Abstract
The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system for recombinant protein production for a variety of applications. It grows rapidly on inexpensive media containing methanol, glucose, glycerol, or ethanol as a sole carbon source. P. pastoris makes many posttranslational modifications and produces recombinant proteins either intracellularly or extracellularly. Because of these properties, P. pastoris has become a highly preferred host organism for biotechnology, pharmaceutical industry, and researchers.
Recombinant protein production is usually performed under the control of the promoter of the alcohol oxidase gene I (AOX1). The AOX1 promoter is induced by methanol and repressed by glucose and ethanol. The regulation mechanisms of the AOX1 promoter have been studied in recent years. Another promoter used in recombinant protein production is derived from glyceraldehyde 3-phosphate dehydrogenase (GAP). It is a constitutive promoter. Recent literature showed that newly identified promoters of P. pastoris are promising as well, in addition to pAOX1 and pGAP.
In this chapter, the regulation mechanisms of inducible pAOX1 and constitutive pGAP promoters are discussed. In addition, here we present an overview about the novel ADH3 promoter and alternative promoters of P. pastoris.
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References
Cregg JM, Tschopp JF, Stillman C, Siegel R, Akong M, Craig WS, Buckholz RG, Madden KR, Kellaris A, Davis GR, Smiley BL, Cruze J, Torregrossa R, Veliçelebi G, Thıll GP (1987) High-level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Nat Biotechnol 5:479–485
Romanos M, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488
Kjeldsen T, Frost-Petterson A, Hach M (1999) Secretory expression and characterization of insulin in Pichia pastoris. Biotechnol Appl Biochem 29:79–86
Wang Y, Liang ZH, Zhang YS, Yao SY, Xu YG, Tang YH, Zhu SQ, Cui DF, Feng YM (2001) Human insulin from a precursor overexpressed in the methylotrophic yeast Pichia pastoris and a simple procedure for purifying the expression product. Biotechnol Bioeng 73:74–79
Pais JM, Varas L, Valde´ s J, Cabello C, Rodriguez L, Mansur M (2003) Modeling of mini-proinsulin production in Pichia pastoris using the AOX promoter. Biotechnol Lett 25:251–255
Gurramkonda C, Polez S, Skoko N, Adnan A, Gabel T, Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U (2010) Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. Microb Cell Factories 9(31):1–11
Raymond CK, Bukowski T, Holderman SD, Ching AFT, Vanaja E, Stamm MR (1998) Development of the methylotrophic yeast Pichia methanolica for the expression of the 65-kilodalton isoform of human glutamate decarboxylase. Yeast 14:11–23
Sakai Y, Rogi T, Takeuchi R, Kato N, Tani Y (1995) Expression of Saccharomyces adenylate kinase gene in Candida boidinii under the regulation of its alcohol oxidase promoter. Appl Microbiol Biotechnol 42:860–864
Sakai Y, Akiyama M, Kondoh H, Shibano Y, Kato N (1996) High-level secretion of fungal glucoamylase using the Candida boidinii gene expression system. Biochim Biophys Acta 1308:81–87
Weydemann U, Keup P, Piontek M, Strasser AWM, Schweden J, Gellissen G, Janowicz ZA (1995) High-level secretion hirudin by Hansenula polymorpha-authentic processing of three different preprohirudins. Appl Microbiol Biotechnol 44:377–385
Gellissen G, Melber K (1996) Methylotrophic yeast Hansenula polymorpha as production organism for recombinant pharmaceuticals. Drug Res 46:943–948
Hagenson MJ (1991) Production of recombinant proteins in the methylotrophic yeast Pichia pastoris. Bioprocess Technol 12:193–212
Lin-Cereghino J, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24:45–66
Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM (2005) Heterologous protein production using the Pichia pastoris expression system. Yeast 22:249–270
