Abstract
The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5′ untranslated region. This region was identified as the full-length promoter and named PromA (− 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (− 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (− 2727 to − 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (− 2727 to − 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baek K, Lee Y, Nam O, Park S, Sim SJ, Jin E (2016) Introducing Dunaliella LIP promoter containing light-inducible motifs improves transgenic expression in Chlamydomonas reinhardtii. Biotechnol J 11(3):384–392. https://doi.org/10.1002/biot.201500269
Butler JEF, Kadonaga JT (2002) The RNA polymerase II corepromoter: a key component in the regulation of gene expression. Genes Dev 16(20):2583–2592. https://doi.org/10.1101/gad.1026202
Barahimipour R, Neupert J, Bock R (2016) Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker. Plant Mol Biol 90(4):403–418. https://doi.org/10.1007/s11103-015-0425-8
Bhullar S, Chakravarthy S, Pental D, Burma PK (2009) Analysis of promoter activity in transgenic plants by normalizing expression with a reference gene: anomalies due to the influence of the test promoter on the reference promoter. J Biosci 34:953–962. https://doi.org/10.1007/s12038-009-0109-0
Bilas R, Katarzyna Szafran K, Hnatuszko-Konka K, Kononowicz AK (2016) Cis-regulatory elements used to control gene expression in plants. Plant Cell Tissue Organ Cult 127(2):269–287. https://doi.org/10.1007/s11240-016-1057-7
Casais-Molina ML, Peraza-Echeverria S, Echevarría-Machado I, Herrrera-Valencia VA (2016) Expression of Chlamydomonas reinhardtii CrGPDH2 and CrGPDH3 cDNAs in yeast reveals that they encode functional glycerol-3-phosphate dehydrogenases involved in glycerol production and osmotic stress tolerance. J Appl Phycol 28(1):219–226. https://doi.org/10.1007/s10811-015-0588-3
Chang KN, Zhong S, Weirauch MT, Hon G, Pelizzola M, Li H, Huang S-sC, Schmitz RJ, Urich MA, Kuo D, Nery JR, Qiao H, Yang A, Jamali A, Chen H, Ideker T, Ren B, Bar-Joseph Z, Hughes TR, Ecker JR (2013) Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis. eLife 2:e00675. https://doi.org/10.7554/eLife.00675
Corchero JL, Gasser B, Resina D, Smith W, Parrilli E, Vázquez F, Abasolo I, Giuliani M, Jäntti J, Ferrer P, Saloheimo M, Mattanovich D, Schwartz S Jr, Tutino ML, Villaverde A (2013) Unconventional microbial systems for the cost-efficient production of high-quality protein therapeutics. Biotechnol Adv 31(2):140–153. https://doi.org/10.1016/j.biotechadv.2012.09.001
Daniell H, Ruiz ON, Dhingra A (2005) Chloroplast genetic engineering to improve agronomic traits. In: Peña L (ed) Transgenic plants: methods and protocols. Methods in molecular biology™, vol 286. Humana Press, New York, pp 111–137. https://doi.org/10.1385/1-59259-827-7:111
Dyo YM, Purton S (2018) The algal chloroplast as a synthetic biology platform for production of therapeutic proteins. Microbiology 164:113–121. https://doi.org/10.1099/mic.0.000599
Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229(4):873–883. https://doi.org/10.1007/s00425-008-0879-x
Ferrante P, Catalanotti C, Bonente G, Giuliano G (2008) An optimized, chemically regulated gene expression system for Chlamydomonas. PLoS One 3(9):e3200. https://doi.org/10.1371/journal.pone.0003200
Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40(D1):D1178–D1186. https://doi.org/10.1093/nar/gkr944
Guo L, Yu Y, Xia X, Yin W (2010) Identification and functional characterisation of the promoter of the calcium sensor gene CBL1 from the xerophyte Ammopiptanthus mongolicus. BMC Plant Biol 10:18. https://doi.org/10.1186/1471-2229-10-18
Harris EH (1989) The Chlamydomonas sourcebook, a comprehensive guide to biology and laboratory use. Academic Press, San Diego
Heitzer M, Zschoernig B (2007) Construction of modular tandem expression vectors for the green alga Chlamydomonas reinhardtii using the Cre/lox-system. Biotechniques 43(3):324–332. https://doi.org/10.2144/000112556
Hernández-García CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217-218:109–119. https://doi.org/10.1016/j.plantsci.2013.12.007
