Abstract
Double-promoter expression system (DPES) design as de novo metabolic engineering strategy enables fine-tuned and enhanced gene expression. We constructed a collection of monodirectional hybrid-architectured DPESs with engineered promoter variants PADH2-Cat8-L2 and PmAOX1 and with the naturally occurring promoter PGAP to enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media. Reporter red fluorescent protein (mApple) and enhanced green fluorescent protein (eGFP) were expressed under PADH2-Cat8-L2 and PmAOX1 or PGAP, respectively, enabling the determination of the transcription period and strength of each constituent in the DPESs. We determined fluorescent protein expressions in batch cultivations on 2% (v/v) ethanol, excess glucose, and excess glycerol, and compared them with single-promoter expression systems constructed with PADH2-Cat8-L2, PmAOX1, and PGAP. The transcription- and expression-upregulation power of bifunctional DPESs was higher than that of twin DPESs (two-copy expression systems). Our findings answer long-standing questions regarding the high- (or multi-) copy clone results in the literature. Our first conclusion is that increasing identical components in the DPES architectures linearly increases the concentrations of cis-acting DNA sites and increases the demand for key transcription factors (TFs) that perturb their good coupling of supply and demand. The next is that the synthesis of some amino acids may create bottleneck(s) as rate-limiting amino acid(s) in recombinant protein synthesis. With bifunctional DPESs, each constituent upregulated the transcription and increased the expression and reduced the demand for the same TF(s) in the generation of novel regulatory circuits, due to the increased number of nonidentical cis-acting DNA sites. We tested superior DPES performances in extracellular human growth hormone (rhGH) production. Thereby, the indications related to the rate-limiting amino acids were verified. Compared with its constituents PADH2-Cat8-L2 and PmAOX1, the bifunctional DPES4 enhanced rhGH production by 1.44- and 2.02-fold, respectively. The DPES design method, with its constraint and parameters, enables the generation of promising r-protein production platforms with high impact on industrial-scale production processes and opens up new avenues for research in yeasts.
Key points
• Design method with the constraint and parameters for the construction of the DPESs is presented.
• Hybrid-architectured de novo DPESs are designed to enhance and fine-tune gene expression.
• Bifunctional DPESs demonstrate enhanced transcription and expression.
• Twin DPESs linearly increase cis-acting DNA sites and consequently increase the demand for the same TFs.
• Bifunctional DPESs enable good coupling of supply and demand to bind with TFs.
• Ethanol - controlled Snf1 pathway and crosstalk enable fine-tuned transcription and enhanced expression.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci 102:12678–12683
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 114:2319–2327. https://doi.org/10.1002/bit.26363
Ata Ö, Rebnegger C, Tatto NE, Valli M, Mairinger T, Hann S, Çalık P, Mattanovich D (2018) A single Gal4- like transcription factor activates the Crabtree effect in Komagataella phaffii. Nat Commun 9(1):4911. https://doi.org/10.1038/s41467-018-07430-4
Benjamin DJ, Berger JO, Johannesson M, Nosek BA, Wagenmakers EJ, Berk R, Cesarini D (2018) Redefine statistical significance. Nat Hum Behav 2(1):6–10. https://doi.org/10.1038/s41562-017-0189-z
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
Blazeck J, Garg R, Reed B, Alper HS (2012) Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng 109(11):2884–2895. https://doi.org/10.1002/bit.24552
Çalık P, Çalık G, Takaç S, Özdamar TH (1999) Metabolic flux analysis for serine alkaline protease fermentation by Bacillus licheniformis in a defined medium: effects of oxygen transfer rate. Biotechnol Bioeng 64:151–167. https://doi.org/10.1002/(SICI)1097-0290(19990720)64:2<151::AID-BIT4>3.0.CO;2-U
