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Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp. Px6-4 gene for ferulic acid decarboxylase

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

Ferulic acid decarboxylase (FADase) can catalyze the transformation of ferulic acid into 4-vinyl guaiacol via decarboxylation in microorganisms. In this study, a gene encoding FADase was first isolated from the bacterium Enterobacter sp. Px6-4 using degenerate primers and a genome walking technique. The putative encoding gene (fad) of FADase consists of 507-bp nucleotides, coding a polypeptide of 168 amino acid residues. In addition, a putative gene encoding the transcriptional regulator was identified from the upstream of the fad gene. The deduced peptide sequence of the FADase from Enterobacter sp. Px6-4 showed a 51.2–53.3% sequence identity to decarboxylases from other bacteria. The gene fad was successfully expressed in Escherichia coli BL21, and the recombinant FADase was purified as a protein of ca. 23 kDa with an optimal activity at pH 4.0 and 28 °C. The purified FADase could convert ferulic acid to 4-vinyl guaiacol effectively, and its hydrolytic activity could be inhibited by Cu2+ (99%) and Hg2+ (99.5%). A phylogenetic analysis of the FADase protein from bacteria revealed several different clades. Our result provided a basis for further studies of the ferulic acid transformation pathway and for enhanced production of vanillin in the future.

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References

  • Akin DE (1988) Biological structure of lignocellulose and its degradation in the rumen. Anim Food Sci Technol 21:295–310

    Article  CAS  Google Scholar 

  • Barthelmebs L, Lecomte B, Divies C, Cavin JF (2000) Inducible metabolism of phenolic acids in Pediococcus pentosaceus is encoded by an autoregulated operon which involves a new class of negative transcriptional regulator. J Bacteriol 182:6724–6731

    Article  CAS  Google Scholar 

  • Barthelmebs L, Diviès C, Cavin JF (2001) Expression in Escherichia coli of native and chimeric phenolic acid decarboxylases with modified enzymatic activities and method for screening recombinant E. coli strains expressing these enzymes. Appl Environ Microbiol 67:1063–1069

    Article  CAS  Google Scholar 

  • Benoit I, Danchin EG, Bleichrodt RJ, de Vries RP (2008) Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity. Biotechnol Lett 30:387–396

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Brezillon C, Kroon PA, Faulds CB, Brett GM, Williamson G (1996) Novel ferulic acid esterases are induced by growth of Aspergillus niger on sugar beet pulp. Appl Microbiol Biotechnol 45:371–376

    Article  CAS  Google Scholar 

  • Brunati M, Marinelli F, Bertolini C, Gandolfi R, Daffonchio D, Molinari F (2004) Biotransformations of cinnamic and ferulic acid with actinomycetes. Enzyme Microb Technol 34:3–9

    Article  CAS  Google Scholar 

  • Cavin JF, Barthelmebs L, Guzzo J, Beeumen JV, Samyn B, Travers JF, Diviès C (1997a) Purification and characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum. FEMS Microbiol Lett 147:291–295

    Article  CAS  Google Scholar 

  • Cavin JF, Barthelmebs L, Diviès C (1997b) Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification and characterization. Appl Environ Microbiol 63:1939–1944

    CAS  Google Scholar 

  • Cavin JF, Dartois V, Diviès C (1998) Gene cloning, transcriptional analysis, purification and characterization of phenolic acid decarboxylase from Bacillus subtilis. Appl Environ Microbiol 64:1466–1471

    CAS  Google Scholar 

  • Degrassi G, De Laureto PP, Bruschi CV (1995) Purification and characterization of ferulate and p-coumarate decarboxylase from Bacillus pumilus. Appl Environ Microbiol 61:326–332

    CAS  Google Scholar 

  • Donaghy J, McKay AM (1997) Purification and characterization of a feruloyl esterase from the fungus, Penicillium expansum. J Appl Microbiol 83:718–726

    Article  CAS  Google Scholar 

  • Edlin DAN, Narbad A, Gasson MJ, Dickinson JR, Lloyd D (1998) Purification and characterization of hydroxycinnamate decarboxylase from Brettanomyces anomalus. Enzyme Microbiol Technol 22:232–239

    Article  CAS  Google Scholar 

  • Gury J, Barthelmebs L, Tran NP, Divies C, Cavin JF (2004) Cloning, deletion, and characterization of PadR, the transcriptional repressor of the phenolic acid decarboxylase-encoding padA gene of Lactobacillus plantarum. Appl Environ Microbiol 70:2146–2153

    Article  CAS  Google Scholar 

  • Huang ZX, Dostal L, Rosazza JPN (1993) Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens. Appl Environ Microbiol 59:2244–2250

    CAS  Google Scholar 

  • Huang Z, Dostal L, Rosazza JPN (1994) Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens. J Bacteriol 176:5912–5918

    CAS  Google Scholar 

  • Ishii T (1997) Structure and function of feruloylated polysaccharides. Plant Sci 127:111–127

