Metagenomic discovery of feruloyl esterases from rumen microflora

  • Dominic W. S. WongEmail author
  • Victor J. Chan
  • Hans Liao
Biotechnologically relevant enzymes and proteins


Feruloyl esterases (FAEs) are a key group of enzymes that hydrolyze ferulic acids ester-linked to plant polysaccharides. The cow’s rumen is a highly evolved ecosystem of complex microbial microflora capable of converting fibrous substances to energy. From direct cloning of the rumen microbial metagenome, we identified seven active phagemids conferring feruloyl esterase activity. The genomic inserts ranged from 1633 to 4143 bp, and the ORFs from 681 to 1359 bp. BLAST search reveals sequence homology to feruloyl esterases and esterases/lipases identified in anaerobes. The seven genes were expressed in Escherichia coli, and the proteins were purified to homogeneity. The FAEs were found to cover types B, C, and D in the feruloyl esterase classification system using model hydroxycinnamic acid esters. The release of ferulic acid (FA) catalyzed by these enzymes was established using natural substrates corn fiber (CF) and wheat insoluble arabinoxylan (WIA). Three of the enzymes were demonstrated to cleave diferulates and hence the capability to break down Araf-FA-FA-Araf cross-links. The wide variation in the sequence, activity, and substrate specificity observed in the FAEs discovered in this study is a confirming evidence that combined actions of a full range of FAE enzymes contribute to the high-efficiency fiber digestion in the rumen microbial ecosystem.


Feruloyl esterase Ferulic acid Diferulate Metagenome Rumen 


Compliance with ethical standards


Reference to a company and/or products is only for purposes of information and does not imply approval of recommendation of the product to the exclusion of others that may also be suitable. All programs and services of the US Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.

