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Recent Advances in Pichia pastoris as Host for Heterologous Expression System for Lipases: A Review

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Lipases and Phospholipases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1835))

Abstract

The production of heterologous lipases is one of the most promising strategies to increase the productivity of the bioprocesses and to reduce costs, with the final objective that more industrial lipase applications could be implemented.

In this chapter, an overview of the new success in synthetic biology, with traditional molecular genetic techniques and bioprocess engineering in the last 5 years in the cell factory Pichia pastoris, the most promising host system for heterologous lipase production, is presented.

The goals get on heterologous Candida antarctica, Rhizopus oryzae, and Candida rugosa lipases, three of the most common lipases used in biocatalysis, are showed. Finally, new cell factories producing heterologous lipases are presented.

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References

  1. Ahmad M, Hirz M, Pichler H et al (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98:5301–5317

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Boettner M, Steffens C, von Mering C et al (2007) Sequence-based factors influencing the expression of heterologous genes in the yeast Pichia pastoris—A comparative view on 79 human genes. J Biotechnol 130:1–10

    Article  CAS  PubMed  Google Scholar 

  3. Valero F (2012) Heterologous expression system for lipases: a review. Methods Mol Biol 861:161–178

    Article  CAS  PubMed  Google Scholar 

  4. Wagner JM, Alper HA (2016) Synthetic biology and molecular genetics in non-conventional yeast: current tools and future advances. Fungal Genet Biol 89:126–136

    Article  CAS  PubMed  Google Scholar 

  5. Potvin G, Ahmad A, Zhang Z (2012) Bioprocess engineering aspects of heterologous protein production in Pichia pastoris: a review. Biochem Eng J 64:91–105

    Article  CAS  Google Scholar 

  6. Gasser B, Steiger MG, Mattanovich D (2015) Methanol regulated yeast promoters. Production vehicles and toolbox for synthetic biology. Microb Cell Fact 14:196

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Mellitzer A, Ruth C, Gustafsson C et al (2014) Synergetics modular promoter and gene optimization to push cellulose secretion by Pichia pastoris beyond existing benchmarks. J Biotechnol 191:187–195

    Article  CAS  PubMed  Google Scholar 

  8. Krainer FW, Dietzsch C, Hajek T et al (2012) Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway. Microb Cell Fact 11:22

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Yang S, Y Kuang Y, Li H et al (2013) Enhanced production of recombinant secretory proteins in Pichia pastoris by optimizing Kex2 P1’site. Plos One 8:1–11

    Article  Google Scholar 

  10. Ruth C, Buchetics M, Vidimce V et al (2014) Pichia pastoris Aft1 a novel transcription factor, enhancing recombinant protein secretion. Microb Cell Fact 13:120

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Wang JR, Li YY, Liu DN et al (2015) Codon optimization significantly improves the expression level of α-amylase gene from Bacillus licheniformis in Pichia pastoris. Biomed Res Int 2015:1–9

    Google Scholar 

  12. Cámara E, Albiol J, Ferrer P (2016) Droplet digital PCR-aided screening and characterization of Pichia pastoris multiple gene copy strains. Biotechnol Bioeng 113:1542–1551

    Article  CAS  PubMed  Google Scholar 

  13. Jiang B, Argyros R, Bukowski J et al (2015) Inactivation of a GAL4-like transcription factor improves cell fitness and product yield in glycoengineered Pichia pastoris strains. Appl Environ Microbiol 81(1):260–271

    Article  CAS  PubMed  Google Scholar 

  14. Puxbaum V, Mattanovich D, Gasser B (2015) Quo vadis? The challenges of recombinant protein folding and secretion in in Pichia pastoris. Appl Microbiol Biotechnol 99:2925–2938

    Article  CAS  PubMed  Google Scholar 

  15. Looser V, Bruhlmann B, Bumbak F et al (2015) Cultivation strategies to enhance productivity of Pichia pastoris: a review. Biotechnol Adv 33:1177–1193

    Article  CAS  PubMed  Google Scholar 

  16. Adelantado N, Tarazona P, Grillitsch K et al (2017) The effect of hypoxia on the lipidome of recombinant Pichia pastoris. Microb Cell Fact 16:86

    Article  PubMed Central  PubMed  Google Scholar 

  17. Pepper LR, Cho YK, Boder ET et al (2008) A decade of yeast surface display technology: where are we now? Comb Chem High Throughput Screen 11:127–134