Cregg JM, Vedvick TS, Raschke WC (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology 11:905–910
Ahmad M, Hirz M, Pichler H, Schwab H (2014) Protein expression in P. pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98:5301–5317
Weinhandl K, Winkler M, Glieder A, Camattari A (2014) Carbon source dependent promoters in yeasts. Microb Cell Factories 13:5
Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnol 30(4):386–404
Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in P. pastoris. Nucleic Acids Res 15:3859–3876
Ellis SB, Brust PF, Koutz PJ, Waters AF, Harpold MM, Gingeras TR (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol Cell Biol 5:1111–1121
Karaoglan M, Erden Karaoglan F, Inan M (2016) Comparison of ADH3 promoter with commonly used promoters for recombinant protein production in Pichia pastoris. Protein Expr Purif 121:112–117
Küberl A, Schneider J, Thallinger GG, Ander I, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, Puhler A, Schwab H, Glieder A, Pichler H (2011) High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol 154:312–320
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
Menendez J, Valdes I, Cabrera N (2003) The ICL1 gene of Pichia pastoris, transcriptional regulation and use of its promoter. Yeast 20:1097–1108
Liu B, Zhang Y, Zhang X, Yan C, Zhang Y, Xu X, Zhang W (2016) Discovery of a rhamnose utilization pathway and rhamnose-inducible promoters in Pichia pastoris. Sci Rep 6:27352. https://doi.org/10.1038/srep27352
Stadlmayr G, Mecklenbraüker A, Rothmüller M, Maurer M, Sauer M, Mattanovich D, Gasser B (2010) Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol 150:519–529
Landes N, Gasser B, Vorauer-Uhl K, Lhota G, Mattanovich D, Maurer M (2016) The vitamin-sensitive promoter PTHI11 enables pre-defined autonomous induction of recombinant protein production in Pichia pastoris. Biotechnol Bioeng 113(12):2633–2643
Prielhofer R, Maurer M, Klein J, Wenger J, Kiziak C, Gasser B, Mattanovich D (2013) Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Factories 12:5
Koller A, Valesco J, Subramani S (2000) The CUP1 promoter of Saccharomyces cerevisiae is inducible by copper in Pichia pastoris. Yeast 16:651–656
Kim S-II, Ha B-S, Kim M-S, Park M, Ro H-S (2016) Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in Pichia pastoris. Biotechnol Bioprocess Eng 21:53–59
Waterham HR, Digan ME, Koutz PJ, Lair SV, Cregg JM (1997) Isolation of the Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase gene and regulation and use of its promoter. Gene 186:37–44
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
Ahn J, Hong J, Lee H, Park M, Lee E, Kim C, Choi E, Jung J (2007) Translation elongation factor 1-alpha gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter. Appl Microbiol Biotechnol 74(3):601–608
Liang S, Zou C, Lin Y, Zhang X, Ye Y (2013) Identification and characterization of P GCW14: a novel, strong constitutive promoter of Pichia pastoris. Biotechnol Lett 35(11):1865–1871
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
Inan M, Meagher M (2001) The effect of ethanol and acetate on protein expression in Pichia pastoris. J Biosci Bioeng 92:337–341
Inan M, Meagher M (2001) Non-repressing carbon sources for alcohol oxidase (AOX1) promoter of Pichia pastoris. J Biosci Bioeng 92:585–589
Eggeling L, Sahm H (1980) Direct enzymatic assay for alcohol oxidase, alcohol dehydrogenase, and formaldehyde dehydrogenase in colonies of Hansenula polymorpha. Appl Environ Microbiol 39:268–269
Bruinenberg PG, Veenhuis M, Van Dijken JP, Duine JA, Harder W (1982) A quantitative analysis of selective inactivation of peroxisomal enzymes in the yeast, Hansenula polymorpha, by high-performance liquid chromatography. FEMS Microbiol Lett 15:45–50
Veenhuis M, Van Dijken JP, Harder W (1983) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol 24:1–82
Cregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9:1316–1323
