Abstract
Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In this study, the promoter of the endogenous intraflagellar transport 25 (IFT25) gene of C. reinhardtii was tested for its ability to drive the expression of green fluorescent protein (GFP), which functions as a readout for target gene expression. IFT25 promoter (IFT25P) alone was not able to drive GFP expression to a detectable level. IFT25P, however, can drive robust IFT25-GFP fusion protein expression when the intron-containing IFT25 gene was inserted between IFT25P and GFP cDNA. When an extended version of foot-and-mouth virus 2A protease (2AE) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2AE-GFP polyprotein via 2A self-cleaving with a cleavage efficacy of approximately 99%. The monomer GFP was accumulated to a level of as high as 0.68% of total soluble proteins. To test whether the newly developed bicistronic IFT25P-IFT25-2AE expression system can be used to overexpress heterologous proteins of different origins and sizes, we inserted codon-optimized cDNAs encoding a Trichoderma reesei xylanase1 (25 kDa) and a Lachnospiraceae bacterium ND2006 type V CRISPR-Cas protein LbCpf1 (147 kDa) to the vector and found that the production of xylanase1 and LbCpf1 was as high as 0.69 and 0.49% of total soluble protein. Our result showed that IFT25P-IFT25-2AE system is more efficient to drive nuclear gene expression in C. reinhardtii than other conventionally used promoters, thus representing a novel efficient recombinant protein expression tool and has the potential to be scaled for commercial production of nuclear-encoded recombinant proteins of different sizes and origins in C. reinhardtii.
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References
Allen MD, del Campo JA, Kropat J, Merchant SS (2007) FEA1, FEA2, and FRE1, encoding two homologous secreted proteins and a candidate ferrireductase, are expressed coordinately with FOX1 and FTR1 in iron-deficient Chlamydomonas reinhardtii . Eukaryot cell 6 (10):1841–52 10.1128/EC.00205-07
Bellyei S, Szigeti A, Boronkai A, Pozsgai E, Gomori E, Melegh B, Janaky T, Bognar Z, Hocsak E, Sumegi B, Gallyas F Jr (2007a) Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway. Apoptosis 12:97–112. doi:10.1007/s10495-006-0486-x
Bellyei S, Szigeti A, Pozsgai E, Boronkai A, Gomori E, Hocsak E, Farkas R, Sumegi B, Gallyas F Jr (2007b) Preventing apoptotic cell death by a novel small heat shock protein. Eur J Cell Biol 86:161–171. doi:10.1016/j.ejcb.2006.12.004
Bhogaraju S, Taschner M, Morawetz M, Basquin C, Lorentzen E (2011) Crystal structure of the intraflagellar transport complex 25/27. EMBO J 30(10):1907–1918. doi:10.1038/emboj.2011.110
Blankenship JE, Kindle KL (1992) Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain. Mol Cell Biol 12(11):5268–5279
Butanaev AM (1994) Use of the hygromycin phosphotransferase gene as the dominant selective marker for Chlamydomonas reinhardtii transformation. Mol Biol (Mosk) 28(5):1061–1068
Cerutti H, Johnson AM, Gillham NW, Boynton JE (1997a) Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas. Plant Cell 9(6):925–945. doi:10.1105/tpc.9.6.925
Cerutti H, Johnson AM, Gillham NW, Boynton JE (1997b) A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression. Genetics 145(1):97–110
Chow KC, Tung WL (1999) Electrotransformation of Chlorella vulgaris. Plant Cell Rep 18(9):778–780. doi:10.1007/s002990050660
Davies JP, Weeks DP, Grossman AR (1992) Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii. Nucleic Acids Res 20(12):2959–2965
de Felipe P, Luke GA, Hughes LE, Gani D, Halpin C, Ryan MD (2006) E unum pluribus: multiple proteins from a self-processing polyprotein. Trends Biotechnol 24(2):68–75. doi:10.1016/j.tibtech.2005.12.006
De Wilde C, Van Houdt H, De Buck S, Angenon G, De Jaeger G, Depicker A (2000) Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol Biol 43(2–3):347–359