Ethical statement

This work has been performed in compliance with ethical standards. Authors declare they have no conflict of interest. The article does not contain any studies with human participation performed by any of the authors. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Andreasen MF, Kroon PA, Williamson G, Garcia-Conesa M-T (2001) Intestinal release and uptake of phenolic antioxidant diferulic acids. Free Radic Biol Med 31:304–314. CrossRefGoogle Scholar
  2. Bartolome B, Faulds CB, Kroon PA, Waldron K, Gilbert HJ, Hazlewood G, Williamson G (1997) An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. Cellulose esterase (XylD) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls. Appl Environ Microbiol 63:208–212Google Scholar
  3. Beloqui A, Pita M, Polaina J, Martinez-Arias A, Golyshina OV, Zumarraga M, Yakimov MM, Garcia-Arellano H, Alcalde M, Fernandez VM, Elborough K, Andreu JM, Ballesteros A, Plou FJ, Timmis KN, Ferrer M, Golyshin PN (2006) Novel polyphenol oxidase mined from a metagenome expression library of bovine rumen: biochemical properties, structural analysis, and phylogenetic relationships. J Biol Chem 281:22933–22942.
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  5. Brenner S (1988) The molecular evolution of genes and proteins: a tale of two serines. Nature 334:528–530CrossRefGoogle Scholar
  6. Biely P, Singh S, Puchart V (2016) Toward enzymatic breakdown of complex plant xylan structures: State of the art. Biotechnol Adv 34:1260-1274Google Scholar
  7. Carmedy WR (1961) An easily prepared wide range buffer series. J Chem Educ 38:559–560CrossRefGoogle Scholar
  8. Crepin VF, Faulds CB, Connerton IF (2003) A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibitor. Biochem J 370:417-427Google Scholar
  9. Crepin VF, Faulds CB, Connerton IF (2004) Functional classification of the microbial feruloyl esterases. Appl Microbiol Biotechnol 63:647–652.
  10. De Vries RP, Kester HCM, Poulsen CH, Benen JAE, Visser J (2000) Synergy between enzymes from Aspergillus involved in the degradation of plant cell wall polysaccharides. Carbohydr Res 327:401–410CrossRefGoogle Scholar
  11. Dilokpimol A, Makela MR, Aguilar-Ponrwa M, Benoit-Gelber I, Hidden KS, deVries RP (2016) Diversity of fungal feruloyl esterases: updated phylogenetic classification, properties, and industrial applications. Biotechnol Biofuels.
  12. Doner LW, Johnston DB, Singh V (2001) Analysis and properties of arbinoxylan from discrete corn wet-milling fiber fractions. J Agric Food Chem 49:1266–1269. CrossRefGoogle Scholar
  13. Faulds CB (2016) What can feruloyl esterases do for us? Phytochem Rev 9:121–132. CrossRefGoogle Scholar
  14. Faulds CB, Molina R, Gonzalez R, Husband F, Juge N, Sanz-Aparicio J, Hermoso JA (2005) Probing the determinant of substrate specificity of a feruloyl esterase, AnFaeA from Aspergillus niger. FEBS J 272:4362-4371.
  15. Ferrer M, Golyshina CV, Chermikova TN, Khachane AN, Reyes-Duarte D, Santos VAPMD, Strompl C, Elborough K, Jarvisl G, Neef A, Yakimov MM, Timmis KN, Golyshin PN (2005) Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora. Eniviron Microbiol 7:1996–2010. CrossRefGoogle Scholar
  16. Gupta Udatha DBRK, Kouskoumvekaki I, Olsson L, Panagiotou G (2011) The interplay of descriptor-based computational analysis with pharmacophore modeling builds the basis for a novel classification scheme for feruloyl esterases. Biotechnol Adv 29:94–110. CrossRefGoogle Scholar
  17. Heald S, Myers S, Walford T, Robbins K, Hill C (2013) Preparation of vanillin from microbial transformation media by extraction by means supercritical fluids or gases. US 8,563,392 B2Google Scholar
  18. Hermoso JA, Sanz-Aparicio J, Molma R, Juge N, Gonzalez R, Faulds CB (2004) The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family. J Mol Biol 338:495–507. CrossRefGoogle Scholar
  19. Hess M, Sczyrba A, Egan R, Kim TW, Chokhawala H, Schroth G, Luo S, Clark DS, Chen F, Zhang T, Mackie RI, Pennacchio LA, Tringe SG, Visel A, Woyke T, Wang Z, Rubin EM (2011) Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331:463–467. CrossRefGoogle Scholar
  20. Hunt CJ, Antonopoulou L, Tanksale A, Rova U, Christakopoulos P, Haritos VS (2017) Insights into substrate binding of ferulic acid esterases by arabinose and methyl hydroxycinnamate esters and molecular docking. Sci Rep 7:17315. CrossRefGoogle Scholar