Nakagawa T, Mizumura T, Mukaiyama H, Miyaji T, Yurimoto H, Kato N (2002) Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica. Yeast 19:1067–1073
Inan M (2000) Studies on the alcohol oxidase (AOX1) promoter of Pichia pastoris. PhD thesis, University of Nebraska, 98 p
Hartner FS, Ruth C, Langeneegger D, Johnson SN, Hyka P, Lin-Cereghino GP, Lin-Cereghino J, Kovar K, Cregg JM, Glieder A (2008) Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res 36(12):e76
Xuan Y, Zhou X, Zhang W, Zhang X, Song Z, Zhang Y (2009) An upstream activation sequence controls the expression of AOX1 gene in Pichia pastoris. FEMS Yeast Res 9:1271–1282
Lin-Cereghino G, Godfrey L, Cruz BD, Johnson S, Khuongsathiene S, Tolstorukov I, Yan M, Lin-Cereghino J, Veenhuis M, Subramani S, Cregg JM (2006) Mxr1p, a key regulator of the methanol utilization pathway and peroxisomal genes in Pichia pastoris. Mol Cell Biol 26:883–897
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
Wang X, Wang Q, Wang J, Bai P, Shi L, Shen W, Zhou M, Zhou X, Zhang Y, Cai M (2016) Mit1 transcription factor mediates methanol signaling and regulates the alcohol oxidase 1 (AOX1) promoter in Pichia pastoris. J Biol Chem 291(12):6245–6261
Yılmaz S (2012) Molecular and functional characterization of Pichia pastoris MIG1 gene. MSc thesis, Akdeniz University, 100 p
Wang J, Wang X, Shi L, Qi F, Zhang P, Zhang Y, Zhou X, Song Z, Cai M (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
Wang X, Cai M, Shi L, Wang Q, Zhu J, Wang J, Zhou M, Zhou X, Zhang Y (2016) PpNrg1 is a transcriptional repressor for glucose and glycerol repression of AOX1 promoter in methylotrophic yeast Pichia pastoris. Biotechnol Lett 38(2):291–298
Qin X, Qian J, Yao G, Zhuang Y, Zhang S, Chu J (2011) GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl Environ Microbiol 77:3600–3608
Ata Ö, Prielhofer R, Gasser B, Mattanovich D, Çalık P (2017) Transcriptional engineering of the glyceraldehyde-3-phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris. Biotechnol Bioeng 114(10):2319–2327. https://doi.org/10.1002/bit.26363
Sohn SB, Graf AB, Kim TY, Gasser B, Maurer M, Ferrer P, Mattanovich D, Lee SY (2010) Genome-scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production. Biotechnol J 5:705–715
Baumann K, Maurer M, Dragosits M, Cos O, Ferrer P, Mattanovich D (2008) Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteins. Biotechnol Bioeng 100:177–183
de Schutter K, Lin YC, Tiels P, van Hecke A, Glinka S, Weber- Lehmann J, Rouze P, Van de Peer Y, Callewaert N (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol 27:561–566
Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B (2009) Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Factories 2:8–29
Karaoglan M, Erden Karaoglan F, Inan M (2016) Functional analysis of alcohol dehydrogenase (ADH) genes in Pichia pastoris. Biotechnol Lett 38:463–469
Erden Karaoglan F (2016) Studies on Pichia pastoris alcohol dehydrogenase (ADH3) promoter. PhD thesis, Akdeniz University, 65 p
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
Resina D, Cos O, Ferrer P, Valero F (2005) Developing high cell density fed-batch cultivation strategies for heterologous protein production in Pichia pastoris using the nitrogen source-regulated FLD1 promoter. Biotechnol Bioeng 91:760–767
Zhang X, Zhang X, Liang S, Ye Y, Lin Y (2013) Key regulatory elements of a strong constitutive promoter, P GCW14, from Pichia pastoris. Biotechnol Lett 35(12):2113–2119
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Türkanoğlu Özçelik, A., Yılmaz, S., Inan, M. (2019). Pichia pastoris Promoters. In: Gasser, B., Mattanovich, D. (eds) Recombinant Protein Production in Yeast. Methods in Molecular Biology, vol 1923. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9024-5_3
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DOI: https://doi.org/10.1007/978-1-4939-9024-5_3
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