysis of metagenomic library DNA; G = in silico analysis of shotgun eDNA (see also Fig. 1). na = data not available.
a
28 from anaerobic digester, 21 from water sample, 11 from sunken shipwreck’s tar, 7 from soil and sediment, 5 from compost, 4 from eukaryote-associated microbiome, 1 from paper mill.
b
1 from sediment, 1 from compost.
c
10 from soil, 3 from rumen.
d
From compost.

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 Berini et al. 9

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Figure 1. Scheme of metagenomic strategies for the identification of novel biocatalysts from environmental DNA. For functional screening: A = phenotypical detection;
B = heterologous complementation; C = induced gene expression. For genetic screening: D = PCR amplification of metagenomic library DNA; E = PCR amplification of
eDNA; F = in silico analysis of metagenomic library DNA; G = in silico analysis of shotgun eDNA.

transcription, heterologous protein misfolding, poor secretion
and inclusion body formation (Binda et al. 2013; Lam and Charles
2015). On average, only 30%–40% of environmental bacterial
genes could be efficiently expressed in E. coli, a value dropping to
7% for high G + C DNA (Gabor, Alkema and Janssen 2004). Ran-
dom insertion of bidirectional promoters into the library (Kim
et al. 2016b) and addition of heterologous σ -factors for increas-
ing transcription proficiency (Gaida et al. 2015) were recently ex-
perimented to increase E. coli performance. A different strategy
is using alternative expression systems, such as Agrobacterium,
Bacillus, Rhodococcus, Streptomyces and Pseudomonas, along with
a few archaea (Liebl et al. 2014). Multihost expression strate-
gies were proposed too, using shuttle vectors with a broad host
range either sequentially or in parallel (Katzke et al. 2017). Recent
examples (Table 1) include an esterase discovered in a fos-
mid library propagated in E. coli and in Thermus thermophilus
(Leis et al. 2015), two dioxygenases from a cosmid metage-
nomic library in Pseudomonas putida (Nagayama et al. 2015) and
a β-galactosidase from a library in Sinorhizobium meliloti (Cheng
et al. 2017). Interestingly, Bouhajja et al. (2017) identified novel
toluene monooxygenase genes by cloning eDNA in three par-
allel hosts, i.e. E. coli, Cupriavidus metallidurans and Edaphobacter
aggregans.
Another critical factor is the sensitivity of commonly used
function-based screening methods, which might be too low
to make gene expression easily detectable (Gabor, Alkema
 10 FEMS Microbiology Letters, 2017, Vol. 364, No. 21
Figure 2. Distribution of the 332 novel industrially relevant enzymes discovered by metagenomics in the time frame since 1 January 2014 to 31 March 2017 (Table 1)
according to (A) DNA source, (B) vector used for library construction, (C) screening method and (D) enzyme class.
and Janssen 2004; Ekkers et al. 2012). Classical phenotypical
detection (A in Table 1, Fig. 1), applied for decades in screen-
ing microbial isolates and based on identifying specific phe-
notypic traits associated with the activity of interest, remains
the election method in metagenomic, too (260 enzymes). Phe-
notypical detection on agar plates is still the most widely
used approach, especially when searching for hydrolytic en-
zymes (such as lipases) forming easy-to-detect degradation
haloes (Placido et al. 2015; Popovic et al. 2017) on appro-
priate substrates. Alternative methods were based on high-
throughput assays in liquid culture (Zottig, Meddeb-Mouelhi and
Beauregard 2016) or high-performance thin-layer chromatog-
raphy (Rabausch et al. 2013). High-throughput screening (HTS)
with multiple substrates, either used in parallel (Ufarté et al.
2016) or as a mixture (Maruthamuthu et al. 2016), significantly
increased hit rate. Proper selection of the screening substrate
is highly recommended. Recently, novel hemicellulases were
discovered also thanks to a new generation of versatile multi-
ple chromogenic substrates mimicking insoluble plant cell wall
components (Maruthamuthu et al. 2016). To minimize redun-
dancy (hits showing the same activities/sequences), Ufarté et al.
(2016) suggested multistep screening of several small libraries
originating from different samples instead of focusing on only
one large library from a unique source. A recent frontier in func-
tional metagenomics is using micro- and picodroplet techniques
for eDNA HTS: each single clone is encapsulated in water-in-oil
droplets together with a suitable substrate and lysis reagents,
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and hits are selected based on the emitted fluorescence or ab-
sorbance (Mair, Gielen and Hollfelder 2017). This method al-
lowed screening over a million clones per day, recovering both
slow and fast biocatalysts, promiscuous enzymes or those en-
coded by rare members of the microbial community. Through
this approach, Colin et al. 2015 identified novel sulfatases and
phosphotriesterases from soil and rumen metagenomic libraries
(Table 1).
In addition to phenotypical detection, two other functional
screening approaches merit mention. Heterologous comple-
mentation (B in Table 1, Fig. 1) relies on the selection of clones
that have acquired the capability to grow under selective con-
ditions, such as in the presence of a specific substrate given as
the sole carbon source. It was used to detect a phytase in E. coli
clones grown on phytate as the sole phosphorus source (Tan et al.
2014), as well as for the identification of a thermostable peni-
cillin G acylase by selecting E. coli clones growing in the pres-
ence of penicillin G (Zhang et al. 2014b). Induced gene expression
(C in Table 1, Fig. 1) includes substrate-induced gene expression
(SIGEX) (Uchiyama et al. 2005), product-induced gene expression
(PIGEX) (Uchiyama and Miyazaki 2010) and metabolite-regulated
expression (METREX) (Williamson et al. 2005), which are all based
on the use of operon-trap green fluorescent protein (GFP) expres-
sion vectors: positive clones, co-expressing the desired enzyme
activity and the GFP upon exposure to a target substrate (SIGEX),
product (PIGEX) or quorum-sensing signal molecule (METREX)
can be isolated by fluorescence-assisted cell sorting. By using