Diaz-Santos E, de la Vega M, Vila M, Vigara J, Leon R (2013) Efficiency of different heterologous promoters in the unicellular microalga Chlamydomonas reinhardtii. Biotechnol Prog 29(2):319–328. doi:10.1002/btpr.1690
Dumas P, Bergdoll M, Cagnon C, Masson JM (1994) Crystal structure and site-directed mutagenesis of a bleomycin resistance protein and their significance for drug sequestering. EMBO J 13(11):2483–2492
Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229(4):873–883. doi:10.1007/s00425-008-0879-x
Fan ZC, Bird RC (2012) An alternative −1/+2 open reading frame exists within viral N(pro)(1-19) region of bovine viral diarrhea virus SD-1. Virus Res 163(1):341–351. doi:10.1016/j.virusres.2011.10.022
Fan ZC, Dennis JC, Bird RC (2008) Bovine viral diarrhea virus is a suitable viral vector for stable expression of heterologous gene when inserted in between N(pro) and C genes. Virus Res 138(1–2):97–104. doi:10.1016/j.virusres.2008.08.015
Fan ZC, Behal RH, Geimer S, Wang ZH, Williamson SM, Zhang HL, Cole DG, Qin HM (2010) Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly. Mol Biol Cell 21(15):2696–2706. doi:10.1091/mbc.E10-03-0191
Fernandez E, Schnell R, Ranum LP, Hussey SC, Silflow CD, Lefebvre PA (1989) Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 86(17):6449–6453
Fischer N, Rochaix JD (2001) The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Gen Genomics 265(5):888–894. doi:10.1007/s004380100485
Fuhrmann M, Oertel W, Hegemann P (1999) A synthetic gene coding for the green fluorescent protein (GFP) is a versatile reporter in Chlamydomonas reinhardtii. Plant J 19(3):353–361
Fuhrmann M, Hausherr A, Ferbitz L, Schodl T, Heitzer M, Hegemann P (2004) Monitoring dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic luciferase reporter gene. Plant Mol Biol 55(6):869–881. doi:10.1007/s11103-004-2150-6
Furler S, Paterna JC, Weibel M, Bueler H (2001) Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons. Gene Ther 8(11):864–873. doi:10.1038/sj.gt.3301469
Gong Y, Hu H, Gao Y, Xu X, Gao H (2011) Microalgae as platforms for production of recombinant proteins and valuable compounds: progress and prospects. J Ind Microbiol Biotechnol 38(12):1879–1890. doi:10.1007/s10295-011-1032-6
Griesbeck C, Kobl I, Heitzer M (2006) Chlamydomonas reinhardtii: a protein expression system for pharmaceutical and biotechnological proteins. Mol Biotechnol 34(2):213–223. doi:10.1385/mb:34:2:213
Hall LM, Taylor KB, Jones DD (1993) Expression of a foreign gene in Chlamydomonas reinhardtii. Gene 124(1):75–81. doi:10.1016/0378-1119(93)90763-S
Halpin C, Cooke SE, Barakate A, El Amrani A, Ryan MD (1999) Self-processing 2A-polyproteins—a system for co-ordinate expression of multiple proteins in transgenic plants. Plant J 17(4):453–459
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):324–326 328 passim
Huang TH, Tran VH, Roufogalis BD, Li Y (2007) Gypenoside XLIX, a naturally occurring gynosaponin, PPAR-alpha dependently inhibits LPS-induced tissue factor expression and activity in human THP-1 monocytic cells. Toxicol Appl Pharm 218(1):30–36. doi:10.1016/j.taap.2006.10.013
Jarvis E, Brown L (1991) Transient expression of firefly luciferase in protoplasts of the green alga Chlorella ellipsoidea. Curr Genet 19(4):317–321. doi:10.1007/BF00355062
Keady Brian T, Samtani R, Tobita K, Tsuchya M, San Agustin Jovenal T, Follit John A, Jonassen Julie A, Subramanian R, Lo Cecilia W, Pazour Gregory J (2012) IFT25 links the signal-dependent movement of hedgehog components to intraflagellar transport. Dev Cell 22(5):940–951. doi:10.1016/j.devcel.2012.04.009
Kim DH, Kim YT, Cho JJ, Bae JH, Hur SB, Hwang I, Choi TJ (2002) Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea. Mar Biotechnol 4(1):63–73. doi:10.1007/s1012601-0070-x
