Enzyme Evolution by Yeast Cell Surface Engineering

  • Natsuko Miura
  • Kouichi Kuroda
  • Mitsuyoshi UedaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1319)


Artificial evolution of proteins with the aim of acquiring novel or improved functionality is important for practical applications of the proteins. We have developed yeast cell surface engineering methods (or arming technology) for evolving enzymes. Here, we have described yeast cell surface engineering coupled with in vivo homologous recombination and library screening as a method for the artificial evolution of enzymes such as firefly luciferases. Using this method, novel luciferases with improved substrate specificity and substrate reactivity were engineered.

Key words

Yeast cell surface engineering Protein evolution In vivo homologous recombination Firefly luciferase Substrate specificity and reactivity 


  1. 1.
    Ueda M, Tanaka A (2000) Genetic immobilization of proteins on the yeast cell surface. Biotechnol Adv 18:121–140PubMedCrossRefGoogle Scholar
  2. 2.
    Miura N, Aoki W, Tokumoto N et al (2009) Cell surface modification for non-GMO without chemical treatment by novel GMO-coupled and -separated co-cultivation method. Appl Microbiol Biotechnol 82:293–301PubMedCrossRefGoogle Scholar
  3. 3.
    Zou W, Ueda M, Yamanaka H et al (2001) Construction of a combinatorial protein library displayed on yeast cell surface using DNA random priming method. J Biosci Biotechnol 92:393–396Google Scholar
  4. 4.
    Shiraga S, Ueda M, Takahashi S et al (2002) Construction of the combinatorial library of Rhizopus oryzae lipase mutated in the lid domain by displaying on yeast cell surface. J Mol Catal B Enzym 17:167–173CrossRefGoogle Scholar
  5. 5.
    Ueda M (2004) Combinatorial bioengineering-development of molecular evolution. J Mol Catal B Enzym 28:4–6Google Scholar
  6. 6.
    Ueda M (2004) Future direction of molecular display by yeast-cell surface engineering. J Mol Catal B Enzym 28:139–144CrossRefGoogle Scholar
  7. 7.
    Shiraga S, Kawakami M, Ueda M (2004) Construction of combinatorial library of the starch-binding domain of Rhizopus oryzae glucoamylase and screening of clones with enhanced activity by yeast display method. J Mol Catal B Enzym 28:229–234CrossRefGoogle Scholar
  8. 8.
    Lin Y, Shiraga S, Tsumuraya T et al (2004) Isolation of novel catalytic antibody clones from combinatorial library displayed on yeast-cell surface. J Mol Catal B Enzym 28:247–252CrossRefGoogle Scholar
  9. 9.
    Fukuda T, Shiraga S, Kato M et al (2005) Construction of novel single cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries. Nanobiotechnology 1:105–111CrossRefGoogle Scholar
  10. 10.
    Shiraga S, Ishiguro M, Fukami H et al (2005) Creation of Rhizopus oryzae lipase having a unique oxyanion hole by combinatorial mutagenesis in the lid domain. Appl Microbiol Biotechnol 68:779–785PubMedCrossRefGoogle Scholar
  11. 11.
    Fukuda T, Shiraga S, Kato M et al (2006) Construction of a cultivation system of a yeast single cell in a cell chip microchamber. Biotechnol Prog 22:944–948PubMedCrossRefGoogle Scholar
  12. 12.
    Fukuda T, Kato M, Suye S et al (2007) Development of high-throughput screening system by single cell reaction using microchamber array chip. J Biosci Bioeng 104:241–243PubMedCrossRefGoogle Scholar
  13. 13.
    Fukuda T, Kato M, Kadonosono T et al (2007) Enhancement of substrate recognition ability by combinatorial mutation of β-glucosidase displayed on the yeast cell surface. Appl Microbiol Biotechnol 76:1027–1033PubMedCrossRefGoogle Scholar