PIGEX, a periplasmic α-amylase was discovered in a clone from
a cow dung metagenomic library that fluoresced on maltose as
substrate (Pooja et al. 2015). By-products from a wide variety of
enzymes (e.g. cellulases, lipases and lyases) may be detected us-
ing a single cell-based reporter system in which GFP expression
is activated by the presence of phenyl compounds (Choi et al.
2014), as in the case of a psychrophilic alkaline phosphatase
from tidal flat sediment (Lee et al. 2015).
Alternatively to functional metagenomics, genetic screening
is gene expression independent. It is traditionally based on the
use of PCR with primers specific for conserved regions of the
genes being targeted, which for enzymes are usually the cat-
alytic domains (Ekkers et al. 2012; Coughlan et al. 2015). It can
be applied for screening either clones of metagenomic libraries
(D in Table 1, Fig. 1) or the eDNA extracted directly from envi-
ronmental samples (E). The limitation is that, being based on
the identification of conserved nucleotide sequences, genetic
screening is biased for members of already known gene families
and additionally it does not guarantee the recovery of full-length
genes. Table 1 indicates that only nine of the recently isolated
metagenome-sourced enzymes were identified by PCR-based
screening. Interestingly, five other enzymes, namely one chiti-
nase (Hjort et al. 2014) and four alcohol dehydrogenases (Itoh
et al. 2014; Itoh, Kariya and Kurokawa 2014), were discovered by
combining PCR- and function-based strategies on the same li-
brary (Fig. 2C). The combination of the two approaches, either
sequentially or in parallel, in fact might increase the screening
hit rate.
In the last few years, the rapid advancement of next-
generation sequencing (NGS) technologies, coupled with a sig-
nificant reduction in sequencing costs, promoted the evolution
of another genetic screening approach that is based on the bioin-
formatic analysis of sequenced DNA (in silico screening). Among
the enzymes listed in Table 1, eight were discovered by in sil-
ico screening of DNA from metagenomic libraries (F in Table 1,
Fig. 1). The real revolution occurred with the birth of ‘shotgun
metagenomics’, i.e. the direct sequencing and analysis of iso-
lated eDNA, bypassing the laborious steps of library construction
(G in Table 1, Fig. 1). Compared to classical PCR-based screening,
this approach has the advantage of potentially uncovering genes
that are more divergent and more interesting than the consen-
sus genes with known sequences. Additionally, the detection of
very rare genes in complex populations is facilitated (Culligan
et al. 2014a; Kumar et al. 2015). In the last three years (Table 1), 37
novel enzymes, including (hemi)cellulases, chitinases, oxidore-
ductases, proteases and nitrilases, were discovered by shotgun
metagenomics and we can expect an increasing contribution
from this approach for the near future. Among them, it is worth
mentioning the esterase discovered by Zarafeta et al. (2016) from
hot spring mud, which, to our knowledge, is the first biocata-
lyst belonging to an uncharacterized enzymatic family to be re-
trieved by sequence-based screening.
INDUSTRIALLY RELEVANT ENZYMES FROM
METAGENOMICS
The most common targets in metagenomic investigations for
industrial enzymes include glycosyl hydrolases (GHs), lipoly-
tic enzymes, oxidoreductases, proteases and phosphatases;
a few other industrially useful enzyme classes, as acylases,
nucleases and nitrilases, were also found by metagenomics
(Coughlan et al. 2015; DeCastro, Rodrı́guez-Belmonte and
González-Siso 2016). Table 2 reports examples of such classes
Berini et al. 11
of enzymes from metagenomes patented in the last decade.
Additionally, Table 3 indicates some successful cases of