Kim S, Sapkota K, Choi B-S, Kim S-J (2010) Expression of human growth hormone gene in Pleurotus eryngii. Cent Eur J Biol 5(6):791–799. doi:10.2478/s11535-010-0071-8
Kong F, Yamasaki T, Kurniasih SD, Hou L, Li X, Ivanova N, Okada S, Ohama T (2015) Robust expression of heterologous genes by selection marker fusion system in improved Chlamydomonas strains. J Biosci Bioeng 120(3):239–245. doi:10.1016/j.jbiosc.2015.01.005
Kozminski KG, Diener DR, Rosenbaum JL (1993) High level expression of nonacetylatable alpha-tubulin in Chlamydomonas reinhardtii. Cell Motil Cytoskel 25(2):158–170. doi:10.1002/cm.970250205
Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV (2004) Genetic transformation of the green alga—Chlamydomonas reinhardtii by Agrobacterium tumefaciens. Plant Sci 166(3):731–738. doi:10.1016/j.plantsci.2003.11.012
Kumar A, Falcao VR, Sayre RT (2013) Evaluating nuclear transgene expression systems in Chlamydomonas reinhardtii. Algal Res 2(4):321–332. doi:10.1016/j.algal.2013.09.002
Lauersen KJ, Berger H, Mussgnug JH, Kruse O (2013) Efficient recombinant protein production and secretion from nuclear transgenes in Chlamydomonas reinhardtii. J Biotechnol 167(2):101–110. doi:10.1016/j.jbiotec.2012.10.010
Lauersen KJ, Kruse O, Mussgnug JH (2015) Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Appl Microbio Biotechnol 99(8):3491–3503. doi:10.1007/s00253-014-6354-7
Lechtreck KF, Luro S, Awata J, Witman GB (2009) HA-tagging of putative flagellar proteins in Chlamydomonas reinhardtii identifies a novel protein of intraflagellar transport complex B. Cell Motil Cytoskel 66:469–482. doi:10.1002/cm.20369
Leon-Banares R, Gonzalez-Ballester D, Galvan A, Fernandez E (2004) Transgenic microalgae as green cell-factories. Trends Biotechno 22(1):45–52. doi:10.1016/j.tibtech.2003.11.003
Lerche K, Hallmann A (2014) Stable nuclear transformation of Pandorina morum. BMC Biotechnol 14:65. doi:10.1186/1472-6750-14-65
Lumbreras V, Purton S (1998) Recent advances in Chlamydomonas transgenics. Protist 149(1):23–27. doi:10.1016/S1434-4610(98)70006-9
Mayfield SP, Manuell AL, Chen S, Wu J, Tran M, Siefker D, Muto M, Marin-Navarro J (2007) Chlamydomonas reinhardtii chloroplasts as protein factories. Curr Opin Biotechnol 18(2):126–133. doi:10.1016/j.copbio.2007.02.001
Meng DM, Dai HX, Gao XF, Zhao JF, Guo YJ, Ling X, Dong B, Zhang ZQ, Fan ZC (2016) Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris. Protein Expr Purif 127:35–43. doi:10.1016/j.pep.2016.07.001
Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Marechal-Drouard L, Marshall WF, Qu LH, Nelson DR, Sanderfoot AA, Spalding MH, Kapitonov VV, Ren Q, Ferris P, Lindquist E, Shapiro H, Lucas SM, Grimwood J, Schmutz J, Cardol P, Cerutti H, Chanfreau G, Chen CL, Cognat V, Croft MT, Dent R, Dutcher S, Fernandez E, Fukuzawa H, Gonzalez-Ballester D, Gonzalez-Halphen D, Hallmann A, Hanikenne M, Hippler M, Inwood W, Jabbari K, Kalanon M, Kuras R, Lefebvre PA, Lemaire SD, Lobanov AV, Lohr M, Manuell A, Meier I, Mets L, Mittag M, Mittelmeier T, Moroney JV, Moseley J, Napoli C, Nedelcu AM, Niyogi K, Novoselov SV, Paulsen IT, Pazour G, Purton S, Ral JP, Riano-Pachon DM, Riekhof W, Rymarquis L, Schroda M, Stern D, Umen J, Willows R, Wilson N, Zimmer SL, Allmer J, Balk J, Bisova K, Chen CJ, Elias M, Gendler K, Hauser C, Lamb MR, Ledford H, Long JC, Minagawa J, Page MD, Pan J, Pootakham W, Roje S, Rose A, Stahlberg E, Terauchi AM, Yang P, Ball S, Bowler C, Dieckmann CL, Gladyshev VN, Green P, Jorgensen R, Mayfield S, Mueller-Roeber B, Rajamani S, Sayre RT, Brokstein P, Dubchak I, Goodstein D, Hornick L, Huang YW, Jhaveri J, Luo Y, Martinez D, Ngau WC, Otillar B, Poliakov A, Porter A, Szajkowski L, Werner G, Zhou K, Grigoriev IV, Rokhsar DS, Grossman AR (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318(5848):245–250. doi:10.1126/science.1143609