Çalık P, Orman MA, Çelik E, Halloran M, Çalık G, Özdamar TH (2008) Expression system for synthesis and purification of recombinant human growth hormone in Pichia pastoris and structural analysis by MALDI-ToF mass spectrometry. Biotechnol Prog 24:221–226. https://doi.org/10.1021/bp070294t
Çalık P, Bayraktar E, Inankur B, Soyaslan EŞ, Şahin M, Taşpınar H, Açik E, Yilmaz R, Özdamar TH (2010) Influence of pH on recombinant human growth hormone production by Pichia pastoris. J Chem Technol Biotechnol 85:1628–1635. https://doi.org/10.1002/jctb.2474
Çalık P, Ata Ö, Güneş H, Massahi A, Boy E, Keskin A, Öztürk S, Zerze GH, Özdamar TH (2015) Recombinant protein production in Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter: from carbon source metabolism to bioreactor operation parameters. Biochem Eng J 95:20–36. https://doi.org/10.1016/j.bej.2014.12.003
Çalık P, Hoxha B, Çalık G, Özdamar TH (2018) Hybrid fed-batch bioreactor operation design: control of substrate uptake enhances recombinant protein production in high-cell-density fermentations. J Chem Technol Biotechnol 93(11):3326–3335. https://doi.org/10.1002/jctb.5696
Cámara E, Landes N, Albiol J, Gasser B, Mattanovich D, Ferrer P (2017) Increased dosage of AOX1 promoter-regulated expression cassettes leads to transcription attenuation of the methanol metabolism in Pichia pastoris. Sci Rep 7:44302. https://doi.org/10.1038/srep44302
Cámara E, Monforte S, Albiol J, Ferrer P (2019) Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter. Biotechnol Bioeng 116:1710–1720. https://doi.org/10.1002/bit.26947
Çelik E, Çalık P (2012) Production of recombinant proteins by yeast cells. Biotechnol Adv 30(5):1108–1118. https://doi.org/10.1016/j.biotechadv.2011.09.011
Çelik E, Çalık P, Oliver SG (2009) Fed-batch methanol feeding strategy for recombinant protein production by Pichia pastoris in the presence of co-substrate sorbitol. Yeast 26(9):473–484. https://doi.org/10.1002/yea.1679
Curran KA, Crook NC, Karim AS, Gupta A, Wagman AM, Alper HS (2014) Design of synthetic yeast promoters via tuning of nucleosome architecture. Nat Commun 5(1):1–8. https://doi.org/10.1038/ncomms5002
de Boer CG, Vaishnav ED, Sadeh R, Abeyta EL, Friedman N, Regev A (2020) Deciphering eukaryotic gene-regulatory logic with 100 million random promoters. Nat Biotechnol 38:56–65. https://doi.org/10.1038/s41587-019-0315-8
Dikicioglu D, Wood V, Rutherford KM, McDowall MD, Oliver SG (2014) Improving functional annotation for industrial microbes: a case study with Pichia pastoris. Trends Biotechnol 32(8):396–399. https://doi.org/10.1016/j.tibtech.2014.05.003
Ergün BG, Gasser B, Mattanovich D, Çalık P (2019) Engineering of alcohol dehydrogenase 2 hybrid-promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol. Biotechnol Bioeng 116:2674–2686. https://doi.org/10.1002/bit.27095
Ergün BG, Demir İ, Özdamar TH, Gasser B, Mattanovich D, Çalık P (2020) Engineered deregulation of expression in yeast with designed hybrid-promoter architectures in coordination with discovered master regulator transcription factor. Adv Biosyst 4(4):1900172. https://doi.org/10.1002/adbi.201900172
Fang Z, Xu L, Pan D, Jiao L, Liu Z, Yan Y (2014) Enhanced production of Thermomyces lanuginosus lipase in Pichia pastoris via genetic and fermentation strategies. J Ind Microbiol Biotechnol 41:1541–1551. https://doi.org/10.1007/s10295-014-1491-7
Fischer JE, Glieder A (2019) Current advances in engineering tools for Pichia pastoris. Curr Opin Biotechnol 59:175–181. https://doi.org/10.1016/j.copbio.2019.06.002
Goffeau A, Barrell BG, Bussey H, Davis RW, Dujon B, Feldmann H, Galibert F, Hoheisel JD, Jacq C, Johnston M, Louis EJ, Mewes HW, Murakami Y, Philippsen P, Tettelin H, Oliver SG (1996) Life with 6000 genes. Science 274(5287):546–567. https://doi.org/10.1126/science.274.5287.546