metagenome-sourced enzymes that were marketed. Herein, we
get insight into the activities and potential applications of few
(the most interesting ones from our point of view) among the 332
metagenome-sourced enzymes recently discovered (Table 1, Fig.
2D). Although the majority of them could be considered useful
variants of already known protein families, some of the biocat-
alysts reported in Table 1 belong to previously uncharacterized
enzyme families or subfamilies, with unconventional structures
and active site architectures pointing to novel catalytic mecha-
nisms.
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Glycosyl hydrolases
The classes of metagenome-sourced enzymes mostly repre-
sented in our 3-year analysis are (hemi)cellulases and es-
terases/lipases (Fig. 2D), the two categories of biocatalysts that
have been extensively targeted since the beginning of the
metagenomic era (Lorenz and Eck 2005). Comprehensive lists of
metagenome-sourced enzymes for lignocellulose saccharifica-
tion and biofuel production are reported in the work of Duan and
Feng (2010), Montella, Amore and Faraco (2016), Batista-Garcı́a
et al. (2016) and Xing, Zhang and Huang (2012). Some of the most
recent papers listed in Table 1 deal with the identification of
versatile (hemi)cellulases with a wide substrate specificity use-
ful for plant biomass saccharification: for example, an endoglu-
canase possessing also xylanase activity was identified by func-
tional metagenomics from goat rumen (Cheng et al. 2016), and
Matsuzawa and Yaoi (2017) reported the characterization from
a soil metagenome of a saccharide-stimulated β-glucosidase
possessing additional β-galactosidase and β-fucosidase activ-
ities. Recent studies disclosed interesting uncommon halotol-
erant (hemi)cellulases: for instance, Garg et al. (2016) retrieved
from soil the most halostable endoglucanase so far known,
which retained 70%–100% activity after 1-year incubation with
2 M NaCl, 3 M LiCl or 1 M KCl. The NGS screening of a library
from an enriched, anaerobic beer lees-converting consortium
allowed the identification of three novel acidophilic endoglu-
canases, one of them highly tolerant to high salt concentrations
and to ionic liquids used in cellulose pre-treatment (Yang et al.
2016). Interestingly, this enzyme and the thermostable xylanase
isolated by Sun et al. (2015) and used in polysaccharide degra-
dation for biofuel production were heterologously produced in
Bacillus spp., where the recombinant enzymes were secreted
into culture broth, thus facilitating subsequent purification and
use. Other industrially valuable (hemi)cellulases include two
β-glucosidases with high tolerance towards detergents and ox-
idants (Biver et al. 2014) or towards high sugar concentrations
(Gomes-Pepe et al. 2016), and a β-galactosidase useful for the in-
dustrial production of dairy lactose-free products thanks to its
superior activity at low temperatures (Erich et al. 2015) (Table 1).
Talking about novel protein structures and unconventional
active site architectures, Pimentel et al. (2017) discovered from
a soil metagenome a modular endoglucanase with a rare Calx-
beta motif at the C-terminal domain, whereas Xiang et al. (2014)
identified a halotolerant, acid-stable endoglucanase possessing
an unusual catalytic triad. Functional screening of metagenomic
libraries from cow rumen, corn field soil and elephant feces
led to the identification of three novel GHs—a β-glucosidase
(Ramı́rez-Escudero et al. 2016), a β-galactosidase (Cheng et al.
2017) and a α-l-rhamnosidase (Rabausch, Ilmberger and Streit
2014), respectively—whose sequences could not be assigned to
any GH family so far known.
 12 FEMS Microbiology Letters, 2017, Vol. 364, No. 21