Herrera-Valencia VA, Macario-González LA, Casais-Molina ML, Beltran-Aguilar AG, Peraza-Echeverria S (2012) In silico cloning and characterization of the glycerol-3-phosphate dehydrogenase (GPDH) gene family in the green microalga Chlamydomonas reinhardtii. Curr Microbiol 64(5):477–485. https://doi.org/10.1007/s00284-012-0095-6
Hou J, Jiang P, Qi S, Zhang K, He Q, Xu C, Ding Z, Zhang K, Li K (2016) Isolation and functional validation of salinity and osmotic stress inducible promoter from the maize type-II H+-pyrophosphatase gene by deletion analysis in transgenic tobacco plants. PLoS One 11(4):e0154041. https://doi.org/10.1371/journal.pone.0154041
Hudson M, Ringli C, Boylan MT, Quail PH (1999) The FAR1 locus encodes a novel nuclear protein specific to phytochrome A signaling. Genes Dev 13(15):2017–2027
Husic DH, Tolbert NE (1986) Effect of osmotic stress on carbon metabolism in Chlamydomonas reinhardtii accumulation of glycerol as an osmoregulatory solute. Plant Physiol 82(2):594–596. https://doi.org/10.1104/pp.82.2.594
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907
Kim EJ, Ma X, Cerutti H (2015) Gene silencing in microalgae: mechanisms and biological roles. Bioresour Technol 184:23–32 doi: 0.1016/j.biortech.2014.10.119
Kucho K, Yoshioka S, Taniguchi F, Ohyama K, Fukuzawa H (2003) Cis-acting elements and DNA-binding proteins involved in CO2-responsive transcriptional activation of Cah1 encoding a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 133(2):783–793. https://doi.org/10.1104/pp.103.026492
Kuhlemeier C, Fluhr R, Green PJ, Chua N-H (1987) Sequences in the pea rbcS-3A gene have homology to constitutive mammalian enhancers but function as negative regulatory elements. Genes Dev 1:247–255
Kumar A, Falcao VR, Sayre RT (2013) Evaluating nuclear transgene expression systems in Chlamydomonas reinhardtii. Algal Res 2(4):321–332. https://doi.org/10.1016/j.algal.2013.09.002
León R, Galván FJ (1994) Halotolerance studies on Chlamydomonas reinhardtii: glycerol excretion by free and immobilized cells. Appl Phycol 6(1):13–20. https://doi.org/10.1007/BF02185898
Li J, Yumin L, Xue L, Xie H (2010) A structurally novel salt-regulated promoter of duplicated carbonic anhydrase gene 1 from Dunaliella salina. Mol Biol Rep 37:1143–1154. https://doi.org/10.1007/s11033-009-9901-z
Liang M-H, Lu Y, Chen H-H, Jian J-G (2017) The salt-regulated element in the promoter of lycopene β-cyclase gene confers a salt regulatory pattern in carotenogenesis of Dunaliella bardawil. Environ Microbiol 19(3):982–989. https://doi.org/10.1111/1462-2920.13539
Loppes R, Radoux M (2001) Identification of short promoter regions involved in the transcriptional expression of the nitrate reductase gene in Chlamydomonas reinhardtii. Plant Mol Biol 45(2):215–227
Lumbreras V, Stevens DR, Purton S (1998) Efficient foreign gene expression in Chlamydomonas reinhardtii mediated by an endogenous intron. Plant J 14(4):441–447. https://doi.org/10.1046/j.1365-313X.1998.00145.x
Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Maréchal-Drouard L, Marshall WF, Qu L-H, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Chlamydomonas annotation team, JGI annotation team, Grigoriev IV, Rokhsar DS, Grossman AR (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318(5848):245–250. https://doi.org/10.1126/science.1143609
Molino JVD, de Carvalho JCM, Mayfield SP (2018) Comparison of secretory signal peptides for heterologous protein expression in microalgae: expanding the secretion portfolio for Chlamydomonas reinhardtii. PLoS One 13(2):e0192433. https://doi.org/10.1371/journal.pone.0192433
Park S, Lee Y, Lee JH, Jin E (2013) Expression of the high light-inducible Dunaliella LIP promoter in Chlamydomonas reinhardtii. Planta 238(6):1147–1156. https://doi.org/10.1007/s00425-013-1955-4
Qin F, Sakuma Y, Li J, Liu Q, Li Y-Q, Shinozaki K, Kazuko Y-S (2004) Cloning and functional analysis of a novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant Cell Physiol 45(8):1042–1052. https://doi.org/10.1093/pcp/pch118
Rosales-Mendoza S, Paz-Maldonado LMT, Soria-Guerra RE (2012) Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep 31(3):479–494. https://doi.org/10.1007/s00299-011-1186-8
