Skip to main content
SpringerLink
Log in

Engineering of Promoters for Gene Expression in Pichia pastoris

  • Protocol
  • First Online:
Yeast Metabolic Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2513))

Abstract

The availability of exceptionally strong and tightly regulated promoters is a key feature of Komagataella phaffii (syn. Pichia pastoris), a widely applied yeast expression system for heterologous protein production. Most commonly, the methanol-inducible promoter of the alcohol oxidase 1 gene (PAOX1) and the constitutive promoter of the glyceraldehyde 3 phosphate dehydrogenase gene (PGAP) have been used. Recently, also promising novel constitutive (PGCW14), regulated (PGTH1, PCAT1), and bidirectional promoters (histone promoters and synthetic hybrid variants) have been reported.

As natural promoters showed so far limited tunability of expression levels and regulatory profiles, various promoter engineering efforts have been undertaken for P. pastoris . PAOX1, PDAS2, PGAP, and PGCW14 have been engineered by systematic deletion studies or random mutagenesis of upstream regulatory sequences. New engineering strategies have focused on PAOX1 core promoter modifications by random or rational approaches and transcriptional regulatory circuits to render PAOX1 independent of methanol induction. These promoter engineering efforts in P. pastoris have resulted in improved, sequence-diversified synthetic promoter variants allowing coordinated fine-tuning of gene expression for a multitude of biotechnological applications.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Blazeck J, Alper HS (2013) Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J 8:46–58. https://doi.org/10.1002/biot.201200120

    Article  CAS  PubMed  Google Scholar 

  2. Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54:741–750

    Article  CAS  Google Scholar 

  3. Hartner FS, Glieder A (2006) Regulation of methanol utilisation pathway genes in yeasts. Microb Cell Factories 5:39–59. https://doi.org/10.1186/1475-2859-5-39

    Article  CAS  Google Scholar 

  4. Yurimoto H, Oku M, Sakai Y (2011) Yeast methylotrophy: metabolism, gene regulation and peroxisome homeostasis. Int J Microbiol 2011:101298. https://doi.org/10.1155/2011/101298

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Vogl T, Hartner FS, Glieder A (2013) New opportunities by synthetic biology for biopharmaceutical production in Pichia pastoris. Curr Opin Biotechnol 24:1094–1101. https://doi.org/10.1016/j.copbio.2013.02.024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lin-Cereghino J, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66

    Article  Google Scholar 

  7. Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. Mol Biotechnol 16:23–52. https://doi.org/10.1385/MB:16:1:23

    Article  CAS  PubMed  Google Scholar 

  8. Tschopp JF, Brust PF, Cregg JM et al (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15:3859–3876

    Article  CAS  Google Scholar 

  9. Ruth C, Zuellig T, Mellitzer A et al (2010) Variable production windows for porcine trypsinogen employing synthetic inducible promoter variants in Pichia pastoris. Syst Synth Biol 4:181–191. https://doi.org/10.1007/s11693-010-9057-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hohenblum H, Gasser B, Maurer M et al (2004) Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris. Biotechnol Bioeng 85:367–375. https://doi.org/10.1002/bit.10904

    Article  CAS  PubMed  Google Scholar 

  11. Gasser B, Saloheimo M, Rinas U et al (2008) Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview. Microb Cell Factories 7:11. https://doi.org/10.1186/1475-2859-7-11

    Article  CAS  Google Scholar 

  12. Mattanovich D, Gasser B, Hohenblum H, Sauer M (2004) Stress in recombinant protein producing yeasts. J Biotechnol 113:121–135. https://doi.org/10.1016/j.jbiotec.2004.04.035

    Article  CAS  PubMed  Google Scholar 

  13. Ruth C, Glieder A (2010) Perspectives on synthetic promoters for biocatalysis and biotransformation. Chembiochem 11:761–765. https://doi.org/10.1002/cbic.200900761

    Article  CAS  PubMed  Google Scholar 

  14. Venter M (2007) Synthetic promoters: genetic control through cis engineering. Trends Plant Sci 12:118–124. https://doi.org/10.1016/j.tplants.2007.01.002

    Article  CAS  PubMed  Google Scholar 

  15. Blazeck J, Garg R, Reed B, Alper HS (2012) Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng 109:2884–2895. https://doi.org/10.1002/bit.24552