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
He X, Liu N, Li W, Zhang Z, Zhang B, Ma Y (2008) Inducible and constitutive expression of a novel thermostable alkaline β-mannanase from alkaliphilic Bacillus sp N16-5 in Pichia pastoris and characterization of the recombinant enzyme. Enzym Microb Technol 43:13–18. https://doi.org/10.1016/j.enzmictec.2008.03.011
He D, Luo W, Wang Z, Lv P, Yuan Z (2015) Combined use of GAP and AOX1 promoters and optimization of culture conditions to enhance expression of Rhizomucor miehei lipase. J Ind Microbiol Biotechnol 42:1175–1182. https://doi.org/10.1007/s10295-015-1633-6
Kalender Ö, Çalık P (2020) Transcriptional regulatory proteins in central carbon metabolism of Pichia pastoris and Saccharomyces cerevisiae. Appl Microbiol Biotechnol 104(17):7273–7311. https://doi.org/10.1007/s00253-020-10680-2
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Li X, Yang Y, Zhan C, Zhang Z, Liu X, Liu H, Bai Z (2018) Transcriptional analysis of impacts of glycerol transporter 1 on methanol and glycerol metabolism in Pichia pastoris. FEMS Yeast Res 18(1):1–14. https://doi.org/10.1093/femsyr/fox081
Lin-Cereghino GP, Godfrey L, de la Cruz BJ, 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(3):883–897. https://doi.org/10.1128/MCB.26.3.883-897 PMID: 16428444; PMCID: PMC1347016
Massahi A, Çalık P (2016) Endogenous signal peptides in recombinant protein production by Pichia pastoris: from in-silico analysis to fermentation. J Theor Biol 408:22–33. https://doi.org/10.1016/j.jtbi.2016.07.039
Massahi A, Çalık P (2018) Naturally occurring novel promoters around pyruvate branch-point for recombinant protein production in Pichia pastoris (Komagataella phaffii): pyruvate decarboxylase- and pyruvate kinase-promoters. Biochem Eng J 138:111–120. https://doi.org/10.1016/j.bej.2018.07.012
Oliver SG, Lock A, Harris MA, Norse P, Wood V (2016) Model organism databases: essential resources that need the support of both funders and users. BMC Biol 14:49. https://doi.org/10.1186/s12915-016-0276-z
Öztürk S, Ergün BG, Çalık P (2017) Double promoter expression systems for recombinant protein production by industrial microorganisms. Appl Microbiol Biotechnol 101:7459–7475. https://doi.org/10.1007/s00253-017-8487-y
Parashar D, Satyanarayana T (2016) Enhancing the production of recombinant acidic α-amylase and phytase in Pichia pastoris under dual promoters [constitutive (GAP) and inducible (AOX)] in mixed fed batch high cell density cultivation. Process Biochem 51:1315–1322. https://doi.org/10.1016/j.procbio.2016.07.027
Peña DA, Gasser B, Zanghellini J, Steiger MG, Mattanovich D (2018) Metabolic engineering of Pichia pastoris. Metab Eng 50:2–15. https://doi.org/10.1016/j.ymben.2018.04.017
Portela RM, Vogl T, Kniely C, Fischer JE, Oliveira R, Glieder A (2017) Synthetic core promoters as universal parts for fine-tuning expression in different yeast species. ACS Synth Biol 6(3):471–484. https://doi.org/10.1021/acssynbio.6b00178
Portela RM, Vogl T, Ebner K, Oliveira R, Glieder A (2018) Pichia pastoris alcohol oxidase 1 (AOX1) core promoter engineering by high resolution systematic mutagenesis. Biotechnol J 13(3):1700340. https://doi.org/10.1002/biot.201700340
Prielhofer R, Reichinger M, Wagner N, Claes K, Kiziak C, Gasser B, Mattanovich D (2018) Superior protein titers in half the fermentation time: promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris. Biotechnol Bioeng 115(10):2479–2488. https://doi.org/10.1002/bit.26800
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(11):3600–3608. https://doi.org/10.1128/AEM.02843-10
Redden H, Alper HS (2015) The development and characterization of synthetic minimal yeast promoters. Nat Commun 6(1):1–9. https://doi.org/10.1038/ncomms8810
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. https://doi.org/10.1038/nmeth.2089
