Abstract
Promoter choice is an important step in recombinant protein production, which directly determines the expression manner as constitutive or inducible and the expression level of the recombinant protein. This study aims to investigate the applicability of heterologous yeast promoters (Kluyveromyces marxianus TPI, Hansenula polymorpha PMA, Candida tropicalis ICL, and Saccharomyces cerevisiae CUP) in Pichia pastoris. The regulation mode of the CtICL and ScCUP promoters in P. pastoris was found to be inducible and that of the KmTPI and HpPMA was constitutive. The carbon sources in which the promoters exhibited the highest activity were determined as glycerol for PMA and TPI, glucose for CUP, and ethanol for ICL. The DNA region showing the highest activity was determined as 1000 bp for all promoters by promoter deletion analysis. Results from the study demonstrate the potential of inducible and constitutive heterologous promoters allowing expression under different conditions in the P. pastoris expression system and offers alternatives to frequently used promoters.
Key points
• Heterologous promoters exhibited similar expression pattern in P. pastoris with its native host.
• HpPMA has the highest promoter activity among the heterologous promoters tested.
• Reporter gene expression with ScCUP is responsive to elevating Cu2+in P. pastoris.
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The authors confirm that the datasets supporting the findings and conclusions of this study are available within the article.
References
Atomi H, Umemura K, Higashijima T, Kanai T, Yotsumoto Y, Teranishi Y, Ueda M, Tanaka A (1995) The upstram region of the isocitrate lyase gene (UPR-ICL) of Candida tropicalis induces gene expression in both Saccharomyces cerevisiae and Escherichia coli by acetate via two distinct promoters. Arch Microbiol 163:322–328. https://doi.org/10.1007/BF00404204
Bakke I, Berg L, Aune TE, Brautaset T, Sletta H, Tøndervik A, Valla S (2009) Random mutagenesis of the Pm promoter as a powerful strategy for improvement of recombinant-gene expression. Appl Environ Microbiol 75(7):2002–2011. https://doi.org/10.1128/aem.02315-08
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
Cai P, Duan X, Wu X, Gao L, Ye M, Zhou YJ (2021) Recombination machinery engineering facilitates metabolic engineering of the industrial yeast Pichia pastoris. Nucleic Acids Res 49:7791–7805. https://doi.org/10.1093/nar/gkab535
Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol Rev 24(1):45–66. https://doi.org/10.1111/j.1574-6976.2000.tb00532.x
Cox H, Mead D, Sudbery P, Eland M, Mannazzu I, Evans L (2000) Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter. Yeast 16:1191–1203. https://doi.org/10.1002/1097-0061(20000930)16:13%3C1191::aid-yea589%3E3.0.co;2-2
Duzenli OF, Okay S (2020) Promoter engineering for the recombinant protein production in prokaryotic systems. AIMS Bioeng 7(2):62–81. https://doi.org/10.3934/bioeng.2020007
Dohner L, Becher D, Salim S, Siekstele R, Sasnauskas K (2004) DNA sequence comprising regulatory regions for expressing proteins. U.S. 20040161841, Google Patents, https://patents.google.com/patent/US20040161841. Accessed 13 May 2022.
Erden-Karaoğlan F, Karaoğlan M, Yılmaz G, Yılmaz S, Inan M (2022) Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters. Biotechnol J 17:e2100332. https://doi.org/10.1002/biot.202100332
Feng X, Marchisio MA (2021) Saccharomyces cerevisiae promoter engineering before and during the Synthetic Biology Era. Biology 10(6):504. https://doi.org/10.3390/biology10060504
Gorman JA, Clark PE, Lee MC, Debouck C, Rosenberg M (1986) Regulation of the yeast metallothionein gene. Gene 48(1):13–22. https://doi.org/10.1016/0378-1119(86)90347-1
Gross C, Kelleher M, Iyer VR, Brown PO, Winge DR (2000) Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays. J Biol Chem 275:32310–32316. https://doi.org/10.1074/jbc.m005946200
Hartner FS, Ruth C, Langenegger 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. https://doi.org/10.1093/nar/gkn369
Karaoğlan M, Erden-Karaoğlan 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. https://doi.org/10.1016/j.pep.2016.01.017
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%3C651::aid-yea580%3E3.0.co;2-f
Kristiansson E, Thorsen M, Tamás MJ, Nerman O (2009) Evolutionary forces act on promoter length: Identification of enriched cis-regulatory elements. Mol Biol Evol 26(6):1299–1307. https://doi.org/10.1093/molbev/msp040
Macreadie IG, Horaitis O, Vaughan PR, Clark-Walker GD (1991) Constitutive expression of the Saccharomyces cerevisiae CUP1 gene in Kluyveromyces lactis. Yeast 7(2):127–135. https://doi.org/10.1002/yea.320070206