  14. 14.
    Okochi N, Kato M, Kadonosono T et al (2007) Design of a serine protease-like catalytic triad on an antibody light chain displayed on the yeast cell surface. Appl Microbiol Biotechnol 77:597–603PubMedCrossRefGoogle Scholar
  15. 15.
    Kadonosono T, Kato M, Ueda M (2008) Alteration of substrate specificity of rat neurolysin from matrix metalloproteinase-2/9-type to -3-type specificity by comprehensive mutation. Protein Eng Des Sel 21:507–513PubMedCrossRefGoogle Scholar
  16. 16.
    Matsui K, Kuroda K, Ueda M (2009) Creation of a novel peptide endowing yeasts with acid tolerance using yeast cell-surface engineering. Appl Microbiol Biotechnol 82:105–113PubMedCrossRefGoogle Scholar
  17. 17.
    Isogawa D, Fukuda T, Kuroda K et al (2009) Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library. Appl Microbiol Biotechnol 85:95–104PubMedCrossRefGoogle Scholar
  18. 18.
    Aoki W, Yoshino Y, Morisaka H et al (2011) High-throughput screening of improved protease inhibitors using a yeast cell surface displaying system and a yeast cell chip. J Biotechnol Bioeng 111:16–18Google Scholar
  19. 19.
    Kuroda K, Nishitani T, Ueda M (2012) Specific adsorption of tungstate by cell surface display of the newly designed ModE mutant. Appl Microbiol Biotechnol 96:153–159PubMedCrossRefGoogle Scholar
  20. 20.
    Fushimi T, Miura N, Shintani H et al (2013) Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by cell surface engineering. Appl Microbiol Biotechnol 97:4003–4011PubMedCrossRefGoogle Scholar
  21. 21.
    Zou W, Ueda M, Tanaka A (2002) Screening of a molecule endowing Saccharomyces cerevisiae with n-nonane-tolerance from a combinatorial random protein library. Appl Microbiol Biotechnol 58:806–812PubMedCrossRefGoogle Scholar
  22. 22.
    Fukuda N, Ishii J, Shibasaki S et al (2007) High-efficiency recovery of target cells using improved yeast display system for detection of protein-protein interactions. Appl Microbiol Biotechnol 76:151–158PubMedCrossRefGoogle Scholar
  23. 23.
    Maeda H, Nagayama M, Kuroda K et al (2009) Purification of inactive precursor of carboxypeptidase Y using selective cleavage method coupled with molecular display. Biosci Biotechnol Biochem 73:753–755PubMedCrossRefGoogle Scholar
  24. 24.
    Shiraga S, Kawakami M, Ishiguro M et al (2005) Enhanced reactivity of Rhizopus oryzae lipase displayed on yeast cell surface in organic solvents: potential as a whole cell biocatalyst in organic solvents. Appl Environ Microbiol 71:4335–4338PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Nakamura Y, Matsumoto T, Nomoto F et al (2006) Enhancement of activity of lipase-displaying yeast cells and their application to optical resolution of (RS)-1-benzyloxy-3-chloro-2-propyl succinate. Biotechnol Prog 22:998–1002PubMedCrossRefGoogle Scholar
  26. 26.
    Fukuda T, Ishikawa T, Ogawa M et al (2006) Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering. Biotechnol Prog 22:933–938PubMedCrossRefGoogle Scholar
  27. 27.
    Kato M, Fuchimoto J, Tanio T et al (2007) Preparation of a whole-cell biocatalyst of mutated Candida antarctica lipase B (mCALB) by a yeast molecular display system and its practical properties. Appl Microbiol Biotechnol 75:549–555PubMedCrossRefGoogle Scholar
  28. 28.
    Kadonosono T, Kato M, Ueda M (2007) Substrate specificity of rat brain neurolysin disclosed by molecular display system and putative substrates in rat tissues. Appl Microbiol Biotechnol 75:353–1360Google Scholar
  29. 29.
