Directed evolution of a highly active Yersinia mollaretii phytase

Abstract

Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).

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References

  1. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201

    Article  CAS  Google Scholar 

  2. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795

    Article  Google Scholar 

  3. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucleic Acids Res 28:235–242

    Article  CAS  Google Scholar 

  4. Billington DC (1993) The Inositol phosphates: chemical synthesis and biological significance. VCH Verlagsgesellschaft, Weinheim

    Google Scholar 

  5. Bitar K, Reinhold JG (1972) Phytase and alkaline phosphatase activities in intestinal mucosae of rat, chicken, calf, and man. Biochim Biophys Acta 268:442–452

    CAS  Google Scholar 

  6. Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U (2010) Phosphorothioate-based ligase-independent gene cloning (PLICing): an enzyme-free and sequence-independent cloning method. Anal Biochem 406:141–146

    Article  CAS  Google Scholar 

  7. Bohm K, Herter T, Muller JJ, Borriss R, Heinemann U (2010) Crystal structure of Klebsiella sp. ASR1 phytase suggests substrate binding to a preformed active site that meets the requirements of a plant rhizosphere enzyme. FEBS J 277:1284–1296

    Article  Google Scholar 

  8. Bohn L, Meyer AS, Rasmussen SK (2008) Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ Sci B 9:165–191

    Article  CAS  Google Scholar 

  9. Cavazza C (2000) Carnitine and inositol phosphate-containing composition useful as dietary supplement or drug. World Intellectual Property Organization. Sigma-tau Healthscience, Italy

    Google Scholar 

  10. Chifflet S, Torriglia A, Chiesa R, Tolosa S (1988) A method for the determination of inorganic phosphate in the presence of labile organic phosphate and high concentrations of protein: application to lens ATPases. Anal Biochem 168:1–4

    Article  CAS  Google Scholar 

  11. Choi YM, Suh HJ, Kim JM (2001) Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J Protein Chem 20:287–292

    Article  CAS  Google Scholar 

  12. Copeland RA (2000) Enzymes: a practical introduction to structure, mechanism, and data analysis. Wiley-VCH, New York

    Google Scholar 

  13. Cowieson A, Cooper R (2010) Introduction to the event and overview of the phytase market. International Phytase Summit, Washington, D.C

    Google Scholar 

  14. Cummings L, Riley L, Black L, Souvorov A, Resenchuk S, Dondoshansky I, Tatusova T (2002) Genomic BLAST: custom-defined virtual databases for complete and unfinished genomes. FEMS Microbiol Lett 216:133–138

    Article  CAS  Google Scholar 

  15. De Groeve MR, Tran GH, Van Hoorebeke A, Stout J, Desmet T, Savvides SN, Soetaert W (2010) Development and application of a screening assay for glycoside phosphorylases. Anal Biochem 401:162–167

    Article  Google Scholar 

  16. Ferre F, Clote P (2005) DiANNA: a web server for disulfide connectivity prediction. Nucleic Acids Res 33:W230–W232

    Article  CAS  Google Scholar 

  17. Fu S, Sun J, Qian L, Li Z (2008) Bacillus phytases: present scenario and future perspectives. Appl Biochem Biotechnol 151:1–8

    Article  CAS  Google Scholar 

  18. Fu D, Huang H, Meng K, Wang Y, Luo H, Yang P, Yuan T, Yao B (2009) Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution. Biotechnol Bioeng 103:857–864

    Article  CAS  Google Scholar 

  19. Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, Hazlewood GP, Short JM, Robertson DE, Gray KA (2004) Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement. Appl Environ Microbiol 70:3041–3046

    Article  CAS  Google Scholar 

  20. Gawronski JD, Benson DR (2004) Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection. Anal Biochem 327:114–118

    Article  CAS  Google Scholar 

  21. Geer LY, Domrachev M, Lipman DJ, Bryant SH (2002) CDART: protein homology by domain architecture. Genome Res 12:1619–1623

    Article  CAS  Google Scholar 

  22. Gonzalez-Romo P, Sanchez-Nieto S, Gavilanes-Ruiz M (1992) A modified colorimetric method for the determination of orthophosphate in the presence of high ATP concentrations. Anal Biochem 200:235–238

    Article  CAS  Google Scholar 

  23. Harland BF, Oberleas D (1999) Phytase in animal nutrition and waste management. BASF Reference Manual 237–240

  24. Huang H, Luo H, Yang P, Meng K, Wang Y, Yuan T, Bai Y, Yao B (2006) A novel phytase with preferable characteristics from Yersinia intermedia. Biochem Biophys Res Commun 350:884–889