    Article  CAS  PubMed  Google Scholar 

  16. Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci U S A 102:12678–12683. https://doi.org/10.1073/pnas.0504604102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Blazeck J, Liu L, Redden H, Alper H (2011) Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol 77:7905–7914. https://doi.org/10.1128/AEM.05763-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Raschmanová H, Weninger A, Glieder A et al (2018) Implementing CRISPR-Cas technologies in conventional and non-conventional yeasts: current state and future prospects. Biotechnol Adv 36:641–665. https://doi.org/10.1016/j.biotechadv.2018.01.006

    Article  CAS  PubMed  Google Scholar 

  19. Weninger A, Killinger M, Vogl T (2016) Key methods for synthetic biology: genome engineering and DNA assembly. In: Glieder A, Kubicek CP, Mattanovich D et al (eds) Synthetic biology. Springer International Publishing, Cham, pp 101–141

    Chapter  Google Scholar 

  20. Weninger A, Glieder A, Vogl T (2015) A toolbox of endogenous and heterologous nuclear localization sequences for the methylotrophic yeast Pichia pastoris. FEMS Yeast Res 15:fov082. https://doi.org/10.1093/femsyr/fov082

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vogl T, Gebbie L, Palfreyman RW, Speight R (2018) Effect of plasmid design and type of integration event on recombinant protein expression in Pichia pastoris. Appl Environ Microbiol 84:AEM.02712–AEM.02717. https://doi.org/10.1128/AEM.02712-17

    Article  Google Scholar 

  22. Krivoruchko A, Siewers V, Nielsen J (2011) Opportunities for yeast metabolic engineering: lessons from synthetic biology. Biotechnol J 6:262–276. https://doi.org/10.1002/biot.201000308

    Article  CAS  PubMed  Google Scholar 

  23. Hartner FS, Ruth C, Langenegger D et al (2008) Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 36:e76. https://doi.org/10.1093/nar/gkn369

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Curran KA, Crook NC, Karim AS et al (2014) Design of synthetic yeast promoters via tuning of nucleosome architecture. Nat Commun 5:4002. https://doi.org/10.1038/ncomms5002

    Article  CAS  PubMed  Google Scholar 

  25. Blount BA, Weenink T, Vasylechko S, Ellis T (2012) Rational diversification of a promoter providing fine-tuned expression and orthogonal regulation for synthetic biology. PLoS One 7:e33279. https://doi.org/10.1371/journal.pone.0033279

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Teo WS, Chang MW (2014) Development and characterization of AND-gate dynamic controllers with a modular synthetic GAL1 core promoter in Saccharomyces cerevisiae. Biotechnol Bioeng 111:144–151. https://doi.org/10.1002/bit.25001

    Article  CAS  PubMed  Google Scholar 

  27. Xuan Y, Zhou X, Zhang W et al (2009) An upstream activation sequence controls the expression of AOX1 gene in Pichia pastoris. FEMS Yeast Res 9:1271–1282. https://doi.org/10.1111/j.1567-1364.2009.00571.x

    Article  CAS  PubMed  Google Scholar 

  28. Berg L, Strand TA, Valla S, Brautaset T (2013) Combinatorial mutagenesis and selection to understand and improve yeast promoters. Biomed Res Int 2013:926985. https://doi.org/10.1155/2013/926985

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Vogl T, Ruth C, Pitzer J et al (2014) Synthetic core promoters for Pichia pastoris. ACS Synth Biol 3:188–191. https://doi.org/10.1021/sb400091p

    Article  CAS  PubMed  Google Scholar 

  30. Qin X, Qian J, Yao G et al (2011) GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl Environ Microbiol 77:3600–3608. https://doi.org/10.1128/AEM.02843-10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang X, Zhang X, Liang S et al (2013) Key regulatory elements of a strong constitutive promoter, P GCW14, from Pichia pastoris. Biotechnol Lett 35:2113–2119. https://doi.org/10.1007/s10529-013-1312-5

    Article  CAS  PubMed  Google Scholar 

  32. Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnol 30:385–404. https://doi.org/10.1016/j.nbt.2012.11.010

    Article  CAS  Google Scholar 

  33. Ellis SB, Brust PF, Koutz PJ et al (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol 5:1111–1121

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Waterham HR, Digan ME, Koutz PJ et al (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44

    Article  CAS  Google Scholar 

  35. Koutz P, Davis GR, Stillman C et al (1989) Structural comparison of the Pichia pastoris alcohol oxidase genes. Yeast 5:167–177. https://doi.org/10.1002/yea.320050306