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Pan X, Xu L, Xiao X et al (2012) Efficient display of active Geotrichum sp. Lipase on Pichia pastoris cell wall and its applications as a whole-cell biocatalyst to enrich EPA and DHA in fish oil. J Agric Food Chem 60:9673–9679

    Article  CAS  PubMed  Google Scholar 

  19. Pittet M, Conzelmann A (2007) Biosynthesis and function of GPI proteins in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1771:405–420

    Article  CAS  PubMed  Google Scholar 

  20. Zhang L, Liang S, Zhou X (2013) Screening for glycosylphosphatidylinositol-modified cell wall proteins in Pichia pastoris and their recombinant expression on the cell surface. Appl Environ Microbiol 79(18):5519–5526

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Li W, Shi H, Ding H et al (2015) Cell surface display and characterization of Rhizopus oryzae lipase in Pichia pastoris using Sed1p as an anchor protein. Curr Microbiol 71:150–155

    Article  CAS  PubMed  Google Scholar 

  22. De Schutter K, Lin Y, Tiels P et al (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566

    Article  CAS  PubMed  Google Scholar 

  23. Mattanovich D, Garf AB, Stadlmann J et al (2009) Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact 8:29

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Bolten CJ, Kiefer P, Letisse F et al (2007) Sampling for metabolome analysis of microorganisms. Anal Chem 79:3843–3849

    Article  CAS  PubMed  Google Scholar 

  25. Van Gulik WM (2010) Fast sampling for quantitative microbial metabolomics. Curr Opin Bioechnol 21:27–34

    Article  CAS  Google Scholar 

  26. Cascante M, Marin S (2008) Metabolomics and fluxomics approaches. Essays Biochem 45:67–81

    Article  CAS  PubMed  Google Scholar 

  27. Sauer U (2006) Metabolomic networks in motion 13C-based flux analysis. Mol Syst Biol 2:62

    Article  PubMed Central  PubMed  Google Scholar 

  28. Wiechert W, Nök F (2013) Isotopically non-stationary metabolic flux analysis: complex yet highly informative. Curr Opin Biotechnol 24:979–986

    Article  CAS  PubMed  Google Scholar 

  29. Cos O, Ramón R, Montesinos JL (2006) A simple model-based control for Pichia pastoris allows a more efficient heterologous protein production bioproces. Biotechnol Bioeng 95(1):145–154

    Article  CAS  PubMed  Google Scholar 

  30. Barrigon JM, Valero F, Montesinos JL (2015) A macrokinetic model-based comparative meta analysis of recombinant protein production by Pichia pastoris under AOX1 promoter. Biotechnol Bioeng 112(6):1132–1145

    Article  CAS  PubMed  Google Scholar 

  31. Looser V, Lüthy D, Straumann M et al (2017) Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris functional characterization of a novel promoter. Appl Microbiol Biotechnol 101(3):3163–3176

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Spadiut O, Rittmann S, Dietzsch C et al (2013) Dynamic process conditions in bioprocess development. Eng Life Sci 13(1):88–101

    Article  CAS  Google Scholar 

  33. Spadiut O, Zalai D, Dietzsch C et al (2014) Quantitative comparison of dynamic physiological feeding profiles for recombinant protein production with Pichia pastoris. Bioprocess Biosyst Eng 37(6):1163–1172

    Article  CAS  PubMed  Google Scholar 

  34. Spadiut O, Zalai D, Dietzsch C, Herwig C et al (2014) Determination of a dynamic feeding strategy for recombinant Pichia pastoris strains. Methods Mol Biol 1152:185–194

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Khatri NN, Hoffmann F (2006) Impact of methanol concentration on secreted protein production in oxygen-limited cultures of recombinant Pichia pastoris. Biotechnol Bioeng 93(5):871–879

    Article  CAS  PubMed  Google Scholar 

  36. Jordá J, de Jesús SS, Peltier P et al (2014) Metabolic flux analysis of recombinant Pichia pastoris growing on different glycerol/methanol mixtures by iterative fitting of NMR 13C-labelling data from proteinogenic amino acids. N Biotechnol 31:120–132

    Article  CAS  PubMed  Google Scholar 

  37. Çalik P, Ata OH, Günes A et al (2015) Recombinant protein production in Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter: from carbon source metabolism to bioreactor operational parameters. Biochem Eng J 95:1611–1619

    Article  CAS  Google Scholar 

  38. Buchetics M, Dragosits M, Maurer C et al (2011) Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth. Biotechnol Bioeng 108:2403–2412

    Article  CAS  PubMed  Google Scholar 

  39. Rebnegger C, Graf AB, Valli MG et al (2014) In Pichia pastoris growth rate regulates protein synthesis and secretion, mating and stress response. Biotechnol J 9:511–525

    Article  CAS  PubMed  Google Scholar 

  40. García-Ortega X, Ferrer P, Montesinos JL et al (2013) Fed-batch operational strategies for recombinant protein production with Pichia pastoris using the constitutive GAP promoter. Biochem Eng J 79:172–181

    Article  CAS  Google Scholar 

  41. Günes H, Çalik P (2016) Oxygen transfer as a tool for fine-tuning recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter. Bioprocess Biosyst Eng 39(7):1061–1072

    Article  CAS  PubMed  Google Scholar 

  42. Kern A, Hartner M, et a FJ (2007) Pichia pastoris just in time alternative respiration. Microbiology 153:1250–1260

    Article  CAS  PubMed  Google Scholar 

  43. García-Ortega X, Adelantado N, Ferrer P et al (2016) a step forward to improve recombinant protein production in Pichia pastoris: from specific growth rate effect on protein secretion to carbon-starving conditions as advanced strategy. Process Biochem 51:681–691

    Article  CAS  Google Scholar 

  44. García-Ortega X, Valero F, Montesinos-Seguí JL (2017) Physiological state as transferable operating criterion to improve recombinant protein production in Pichia pastoris through oxygen limitation. J Chem Technol Biotechnol 92:2573–2582

    Article  CAS  Google Scholar 

  45. De Almeida JRM, de Moraes LMP, Torres FAG (2005) Molecular characterization of the 3-phosphoglycerate kinase gene (PGK1) from the methylotrophic yeast Pichia pastoris. Yeast 22:725–737

    Article  CAS  PubMed  Google Scholar 

  46. Robert JM, Lattari FS, Machado AC et al (2017) Production of recombinant lipase B from Candida antarctica in Pichia pastoris under the control of the promoter PGK using crude glycerol from biodiesel production as carbon source. Biochem Eng J 118:123–111

    Article  CAS  Google Scholar 

  47. Samuel P, Vadhana AKM, Kamatchi R et al (2013) Effect of molecular chaperones on the expression of Candida antarctica lipase B in Pichia pastoris. Microbiol Res 168:615–620

    Article  CAS  PubMed  Google Scholar 

  48. Yang J-K, Liu L-Y, Dai J-H et al (2013) de novo design and synthesis of Candida antarctica lipase B gene an α-factor leads to high level expression in Pichia pastoris. Plos one 8:e53939

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Li X, He X, Li Z et al (2013) Combined strategies for improving the production of recombinant Rhizopus oryzae lipase in Pichia pastoris. Bioresources 8(2):2867–2880

    Google Scholar 

  50. Eom GT, Lee SH, Song BK et al (2013) High-level extracellular production and characterization of Candida antarctica lipase B in Pichia pastoris. J Biosci Bioeng 116(2):165–170

    Article  CAS  PubMed  Google Scholar 

  51. Cámara E, Landes N, Albiol J et al (2017) Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris. Sci Rep 7:44302 www.nature.con/scientificreports

    Article  PubMed Central  PubMed  Google Scholar 

  52. Jordá J, Jouthen P, Cámara E et al (2012) Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose: methanol mixtures. Microb Cell Fact 11:57

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Jordá J, Suarez C, Carnicer M et al (2013) Glucose-methanol co-utilization in Pichia pastoris studied by metabolomics and instationary 13C flux analysis. BMC Syst Biol 7:17

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  54. Jordá J, Cueto-Rojas H, Carnicer M et al (2013) Quantitative metabolomics and instationary 13C-metabolic flux analysis reveals impact of recombinant protein production on trehalose and energy metabolism in Pichia pastoris. Metabolites 4:281–299

    Article  CAS  Google Scholar 

  55. Carnicer M, ten Pierik A, van Dam J et al (2012) Quantitative metabolomics analysis of amino acid metabolism in recombinant Pichia pastoris under different oxygen availability conditions. Microb Cell Fact 11:83

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  56. Carnicer M, Canelas AB, ten Pierik A et al (2012) Development of quantitative metabolomics for Pichia pastoris. Metabolomics 8:284–298