  21. Lai KK, Stogios PJ, Vu C, Xu X, Cui H, Molloy S, Savchenko A, Yakunin A, Gonzalez CF (2011) An inserted α/β subdomain shapes the catalytic pocket of Lactobacillus johnsonii cinnamoyl esterase. PLoS One 6(8):e23269. CrossRefGoogle Scholar
  22. LaVallie ER, DiBlasio EA, Kovacic S, Grant KL, Schendel PF, McCoy JM (1993) A thioredoxin gene fusion expression system that circumvent inclusion body formation in the E. coli cytoplasm. BioTechnology 11:187–193. Google Scholar
  23. Morgavi DP, Kelly WJ, Janssen PH, Attwood GT (2013) Rumen microbial genomics and its application to ruminant production. Animal 7(suppl. 1):184–201. CrossRefGoogle Scholar
  24. Ribeiro GO, Gruninger RJ, Badhan A, McAllister TA (2016) Mining the rumen for fibrolytic feed enzymes. Anim Front 6:20–26. CrossRefGoogle Scholar
  25. Schubot FD, Kataeva IA, Blum DL, Shah AK, Ljungdahl IG, Rose JP, Wang B-C (2001) Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum. Biochemistry 40:12524–12532. CrossRefGoogle Scholar
  26. Shang M, Chan VJ, Wong DWS, Liao H (2018) A novel method for rapid and sensitive metagenomic activity screening. MethodsX 5:669–675. CrossRefGoogle Scholar
  27. Sigoillot C, Camarero S, Vidal T, Record E, Asther M, Perez-Boada M, Martinez KJ, Sigoillot J-C, Asther M, Colom JF, Martinez AT (2005) Comparison of different fungal enzymes for bleaching high-quality paper pulps. J Biotechnol 115:333–343. CrossRefGoogle Scholar
  28. Suzuki K, Hori A, Kawamoto K, Thangudu RR, Ishida T, Igarashi K, Samejima M, Yamada C, Arakawa T, Wakagi T, Koseki T, Fushinobu S (2014) Crystal structure of a feruloyl esterase belonging to the tannase family: a disulfide bond near a catalytic triad. Proteins 82:2857–2867. CrossRefGoogle Scholar
  29. Tabka MD, Herpoel-Gimberta I, Monod F, Asther M, Sigoillot JC (2006) Enzymatic saccharification of wheat straw for bioethanol production by a combined cellulase-xylanase and feruloyl esterase treatment. Enzym Microb Technol 39:897–902. CrossRefGoogle Scholar
  30. Uraji M, Tamura H, Mizohata E, Arima J, Wan K, Ogawa K, Inoue T, Natanaka T (2018) Loop of Streptomyces feruloyl esterase plays an important role in the enzyme's catalyzing the release of ferulic acid from Biomass. Appl Environ Microbiol 84:e02300–e02317. Google Scholar
  31. Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T (2018) SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 46(W1):W296–W303. CrossRefGoogle Scholar
  32. Wong DWS (2006) Feruloyl esterase-a key enzyme in biomass degradation. Appl Biochem Biotechnol 133:87–112. CrossRefGoogle Scholar
  33. Wong DWS, Chan VJ, Batt SB (2008) Cloning and characterization of a novel exo-α-1,5-L-arabinanase gene and the enzyme. Appl Microbiol Biotechnol 79:941–949. CrossRefGoogle Scholar
  34. Wong DWS, Chan VJ, McCormack AA, Batt SB (2010) A novel xyloglucan-specific endo-β-1,4-glucanase: biochemical properties and inhibition studies. Appl Microbiol Biotechnol 86:1463–1471. CrossRefGoogle Scholar
  35. Wong DWS, Chan VJ, Batt SB, Gauttam S, Liao H (2011) Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass. J Ind Microbiol Biotechnol 38:1961–1967. CrossRefGoogle Scholar
  36. Wong DWS, Chan VJ, Liao H, Zidwick MJ (2013a) Cloning of a novel feruloyl esterase gene from rumen microbial metagenome and enzyme characterization in synergism with endoxylanases. J Ind Microbiol Biotechnol 40:287–295. CrossRefGoogle Scholar
  37. Wong DWS, Chan VJ, McCormack AA (2013b) Comparative characterization of a bifunctional endo-1,4-β-mannanase/1,3-1,4-β-glucanase and its individual domains. Protein Pept Lett 20:517–523CrossRefGoogle Scholar
  38. Wong DWS, Takeoka G, Chan VJ, Liao H, Murakami MT (2015) Cloning of a novel feruloyl esterase from rumen microbial metagenome for substantial yield of mono- and di-ferulic acids from natural substrates. Protein Pept Lett 22:681–688CrossRefGoogle Scholar
  39. Zhao S, Wang J, Bu D, Liu K, Zhu Y, Dong Z, Yu Z (2010) Novel glycoside hydrolases identified by screening a Chinese Hoistein dairy cow rumen-derived metagenome library. Appl Environ Microbiol 76:6701–6705. CrossRefGoogle Scholar

Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Western Regional Research Center, USDA-ARSAlbanyUSA
  2. 2.Cargill Biotechnology Development CenterMinneapolisUSA

Personalised recommendations