TY - JOUR
AU1 - Berini,, Francesca
AU2 - Casciello,, Carmine
AU3 - Marcone, Giorgia, Letizia
AU4 - Marinelli,, Flavia
AB - 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. metagenomics, environmental libraries, screening, heterologous expression, industrial enzymes INTRODUCTION Microbial enzymes are employed in almost all industrial sectors, from the chemical, pharmaceutical and food industries to the manufacturing of detergents, textiles, leather, pulp and paper (Demain and Adrio 2008). In 2015, the application of industrial enzymes generated a turnover of about 5 billion USD, a value that is forecasted to rise to 6.9 billion USD in 2017 and to 10.7 billion USD in 2024 (World Enzyme to 2017, https://www.gminsights.com/industry-analysis/enzymes-market), thanks to the increasing demand for novel biocatalysts with high process performances to replace traditional chemistry. Enzyme-based processes are favored by reduced costs, increased efficiency, improved product recovery, and reduced use of toxic compounds and harsh conditions. Microorganisms represent the largest proportion of biomass on Earth, with a total number of prokaryotic cells estimated to be 4–6 × 1030 (Bunge, Willis and Walsh 2014), inhabiting a widest variety of ecosystems, including the less-hospitable habitats, such as hot springs, nearly saturated salt brines, acid mine waters at pH near zero, deep-sea hydrothermal vents and Antarctic ices (Mirete, Morgante and Gonzàlez-Pastor 2016). Environmental DNA (eDNA) analysis of microbial communities currently estimates that only from 0.1% to 1% of these prokaryotes are culturable by using traditional microbiological methods (Culligan et al.2014a). Also part of eukaryotic microbes is unculturable (Hirst, Kita and Dawson 2011), although the extent of this phenomenon is less clear. Magnuson and Lasure (2002) reported that 10% to 30% of fungi from different soils can be cultivated. In the last two decades, the development of culture-independent methods based on collecting biological material from environmental samples (metagenomics, metatranscriptomics 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 advances 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 metagenomic 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 innovative examples in mining metagenomes for discovering potential 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 criteria 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 bacterial origin. Table 1. Industrially relevant enzymes discovered from metagenomics since 1 January 2014 to 31 March 2017. Target . DNA source . Library type . Heterologous host . Screening approach . Number of screened clones . Number of positive clones . Hit frequency . Number of characterized enzymes . Reference . Activity . EC number . . . . . . . . . . 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 sediment / / Sequence-based (G) / / / 1 Zhao et al. (2017) 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 Ramírez-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 and B) 1.0 × 104 7 1:1450 1 Uchiyama, Yaoi and 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 spring Plasmid E. coli Function-based (A) na na na 1 Schröder et al. (2014) 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/ arabinofuranosidase 3.2.1.37 Bovine rumen Phagemid E. coli Function-based (A) na na na 1 Jordan et al. (2016) α-Xylosidase/ arabinofuranosidase 3.2.1.177 Soil Fosmid E. coli Function-based (A) 5.0 × 104 1 1:50 000 1 Matsuzawa et al. (2016) β-Xylosidase/ arabinofuranosidase 3.2.1.21 Compost Plasmid E. coli Function-based (A) 3.0 × 104 40 1:750 1 Matsuzawa, Kaneko and 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) β-Galactosidase 3.2.1.23 Soil Cosmid E. coli and Sinorhizobium melitoti Function-based (B) 7.9 × 104 na na 3 Cheng et al. (2017) α-Galactosidase 3.2.1.23 Hot spring water and sediment / / Sequence-based (G) / / / 1 Schröder et al. (2017) 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-Acetylhexosaminidase 3.2.1.52 Soil Fosmid E. coli Function-based (A) 1.0 × 105 30 1:3300 2 Nyffenegger et al. (2015) α-Rhamnosidase 3.2.1.40 Feces Fosmid E. coli Function-based (A) 2.0 × 104 na na 1 Rabausch, Ilmberger and Streit (2014) Lichenase (endoglucanase) 3.2.1.73 Soil Plasmid E. coli Function-based (A) 2.0 × 104 1 1:20 000 1 Kim, Oh and Kwon (2014) 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 multifunctional activity 3.2.1.- Ovine rumen Fosmid E. coli Function-based (A) 1.2 × 105 155 1:750 1 Song et al. (2017) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen / / Sequence-based (G) / 2597 / 1 Patel et al. (2016) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen BAC E. coli Function-based (A) na na na 1 Gruninger et al. (2014) Glycosyl hydrolase with multifunctional activity 3.2.1.- Compost Fosmid E. coli Function-based (A) 2.5 × 102 5 1:50 1 Sae-Lee and Boonmee (2014) β-Xylanase, cellulase, α-fucosidase 3.2.1.8, 3.2.1.51 Insect gut Fosmid E. coli Function-based (A) 4.0 × 104 31 1:1300 8 Rashamuse et al. (2017) β-Galactosidase, β-xylosidase, α-glucosidase 3.2.1.23, 3.2.1.37, 3.2.1.20 Wheat straw Fosmid E. coli Function-based (A) 4.4 × 104 71 1:600 7 Maruthamuthu et al. (2016) Endoglucanase, endoxylanase 3.2.1.4, 3.2.1.8 Sugarcane bagasse Fosmid E. coli Function-based (A) 1.0 × 105 7 1:14 300 2 Kanokratana et al. (2015) β-Xylosidase, xylanase, β-glucosidase, cellulase 3.2.1.37, 3.2.1.8, 3.2.1.21 Digester Fosmid E. coli Function-based (A) 9.7 × 103 178 1:54 4 Wang et al. (2015a) Glycosyl hydrolases belonging to various families 3.2.1.