Table 2. Examples of metagenome-sourced enzymes patented in the last decade and their suggested applications.

Patent
Enzyme Source Patent number filling date Suggested applications

Cellulase/hemicellulase
Xylanase Hot spring EP2990482 A1 2014 Biofuel production from
lignocellulosic biomasses
Cellobiohydrolase Hot spring EP2980212 A1 2013 Biofuel production from
lignocellulosic biomasses
Cellulase Bovine WO2014142529 A1 2013 Biofuel production from
rumen lignocellulosic biomasses; fiber,
detergent, feed, food, pulp, paper
production

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β-Galactosidase na EP2530148 A1 2011 Food processing (lactose depletion)
Xylose isomerase Termite US8772012 B2 2009 Biofuel production from
intestine lignocellulosic biomasses
Amylase
α-Amylase Animal CN103290039 B 2013 Animal feed, starch processing
manure industries
Chitinase
Chitosanase Soil CN103361374 A 2013 Industrial chitosan production
Esterase/lipase
Esterase Brine pool US20160053239 A1 2013 Industrial processes for
lipases/esterases (e.g. leather
manufacture, oil biodegradation,
synthesis of pharmaceuticals and
chemicals) under harsh conditions
Lipase Soil CN103834626 A 2013 Industrial applications that require
high temperature-resistant lipases
Lipase/phospholipase Tidal flat EP2784160 A1 2011 Oil and fat purification and
sediment conversion, bio-medicine, fine
chemistry
Protease
Peptidase Compost CN103409443 A 2013 Food processes, life science research,
protein waste treatment
Alkaline protease Seabed mud CN103409398 A 2013 Supplement to liquid detergents
Phosphatase/phytase
Alkaline phosphatase na US8647854 B2 2009 Genetic cloning, enzyme
immunoassays
Other
Alginate lyase Abalone- CN102971426 B 2011 Seaweed processing
associated
microbiome
DNA polymerase Water WO2012173905 A1 2011 DNA polymerization in high salt
conditions
l-Methionine γ -lyase Deep sea CN101962651 B 2010 Clinical detection, food flavor
sediment production, cancer therapy
Muramidase (Lysozyme) Soil CN101892252 B 2010 Antibiosis, bacteriolysis

na = data not available

Among other industrially relevant GHs, a handful of α-
amylases (Table 1, Fig. 2D) with a potential for applications in
starch processing and detergent, food and pharmaceutical pro-
duction were discovered by functional screening of metage-
nomic libraries from cow and pygmi loris dung (Xu et al. 2014;
Pooja et al. 2015), Greenland submarine ikaite columns (Vester,
Glaring and Stougaard 2014) and Hermetia illucens gut microflora
(Lee et al. 2016b), this last being stable also in polar organic sol-
vents and non-ionic detergents.
Chitinases, which are active on chitin—the second most
abundant biopolymer after lignocellulose—recently attracted
attention for the production of chitin derivatives (chitosan
and chitooligosaccharides) with high pharmaceutical and nu-
tritional potential (Cretoiu et al. 2015). In 2014, we identified
a chitobiosidase by combining genetic and functional screen-
ings applied to a suppressive soil metagenome, now under de-
velopment as a potential biocontrol agent thanks to its an-
tifungal activity (Hjort et al. 2014; Berini et al. 2016, 2017).
In 2015, the PCR-based analysis of a library from a chitin-
amended agricultural soil led us to discover a novel halophilic
chitinase, with the potential to be exploited for the treat-
ment and valorization of seafood wastes (Cretoiu et al. 2015).
An interesting enzyme for its potential application in pro-
ducing chitosan from chitin is the chitin deacetylase that
has been identified in Arctic Ocean deep-sea sediments (Liu
et al. 2016).