    Article  CAS  PubMed  Google Scholar 

  57. Barrigon JM, Montesinos JL, Valero F (2013) Searching the best operational strategies for Rhizopus oryzae lipase production in Pichia pastoris Mut+ phenotype: methanol limited or non-limited cultures? Biocehm Eng J 75:47–54

    Article  CAS  Google Scholar 

  58. Canales C, Altamirano C, Berrios J (2015) Effect of dilution rate and methanol-glycerol mixed feeding on heterologous Rhizopus oryzae lipase production with Pichia pastoris Mut+ phenotype in continuous culture. Biotechnol Prog 31(3):707–714

    Article  CAS  PubMed  Google Scholar 

  59. Ponte X, Montesinos-Seguí JL, Valero F (2016) Bioprocess efficiency in Rhizopus oryzae lipase production by Pichia pastoris under the control of PAOX1 is oxygen tension dependent. Process Biochem 51:1954–1193

    Article  CAS  Google Scholar 

  60. Wang JR, Li YY, Xu SD et al (2014) High-level expression of pro-form lipase from Rhizopus oryzae in Pichia pastoris and its purification and characterization. Int J Mol Sci 15:203–217

    Article  CAS  Google Scholar 

  61. Beer HD, Wohlfahrt G, Schmid RD et al (1996) The folding and activity of the extracellular lipase of Rhizopus oryzae are modulated by a prosequence. Biochem J 319:351–359

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  62. Xu L, Liu Z, Wang G et al (2016) Overexpression of Candida rugosa lipase Lip1 via combined strategies in Pichia pastoris. Enzyme Microb Technol 82:115–124

    Article  CAS  Google Scholar 

  63. Lotti M, Grandori D, Fusseti F et al (1993) Cloning and analysis of Candida cylindracea lipase sequences. Gene 124:45–55

    Article  CAS  PubMed  Google Scholar 

  64. Ferrer P, Alarcón M, Ramón R et al (2009) Recombinant Candida rugose LIP2 expression in P. pastoris under the control of the AOX1 promoter. Biochem Eng J 46:271–277

    Article  CAS  Google Scholar 

  65. Kuo TC, Shaw JF, Lee GC (2015) Improvement in the secretory expression of recombinant Candida rugosa lipase in Pichia pastoris. Process Biochem 50:2137–2143

    Article  CAS  Google Scholar 

  66. Qin L-N, Cai F-R, Huang Z-B et al (2012) Improved production of heterologous lipase in Trichoderma reesei by RNAi mediated gene silencing of an endogenic highly expressed gen. Bioresour Technol 109:116–122

    Article  CAS  PubMed  Google Scholar 

  67. Stergiou PY, Foukis A, Filippou M et al (2013) Advances in lipase-catalyzed esterification reactions. Biotechnol Adv 31:1846–1859

    Article  CAS  PubMed  Google Scholar 

  68. Zhang X, Li X, Xia L (2015) Heterologous expression of an alkali and thermotolerant lipase from Talaromyces thermophilus in Trichoderma reesei. Appl Biochem Biotechnol 176:1722–1735

    Article  CAS  PubMed  Google Scholar 

  69. Zhang X, Xia L (2017) Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus. J Ind Microbiol Biotechnol 44(3):377–385

    Article  CAS  PubMed  Google Scholar 

  70. Kunigo M, Buerth C, Tielker D et al (2013) Heterologous protein secretion by Candida utilis. Appl Microbiol Biotechnol 97(16):7357–7368

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the project CTQ2016-74959-R (MINECO/FEDER,UE). The group is a member of 2014-SGR-452 and the Reference Network in Biotechnology (XRB) (Generalitat de Catalunya).

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Authors and Affiliations

  1. Departament d’Enginyeria Química, Biològica i Ambiental. EE, Universitat Autònoma de Barcelona, Barcelona, Spain

    Francisco Valero

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  1. Francisco Valero
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Correspondence to Francisco Valero .

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  1. CIATEJ, Guadalajara, Jalisco, Mexico

    Georgina Sandoval

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Valero, F. (2018). Recent Advances in Pichia pastoris as Host for Heterologous Expression System for Lipases: A Review. In: Sandoval, G. (eds) Lipases and Phospholipases. Methods in Molecular Biology, vol 1835. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8672-9_11

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  • DOI: https://doi.org/10.1007/978-1-4939-8672-9_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8671-2

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