    Article  CAS  Google Scholar 

  25. Huang H, Luo H, Wang Y, Fu D, Shao N, Wang G, Yang P, Yao B (2008) A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Appl Microbiol Biotechnol 80:417–426

    Article  CAS  Google Scholar 

  26. Huang H, Shi P, Wang Y, Luo H, Shao N, Wang G, Yang P, Yao B (2009) Diversity of beta-propeller phytase genes in the intestinal contents of grass carp provides insight into the release of major phosphorus from phytate in nature. Appl Environ Microbiol 75:1508–1516

    Article  CAS  Google Scholar 

  27. Jermutus L, Tessier M, Pasamontes L, van Loon AP, Lehmann M (2001) Structure-based chimeric enzymes as an alternative to directed enzyme evolution: phytase as a test case. J Biotechnol 85:15–24

    Article  CAS  Google Scholar 

  28. Kim MS, Lei XG (2008) Enhancing thermostability of Escherichia coli phytase App A2 by error-prone PCR. Appl Microbiol Biotechnol 79:69–75

    Article  CAS  Google Scholar 

  29. Kim YO, Kim HW, Lee JH, Kim KK, Lee SJ (2006) Molecular cloning of the phytase gene from Citrobacter braakii and its expression in Saccharomyces cerevisiae. Biotechnol Lett 28:33–38

    Article  CAS  Google Scholar 

  30. Kim MS, Weaver JD, Lei XG (2008) Assembly of mutations for improving thermostability of Escherichia coli App A2 phytase. Appl Microbiol Biotechnol 79:751–758

    Article  CAS  Google Scholar 

  31. Kostrewa D, Wyss M, D’Arcy A, van Loon AP (1999) Crystal structure of Aspergillus niger pH 2.5 acid phosphatase at 2. 4 A resolution. J Mol Biol 288:965–974

    Article  CAS  Google Scholar 

  32. Kumar S, Tsai CJ, Nussinov R (2000) Factors enhancing protein thermostability. Protein Eng 13:179–191

    Article  CAS  Google Scholar 

  33. Lehmann M, Kostrewa D, Wyss M, Brugger R, D’Arcy A, Pasamontes L, van Loon AP (2000) From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng 13:49–57

    Article  CAS  Google Scholar 

  34. Lei XG, Porres JM, Mullaney EJ, Brinch-Pedersen H (2007) Phytase: source, structure and application. Industrial enzymes: structure, function and applications. J. Polaina and A. P. MacCabe, Springer Netherlands 505–529

  35. Lim D, Golovan S, Forsberg CW, Jia Z (2000) Crystal structures of Escherichia coli phytase and its complex with phytate. Nat Struct Biol 7:108–113

    Article  CAS  Google Scholar 

  36. Lindqvist Y, Schneider G, Vihko P (1993) Three-dimensional structure of rat acid phosphatase in complex with L(+)-tartrate. J Biol Chem 268:20744–20746

    CAS  Google Scholar 

  37. Liu Q, Huang Q, Lei XG, Hao Q (2004) Crystallographic snapshots of Aspergillus fumigatus phytase, revealing its enzymatic dynamics. Structure 12:1575–1583

    Article  CAS  Google Scholar 

  38. Luo HY, Yao B, Yuan TZ, Wang YR, Shi XY, Wu NF, Fan YL (2004) Overexpression of Escherchia coli phytase with high specific activity. Sheng Wu Gong Cheng Xue Bao 20:78–84

    CAS  Google Scholar 

  39. Luthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with three-dimensional profiles. Nature 356:83–85

    Article  CAS  Google Scholar 

  40. Miksch G, Kleist S, Friehs K, Flaschel E (2002) Overexpression of the phytase from Escherichia coli and its extracellular production in bioreactors. Appl Microbiol Biotechnol 59:685–694

    Article  CAS  Google Scholar 

  41. Oakley AJ (2010) The structure of Aspergillus niger phytase PhyA in complex with a phytate mimetic. Biochem Biophys Res Commun 397:745–749

    Article  CAS  Google Scholar 

  42. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  Google Scholar 

  43. Ragon M, Hoh F, Aumelas A, Chiche L, Moulin G, Boze H (2009) Structure of Debaryomyces castellii CBS 2923 phytase. Acta Crystallogr Sect F Struct Biol Cryst Commun 65:321–326

    Article  CAS  Google Scholar 

  44. Ramachandran GN, Ramakrishnan C, Sasisekharan V (1963) Stereochemistry of polypeptide chain configurations. J Mol Biol 7:95–99

    Article  CAS  Google Scholar 

  45. Rodriguez E, Wood ZA, Karplus PA, Lei XG (2000) Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris. Arch Biochem Biophys 382:105–112

    Article  CAS  Google Scholar 

  46. Sajidan A, Farouk A, Greiner R, Jungblut P, Muller EC, Borriss R (2004) Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1. Appl Microbiol Biotechnol 65:110–118

    Article  CAS  Google Scholar 

  47. Sandberg A, Brune M, Carlsson N, Hallberg L, Skoglund E, Rossander-Hulthen L (1999) Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. Am J Clin Nutr 70:240–246

    CAS  Google Scholar 

  48. Sebastian S, Touchburn SP, Chavez ER (1998) Implications of phytic acid and supplemental microbial phytase in poultry nutrition: a review. World Poultry Sci J 54:27–47

    Article  Google Scholar 

  49. Senn AM, Wolosiuk RA (2005) A high-throughput screening for phosphatases using specific substrates. Anal Biochem 339:150–156

    Article  CAS  Google Scholar 

  50. Shivange AV, Marienhagen J, Mundhada H, Schenk A, Schwaneberg U (2009) Advances in generating functional diversity for directed protein evolution. Curr Opin Chem Biol 13:19–25

    Article  CAS  Google Scholar 

  51. Shivange AV, Schwaneberg U, Roccatano D (2010) Conformational dynamics of active site loop in Escherichia coli phytase. Biopolymers 93:994–1002

    Article  CAS  Google Scholar 

  52. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234

    Article  CAS  Google Scholar 

  53. Tee KL, Schwaneberg U (2007) Directed evolution of oxygenases: screening systems, success stories and challenges. Comb Chem High Throughput Screen 10:197–217

    Article  CAS  Google Scholar 

  54. Varshavsky A (1996) The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci U S A 93:12142–12149

    Article  CAS  Google Scholar 

  55. Vats P, Bhattacharyya MS, Banerjee UC (2005) Use of phytases myo-inositolhexakisphosphate phosphohydrolases for combatting environmental pollution: a biological approach. Crit Rev Environ Sci Technol 35:469–486

    Article  CAS  Google Scholar 

  56. Viader-Salvado JM, Gallegos-Lopez JA, Carreon-Trevino JG, Castillo-Galvan M, Rojo-Dominguez A, Guerrero-Olazaran M (2010) Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris. Appl Environ Microbiol 76:6423–6430

    Article  CAS  Google Scholar 

  57. Wodzinski RJ, Ullah AH (1996) Phytase. Adv Appl Microbiol 42:263–302

    Article  CAS  Google Scholar 

  58. Wong TS, Roccatano D, Zacharias M, Schwaneberg U (2006a) A statistical analysis of random mutagenesis methods used for directed protein evolution. J Mol Biol 355:858–871

    Article  CAS  Google Scholar 

  59. Wong TS, Zhurina D, Schwaneberg U (2006b) The diversity challenge in directed protein evolution. Comb Chem High Throughput Screen 9:271–288

    Article  CAS  Google Scholar 

  60. Wong TS, Roccatano D, Schwaneberg U (2007) Are transversion mutations better? A mutagenesis assistant program analysis on P450 BM-3 heme domain. Biotechnol J 2:133–142

    Article  CAS  Google Scholar 

  61. Wong TS, Roccatano D, Loakes D, Tee KL, Schenk A, Hauer B, Schwaneberg U (2008) Transversion-enriched sequence saturation mutagenesis (SeSaM-Tv+): a random mutagenesis method with consecutive nucleotide exchanges that complements the bias of error-prone PCR. Biotechnol J 3:74–82

    Article  CAS  Google Scholar 

  62. Wyss M, Brugger R, Kronenberger A, Remy R, Fimbel R, Oesterhelt G, Lehmann M, van Loon AP (1999) Biochemical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): catalytic properties. Appl Environ Microbiol 65:367–373

    CAS  Google Scholar 

  63. Zhang W, Mullaney EJ, Lei XG (2007) Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase. Appl Environ Microbiol 73:3069–3076

    Article  CAS  Google Scholar 

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Acknowledgments

We thank BASF SE for financial support and Dr. Alexander Schenk for providing pALXtreme-5b vector and E. coli BL21-Gold(DE3)laqIQ1 expression strain.

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Correspondence to Ulrich Schwaneberg.

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Shivange, A.V., Serwe, A., Dennig, A. et al. Directed evolution of a highly active Yersinia mollaretii phytase. Appl Microbiol Biotechnol 95, 405–418 (2012). https://doi.org/10.1007/s00253-011-3756-7

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Keywords

  • Directed evolution
  • High-throughput screening
  • SeSaM
  • Phytase
  • Thermostability