Table 3. Examples of metagenome-sourced enzymes commercialized
by BASF Enzymes LLC (https://www.basf.com).
Commercial
name Enzyme class Applications
Luminase Xylanase Pulp biobleaching in paper
production
Fuelzyme α-Amylase Fuels and industrial-use alcohols
production
Pyrolase 160, Cellulase Secondary oil and gas recovery
Pyrolase 200
Phyzyme XP Phytase Additive for livestock feed
Lipases and esterase
Dozens of eDNA libraries were screened in the past to
identify novel esterases/lipases for the hydrolysis or the
synthesis of ester bonds in a variety of applications, including
detergent, food, pulp and paper industries, diagnostics and ther-
apeutics, as well as biodiesel production and biopolymer syn-
thesis. Particular attention was directed to extreme environ-
ments, as reviewed in López-López, Cerdán and González Siso
(2014). Popovic et al. (2017) recently made an impressive con-
tribution to this class of enzymes: they discovered 77 esterases
by functional screening of over one million fosmid clones from
16 terrestrial and marine environments, including a metal-ion-
dependent esterase with novel active site architecture (Table 1).
Functional metagenomics led to the discovery of a hormone-
sensitive lipase from permafrost with an unusual catalytic mo-
tif (Petrovskaya et al. 2016). Enzymes belonging to novel fam-
ilies of esterases were recovered from cow rumen (Rodrı́guez
et al. 2015) and hot spring mud (Zarafeta et al. 2016). Addition-
ally, many other metagenome-sourced enzymes have peculiar
biochemical properties that make them valuable for industrial
application. Examples are the solvent- and detergent-resistant
enzymes found in a soil contaminated with petroleum hydro-
carbons (Pereira et al. 2015) and the alkaline, thermostable and
solvent-tolerant carboxylesterase from marine mud (Gao et al.
2016b). Peng et al. (2014) described an alkaline-stable lipase from
marine sediments with a potential application in milkfat flavor
production, whereas Su et al. (2015) and Kim et al. (2015) iden-
tified two novel lipases from marine sponge-associated micro-
biome and oil-polluted mud flats, respectively, which might be
applied in detergent industry and organic synthesis. Alnoch et al.
(2015) and Kumar et al. (2017) described two regio- and enantios-
elective lipases from soil samples to be used pharmaceutically
for the production of racemic intermediates.
Oxidoreductases
Oxidoreductases represent a heterogeneous group of enzymes
with multiple applications in pharmaceutical and food in-
dustries and in bioremediation. Recently discovered oxidore-
ductases (Table 1, Fig. 2D) include five soil-sourced dioxyge-
nases with bioremediation potential (Chemerys et al. 2014; dos
Santos et al. 2015) and the first metagenome-sourced d-amino
acid oxidase with potential application in the biosynthesis of the
antibiotic intermediate of 7-aminocephalosporanic acid from
cephalosporin C (Ou et al. 2015). Belonging to this enzymatic
class are multi-copper oxidases (MCOs), enzymes with a broad
range of activity on both phenolic and non-phenolic substrates,
which attract attention due to their involvement in degrading
lignocellulose biomasses. Table 1 includes two MCOs from coal-
Berini et al. 13
bed bacterial community (Strachan et al. 2014) and activated
sludge-associated microbiome (Kimura and Kamagata 2016).
More recently, we reported on the isolation and characterization
of the first metagenome-sourced acidobacterial MCO, with high
tolerance to salt and heat (Ausec et al. 2017).
Proteases
Five metagenomic studies led to the identification of novel pro-
teases, one of the most widely used industrial classes of en-
zymes in detergent, leather and food processing, peptide and
drug synthesis, brewing and wastewater treatment (Table 1,
Fig. 2D). Among them, two serine proteases from Yucatán under-
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ground water belong to a novel, still uncharacterized subfamily