- Digester / / Sequence-based (G) / 163 / 6 Wei et al. (2015) β-Glucosidase, endomannanase, endoxylanase, β-xylosidase 3.2.1.21, 3.2.1.25, 3.2.1.8, 3.2.1.37 Subseafloor sediments / / Sequence-based (G) / 60 / 10 Klippel et al. (2014) 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, glycosyltransferases 3.2.1.21 Soil Plasmid E. coli Function-based (A) 5.0 × 105 9 1:55 600 9 Stroobants, Portetelle, Vandenbol (2014) Amylase Amylopullulanase (α-amylase) 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 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 column BAC E. coli Function-based (A) 2.8 × 103 3 1:900 3 Vester, Glaring and 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 function-based (A) 7.8 × 103 1 1:7800 1 Hjort et al. (2014); Berini 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, 3.5.1.41 Soil Fosmid E. coli Sequence-based (D) 1.45 × 105 8 1:18 100 1 Cretoiu et al. (2015) Chitinase 3.2.1.14 Pig feces / / Sequence-based (E) / 1 / 1 Liu et al. (2015a) Chitinase 3.2.1.14, 3.5.1.41 Soil / / Sequence-based(G) / 10 / 1 Stöveken et al. (2015) 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 digester, sunken shipwreck's tar, water, soil etc. Fosmid E. coli Function-based (A) 1.1 × 106 714 1:1500 77a Popovic et al. (2017) 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 sediment Fosmid E. coli Function-based (A) 9.6 × 103 120 1:80 3 Placido et al. (2015) 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) Esterase 3.1.1.1 Hot spring water, sediment and compost Fosmid E. coli and Thermus thermophilus Function-based (A for E. coli, B for T. thermophilus) 8.0 × 103 8 1:2650 2b Leis et al. (2015) 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 Rodríguez 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 sediment Plasmid E. coli Function-based (A) 1.3 × 104 7 1:1900 1 Sahoo et al. (2017) 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) 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 microbiome Plasmid E. coli Function-based (A) 4.0 × 104 20 1:2000 1 Preeti et al. (2014) 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) Esterase/lipase 3.1.1.- Mud flat Plasmid E. coli Function-based (A) 3.0 × 103 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/phospholipase 3.1.1.- Bovine rumen Fosmid E. coli Function-based (A) 2.4 × 104 14 1:1700 5 Privé et al. (2015) 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, sulfotransferase 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) Feruloyl esterase 3.1.1.73 Hindgut symbiont Fosmid E. coli Function-based (A) 4.0 × 104 7 1:5700 6 Rashamuse et al. (2014) Phospholipase (patatin-like) 3.1.1.- Hydrothermal vent Fosmid E. coli Function-based (A) 1.8 × 104 7 1:2600 1 Fu et al. (2015) 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 function-based (A) 1.2 × 102 23 1:5 2 Itoh et al. (2014) Alcohol dehydrogenase 1.1.1.1 Soil and compost Plasmid E. coli Sequence- (D) and function-based (A) 2.0 × 103 40 1:50 2d Itoh, Kariya and Kurokawa (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, dioxygenase 1.14.-, 1.13.11.- Soil Cosmid E. coli and P. putida Function-based (B) 2.2 × 105 29 1:7600 2 Nagayama et al. (2015) β-Carotene monooxygenase 1.14.99.36 Human gut microbiome Fosmid E. coli Function-based (B) 2.3 × 104 53 1:450 1 Culligan et al. (2014b) Glucose dehydrogenase 1.1.99.10 Hay infusion Phagemid E. coli Function-based (A) 2.0 × 104 13 1:1550 1 Basner and Antranikian (2014) Aldehyde dehydrogenase 1.2.1.3 Hot spring water and soil / / Sequence-based (E) / na / 1 Chen et al. (2014) 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 sediment Plasmid E. coli Function-based (A) 9.0 × 103 1 1:9000 1 Singh et al. (2015b) Subtilisin-like serine protease 3.4.21.62 Underground water Fosmid E. coli Function-based (A) 2.1 × 104 23 1:900 2 Apolinar–Hernández et al. (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) 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 transferase) 2.8.1.1 Soil Plasmid E. coli Function-based (A) 8.5 × 103 5 1:1700 1 Bhat et al. (2015) Target . DNA source . Library type . Heterologous host . Screening approach . Number of screened clones . Number of positive clones . Hit frequency . Number of characterized enzymes . Reference . Activity . EC number . . . . . . . . . . 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 sediment / / Sequence-based (G) / / / 1 Zhao et al. (2017) 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 Ramírez-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 and B) 1.0 × 104 7 1:1450 1 Uchiyama, Yaoi and 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 spring Plasmid E. coli Function-based (A) na na na 1 Schröder et al. (2014) 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/ arabinofuranosidase 3.2.1.37 Bovine rumen Phagemid E. coli Function-based (A) na na na 1 Jordan et al. (2016) α-Xylosidase/ arabinofuranosidase 3.2.1.177 Soil Fosmid E. coli Function-based (A) 5.0 × 104 1 1:50 000 1 Matsuzawa et al. (2016) β-Xylosidase/ arabinofuranosidase 3.2.1.21 Compost Plasmid E. coli Function-based (A) 3.0 × 104 40 1:750 1 Matsuzawa, Kaneko and 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) β-Galactosidase 3.2.1.23 Soil Cosmid E. coli and Sinorhizobium melitoti Function-based (B) 7.9 × 104 na na 3 Cheng et al. (2017) α-Galactosidase 3.2.1.23 Hot spring water and sediment / / Sequence-based (G) / / / 1 Schröder et al. (2017) 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-Acetylhexosaminidase 3.2.1.52 Soil Fosmid E. coli Function-based (A) 1.0 × 105 30 1:3300 2 Nyffenegger et al. (2015) α-Rhamnosidase 3.2.1.40 Feces Fosmid E. coli Function-based (A) 2.0 × 104 na na 1 Rabausch, Ilmberger and Streit (2014) Lichenase (endoglucanase) 3.2.1.73 Soil Plasmid E. coli Function-based (A) 2.0 × 104 1 1:20 000 1 Kim, Oh and Kwon (2014) 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 multifunctional activity 3.2.1.- Ovine rumen Fosmid E. coli Function-based (A) 1.2 × 105 155 1:750 1 Song et al. (2017) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen / / Sequence-based (G) / 2597 / 1 Patel et al. (2016) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen BAC E. coli Function-based (A) na na na 1 Gruninger et al. (2014) Glycosyl hydrolase with multifunctional activity 3.2.1.