    Article  CAS  PubMed  Google Scholar 

  36. Cregg JM, Madden KR, Barringer KJ et al (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9:1316–1323. https://doi.org/10.1128/MCB.9.3.1316.Updated

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hasslacher M, Schall M, Hayn M et al (1997) High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts. Protein Expr Purif 11:61–71. https://doi.org/10.1006/prep.1997.0765

    Article  CAS  PubMed  Google Scholar 

  38. Werten MW, van den Bosch TJ, Wind RD et al (1999) High-yield secretion of recombinant gelatins by Pichia pastoris. Yeast 15:1087–1096. https://doi.org/10.1002/(SICI)1097-0061(199908)15:11<1087::AID-YEA436>3.0.CO;2-F

    Article  CAS  PubMed  Google Scholar 

  39. Couderc R, Baratti J (1980) Oxidation of methanol by the yeast, Pichia pastoris. Purification and properties of the alcohol oxidase. Agric Biol Chem 44:2279–2289

    CAS  Google Scholar 

  40. Lin-Cereghino GP, Godfrey L, de la Cruz BJ et al (2006) Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris. Mol Cell Biol 26:883–897. https://doi.org/10.1128/MCB.26.3.883-897.2006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kranthi BV, Kumar R, Kumar NV et al (2009) Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris. Biochim Biophys Acta 1789:460–468. https://doi.org/10.1016/j.bbagrm.2009.05.004

    Article  CAS  PubMed  Google Scholar 

  42. Kranthi BV, Kumar HRV, Rangarajan PN (2010) Identification of Mxr1p-binding sites in the promoters of genes encoding dihydroxyacetone synthase and peroxin 8 of the methylotrophic yeast Pichia pastoris. Yeast 27:705–711. https://doi.org/10.1002/yea.1766

    Article  CAS  PubMed  Google Scholar 

  43. Kumar NV, Rangarajan PN (2012) The zinc finger proteins Mxr1p and repressor of phosphoenolpyruvate carboxykinase (ROP) have the same DNA binding specificity but regulate methanol metabolism antagonistically in Pichia pastoris. J Biol Chem 287:34465–34473. https://doi.org/10.1074/jbc.M112.365304

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Parua PK, Ryan PM, Trang K, Young ET (2012) Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction. Mol Microbiol 85:282–298. https://doi.org/10.1111/j.1365-2958.2012.08112.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kim S, Warburton S, Boldogh I et al (2013) Regulation of alcohol oxidase 1 (AOX1) promoter and peroxisome biogenesis in different fermentation processes in Pichia pastoris. J Biotechnol 166:174–181. https://doi.org/10.1016/j.jbiotec.2013.05.009

    Article  CAS  PubMed  Google Scholar 

  46. Polupanov AS, Nazarko VY, Sibirny AA (2012) Gss1 protein of the methylotrophic yeast Pichia pastoris is involved in glucose sensing, pexophagy and catabolite repression. Int J Biochem Cell Biol 44:1906–1918. https://doi.org/10.1016/j.biocel.2012.07.017

    Article  CAS  PubMed  Google Scholar 

  47. Polupanov AS, Sibirny AA (2013) Cytoplasmic extension peptide of Pichia pastoris glucose sensor Gss1 is not compulsory for glucose signalling. Cell Biol Int. https://doi.org/10.1002/cbin.10189

  48. Kumar NV, Rangarajan PN (2011) Catabolite repression of phosphoenolpyruvate carboxykinase by a zinc finger protein under biotin- and pyruvate carboxylase-deficient conditions in Pichia pastoris. Microbiology (Reading) 157:3361–3369. https://doi.org/10.1099/mic.0.053488-0

    Article  CAS  Google Scholar 

  49. Liu H, Tan X, Russell KA et al (1995) PER3, a gene required for peroxisome biogenesis in Pichia pastoris, encodes a peroxisomal membrane protein involved in protein import. J Biol Chem 270:10940–10951

    Article  CAS  Google Scholar 

  50. Sears IB, O’Connor J, Rossanese OW, Glick BS (1998) A versatile set of vectors for constitutive and regulated gene expression in Pichia pastoris. Yeast 14:783–790. https://doi.org/10.1002/(SICI)1097-0061(19980615)14:8<783::AID-YEA272>3.0.CO;2-Y