McMillan J, Lu Z, Rodriguez JS, Ahn TH, Lin Z (2019) YeasTSS: an integrative web database of yeast transcription start sites. Database 2019:baz048 (https://dx.doi.org/10.1093%2Fdatabase%2Fbaz048)
Mombeni M, Arjmand S, Siadat SOR, Alizadeh H, Abbasi A (2020) PMOX: A new powerful promoter for recombinant protein production in yeast Pichiapastoris. Enzym Microb Technol 139:109582. https://doi.org/10.1016/j.enzmictec.2020.109582
Qin X, Qian J, Yao G, Zhuang Y, Zhang S, Chu J (2011) GAP promoter library for fine-tuning of gene expressionin Pichia pastoris. Appl Environ Microbiol 77(11):3600–3608. https://doi.org/10.1128/aem.02843-10
Raschke WC, Neiditch BR, Hendricks M, Cregg JM (1996) Inducible expression of a heterologous protein in Hansenula polymorpha using the alcohol oxidase 1 promoter of Pichia pastoris. Gene 177:163–167. https://doi.org/10.1016/0378-1119(96)00293-4
Redden H, Alper H (2015) The development and characterization of synthetic minimal yeast promoters. Nat Commun 6:7810. https://doi.org/10.1038/ncomms8810
Rodriguez L, Narciandi RE, Roca H, Cremata J, Montesinos R, Rodriguez E, Grillo JM, Muzio V, Herrera LS, Delgado JM (1996) Invertase secretion in Hansenula polymorpha under the AOX1 promoter from Pichia pastoris. Yeast 12:815–822. https://doi.org/10.1002/(sici)1097-0061(199607)12:9%3c815::aid-yea916%3e3.0.co;2-h
Shen Q, Yu Z, Zhou XT, Zhang SJ, Zou SP, Xiong N, Xue YP, Liu ZQ, Zhen YG (2021) Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichiapastoris. Appl Microbiol Biotechnol 105:3211–3223. https://doi.org/10.1007/s00253-021-11195-0
Stadlmayr G, Mecklenbräuker 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(4):519–529. https://doi.org/10.1016/j.jbiotec.2010.09.957
Tang H, Wu Y, Deng J, Chen N, Zheng Z, Wei Y, Luo X, Keasling JD (2020) Promoter architecture and promoter engineering in Saccharomyces cerevisiae. Metabolites 6–10(8):320. https://doi.org/10.3390/metabo10080320
Tomàs-Gamisans M, Ødum ASR, Workman M, Ferrer P, Albiol J (2019) Glycerol metabolism of Pichia pastoris (Komagataella spp.) characterised by 13C-based metabolic flux analysis. N Biotechnol 50:52–59. https://doi.org/10.1016/j.nbt.2019.01.005
Umemura K, Atomi H, Kanai T, Teranishi Y, Ueda M, Tanaka A (1995) A novel promoter, derived from the isocitrate lyase gene of Candida tropicalis, inducible with acetate in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 43(3):489–492. https://doi.org/10.1007/BF00218454
Vogl T, Glieder A (2013) Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnol 30(4):385–404. https://doi.org/10.1016/j.nbt.2012.11.010
Vogl T, Fischer JE, Hyden P, Wasmayer R, Sturmberger L, Glieder A (2020) Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris. AMB Express 10(1):38. https://doi.org/10.1186/s13568-020-00972-1
Xu L, Liu P, Dai Z, Fan F, Zhang X (2021) Fine-tuning the expression of pathway gene in yeast using a regulatory library formed by fusing a synthetic minimal promoter with different Kozak variants. Microb Cell Fact 20:148. https://doi.org/10.1186/s12934-021-01641-z
Yan C, Yu W, Yao L, Guo X, Zhou YJ, Gao J (2022) Expanding the promoter toolbox for metabolic engineering of methylotrophic yeasts. Appl Microbiol Biotechnol. https://doi.org/10.1007/s00253-022-11948-5
Yan C, Yu W, Zhai X, Yao L, Guo X, Gao J, Zhou YJ (2022) Characterizing and engineering promoters for metabolic engineering of Ogataea polymorpha. Synth Syst Biotechnol 7(1):498–505. https://doi.org/10.1016/j.synbio.2021.12.005
Zepeda AB, Pessoa A, Farías JG (2018) Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast. Braz J Microbiol 49:119–127. https://doi.org/10.1016/j.bjm.2018.03.010
Zhao Y, Liu S, Lu Z, Zhao B, Wang S, Zhang C, Xiao D, Foo JL, Yu A (2021) Hybrid promoter engineering strategies in Yarrowia lipolytica: isoamyl alcohol production as a test study. Biotechnol Biofuels 14:149. https://doi.org/10.1186/s13068-021-02002-z
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This study was funded by the Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project Number117Z580).
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FEK and MK designed the study and conducted experiments, analyzed data, and wrote the manuscript. All authors have read and approved the manuscript.
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Erden-Karaoğlan, F., Karaoğlan, M. Applicability of the heterologous yeast promoters for recombinant protein production in Pichia pastoris. Appl Microbiol Biotechnol 106, 7073–7083 (2022). https://doi.org/10.1007/s00253-022-12183-8
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DOI: https://doi.org/10.1007/s00253-022-12183-8