- Compost Fosmid E. coli Function-based (A) 2.5 × 102 5 1:50 1 Sae-Lee and Boonmee (2014) β-Xylanase, cellulase, α-fucosidase 3.2.1.8, 3.2.1.51 Insect gut Fosmid E. coli Function-based (A) 4.0 × 104 31 1:1300 8 Rashamuse et al. (2017) β-Galactosidase, β-xylosidase, α-glucosidase 3.2.1.23, 3.2.1.37, 3.2.1.20 Wheat straw Fosmid E. coli Function-based (A) 4.4 × 104 71 1:600 7 Maruthamuthu et al. (2016) Endoglucanase, endoxylanase 3.2.1.4, 3.2.1.8 Sugarcane bagasse Fosmid E. coli Function-based (A) 1.0 × 105 7 1:14 300 2 Kanokratana et al. (2015) β-Xylosidase, xylanase, β-glucosidase, cellulase 3.2.1.37, 3.2.1.8, 3.2.1.21 Digester Fosmid E. coli Function-based (A) 9.7 × 103 178 1:54 4 Wang et al. (2015a) Glycosyl hydrolases belonging to various families 3.2.1.- Digester / / Sequence-based (G) / 163 / 6 Wei et al. (2015) β-Glucosidase, endomannanase, endoxylanase, β-xylosidase 3.2.1.21, 3.2.1.25, 3.2.1.8, 3.2.1.37 Subseafloor sediments / / Sequence-based (G) / 60 / 10 Klippel et al. (2014) 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, glycosyltransferases 3.2.1.21 Soil Plasmid E. coli Function-based (A) 5.0 × 105 9 1:55 600 9 Stroobants, Portetelle, Vandenbol (2014) Amylase Amylopullulanase (α-amylase) 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 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 column BAC E. coli Function-based (A) 2.8 × 103 3 1:900 3 Vester, Glaring and 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 function-based (A) 7.8 × 103 1 1:7800 1 Hjort et al. (2014); Berini 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, 3.5.1.41 Soil Fosmid E. coli Sequence-based (D) 1.45 × 105 8 1:18 100 1 Cretoiu et al. (2015) Chitinase 3.2.1.14 Pig feces / / Sequence-based (E) / 1 / 1 Liu et al. (2015a) Chitinase 3.2.1.14, 3.5.1.41 Soil / / Sequence-based(G) / 10 / 1 Stöveken et al. (2015) 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 digester, sunken shipwreck's tar, water, soil etc. Fosmid E. coli Function-based (A) 1.1 × 106 714 1:1500 77a Popovic et al. (2017) 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 sediment Fosmid E. coli Function-based (A) 9.6 × 103 120 1:80 3 Placido et al. (2015) 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) Esterase 3.1.1.1 Hot spring water, sediment and compost Fosmid E. coli and Thermus thermophilus Function-based (A for E. coli, B for T. thermophilus) 8.0 × 103 8 1:2650 2b Leis et al. (2015) 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 Rodríguez 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 sediment Plasmid E. coli Function-based (A) 1.3 × 104 7 1:1900 1 Sahoo et al. (2017) 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) 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 microbiome Plasmid E. coli Function-based (A) 4.0 × 104 20 1:2000 1 Preeti et al. (2014) 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) Esterase/lipase 3.1.1.- Mud flat Plasmid E. coli Function-based (A) 3.0 × 103 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/phospholipase 3.1.1.- Bovine rumen Fosmid E. coli Function-based (A) 2.4 × 104 14 1:1700 5 Privé et al. (2015) 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, sulfotransferase 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) Feruloyl esterase 3.1.1.73 Hindgut symbiont Fosmid E. coli Function-based (A) 4.0 × 104 7 1:5700 6 Rashamuse et al. (2014) Phospholipase (patatin-like) 3.1.1.- Hydrothermal vent Fosmid E. coli Function-based (A) 1.8 × 104 7 1:2600 1 Fu et al. (2015) 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 function-based (A) 1.2 × 102 23 1:5 2 Itoh et al. (2014) Alcohol dehydrogenase 1.1.1.1 Soil and compost Plasmid E. coli Sequence- (D) and function-based (A) 2.0 × 103 40 1:50 2d Itoh, Kariya and Kurokawa (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, dioxygenase 1.14.-, 1.13.11.- Soil Cosmid E. coli and P. putida Function-based (B) 2.2 × 105 29 1:7600 2 Nagayama et al. (2015) β-Carotene monooxygenase 1.14.99.36 Human gut microbiome Fosmid E. coli Function-based (B) 2.3 × 104 53 1:450 1 Culligan et al. (2014b) Glucose dehydrogenase 1.1.99.10 Hay infusion Phagemid E. coli Function-based (A) 2.0 × 104 13 1:1550 1 Basner and Antranikian (2014) Aldehyde dehydrogenase 1.2.1.3 Hot spring water and soil / / Sequence-based (E) / na / 1 Chen et al. (2014) 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 sediment Plasmid E. coli Function-based (A) 9.0 × 103 1 1:9000 1 Singh et al. (2015b) Subtilisin-like serine protease 3.4.21.62 Underground water Fosmid E. coli Function-based (A) 2.1 × 104 23 1:900 2 Apolinar–Hernández et al. (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) 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 transferase) 2.8.1.1 Soil Plasmid E. coli Function-based (A) 8.5 × 103 5 1:1700 1 Bhat et al. (2015) 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. Open in new tab Table 1. Industrially relevant enzymes discovered from metagenomics since 1 January 2014 to 31 March 2017. Target . DNA source . Library type . Heterologous host . Screening approach . Number of screened clones . Number of positive clones . Hit frequency . Number of characterized enzymes . Reference . Activity . EC number . . . . . . . . . . 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 sediment / / Sequence-based (G) / / / 1 Zhao et al. (2017) 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 Ramírez-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 and B) 1.0 × 104 7 1:1450 1 Uchiyama, Yaoi and 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 spring Plasmid E. coli Function-based (A) na na na 1 Schröder et al. (2014) 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/ arabinofuranosidase 3.2.1.37 Bovine rumen Phagemid E. coli Function-based (A) na na na 1 Jordan et al. (2016) α-Xylosidase/ arabinofuranosidase 3.2.1.177 Soil Fosmid E. coli Function-based (A) 5.0 × 104 1 1:50 000 1 Matsuzawa et al. (2016) β-Xylosidase/ arabinofuranosidase 3.2.1.21 Compost Plasmid E. coli Function-based (A) 3.0 × 104 40 1:750 1 Matsuzawa, Kaneko and 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) β-Galactosidase 3.2.1.23 Soil Cosmid E. coli and Sinorhizobium melitoti Function-based (B) 7.9 × 104 na na 3 Cheng et al. (2017) α-Galactosidase 3.2.1.23 Hot spring water and sediment / / Sequence-based (G) / / / 1 Schröder et al. (2017) 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-Acetylhexosaminidase 3.2.1.52 Soil Fosmid E. coli Function-based (A) 1.0 × 105 30 1:3300 2 Nyffenegger et al. (2015) α-Rhamnosidase 3.2.1.40 Feces Fosmid E. coli Function-based (A) 2.0 × 104 na na 1 Rabausch, Ilmberger and Streit (2014) Lichenase (endoglucanase) 3.2.1.73 Soil Plasmid E. coli Function-based (A) 2.0 × 104 1 1:20 000 1 Kim, Oh and Kwon (2014) 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 multifunctional activity 3.2.1.