    Article  CAS  PubMed  Google Scholar 

  51. Vogl T, Sturmberger L, Kickenweiz T et al (2016) A toolbox of diverse promoters related to methanol utilization: functionally verified parts for heterologous pathway expression in Pichia pastoris. ACS Synth Biol 5:172–186. https://doi.org/10.1021/acssynbio.5b00199

    Article  CAS  PubMed  Google Scholar 

  52. Ahn J, Hong J, Lee H et al (2007) Translation elongation factor 1-alpha gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. Appl Microbiol Biotechnol 74:601–608. https://doi.org/10.1007/s00253-006-0698-6

    Article  CAS  PubMed  Google Scholar 

  53. Stadlmayr G, Mecklenbräuker A, Rothmüller M et al (2010) Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 150:519–529. https://doi.org/10.1016/j.jbiotec.2010.09.957

    Article  CAS  PubMed  Google Scholar 

  54. 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. https://doi.org/10.1002/yea.1243

    Article  CAS  PubMed  Google Scholar 

  55. Shen S, Sulter G, Jeffries TW, Cregg JM (1998) A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene 216:93–102

    Article  CAS  Google Scholar 

  56. Ahn J, Hong J, Park M et al (2009) Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter. Appl Microbiol Biotechnol 75:3528–3534. https://doi.org/10.1128/AEM.02913-08

    Article  CAS  Google Scholar 

  57. Delic M, Mattanovich D, Gasser B (2013) Repressible promoters -- a novel tool to generate conditional mutants in Pichia pastoris. Microb Cell Factories 12:6. https://doi.org/10.1186/1475-2859-12-6

    Article  CAS  Google Scholar 

  58. Koller A, Valesco J, Subramani S (2000) The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris. Yeast 16:651–656. https://doi.org/10.1002/(SICI)1097-0061(200005)16:7<651::AID-YEA580>3.0.CO;2-F

    Article  CAS  PubMed  Google Scholar 

  59. Paddon CJ, Westfall PJ, Pitera DJ et al (2013) High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496:528–532. https://doi.org/10.1038/nature12051

    Article  CAS  PubMed  Google Scholar 

  60. Zhang A-L, Luo J-X, Zhang T-Y et al (2009) Recent advances on the GAP promoter derived expression system of Pichia pastoris. Mol Biol Rep 36:1611–1619. https://doi.org/10.1007/s11033-008-9359-4

    Article  CAS  PubMed  Google Scholar 

  61. Baumann K, Maurer M, Dragosits M et al (2008) Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 100:177–183. https://doi.org/10.1002/bit.21763

    Article  CAS  PubMed  Google Scholar 

  62. Kern A, Hartner FS, Freigassner M et al (2007) Pichia pastoris “just in time” alternative respiration. Microbiology (Reading) 153:1250–1260. https://doi.org/10.1099/mic.0.2006/001404-0

    Article  CAS  Google Scholar 

  63. Liang S, Zou C, Lin Y et al (2013) Identification and characterization of P GCW14 : a novel, strong constitutive promoter of Pichia pastoris. Biotechnol Lett 35:1865–1871. https://doi.org/10.1007/s10529-013-1265-8

    Article  CAS  PubMed  Google Scholar 

  64. Liang S, Wang B, Pan L et al (2012) Comprehensive structural annotation of Pichia pastoris transcriptome and the response to various carbon sources using deep paired-end RNA sequencing. BMC Genomics 13:738. https://doi.org/10.1186/1471-2164-13-738

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Prielhofer R, Maurer M, Klein J et al (2013) Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Factories 12:5. https://doi.org/10.1186/1475-2859-12-5

    Article  CAS  Google Scholar 

  66. Hobl B, Hock B, Schneck S et al (2013) Bacteriophage T7 RNA polymerase-based expression in Pichia pastoris. Protein Expr Purif 92:100–104. https://doi.org/10.1016/j.pep.2013.09.004

    Article  CAS  PubMed  Google Scholar 

  67. Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83:8122–8126

    Article  CAS  Google Scholar 

  68. Yu J-Y, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A 99:6047–6052. https://doi.org/10.1073/pnas.092143499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Khalil AS, Lu TK, Bashor CJ et al (2012) A synthetic biology framework for programming eukaryotic transcription functions. Cell 150:647–658. https://doi.org/10.1016/j.cell.2012.05.045

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Alper H, Moxley J, Nevoigt E et al (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314:1565–1568. https://doi.org/10.1126/science.1131969

    Article  CAS  PubMed  Google Scholar 

  71. Inan M (2000) Studies on the alcohol oxidase (AOX1) promoter of Pichia pastoris. Dissertation, University of Nebraska, Lincoln, Nebraska. 101 pp