    Kadonosono T, Kato M, Ueda M (2007) Metallopeptidase, neurolysin, as a novel molecular tool for analysis of properties of cancer-producing matrix metalloproteinases-2 and 9. Appl Microbiol Biotechnol 75:1285–1291PubMedCrossRefGoogle Scholar
  30. 30.
    Fukuda T, Kato M, Kuroda K et al (2008) Improvement in enzymatic desizing of starched cotton cloth using yeast co-displaying glucoamylase and cellulose-binding domain. Appl Microbiol Biotechnol 77:1225–1232PubMedCrossRefGoogle Scholar
  31. 31.
    Nishitani T, Shimada M, Kuroda K et al (2010) Molecular design of yeast cell surface for adsorption and recovery of molybdenum, one of rare metals. Appl Microbiol Biotechnol 86:641–648PubMedCrossRefGoogle Scholar
  32. 32.
    Nagayama M, Maeda H, Kuroda K et al (2012) Mutated intramolecular chaperones generate high-activity isomers of mature enzymes. Biochemistry 51:3547–3553PubMedCrossRefGoogle Scholar
  33. 33.
    Nakanishi A, Bae J, Kuroda K et al (2012) Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering. AMB Express 2:56PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Matsui K, Hirayama T, Kuroda K et al (2006) Screening for candidate genes involved in tolerance to organic solvents in yeast. Appl Microbiol Biotechnol 71:75–79PubMedCrossRefGoogle Scholar
  35. 35.
    Matsui K, Teranishi S, Kamon S et al (2008) Discovery of a modified transcription factor endowing yeasts with organic-solvent tolerance and reconstruction of an organic-solvent-tolerant yeast. Appl Environ Microbiol 74:4222–4225PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Ueda M (2011) Revolutionary protein engineering using molecular display. In: Sheehan MN (ed) Protein engineering: design, selection, and applications. Nova Science Publisher, New York, pp 73–80Google Scholar
  37. 37.
    Isogawa D, Kuroda K, Ueda M (2011) Whole-cell biocatalyst for utilization of chitosan by yeast cell surface engineering of chitosanase. In: MacKay RG, Tait JM (eds) Handbook of chitosan research and application. Nova Science Publisher, New York, pp 425–434Google Scholar
  38. 38.
    Branchini BR, Magyar RA, Murtiashaw MH et al (1999) Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color. Biochemistry 38:13223–13230PubMedCrossRefGoogle Scholar
  39. 39.
    Branchini BR, Murtiashaw MH, Magyar RA et al (2000) The role of lysine 529, a conserved residue of the acyl-adenylate-forming enzyme superfamily, in firefly luciferase. Biochemistry 39:5433–5440PubMedCrossRefGoogle Scholar
  40. 40.
    Shimoi H, Kitagaki H, Ohmori H et al (1998) Sed1p is a major cell wall protein of Saccharomyces cerevisiae in the stationary phase and is involved in lytic enzyme resistance. J Bacteriol 180:3381–3387PubMedCentralPubMedGoogle Scholar
  41. 41.
    Kuroda K, Matsui K, Higuchi S et al (2009) Enhancement of display efficiency in yeast display system by vector engineering and gene disruption. Appl Microbiol Biotechnol 82:713–719PubMedCrossRefGoogle Scholar
  42. 42.
    Miura N, Kirino A, Endo S et al (2012) Tracing putative trafficking of the glycolytic enzyme enolase via SNARE-driven unconventional secretion. Eukaryot Cell 11:1075–1082PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Ito H, Fukuda Y, Murata K et al (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:163–168PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Natsuko Miura
    • 1
    • 2
  • Kouichi Kuroda
    • 1
  • Mitsuyoshi Ueda
    • 1
    Email author
  1. 1.Division of Applied Life Sciences, Graduate School of AgricultureKyoto UniversitySakyo-ku, KyotoJapan
  2. 2.Radiation Oncology BranchNational Cancer Institute, National Institutes of HealthBethesdaUSA

Personalised recommendations