- Ovine rumen Fosmid E. coli Function-based (A) 1.2 × 105 155 1:750 1 Song et al. (2017) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen / / Sequence-based (G) / 2597 / 1 Patel et al. (2016) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen BAC E. coli Function-based (A) na na na 1 Gruninger et al. (2014) Glycosyl hydrolase with multifunctional activity 3.2.1.- Compost Fosmid E. coli Function-based (A) 2.5 × 102 5 1:50 1 Sae-Lee and Boonmee (2014) β-Xylanase, cellulase, α-fucosidase 3.2.1.8, 3.2.1.51 Insect gut Fosmid E. coli Function-based (A) 4.0 × 104 31 1:1300 8 Rashamuse et al. (2017) β-Galactosidase, β-xylosidase, α-glucosidase 3.2.1.23, 3.2.1.37, 3.2.1.20 Wheat straw Fosmid E. coli Function-based (A) 4.4 × 104 71 1:600 7 Maruthamuthu et al. (2016) Endoglucanase, endoxylanase 3.2.1.4, 3.2.1.8 Sugarcane bagasse Fosmid E. coli Function-based (A) 1.0 × 105 7 1:14 300 2 Kanokratana et al. (2015) β-Xylosidase, xylanase, β-glucosidase, cellulase 3.2.1.37, 3.2.1.8, 3.2.1.21 Digester Fosmid E. coli Function-based (A) 9.7 × 103 178 1:54 4 Wang et al. (2015a) Glycosyl hydrolases belonging to various families 3.2.1.- Digester / / Sequence-based (G) / 163 / 6 Wei et al. (2015) β-Glucosidase, endomannanase, endoxylanase, β-xylosidase 3.2.1.21, 3.2.1.25, 3.2.1.8, 3.2.1.37 Subseafloor sediments / / Sequence-based (G) / 60 / 10 Klippel et al. (2014) 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, glycosyltransferases 3.2.1.21 Soil Plasmid E. coli Function-based (A) 5.0 × 105 9 1:55 600 9 Stroobants, Portetelle, Vandenbol (2014) Amylase Amylopullulanase (α-amylase) 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 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 column BAC E. coli Function-based (A) 2.8 × 103 3 1:900 3 Vester, Glaring and 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 function-based (A) 7.8 × 103 1 1:7800 1 Hjort et al. (2014); Berini 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, 3.5.1.41 Soil Fosmid E. coli Sequence-based (D) 1.45 × 105 8 1:18 100 1 Cretoiu et al. (2015) Chitinase 3.2.1.14 Pig feces / / Sequence-based (E) / 1 / 1 Liu et al. (2015a) Chitinase 3.2.1.14, 3.5.1.41 Soil / / Sequence-based(G) / 10 / 1 Stöveken et al. (2015) 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 digester, sunken shipwreck's tar, water, soil etc. Fosmid E. coli Function-based (A) 1.1 × 106 714 1:1500 77a Popovic et al. (2017) 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 sediment Fosmid E. coli Function-based (A) 9.6 × 103 120 1:80 3 Placido et al. (2015) 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) Esterase 3.1.1.1 Hot spring water, sediment and compost Fosmid E. coli and Thermus thermophilus Function-based (A for E. coli, B for T. thermophilus) 8.0 × 103 8 1:2650 2b Leis et al. (2015) 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 Rodríguez 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 sediment Plasmid E. coli Function-based (A) 1.3 × 104 7 1:1900 1 Sahoo et al. (2017) 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) 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 microbiome Plasmid E. coli Function-based (A) 4.0 × 104 20 1:2000 1 Preeti et al. (2014) 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) Esterase/lipase 3.1.1.- Mud flat Plasmid E. coli Function-based (A) 3.0 × 103 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/phospholipase 3.1.1.- Bovine rumen Fosmid E. coli Function-based (A) 2.4 × 104 14 1:1700 5 Privé et al. (2015) 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, sulfotransferase 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) Feruloyl esterase 3.1.1.73 Hindgut symbiont Fosmid E. coli Function-based (A) 4.0 × 104 7 1:5700 6 Rashamuse et al. (2014) Phospholipase (patatin-like) 3.1.1.- Hydrothermal vent Fosmid E. coli Function-based (A) 1.8 × 104 7 1:2600 1 Fu et al. (2015) 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 function-based (A) 1.2 × 102 23 1:5 2 Itoh et al. (2014) Alcohol dehydrogenase 1.1.1.1 Soil and compost Plasmid E. coli Sequence- (D) and function-based (A) 2.0 × 103 40 1:50 2d Itoh, Kariya and Kurokawa (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, dioxygenase 1.14.-, 1.13.11.- Soil Cosmid E. coli and P. putida Function-based (B) 2.2 × 105 29 1:7600 2 Nagayama et al. (2015) β-Carotene monooxygenase 1.14.99.36 Human gut microbiome Fosmid E. coli Function-based (B) 2.3 × 104 53 1:450 1 Culligan et al. (2014b) Glucose dehydrogenase 1.1.99.10 Hay infusion Phagemid E. coli Function-based (A) 2.0 × 104 13 1:1550 1 Basner and Antranikian (2014) Aldehyde dehydrogenase 1.2.1.3 Hot spring water and soil / / Sequence-based (E) / na / 1 Chen et al. (2014) 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 sediment Plasmid E. coli Function-based (A) 9.0 × 103 1 1:9000 1 Singh et al. (2015b) Subtilisin-like serine protease 3.4.21.62 Underground water Fosmid E. coli Function-based (A) 2.1 × 104 23 1:900 2 Apolinar–Hernández et al. (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) 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 transferase) 2.8.1.1 Soil Plasmid E. coli Function-based (A) 8.5 × 103 5 1:1700 1 Bhat et al. (2015) Target . DNA source . Library type . Heterologous host . Screening approach . Number of screened clones . Number of positive clones . Hit frequency . Number of characterized enzymes . Reference . Activity . EC number . . . . . . . . . . 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 sediment / / Sequence-based (G) / / / 1 Zhao et al. (2017) 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 Ramírez-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 and B) 1.0 × 104 7 1:1450 1 Uchiyama, Yaoi and 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 spring Plasmid E. coli Function-based (A) na na na 1 Schröder et al. (2014) 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/ arabinofuranosidase 3.2.1.37 Bovine rumen Phagemid E. coli Function-based (A) na na na 1 Jordan et al. (2016) α-Xylosidase/ arabinofuranosidase 3.2.1.177 Soil Fosmid E. coli Function-based (A) 5.0 × 104 1 1:50 000 1 Matsuzawa et al. (2016) β-Xylosidase/ arabinofuranosidase 3.2.1.21 Compost Plasmid E. coli Function-based (A) 3.0 × 104 40 1:750 1 Matsuzawa, Kaneko and 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) β-Galactosidase 3.2.1.23 Soil Cosmid E. coli and Sinorhizobium melitoti Function-based (B) 7.9 × 104 na na 3 Cheng et al. (2017) α-Galactosidase 3.2.1.23 Hot spring water and sediment / / Sequence-based (G) / / / 1 Schröder et al. (2017) 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-Acetylhexosaminidase 3.2.1.52 Soil Fosmid E. coli Function-based (A) 1.0 × 105 30 1:3300 2 Nyffenegger et al. (2015) α-Rhamnosidase 3.2.1.40 Feces Fosmid E. coli Function-based (A) 2.0 × 104 na na 1 Rabausch, Ilmberger and Streit (2014) Lichenase (endoglucanase) 3.2.1.73 Soil Plasmid E. coli Function-based (A) 2.0 × 104 1 1:20 000 1 Kim, Oh and Kwon (2014) 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 multifunctional activity 3.2.1.- Ovine rumen Fosmid E. coli Function-based (A) 1.2 × 105 155 1:750 1 Song et al. (2017) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen / / Sequence-based (G) / 2597 / 1 Patel et al. (2016) Glycosyl hydrolase with multifunctional activity 3.2.1.- Bovine rumen BAC E. coli Function-based (A) na na na 1 Gruninger et al. (2014) Glycosyl hydrolase with multifunctional activity 3.2.1.- Compost Fosmid E. coli Function-based (A) 2.5 × 102 5 1:50 1 Sae-Lee and Boonmee (2014) β-Xylanase, cellulase, α-fucosidase 3.2.1.8, 3.2.1.51 Insect gut Fosmid E. coli Function-based (A) 4.0 × 104 31 1:1300 8 Rashamuse et al. (2017) β-Galactosidase, β-xylosidase, α-glucosidase 3.2.1.23, 3.2.1.37, 3.2.1.20 Wheat straw Fosmid E. coli Function-based (A) 4.4 × 104 71 1:600 7 Maruthamuthu et al. (2016) Endoglucanase, endoxylanase 3.2.1.4, 3.2.1.8 Sugarcane bagasse Fosmid E. coli Function-based (A) 1.0 × 105 7 1:14 300 2 Kanokratana et al. (2015) β-Xylosidase, xylanase, β-glucosidase, cellulase 3.2.1.37, 3.2.1.8, 3.2.1.21 Digester Fosmid E. coli Function-based (A) 9.7 × 103 178 1:54 4 Wang et al. (2015a) Glycosyl hydrolases belonging to various families 3.2.1.- Digester / / Sequence-based (G) / 163 / 6 Wei et al. (2015) β-Glucosidase, endomannanase, endoxylanase, β-xylosidase 3.2.1.21, 3.2.1.25, 3.2.1.8, 3.2.1.37 Subseafloor sediments / / Sequence-based (G) / 60 / 10 Klippel et al. (2014) 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, glycosyltransferases 3.2.1.21 Soil Plasmid E. coli Function-based (A) 5.0 × 105 9 1:55 600 9 Stroobants, Portetelle, Vandenbol (2014) Amylase Amylopullulanase (α-amylase) 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 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 column BAC E. coli Function-based (A) 2.8 × 103 3 1:900 3 Vester, Glaring and 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 function-based (A) 7.8 × 103 1 1:7800 1 Hjort et al. (2014); Berini 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, 3.5.1.41 Soil Fosmid E. coli Sequence-based (D) 1.45 × 105 8 1:18 100 1 Cretoiu et al. (2015) Chitinase 3.2.1.14 Pig feces / / Sequence-based (E) / 1 / 1 Liu et al. (2015a) Chitinase 3.2.1.14, 3.5.1.41 Soil / / Sequence-based(G) / 10 / 1 Stöveken et al. (2015) 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 digester, sunken shipwreck's tar, water, soil etc. Fosmid E. coli Function-based (A) 1.1 × 106 714 1:1500 77a Popovic et al. (2017) 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 sediment Fosmid E. coli Function-based (A) 9.6 × 103 120 1:80 3 Placido et al. (2015) 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) Esterase 3.1.1.1 Hot spring water, sediment and compost Fosmid E. coli and Thermus thermophilus Function-based (A for E. coli, B for T. thermophilus) 8.0 × 103 8 1:2650 2b Leis et al. (2015) 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 Rodríguez 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 sediment Plasmid E. coli Function-based (A) 1.3 × 104 7 1:1900 1 Sahoo et al. (2017) 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) 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 microbiome Plasmid E. coli Function-based (A) 4.0 × 104 20 1:2000 1 Preeti et al. (2014) 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) Esterase/lipase 3.1.1.- Mud flat Plasmid E. coli Function-based (A) 3.0 × 103 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/phospholipase 3.1.1.- Bovine rumen Fosmid E. coli Function-based (A) 2.4 × 104 14 1:1700 5 Privé et al. (2015) 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, sulfotransferase 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) Feruloyl esterase 3.1.1.73 Hindgut symbiont Fosmid E. coli Function-based (A) 4.0 × 104 7 1:5700 6 Rashamuse et al. (2014) Phospholipase (patatin-like) 3.1.1.- Hydrothermal vent Fosmid E. coli Function-based (A) 1.8 × 104 7 1:2600 1 Fu et al. (2015) 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 function-based (A) 1.2 × 102 23 1:5 2 Itoh et al. (2014) Alcohol dehydrogenase 1.1.1.1 Soil and compost Plasmid E. coli Sequence- (D) and function-based (A) 2.0 × 103 40 1:50 2d Itoh, Kariya and Kurokawa (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, dioxygenase 1.14.-, 1.13.11.- Soil Cosmid E. coli and P. putida Function-based (B) 2.2 × 105 29 1:7600 2 Nagayama et al. (2015) β-Carotene monooxygenase 1.14.99.36 Human gut microbiome Fosmid E. coli Function-based (B) 2.3 × 104 53 1:450 1 Culligan et al. (2014b) Glucose dehydrogenase 1.1.99.10 Hay infusion Phagemid E. coli Function-based (A) 2.0 × 104 13 1:1550 1 Basner and Antranikian (2014) Aldehyde dehydrogenase 1.2.1.3 Hot spring water and soil / / Sequence-based (E) / na / 1 Chen et al. (2014) 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 sediment Plasmid E. coli Function-based (A) 9.0 × 103 1 1:9000 1 Singh et al. (2015b) Subtilisin-like serine protease 3.4.21.62 Underground water Fosmid E. coli Function-based (A) 2.1 × 104 23 1:900 2 Apolinar–Hernández et al. (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) 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 transferase) 2.8.1.1 Soil Plasmid E. coli Function-based (A) 8.5 × 103 5 1:1700 1 Bhat et al. (2015) 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. Open in new tab HOW TO MINE eDNA IN THE SEARCH FOR NOVEL ENZYMES The term ‘metagenomics’ is defined as the analysis of the genetic complement of an entire habitat by direct extraction and subsequent cloning of DNA from an assemblage of microorganisms (Handelsman et al.1998). Figure 1 summarizes the general workflow of metagenomics in discovering novel enzymes, outlining the multiplicity of possible screening paths from eDNA extraction to heterologous expression of selected protein sequences. 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, although still posing some technical challenges, might be considered 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 assembled from widely diverse ecological sources (Fig. 2A). Since a low hit rate of positive clones in screening still represents a persisting 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 phytopathogens (Hjort et al.2014), whereas two esterases were discovered 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 targeted metagenomics), by which microbial communities are artificially manipulated prior to DNA extraction in order to increase 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, increasing 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 oxidoreductases (Nagayama et al.2015) from cellulose-amended samples or artificially polluted soils. A hormone-sensitive lipase was recovered from an olive oil-fed permafrost-derived microbiome (Petrovskaya et al.2016). Incorporating stable-isotope probing is another tool for innovating target library construction: for example, soil sample incubation with stable-isotope-labeled carbohydrates enriched the eDNA of those bacteria actively engaged in degrading plant-derived biomasses (Verastegui et al.2014). Figure 1. Open in new tabDownload slide 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. Figure 1. Open in new tabDownload slide 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. Figure 2. Open in new tabDownload slide 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. Figure 2. Open in new tabDownload slide 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. Analysis of Table 1 focusing on how metagenomic libraries are preferentially built up indicates that the majority of the recently isolated metagenome-sourced enzymes (194) were discovered 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, fosmids or bacterial artificial chromosomes) were recently preferred 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 example, Cretoiu et al. (2015), Maruthamuthu et al. (2016) and Matsuzawa and Yaoi (2017) reported gene annotation of fosmid inserts containing the targeted open reading frames (ORFs) when searching for novel chitinases and hemicellulases (Table 1). Using large-insert libraries also permitted the discovery of complex gene clusters encoding for natural products, 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 readers 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 nucleotide sequence analysis (also indicated as genetic screening) (Table 1, Figs 1 and 2C). Actually, functional screening is considered the best strategy for identifying genes encoding functional products and for potentially discovering completely new classes of enzymes that lack homologies to known sequences (Culligan et al.2014a; Coughlan et al.2015; Batista-García 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 discovered by functional metagenomics. Our analysis—focusing on enzymes that had been (at least partially) characterized at biochemical/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 metagenomics, 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 constructing 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 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). Random insertion of bidirectional promoters into the library (Kim et al.2016b) and addition of heterologous σ-factors for increasing transcription proficiency (Gaida et al.2015) were recently experimented 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 strategies 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 fosmid library propagated in E. coli and in Thermus thermophilus (Leis et al.2015), two dioxygenases from a cosmid metagenomic library in Pseudomonas putida (Nagayama et al.2015) and a β-galactosidase from a library in Sinorhizobiummeliloti (Cheng et al.2017). Interestingly, Bouhajja et al. (2017) identified novel toluene monooxygenase genes by cloning eDNA in three parallel 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 and Janssen 2004; Ekkers et al.2012). Classical phenotypical detection (A in Table 1, Fig. 1), applied for decades in screening microbial isolates and based on identifying specific phenotypic traits associated with the activity of interest, remains the election method in metagenomic, too (260 enzymes). Phenotypical detection on agar plates is still the most widely used approach, especially when searching for hydrolytic enzymes (such as lipases) forming easy-to-detect degradation haloes (Placido et al.2015; Popovic et al.2017) on appropriate substrates. Alternative methods were based on high-throughput assays in liquid culture (Zottig, Meddeb-Mouelhi and Beauregard 2016) or high-performance thin-layer chromatography (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 multiple chromogenic substrates mimicking insoluble plant cell wall components (Maruthamuthu et al.2016). To minimize redundancy (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 functional 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, and hits are selected based on the emitted fluorescence or absorbance (Mair, Gielen and Hollfelder 2017). This method allowed screening over a million clones per day, recovering both slow and fast biocatalysts, promiscuous enzymes or those encoded 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 complementation (B in Table 1, Fig. 1) relies on the selection of clones that have acquired the capability to grow under selective conditions, 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 penicillin G acylase by selecting E. coli clones growing in the presence 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) expression 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 using 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 catalytic 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 environmental 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 chitinase (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 library (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 significant reduction in sequencing costs, promoted the evolution of another genetic screening approach that is based on the bioinformatic analysis of sequenced DNA (in silico screening). Among the enzymes listed in Table 1, eight were discovered by in silico 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 isolated 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 consensus 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, oxidoreductases, 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 biocatalyst belonging to an uncharacterized enzymatic family to be retrieved by sequence-based screening. INDUSTRIALLY RELEVANT ENZYMES FROM METAGENOMICS The most common targets in metagenomic investigations for industrial enzymes include glycosyl hydrolases (GHs), lipolytic 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, Rodríguez-Belmonte and González-Siso 2016). Table 2 reports examples of such classes 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 biocatalysts reported in Table 1 belong to previously uncharacterized enzyme families or subfamilies, with unconventional structures and active site architectures pointing to novel catalytic mechanisms. Table 2. Examples of metagenome-sourced enzymes patented in the last decade and their suggested applications. Enzyme . Source . Patent number . Patent 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 rumen WO2014142529 A1 2013 Biofuel production from lignocellulosic biomasses; fiber, detergent, feed, food, pulp, paper production β-Galactosidase na EP2530148 A1 2011 Food processing (lactose depletion) Xylose isomerase Termite intestine US8772012 B2 2009 Biofuel production from lignocellulosic biomasses Amylase α-Amylase Animal manure CN103290039 B 2013 Animal feed, starch processing 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 sediment EP2784160 A1 2011 Oil and fat purification and 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-associated microbiome CN102971426 B 2011 Seaweed processing DNA polymerase Water WO2012173905 A1 2011 DNA polymerization in high salt conditions l-Methionine γ-lyase Deep sea sediment CN101962651 B 2010 Clinical detection, food flavor production, cancer therapy Muramidase (Lysozyme) Soil CN101892252 B 2010 Antibiosis, bacteriolysis Enzyme . Source . Patent number . Patent 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 rumen WO2014142529 A1 2013 Biofuel production from lignocellulosic biomasses; fiber, detergent, feed, food, pulp, paper production β-Galactosidase na EP2530148 A1 2011 Food processing (lactose depletion) Xylose isomerase Termite intestine US8772012 B2 2009 Biofuel production from lignocellulosic biomasses Amylase α-Amylase Animal manure CN103290039 B 2013 Animal feed, starch processing 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 sediment EP2784160 A1 2011 Oil and fat purification and 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-associated microbiome CN102971426 B 2011 Seaweed processing DNA polymerase Water WO2012173905 A1 2011 DNA polymerization in high salt conditions l-Methionine γ-lyase Deep sea sediment CN101962651 B 2010 Clinical detection, food flavor production, cancer therapy Muramidase (Lysozyme) Soil CN101892252 B 2010 Antibiosis, bacteriolysis na = data not available Open in new tab Table 2. Examples of metagenome-sourced enzymes patented in the last decade and their suggested applications. Enzyme . Source . Patent number . Patent 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 rumen WO2014142529 A1 2013 Biofuel production from lignocellulosic biomasses; fiber, detergent, feed, food, pulp, paper production β-Galactosidase na EP2530148 A1 2011 Food processing (lactose depletion) Xylose isomerase Termite intestine US8772012 B2 2009 Biofuel production from lignocellulosic biomasses Amylase α-Amylase Animal manure CN103290039 B 2013 Animal feed, starch processing 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 sediment EP2784160 A1 2011 Oil and fat purification and 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-associated microbiome CN102971426 B 2011 Seaweed processing DNA polymerase Water WO2012173905 A1 2011 DNA polymerization in high salt conditions l-Methionine γ-lyase Deep sea sediment CN101962651 B 2010 Clinical detection, food flavor production, cancer therapy Muramidase (Lysozyme) Soil CN101892252 B 2010 Antibiosis, bacteriolysis Enzyme . Source . Patent number . Patent 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 rumen WO2014142529 A1 2013 Biofuel production from lignocellulosic biomasses; fiber, detergent, feed, food, pulp, paper production β-Galactosidase na EP2530148 A1 2011 Food processing (lactose depletion) Xylose isomerase Termite intestine US8772012 B2 2009 Biofuel production from lignocellulosic biomasses Amylase α-Amylase Animal manure CN103290039 B 2013 Animal feed, starch processing 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 sediment EP2784160 A1 2011 Oil and fat purification and 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-associated microbiome CN102971426 B 2011 Seaweed processing DNA polymerase Water WO2012173905 A1 2011 DNA polymerization in high salt conditions l-Methionine γ-lyase Deep sea sediment CN101962651 B 2010 Clinical detection, food flavor production, cancer therapy Muramidase (Lysozyme) Soil CN101892252 B 2010 Antibiosis, bacteriolysis na = data not available Open in new tab 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, Pyrolase 200 Cellulase Secondary oil and gas recovery Phyzyme XP Phytase Additive for livestock feed Commercial name . Enzyme class . Applications . Luminase Xylanase Pulp biobleaching in paper production Fuelzyme α-Amylase Fuels and industrial-use alcohols production Pyrolase 160, Pyrolase 200 Cellulase Secondary oil and gas recovery Phyzyme XP Phytase Additive for livestock feed Open in new tab 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, Pyrolase 200 Cellulase Secondary oil and gas recovery Phyzyme XP Phytase Additive for livestock feed Commercial name . Enzyme class . Applications . Luminase Xylanase Pulp biobleaching in paper production Fuelzyme α-Amylase Fuels and industrial-use alcohols production Pyrolase 160, Pyrolase 200 Cellulase Secondary oil and gas recovery Phyzyme XP Phytase Additive for livestock feed Open in new tab Glycosyl hydrolases The classes of metagenome-sourced enzymes mostly represented in our 3-year analysis are (hemi)cellulases and esterases/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 saccharification and biofuel production are reported in the work of Duan and Feng (2010), Montella, Amore and Faraco (2016), Batista-García 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 useful for plant biomass saccharification: for example, an endoglucanase possessing also xylanase activity was identified by functional 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 activities. Recent studies disclosed interesting uncommon halotolerant (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 endoglucanases, 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 degradation 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 oxidants (Biver et al.2014) or towards high sugar concentrations (Gomes-Pepe et al.2016), and a β-galactosidase useful for the industrial 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 (Ramírez-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. 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 production were discovered by functional screening of metagenomic 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 solvents 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 nutritional potential (Cretoiu et al.2015). In 2014, we identified a chitobiosidase by combining genetic and functional screenings applied to a suppressive soil metagenome, now under development as a potential biocontrol agent thanks to its antifungal 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 treatment and valorization of seafood wastes (Cretoiu et al.2015). An interesting enzyme for its potential application in producing chitosan from chitin is the chitin deacetylase that has been identified in Arctic Ocean deep-sea sediments (Liu et al.2016). 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 therapeutics, as well as biodiesel production and biopolymer synthesis. Particular attention was directed to extreme environments, as reviewed in López-López, Cerdán and González Siso (2014). Popovic et al. (2017) recently made an impressive contribution 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 motif (Petrovskaya et al.2016). Enzymes belonging to novel families of esterases were recovered from cow rumen (Rodríguez et al.2015) and hot spring mud (Zarafeta et al.2016). Additionally, 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 hydrocarbons (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) identified two novel lipases from marine sponge-associated microbiome 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 enantioselective 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 industries and in bioremediation. Recently discovered oxidoreductases (Table 1, Fig. 2D) include five soil-sourced dioxygenases 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-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 proteases, one of the most widely used industrial classes of enzymes 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 underground 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 phosphatases 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 exceedingly 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 heterologous 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 synthesis 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 unconventional E. coli Arctic Express RIL cells favored the soluble production 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 described. CONCLUSIONS Almost unheard of only 10/15 years ago in enzyme biotechnology, 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 metagenomic libraries is becoming a common shared knowledge in the scientific community aiming at developing novel biocatalysts 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 directed 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 acknowledged. 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 Internazionale) 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. REFERENCES Alcaide M , Tchigvintsev A, Martínez-Martínez Met al. . Identification and characterization of carboxyl esterases of grill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics . Appl Environ Microb 2015 ; 81 : 2125 – 36 . Google Scholar Crossref Search ADS WorldCat Alnoch RC , Martini VP, Glogauer Aet al. . Immobilization and characterization of a new regioselective and enantioselective lipase obtained from a metagenomic library . PLoS One 2015 ; 10 : e0114945 . 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TI - Metagenomics: novel enzymes from non-culturable microbes
JF - FEMS Microbiology Letters
DO - 10.1093/femsle/fnx211
DA - 2017-11-15
UR - https://www.deepdyve.com/lp/oxford-university-press/metagenomics-novel-enzymes-from-non-culturable-microbes-CvsR4VjNB3
VL - 364
IS - 21
DP - DeepDyve
ER -