    Google Scholar 

  72. Tsutsumi N, Takagi S, inventors; Novozymes A/S, Bagsvaerd (DK), assignee. Pichia pastoris DAS promoter variants. United States patent application publication US 2011/0129874 A1. 2011 Jun 2

    Google Scholar 

  73. Allison LA (2007) Transcription in eukaryotes. In: Allison LA (ed) Fundamental molecular biology. Blackwell, London, pp 312–391

    Google Scholar 

  74. Hahn S, Young ET (2011) Transcriptional regulation in Saccharomyces cerevisiae: transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators. Genetics 189:705–736. https://doi.org/10.1534/genetics.111.127019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Lubliner S, Keren L, Segal E (2013) Sequence features of yeast and human core promoters that are predictive of maximal promoter activity. Nucleic Acids Res 41:5569–5581. https://doi.org/10.1093/nar/gkt256

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Ohi H, Miura M, Hiramatsu R, Ohmura T (1994) The positive and negative cis-acting elements for methanol regulation in the Pichia pastoris AOX2 gene. Mol Gen Genet 243:489–499

    Article  CAS  Google Scholar 

  77. Inan M, Meagher M, Benson AK, inventors; The Board of Regents of the University of Nebraska (Lincoln, NE), assignee (2004) Alcohol oxidase 1 regulatory nucleotide sequences for heterologous gene expression in yeast. United States patent US 6,699,691 B2. 2 Mar 2004

    Google Scholar 

  78. Inan M, Meagher MM (2001) Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 92:585–589

    Article  CAS  Google Scholar 

  79. Mellitzer A, Weis R, Glieder A, Flicker K (2012) Expression of lignocellulolytic enzymes in Pichia pastoris. Microb Cell Factories 11:61. https://doi.org/10.1186/1475-2859-11-61

    Article  CAS  Google Scholar 

  80. Higuchi R, Krummel B, Saiki RK (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16:7351–7367

    Article  CAS  Google Scholar 

  81. Bryksin A, Matsumura I (2013) Overlap extension PCR cloning. Methods Mol Biol 1073:31–42. https://doi.org/10.1007/978-1-62703-625-2_4

    Article  CAS  PubMed  Google Scholar 

  82. Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950. https://doi.org/10.1038/nbt.1568

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Portela RMC, Vogl T, Ebner K et al (2018) Pichia pastoris alcohol oxidase 1 (AOX1) core promoter engineering by high resolution systematic mutagenesis. Biotechnol J 13:e1700340. https://doi.org/10.1002/biot.201700340

    Article  CAS  PubMed  Google Scholar 

  84. Lee CC, Williams TG, Wong DWS, Robertson GH (2005) An episomal expression vector for screening mutant gene libraries in Pichia pastoris. Plasmid 54:80–85. https://doi.org/10.1016/j.plasmid.2004.12.001

    Article  CAS  PubMed  Google Scholar 

  85. Daly R, Hearn MTW (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138. https://doi.org/10.1002/jmr.687

    Article  CAS  PubMed  Google Scholar 

  86. Cregg JM, Tolstorukov I, Kusari A et al (2009) Expression in the yeast Pichia pastoris. Methods Enzymol 463:169–189. https://doi.org/10.1016/S0076-6879(09)63013-5

    Article  CAS  PubMed  Google Scholar 

  87. Staley CA, Huang A, Nattestad M et al (2012) Analysis of the 5’ untranslated region (5’UTR) of the alcohol oxidase 1 (AOX1) gene in recombinant protein expression in Pichia pastoris. Gene 496:118–127. https://doi.org/10.1016/j.gene.2012.01.006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  88. Takagi S, Tsutsumi N, Terui Y, Kong XY, inventors; Novozymes A/S, Bagsvaerd (DK), assignee (2012) Method for methanol independent induction from methanol inducible promoters in Pichia. United States patent US 8,143,023. 28 June 2012

    Google Scholar 

  89. Vogl T, Sturmberger L, Fauland PC et al (2018) Methanol independent induction in Pichia pastoris by simple derepressed overexpression of single transcription factors. Biotechnol Bioeng 115:1037–1050. https://doi.org/10.1002/bit.26529

    Article  CAS  PubMed  Google Scholar 

  90. Shen W, Xue Y, Liu Y et al (2016) A novel methanol-free Pichia pastoris system for recombinant protein expression. Microb Cell Factories 15:178. https://doi.org/10.1186/s12934-016-0578-4