Shaner NC (2008) Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 5:545–551. https://doi.org/10.1038/nmeth.1209
Shashkova S, Welkenhuysen N, Hohmann S (2015) Molecular communication: crosstalk between the Snf1 and other signalling pathways. FEMS Yeast Res 15(4):fov026. https://doi.org/10.1093/femsyr/fov026
Shen W, Kong C, Xue Y, Liu Y, Cai M, Zhang Y, Jiang T, Zhou X, Zhou M (2016) Kinase screening in Pichia pastoris identified promising targets involved in cell growth and alcohol oxidase 1 promoter (PAOX1) regulation. PLoS One 11(12):e0167766. https://doi.org/10.1371/journal.pone.0167766
Tang CD, Guo J, Li JF, Wei XH, Gao SJ, Yin X, Wu MC (2014) Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system. Ann Microbiol 64(2):561–569. https://doi.org/10.3906/biy-1408-33
Theron CW, Berrios J, Delvigne F, Fickers P (2018) Integrating metabolic modeling and population heterogeneity analysis into optimizing recombinant protein production by Komagataella (Pichia) pastoris. Appl Microbiol Biotechnol 102(1):63–80. https://doi.org/10.1007/s00253-017-8612-y
Tschopp JF, Brust PF, Cregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15:3859–3876. https://doi.org/10.1093/nar/15.9.3859
Vogl T, Ruth C, Pitzer J, Kickenweiz T, Glieder A (2014) Synthetic core promoters for Pichia pastoris. ACS Synth Biol 3(3):188–191. https://doi.org/10.1021/sb400091p
Vogl T, Kickenweiz T, Pitzer J, Sturmberger L, Weninger A, Biggs BW, Köhler E-M, Baumschlager A, Fischer JE, Hyden P, Wagner M, Baumann M, Borth N, Geier M, Ajikumar PK, Glieder A (2018) Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat Commun 9:3589. https://doi.org/10.1038/s41467-018-05915-w
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. https://doi.org/10.1016/s0378-1119(96)00675-0
Wu J, Lin J, Chieng L, Lee C, Hsu T (2003) Combined use of GAP and AOX1 promoter to enhance the expression of human granulocyte-macrophage colony-stimulating factor in Pichia pastoris. Enzym Microb Technol 33:453–459. https://doi.org/10.1016/S0141-0229(03)00147-9
Yang Z, Zhang Z (2018) Engineering strategies for enhanced production of protein and bio-products in Pichia pastoris: a review. Biotechnol Adv 36(1):182–195. https://doi.org/10.1016/j.biotechadv.2017.11.002
Zavec D, Gasser B, Mattanovich D (2020) Characterization of methanol utilisation negative Pichia pastoris for secreted protein production: new cultivation strategies for current and future applications. Biotechnol Bioeng 117:1394–1405. https://doi.org/10.1002/bit.27303
Acknowledgements
Professor Tuli Mukhopadhyay (Indiana University, USA) is gratefully acknowledged for providing the mApple gene.
Funding
This work received funding from the Scientific and Technological Research Council of Turkey (TÜBİTAK) through the projects 116Z215 and 119Z435, and Middle East Technical University (METU) Research Fund. İD was awarded scholarship by the Scientific and Technological Research Council of Turkey (TÜBİTAK-BİDEB).
Author information
Authors and Affiliations
Contributions
İD designed and constructed the DPESs and SPESs, constructed P. pastoris strains, carried out the bioreactor and analytical experiments, contributed to data analyses, and made a first draft of the manuscript. PÇ conceived and designed the study, analyzed the data, and wrote and revised the manuscript. The authors read and approved the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethics statement
The research described in this article did not involve human participants or animals.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(PDF 626 kb)
Rights and permissions
About this article
Cite this article
Demir, İ., Çalık, P. Hybrid-architectured double-promoter expression systems enhance and upregulate-deregulated gene expressions in Pichia pastoris in methanol-free media. Appl Microbiol Biotechnol 104, 8381–8397 (2020). https://doi.org/10.1007/s00253-020-10796-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-020-10796-5