    Article  CAS  Google Scholar 

  • Li XM, Yang JK, Li X, Gu W, Huang JW, Zhang KQ (2008) The metabolism of ferulic acid via 4-vinylguaiacol to vanillin by Enterobacter sp. Px6-4 isolated from Vanilla root. Process Biochem 43:1032–1037

    CAS  Google Scholar 

  • Mathew S, Abraham TE (2004) Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications. Crit Rev Biotechnol 24:59–83

    Article  CAS  Google Scholar 

  • Mathew S, Abraham TE (2006) Bioconversions of ferulic acid, an hydroxycinnamic acid. Crit Rev Microbiol 32:115–125

    Article  CAS  Google Scholar 

  • Muller-Harvey I, Hartley RD (1986) Linkage of p-coumaroyl and feruloyl groups to cell-wall polysaccharides of barley straw. Carbohydr Res 148:71–85

    Article  Google Scholar 

  • Oosterveld A, Beldman G, Schols HA, Voragen AGJ (2000) Characterization of arabinose and ferulic acid rich pectic polysaccharides and hemicelluloses from sugar beet pulp. Carbohydr Res 328:185–197

    Article  CAS  Google Scholar 

  • Priefert H, Rabenhorst J, Steinbüchel A (2001) Biotechnological production of vanillin. Appl Microbiol Biotechnol 56:296–314

    Article  CAS  Google Scholar 

  • Prim N, Pastor FI, Diaz P (2003) Biochemical studies on cloned Bacillus sp. BP-7 phenolic acid decarboxylase PadA. Appl Microbiol Biotechnol 63:51–56

    Article  CAS  Google Scholar 

  • Ramachandra RS, Ravishankar GA (2000) Vanilla flavour: production by conventional and biotechnological routes. J Sci Food Agric 80:289–304

    Article  Google Scholar 

  • Rosazza JPN, Huang Z, Dostal L, Volm T, Rousseau B (1995) Biocatalytic transformations of ferulic acid: an abundant aromatic natural product. J Ind Microbiol 15:457–471

    Article  CAS  Google Scholar 

  • Saulnier L, Thibault JF (1999) Ferulic acid and diferulic acids as components of sugar-beet pectins and maize bran heteroxylans. J Sci Food Agric 79:396–402

    Article  CAS  Google Scholar 

  • Smith MM, Hartley RD (1983) Occurrence and nature of ferulic acid substitution of cell-wall polysaccharides in graminaceous plants. Carbohydr Res 118:65–80

    Article  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The Clustal_X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 24:4876–4882

    Article  Google Scholar 

  • Torres JL, Rosazza JPN (2001) Microbial transformation of p-coumaric acid by Bacillus megaterium and Curvularia lunata. J Nat Prod 64:1408–1414

    Article  CAS  Google Scholar 

  • Yang JK, Huang XW, Tian BY, Wang M, Niu QH, Zhang KQ (2005) Isolation and characterization of a serine protease from the nematophagous fungus Lecanicillium psalliotae, displaying nematicidal activity. Biotechnol Lett 27:1123–1128

    Article  CAS  Google Scholar 

  • Zago A, Degrassi G, Bruschi CV (1995) Cloning, sequencing, and expression in Escherichia coli of the Bacillus pumilus gene for ferulic acid decarboxylase. Appl Environ Microbiol 61:4484–4486

    CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by the National Natural Science Foundation of China (Approval No. 31060012) and the Department of Science and Technology of Yunnan Province (Approval Nos. 2007C170M, 2006GG22, and 2009CI052).

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Authors and Affiliations

  1. Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, 650091, People’s Republic of China

    Wen Gu, Jingwen Huang, Zhaohui Meng, Ke-Qin Zhang & Jinkui Yang

  2. Key Laboratory for Microbial Resources of the Ministry of Education, Yunnan University, Kunming, 650091, People’s Republic of China

    Wen Gu, Jingwen Huang, Zhaohui Meng, Ke-Qin Zhang & Jinkui Yang

  3. Yunnan Academy of Tobacco Science, Kunming, 650106, People’s Republic of China

    Xuemei Li

  4. Technology Centre of Hongyun Honghe Tobacco (Group) Co, Ltd., Kunming, 650202, People’s Republic of China

    Yanqing Duan

  5. Department of Cardiology, No. 1 Affiliated Hospital, Kunming Medical College, Kunming, 650032, People’s Republic of China

    Zhaohui Meng

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  1. Wen Gu
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  2. Xuemei Li
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Correspondence to Jinkui Yang.

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Gu, W., Li, X., Huang, J. et al. Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp. Px6-4 gene for ferulic acid decarboxylase. Appl Microbiol Biotechnol 89, 1797–1805 (2011). https://doi.org/10.1007/s00253-010-2978-4

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  • DOI: https://doi.org/10.1007/s00253-010-2978-4

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