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Sawyer AL, Hankamer BD, Ross IL (2015) Sulphur responsiveness of the Chlamydomonas reinhardtii LHCBM9 promoter. Planta 241(5):1287–1302. https://doi.org/10.1007/s00425-015-2249-9
Scranton MA, Ostrand JT, Fields FJ, Mayfield SP (2015) Chlamydomonas as a model for biofuels and bio-products production. Plant J 82(3):523–531. https://doi.org/10.1111/tpj.12780
Simpson J, Schell J, Van Montagu M, Herrera-Estrella L (1986) Light-inducible and tissue-specific pea lhcp gene expression involves an upstream element combining enhancer- and silencer-like properties. Nature 323:551–554
Specht E, Miyake-Stoner S, Mayfield S (2010) Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32(10):1373–1383. https://doi.org/10.1007/s10529-010-0326-5
Surzycki R, Greenham, Kitayama K, Dibal F, Wagner R, Rochaix J-D, Ajam T, Surzycki S (2009) Factors effecting expression of vaccines in microalga. Biologicals 37:133–138. https://doi.org/10.1016/j.biologicals.2009.02.005
Traewachiwiphak S, Yokthongwattana C, Ves-Urai P, Charoensawan V, Yokthongwattana K (2018) Gene expression and promoter characterization of heat-shock protein 90B gene (HSP90B) in the model unicellular green alga Chlamydomonas reinhardtii. Plant Sci 272:107–116. https://doi.org/10.1016/j.plantsci.2018.04.010
Vaahtera L, Brosché M (2011) More than the sum of its parts – how to achieve a specific transcriptional response to abiotic stress. Plant Sci 180:421–430. https://doi.org/10.1016/j.plantsci.2010.11.009
von Gromoff ED, Schroda M, Oster U, Beck CF (2006) Identification of a plastid response element that acts as an enhancer within the Chlamydomonas HSP70A promoter. Nucleic Acids Res 34(17):4767–4779. https://doi.org/10.1093/nar/gkl602
Wang C, Peng X, Wang J, Lei A, Li H, Hu Z (2016) A β-carotene ketolase gene (bkt1) promoter regulated by sodium acetate and light in a model green microalga Chlamydomonas reinhardtii. Algal Res 20:61–69. https://doi.org/10.1016/j.algal.2016.09.020
Weber B, Zicola J, Oka R, Stam M (2016) Plant enhancers: a call for discovery. Trends Plant Sci 21(11):974–986. https://doi.org/10.1016/j.tplants.2016.07.013
Weirauch MT, Yang A, Albu M, Cote AG, Montenegro-Montero A, Drewe P, Najafabadi HS, Lambert SA, Mann I, Cook K, Zheng H, Goity A, van Bakel H, Lozano JC, Galli M, Lewsey MG, Huang E, Mukherjee T, Chen X, Reece-Hoyes JS, Govindarajan S, Shaulsky G, Walhout A, Bouget FY, Ratsch G, Larrondo LF, Ecker JR, Hughes TR (2014) Determination and inference of eukaryotic transcription factor sequence specificity. Cell 158(6):1431–1443. https://doi.org/10.1016/j.cell.2014.08.009
Yan N, Fan C, Chen Y, Hu Z (2016) The potential for microalgae as bioreactors to produce pharmaceuticals. Int J Mol Sci 17(6):962. https://doi.org/10.3390/ijms17060962
Zhang H, Jing R, Mao X (2017) Functional characterization of TaSnRK2.8 promoter in response to abiotic stresses by deletion analysis in transgenic Arabidopsis. Front Plant Sci 8:1198. https://doi.org/10.3389/fpls.2017.01198
Acknowledgements
The authors would like to thank the editor and reviewers of this manuscript for their critical review and their valuable comments. The authors thank Edanz (www.edanzediting.com) for editing the English text of a draft of this manuscript.
Funding
This research was funded by Consejo Nacional de Ciencia y Tecnologia (CONACYT, Mexico) (Grant number 169217). Anayeli Guadalupe Beltran-Aguilar received funding from CONACYT (Scholarship number 426890).
Author information
Authors and Affiliations
Contributions
VAH-V and SP-E conceived the study. VAH-V, SP-E, and LAL-O designed the experiments. AGB-A and ICB-A performed the experiments. AGB-A, VAH-V, SP-E, and LAL-O analyzed the results. AGB-A and VAH-V wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
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 734 kb)
Rights and permissions
About this article
Cite this article
Beltran-Aguilar, A.G., Peraza-Echeverria, S., López-Ochoa, L.A. et al. A novel salt-inducible CrGPDH3 promoter of the microalga Chlamydomonas reinhardtii for transgene overexpression. Appl Microbiol Biotechnol 103, 3487–3499 (2019). https://doi.org/10.1007/s00253-019-09733-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-019-09733-y