(Apolinar-Hernández et al. 2016) and one serine protease isolated
from a tannery activated sludge looks promising for its unusual
high stability in anionic detergents and organic solvents (Devi
et al. 2016).
Phosphatases
Five novel phytases (Table 1, Fig. 2D) (histidine acid phos-
phatases with applications in agriculture and breeding as an
additive to monogastric animal feed) were recently identified
and characterized. Tan et al. (2014) reported the discovery of two
phytases with unusual sequences from agricultural soils. Two
years later, the same researchers described the characterization
of a phytase from a fungus garden metagenome with an exceed-
ingly long half-life at high temperatures (Tan et al. 2016b). Finally,
a novel psychrophilic alkaline phosphatase was discovered by
induced gene expression screening in tidal flat sediments (Lee
et al. 2015).
Others
Since 2014, seven metagenomic studies have reported heterolo-
gous expression and characterization of other potentially useful
enzymes (Table 1, Fig. 2D), including a thermostable and heavy
metal-resistant nitrilase from Red Sea to be used in the syn-
thesis of cyanocarboxylic acids (Sonbol, Ferreira and Siam 2016)
and a β-agarase from mangrove soil applicable in the cosmetic,
pharmaceutical and food industries (Mai, Su and Zhang 2016). In
Ferrandi et al. (2017), the co-expression of metagenome-sourced
amine-transferases with cold-adapted chaperones in the uncon-
ventional E. coli Arctic Express RIL cells favored the soluble pro-
duction of the enzymes up to several mg per liter of culture. The
subsequent characterization proved that one of these enzymes
is the most thermostable natural amine transferase so far de-
scribed.
CONCLUSIONS
Almost unheard of only 10/15 years ago in enzyme biotech-
nology, metagenomics is now rapidly developing as a mean
of encrypting novel biocatalysts from environmental samples.
Technical challenges still exist, though, that are connected
to screening procedures and heterologous expression of
metagenome-derived enzymes, limiting the great potential
of functional metagenomics. Advances in HTS and NGS pave
the way to make metagenomics more efficient and efficacious
in discovering potential industrially valuable biocatalysts.
We envisage that very soon, metagenome approaches will be
mature enough to substantially impact biobased industrial
production. The experience on how to build and screen metage-
nomic libraries is becoming a common shared knowledge in
 14 FEMS Microbiology Letters, 2017, Vol. 364, No. 21
the scientific community aiming at developing novel biocat-
alysts to replace chemical processes. A proof of this is the
list of the 332 metagenome-sourced enzymes identified and
characterized in the past three years. Scientists and industries
may select from this pool enzymes that may be translated to
industrial development for an incredible variety of industrial
applications. Interestingly, only very few metagenome-sourced
enzymes have already undergone optimization through di-
rected evolution strategies, and this means that there is room
for further improving their protein backbones to fulfill industrial
requirements.
ACKNOWLEDGEMENTS
MIUR (Ministero italiano dell’Istruzione, dell’Università e della
Ricerca) fellowship to CC and CIB (Consorzio Interuniversitario
per le Biotecnologie) contributions to FB and CC are acknowl-
edged.
FUNDING
This work was supported by the Seventh Framework European
Union grant agreement No. 222625 for the MetaExplore project
(Metagenomics for bioexploration – tools and application) and
by MAECI (Ministero Affari Esteri e della Cooperazione Inter-
nazionale) for the CHITOBIOCONTROL project (Israel-Italy Joint
Innovation Program for Industrial, Scientific and Technological
Cooperation in R&D, Industrial Track, call 2016).
Conflict of interest. None declared.
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