    Article  CAS  Google Scholar 

  91. Shen W, Kong C, Xue Y et al (2016) Kinase screening in Pichia pastoris identified promising targets involved in cell growth and alcohol oxidase 1 promoter (PAOX1) regulation. PLoS One 11:e0167766. https://doi.org/10.1371/journal.pone.0167766

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Wang J, Wang X, Shi L et al (2017) Methanol-independent protein expression by AOX1 promoter with trans-acting elements engineering and glucose-glycerol-shift induction in Pichia pastoris. Sci Rep 7:41850. https://doi.org/10.1038/srep41850

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  93. De Schutter K, Lin Y-C, Tiels P et al (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566. https://doi.org/10.1038/nbt.1544

    Article  CAS  PubMed  Google Scholar 

  94. Küberl A, Schneider J, Thallinger GG et al (2011) High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol 154:312–320. https://doi.org/10.1016/j.jbiotec.2011.04.014

    Article  CAS  PubMed  Google Scholar 

  95. Lelli KM, Slattery M, Mann RS (2012) Disentangling the many layers of eukaryotic transcriptional regulation. Annu Rev Genet 46:43–68. https://doi.org/10.1146/annurev-genet-110711-155437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Portela RMC, Vogl T, Kniely C et al (2017) Synthetic core promoters as universal parts for fine-tuning expression in different yeast species. ACS Synth Biol 6:471–484. https://doi.org/10.1021/acssynbio.6b00178

    Article  CAS  PubMed  Google Scholar 

  97. Vogl T, Kickenweiz T, Pitzer J et al (2018) Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun 9:3589. https://doi.org/10.1038/s41467-018-05915-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Vogl T, Ahmad M, Krainer FW et al (2015) Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris. Microb Cell Factories 14:103. https://doi.org/10.1186/s12934-015-0293-6

    Article  CAS  Google Scholar 

  99. Weninger A, Fischer JE, Raschmanová H et al (2018) Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers. J Cell Biochem 119:3183–3198. https://doi.org/10.1002/jcb.26474

    Article  CAS  PubMed  Google Scholar 

  100. Weninger A, Hatzl A-M, Schmid C et al (2016) Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. J Biotechnol 235:139–149. https://doi.org/10.1016/j.jbiotec.2016.03.027

    Article  CAS  PubMed  Google Scholar 

  101. Geier M, Fauland P, Vogl T, Glieder A (2015) Compact multi-enzyme pathways in P. pastoris. Chem Commun 51:1643–1646. https://doi.org/10.1039/C4CC08502G

    Article  CAS  Google Scholar 

  102. Krainer FW, Capone S, Jäger M et al (2015) Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris. Microb Cell Factories 14:4. https://doi.org/10.1186/s12934-014-0187-z

    Article  CAS  Google Scholar 

  103. Krainer FW, Gerstmann MA, Darnhofer B et al (2016) Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris. J Biotechnol 233:181–189. https://doi.org/10.1016/j.jbiotec.2016.07.012

    Article  CAS  PubMed  Google Scholar 

  104. Kickenweiz T, Glieder A, Wu JC (2017) Construction of a cellulose-metabolizing Komagataella phaffii (Pichia pastoris) by co-expressing glucanases and β-glucosidase. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-017-8656-z

  105. Rajamanickam V, Metzger K, Schmid C, Spadiut O (2017) A novel bi-directional promoter system allows tunable recombinant protein production in Pichia pastoris. Microb Cell Factories 16:152. https://doi.org/10.1186/s12934-017-0768-8

    Article  CAS  Google Scholar 

Download references

Acknowledgments

T.V. gratefully acknowledges support from the Austrian Science Fund (Erwin Schrödinger fellowship J 4256-B21).

Author information

Authors and Affiliations

  1. Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University Graz, Graz, Austria

    Thomas Vogl

Authors
  1. Thomas Vogl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Vogl .

Editor information

Editors and Affiliations

  1. Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

    Valeria Mapelli

  2. Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

    Maurizio Bettiga

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Vogl, T. (2022). Engineering of Promoters for Gene Expression in Pichia pastoris. In: Mapelli, V., Bettiga, M. (eds) Yeast Metabolic Engineering. Methods in Molecular Biology, vol 2513. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2399-2_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2399-2_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2398-5

  • Online ISBN: 978-1-0716-2399-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation