Functionalization and Modification of Hydrocarbon-Like Molecules Guided by Metagenomics: Enzymes Most Requested at the Industrial Scale for Chemical Synthesis as Study Cases

  • Mónica Martínez-Martínez
  • Rafael Bargiela
  • Cristina Coscolín
  • José Navarro-Fernández
  • Peter N. Golyshin
  • Manuel Ferrer
Reference work entry
Part of the Handbook of Hydrocarbon and Lipid Microbiology book series (HHLM)


The end of the twentieth century has experienced a revolution in the life sciences and, specifically, in enzymology. In this sense, the relation between chemistry and biology is currently under a deep transformation influenced by the implementation of OMICS tools. We can now access uncultured bacteria, whose genomic material can further be a resource of enzymes for novel enzymology. Among enzymes of interest are esterases and lipases from the α/β-hydrolase fold superfamily, transaminases, and oxidoreductases such as aldo-keto reductases. These enzymes are the first-choice items from the toolbox for functionalization and modification of low-reactive hydrocarbon-like blocks, oils, and fats. Through a series of hydrolytic or synthetic reactions, they can be used for the economic and sustainable production of highly valuable customized and functionalized hydrocarbon-based materials. Through metagenomic studies, about 250 novel esterases and lipases, one β-transaminase, and about 60 alcohol dehydrogenases and aldo-keto reductases have been discovered and their properties described. Research on esterases, lipases, β-transaminases, and oxidoreductases from uncultivated bacteria has revealed unprecedented transformations. The study of their potential in the modification of about 200 different hydrocarbon-like molecules is analyzed herein. This chapter also highlights the catalog of the representative molecules whose functionalization and modification has been successfully achieved using esterases, lipases, β-transaminases, alcohol dehydrogenases, and aldo-keto reductases from uncultivated bacteria discovered through metagenomic approaches. Notably, these groups of enzymes are, at the industrial scale, the most desired means and tools for chemical synthesis.



This project has received funding from the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement No [634486]. This work was further funded by the European Community project KILL-SPILL (FP7-KBBE-2012-312139) and grants PCIN-2014-107 and BIO2014-54494-R from the Spanish Ministry of Economy, Industry and Competitiveness. The present investigation was funded by the Spanish Ministry of Economy, Industry and Competitiveness, the UK Biotechnology and Biological Sciences Research Council (BBSRC) within the ERA NET-IB2 program, grant number ERA-IB-14-030. The authors gratefully acknowledge the financial support provided by the European Regional Development Fund (ERDF). C. Coscolín thanks the Spanish Ministry of Economy and Competitiveness for a PhD fellowship (Grant BES-2015-073829).


  1. Alcaide M, Tornés J, Stogios Peter J, Xu X, Gertler C, Di Leo R, Bargiela R, Lafraya Á, Guazzaroni M-E, López-Cortés N, Chernikova TN, Golyshina OV, Nechitaylo TY, Plumeier I, Pieper DH, Yakimov MM, Savchenko A, Golyshin PN, Ferrer M (2013) Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/β hydrolase family. Biochem J 454:157–166CrossRefPubMedGoogle Scholar
  2. Alcaide M, Stogios PJ, Lafraya A, Tchigvintsev A, Flick R, Bargiela R, Chernikova TN, Reva ON, Hai T, Leggewie CC, Katzke N, La Cono V, Matesanz R, Jebbar M, Jaeger KE, Yakimov MM, Yakunin AF, Golyshin PN, Golyshina OV, Savchenko A, Ferrer M (2015a) Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats. Environ Microbiol 17:332–345CrossRefPubMedGoogle Scholar
  3. Alcaide M, Tchigvintsev A, Martinez-Martinez M, Popovic A, Reva ON, Lafraya A, Bargiela R, Nechitaylo TY, Matesanz R, Cambon-Bonavita MA, Jebbar M, Yakimov MM, Savchenko A, Golyshina OV, Yakunin AF, Golyshin PN, Ferrer M (2015b) Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics. Appl Environ Microbiol 81:2125–2136CrossRefPubMedPubMedCentralGoogle Scholar
  4. Baud D, Ladkau N, Moody TS, Ward JM, Hailes HC (2015) A rapid, sensitive colorimetric assay for the high-throughput screening of transaminases in liquid or solid-phase. Chem Commun (Camb) 51:17225–17228CrossRefGoogle Scholar
  5. Bayer S, Birkemeyer C, Ballschmiter M (2011) A nitrilase from a metagenomic library acts regioselectively on aliphatic dinitriles. Appl Microbiol Biotechnol 89:91–98CrossRefPubMedGoogle Scholar
  6. Beloqui A, de María PD, Golyshin PN, Ferrer M (2008) Recent trends in industrial microbiology. Curr Opin Microbiol 11:240–248CrossRefPubMedGoogle Scholar
  7. Bommarius AS (2015) Biocatalysis: a status report. Annu Rev Chem Biomol Eng 6:319–345CrossRefPubMedGoogle Scholar
  8. Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Stürmer R, Zelinski T (2004) Industrial methods for the production of optically active intermediates. Angew Chem Int Ed Engl 43:788–824CrossRefPubMedGoogle Scholar
  9. Chandrasekharaiah M, Thulasi A, Vijayarani K, Kumar DP, Santosh SS, Palanivel C, Jose VL, Sampath KT (2012) Expression and biochemical characterization of two novel feruloyl esterases derived from fecal samples of Rusa unicolor and Equus burchelli. Gene 500:134–139CrossRefPubMedGoogle Scholar
  10. Chistoserdova L (2014) Is metagenomics resolving identification of functions in microbial communities? Microb Biotechnol 7:1–4CrossRefPubMedGoogle Scholar
  11. Chow J, Kovacic F, Dall Antonia Y, Krauss U, Fersini F, Schmeisser C, Lauinger B, Bongen P, Pietruszka J, Schmidt M, Menyes I, Bornscheuer UT, Eckstein M, Thum O, Liese A, Mueller-Dieckmann J, Jaeger KE, Streit WR (2012) The Metagenome-derived enzymes LipS and LipT increase the diversity of known lipases. PLoS One 7:E47665CrossRefPubMedPubMedCentralGoogle Scholar
  12. Elend C, Schmeisser C, Leggewie C, Babiak P, Carballeira JD, Steele HL, Reymond JL, Jaeger KE, Streit WR (2006) Isolation and biochemical characterization of two novel metagenome-derived esterases. Appl Environ Microbiol 72:3637–3645CrossRefPubMedPubMedCentralGoogle Scholar
  13. Elend C, Schmeisser C, Hoebenreich H, Steele HL, Streit WR (2007) Isolation and characterization of a metagenome-derived and cold-active lipase with high stereospecificity for (R)-ibuprofen esters. J Biotechnol 130:370–377CrossRefPubMedGoogle Scholar
  14. Fernández-Álvaro E, Kourist R, Winter J, Böttcher D, Liebeton K, Naumer C, Eck J, Leggewie C, Jaeger KE, Streit W, Bornscheuer UT (2010) Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome-derived esterases. Microb Biotechnol 3:59–64CrossRefPubMedGoogle Scholar
  15. Fernández-Arrojo L, Guazzaroni ME, López-Cortés N, Beloqui A, Ferrer M (2010) Metagenomic era for biocatalyst identification. Curr Opin Biotechnol 21:725–733CrossRefPubMedGoogle Scholar
  16. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Martins dos Santos VAP, Yakimov MM, Timmis KN, Golyshin PN (2005a) Microbial enzymes mined from the Urania deep-sea hypersaline anoxic basin. Chem Biol 12:895–904CrossRefPubMedGoogle Scholar
  17. Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov MM, Timmis KN, Golyshin PN (2005b) Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Environ Microbiol 7:1996–2010CrossRefPubMedGoogle Scholar
  18. Ferrer M, Bargiela R, Martínez-Martínez M, Mir J, Koch R, Golyshina OV, Golyshin PN (2015) Biodiversity for biocatalysis: a review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes. Biocatal Biotransformation 33:235–249CrossRefGoogle Scholar
  19. Ferrer M, Martinez-Martinez M, Bargiela R, Streit WR, Golyshina OV, Golyshin PN (2016) Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends. Microb Biotechnol 9:22–34CrossRefPubMedGoogle Scholar
  20. Gao W, Wu K, Chen L, Fan H, Zhao Z, Gao B, Wang H, Wei D (2016) A novel esterase from a marine mud metagenomic library for biocatalytic synthesis of short-chain flavor esters. Microb Cell Factories 15:41CrossRefGoogle Scholar
  21. Gong JS, Lu ZM, Li H, Zhou ZM, Shi JS, Xu ZH (2013) Metagenomic technology and genome mining: emerging areas for exploring novel nitrilases. Appl Microbiol Biotechnol 97:6603–6611CrossRefPubMedGoogle Scholar
  22. Hajighasemi M, Nocek BP, Tchigvintsev A, Brown G, Flick R, Xu X, Cui H, Hai T, Joachimiak A, Golyshin PN, Savchenko A, Edwards EA, Yakunin AF (2016) Biochemical and structural insights into enzymatic depolymerization of polylactic acid and other polyesters by microbial carboxylesterases. Biomacromolecules 17:2027–2039CrossRefPubMedGoogle Scholar
  23. Hardeman F, Sjoling S (2007) Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol 59:524–534CrossRefPubMedGoogle Scholar
  24. Itoh N, Isotani K, Makino Y, Kato M, Kitayama K, Ishimota T (2014) PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis. Enzym Microb Technol 55:140–150CrossRefGoogle Scholar
  25. Jeon JH, Kim JT, Kang SG, Lee JH, Kim SJ (2009) Characterization and its potential application of two esterases derived from the arctic sediment metagenome. Mar Biotechnol 11:307–316CrossRefPubMedGoogle Scholar
  26. Jiang Z, Liu J (2010) Lipase-catalyzed copolymerization of ω-pentadecalactone (pdl) and alkyl glycolate: synthesis of poly(PDL-co-GA). ACS Symp Ser 1043:213–225CrossRefGoogle Scholar
  27. Jiao Y, Chen X, Wang X, Liao X, Xiao L, Miao A, Wu J, Yang L (2013) Identification and characterization of a cold-active phthalate esters hydrolase by screening a metagenomic library derived from biofilms of a wastewater treatment plant. PLoS One 8:E75977CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kim YJ, Choi GS, Kim SB, Yoon GS, Kim YS, Ryu YW (2006) Screening and characterization of a novel esterase from a metagenomic library. Protein Expr Purif 45:315–323CrossRefPubMedGoogle Scholar
  29. Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R (2003a) Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli. Appl Environ Microbiol 69:1408–1416CrossRefPubMedPubMedCentralGoogle Scholar
  30. Knietsch A, Waschkowitz T, Bowien S, Henne A, Daniel R (2003b) Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli. J Mol Microbiol Biotechnol 5:46–56CrossRefPubMedGoogle Scholar
  31. Kourist R, Hari Krishna S, Patel JS, Bartnek F, Hitchman TS, Weiner DP, Bornscheuer UT (2007) Identification of a metagenome-derived esterase with high enantioselectivity in the kinetic resolution of arylaliphatic tertiary alcohols. Org Biomol Chem 5:3310–3313CrossRefPubMedGoogle Scholar
  32. Kyrpides NC, Hugenholtz P, Eisen JA, Woyke T, Göker M, Parker CT, Amann R, Beck BJ, Chain PS, Chun J, Colwell RR, Danchin A, Dawyndt P, Dedeurwaerdere T, DeLong EF, Detter JC, De Vos P, Donohue TJ, Dong XZ, Ehrlich DS, Fraser C, Gibbs R, Gilbert J, Gilna P, Glöckner FO, Jansson JK, Keasling JD, Knight R, Labeda D, Lapidus A, Lee JS, Li WJ, Ma J, Markowitz V, Moore ER, Morrison M, Meyer F, Nelson KE, Ohkuma M, Ouzounis CA, Pace N, Parkhill J, Qin N, Rossello-Mora R, Sikorski J, Smith D, Sogin M, Stevens R, Stingl U, Suzuki K, Taylor D, Tiedje JM, Tindall B, Wagner M, Weinstock G, Weissenbach J, White O, Wang J, Zhang L, Zhou YG, Field D, Whitman WB, Garrity GM, Klenk HP (2014) Genomic encyclopedia of bacteria and archaea: sequencing a myriad of type strains. PLoS Biol 12:e1001920CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lee MH, Lee CH, Oh TK, Song JK, Yoon JH (2006) Isolation and characterization of a novel lipase from a metagenomic library of tidal flat sediments: evidence for a new family of bacterial lipases. Appl Environ Microbiol 72:7406–7409CrossRefPubMedPubMedCentralGoogle Scholar
  34. Li G, Wang K, Liu YH (2008) Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the metagenome. Microb Cell Factories 7:388CrossRefGoogle Scholar
  35. Martínez-Martínez M, Alcaide M, Tchigvintsev A, Reva O, Polaina J, Bargiela R, Guazzaroni M-E, Chicote Á, Canet A, Valero F, Rico Eguizabal E, Guerrero Mdel C, Yakunin AF, Ferrer M (2013) Biochemical diversity of carboxyl esterases and lipases from Lake Arreo (Spain): a metagenomic approach. Appl Environ Microbiol 79:3553–3562CrossRefPubMedPubMedCentralGoogle Scholar
  36. Martínez-Martínez M, Lores I, Peña-García C, Bargiela R, Reyes-Duarte D, Guazzaroni ME, Peláez AI, Sánchez J, Ferrer M (2014) Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters. Microb Biotechnol 7:184–191CrossRefPubMedPubMedCentralGoogle Scholar
  37. Martini V, Glogauer A, Muller-Santos M, Lulek J, de Souza E, Mitchell D, Pedrosa F, Krieger N (2014) First co-expression of a lipase and its specific foldase obtained by metagenomics. Microb Cell Factories 13:171CrossRefGoogle Scholar
  38. Mayumi D, Akutsu-Shigeno Y, Uchiyama H, Nomura N, Nakajima-Kambe T (2008) Identification and characterization of novel poly(DL: -lactic acid) depolymerases from metagenome. Appl Microbiol Biotechnol 79:743–750CrossRefPubMedGoogle Scholar
  39. Meilleur C, Hupé JF, Juteau P, Shareck F (2009) Isolation and characterization of a new alkali-thermostable lipase cloned from a metagenomic library. J Ind Microbiol Biotechnol 36:853–861CrossRefPubMedGoogle Scholar
  40. Mende DR, Waller AS, Sunagawa S, Järvelin AI, Chan MM, Arumugam M, Raes J, Bork P (2012) Assessment of metagenomic assembly using simulated next generation sequencing data. PLoS One 7:e31386CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nagarajan S (2012) New tools for exploring “old friends-microbial lipases”. Appl Biochem Biotechnol 168:1163–1196CrossRefPubMedGoogle Scholar
  42. Ngo TD, Ryu BH, Ju H, Jang EJ, Kim KK, Kim TD (2014) Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/beta-lactamase homologue from a metagenomic library. Acta Crystallogr D Biol Crystallogr 70:2455–2466CrossRefPubMedGoogle Scholar
  43. Niehaus F, Gabor E, Wieland S, Siegert P, Maurer KH, Eck J (2011) Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases. Microb Biotechnol 4:767–776CrossRefPubMedPubMedCentralGoogle Scholar
  44. Ouyang LM, Liu JY, Qiao M, Xu JH (2013) Isolation and biochemical characterization of two novel metagenome-derived esterases. Appl Biochem Biotechnol 169:15–28CrossRefPubMedGoogle Scholar
  45. Peña-García C, Martínez-Martínez M, Reyes-Duarte D, Ferrer M (2016) High throughput screening of esterases, lipases and phospholipases in mutant and metagenomic libraries: a review. Comb Chem High Throughput Screen 19:605–615CrossRefPubMedGoogle Scholar
  46. Penning TM (2015) The aldo-keto reductases (AKRs): overview. Chem Biol Interact 234:236–246CrossRefPubMedGoogle Scholar
  47. Perret A, Lechaplais C, Tricot S, Perchat N, Vergne C, Pelle C, Bastard K, Kreimeyer A, Vallenet D, Zaparucha A, Weissenbach J, Salanoubat M (2011) A novel acyl-CoA beta-transaminase characterized from a metagenome. PLoS One 6:e22918CrossRefPubMedPubMedCentralGoogle Scholar
  48. Placido A, Hai T, Ferrer M, Chernikova TN, Distaso M, Armstrong D, Yakunin AF, Toshchakov SV, Yakimov MM, Kublanov IV, Golyshina OV, Pesole G, Ceci LR, Golyshin PN (2015) Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase. Appl Microbiol Biotechnol 99:10031–10046CrossRefPubMedPubMedCentralGoogle Scholar
  49. Plou FJ, Cruces MA, Ferrer M, Fuentes G, Pastor E, Bernabé M, Christensen M, Comelles F, Parra JL, Ballesteros A (2002) Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent. J Biotechnol 96:55–66CrossRefPubMedGoogle Scholar
  50. Singh BK (2010) Exploring microbial diversity for biotechnology: the way forward. Trends Biotechnol 28:111–116CrossRefPubMedGoogle Scholar
  51. Sulaiman S, Yamato S, Kanaya E, Kim JJ, Koga Y, Takano K, Kanaya S (2012) Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach. Appl Environ Microbiol 78:1556–1562CrossRefPubMedPubMedCentralGoogle Scholar
  52. Tao W, Lee MH, Yoon MY, Kim JC, Malhotra S, Wu J, Hwang EC, Lee SW (2011) Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase. J Microbiol Biotechnol 21:1203–1210CrossRefPubMedGoogle Scholar
  53. Tchigvintsev A, Tran H, Popovic A, Kovacic F, Brown G, Flick R, Hajighasemi M, Egorova O, Somody JC, Tchigvintsev D, Khusnutdinova A, Chernikova TN, Golyshina OV, Yakimov MM, Savchenko A, Golyshin PN, Jaeger KE, Yakunin AF (2015) The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes. Appl Microbiol Biotechnol 99:2165–2178CrossRefPubMedGoogle Scholar
  54. Tirawongsaroj P, Sriprang R, Harnpicharnchai P, Thongaram T, Champreda V, Tanapongpipat S, Pootanakit K, Eurwilaichitr L (2008) Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library. J Biotechnol 133:42–49CrossRefPubMedGoogle Scholar
  55. Turner NJ, Truppo MD (2013) Biocatalysis enters a new era. Curr Opin Chem Biol 17:212–214CrossRefPubMedGoogle Scholar
  56. Vergne-Vaxelaire C, Bordier F, Fossey A, Besnard-Gonnet M, Debard A, Mariage A, Pellouin V, Perret A, Petit J-L, Stam M, Salanoubat M, Wissenbach J, De Berardinis V, Zaparucha A (2013) Nitrilase activity screening on structurally diverse substrates: providing biocatalytic tools for organic synthesis. Adv Synth Catal 355:1763–1777CrossRefGoogle Scholar
  57. Wexler M, Bond PL, Richardson DJ, Johnston AW (2005) A wide host-range metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase. Environ Microbiol 7:1917–1926CrossRefPubMedGoogle Scholar
  58. Wilson MC, Piel J (2013) Metagenomic approaches for exploiting uncultivated bacteria as a resource for novel biosynthetic enzymology. Chem Biol 20:636–647CrossRefPubMedGoogle Scholar
  59. Wong DW, Chan VJ, Liao H, Zidwick MJ (2013) Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases. J Ind Microbiol Biotechnol 40:287–295CrossRefPubMedGoogle Scholar
  60. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W, Schleifer KH, Whitman WB, Euzéby J, Amann R, Rosselló-Móra R (2014) Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 12:635–645CrossRefPubMedGoogle Scholar
  61. Yhaya F, Sutinah A, Gregory AM, Liang M, Astenzel MH (2012) RAFT polymerization of vinyl methacrylate and subsequent conjugation via enzymatic thiol-ene chemistry. J Polym Sci A 50:4085–4093CrossRefGoogle Scholar
  62. Yoon S, Kim S, Ryu Y, Kim TD (2007) Identification and characterization of a novel (S)-ketoprofen-specific esterase. Int J Biol Macromol 41:1–7CrossRefPubMedGoogle Scholar
  63. Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, Toyohara K, Miyamoto K, Kimura Y, Oda K (2016) A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351:1196–1199CrossRefPubMedGoogle Scholar
  64. Zhao Z, Zong M, Li N (2009) Efficient regioselective synthesis of 3′-O-crotonylfloxuridine catalysed by Pseudomonas cepacia lipase. Biotechnol Appl Biochem 52:45–51CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  • Mónica Martínez-Martínez
    • 1
  • Rafael Bargiela
    • 3
  • Cristina Coscolín
    • 1
  • José Navarro-Fernández
    • 1
  • Peter N. Golyshin
    • 2
    • 4
  • Manuel Ferrer
    • 1
  1. 1.CSIC, Institute of Catalysis, Dept. of Applied BiocatalysisMadridSpain
  2. 2.Immanuel Kant Baltic Federal UniversityKaliningradRussia
  3. 3.Institute of Catalysis, Consejo Superior de Investigaciones Científicas (CSIC)MadridSpain
  4. 4.School of Biological Sciences, University of BangorBangorUK

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