Moellering ER, Benning C (2010) RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii. Eukaryot Cell 9(1):97–106
Plucinak TM, Horken KM, Jiang W, Fostvedt J, Nguyen ST, Weeks DP (2015) Improved and versatile viral 2A platforms for dependable and inducible high-level expression of dicistronic nuclear genes in Chlamydomonas reinhardtii. Plant J 82(4):717–729. doi:10.1111/tpj.12844
Posten C, Schaub G (2009) Microalgae and terrestrial biomass as source for fuels—a process view. J Biotechnol 142(1):64–69. doi:10.1016/j.jbiotec.2009.03.015
Qin H, Wang Z, Diener D, Rosenbaum J (2007) Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control. Curr Biol 17(3):193–202
Randolph-Anderson B, Boynton J, Gillham N, Harris E, Johnson A, Dorthu M-P, Matagne R (1993) Further characterization of the respiratory deficient dum-1 mutation of Chlamydomonas reinhardtii and its use as a recipient for mitochondrial transformation. Mol Gen Genet 236(2–3):235–244. doi:10.1007/BF00277118
Rasala BA, Mayfield SP (2011) The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs 2(1):50–54. doi:10.4161/bbug.2.1.13423
Rasala BA, Muto M, Lee PA, Jager M, Cardoso RM, Behnke CA, Kirk P, Hokanson CA, Crea R, Mendez M, Mayfield SP (2010) Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 8(6):719–733. doi:10.1111/j.1467-7652.2010.00503.x
Rasala BA, Lee PA, Shen Z, Briggs SP, Mendez M, Mayfield SP (2012) Robust expression and secretion of xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS One 7(8):e43349. doi:10.1371/journal.pone.0043349
Roberts LO, Seamons RA, Belsham GJ (1998) Recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins. RNA 4(5):520–529
Ruecker O, Zillner K, Groebner-Ferreira R, Heitzer M (2008) Gaussia-luciferase as a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga Chlamydomonas reinhardtii. Mol Genet Genomics: MGG 280(2):153–162. doi:10.1007/s00438-008-0352-3
Ryan MD, Drew J (1994) Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial protein. EMBO J 13:928–933
Ryan MD, King AM, Thomas GP (1991) Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J Gen Virol 72(Pt 11):2727–2732
Schroda M, Blöcker D, Beck CF (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J 21(2):121–131. doi:10.1046/j.1365-313x.2000.00652.x
Shao N, Bock R (2008) A codon-optimized luciferase from Gaussia princeps facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas reinhardtii. Curr Genet 53(6):381–388. doi:10.1007/s00294-008-0189-7
Shaver S, Casas-Mollano JA, Cerny RL, Cerutti H (2010) Origin of the polycomb repressive complex 2 and gene silencing by an E(z) homolog in the unicellular alga Chlamydomonas. Epigenetics 5(4):301–312
Shimogawara K, Fujiwara S, Grossman A, Usuda H (1998) High-efficiency transformation of Chlamydomonas reinhardtii by electroporation. Genetics 148(4):1821–1828
Sizova I, Fuhrmann M, Hegemann P (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277(1–2):221–229
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. doi:10.1007/s10529-010-0326-5
Specht EA, Nour-Eldin HH, Hoang KTD, Mayfield SP (2015) An improved ARS2-derived nuclear reporter enhances the efficiency and ease of genetic engineering in Chlamydomonas. Biotechnol J 10(3):473–479. doi:10.1002/biot.201400172
Tang DK, Qiao SY, Wu M (1995) Insertion mutagenesis of Chlamydomonas reinhardtii by electroporation and heterologous DNA. Biochem Mol Biol Int 36(5):1025–1035
Walker TL, Purton S, Becker DK, Collet C (2005) Microalgae as bioreactors. Plant Cell Rep 24(11):629–641. doi:10.1007/s00299-005-0004-6
Wang Z, Fan ZC, Williamson SM, Qin H (2009) Intraflagellar transport (IFT) protein IFT25 is a phosphoprotein component of IFT complex B and physically interacts with IFT27 in Chlamydomonas. PLoS One 4(5):e5384. doi:10.1371/journal.pone.0005384
Wu-Scharf D, Jeong B, Zhang C, Cerutti H (2000) Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Science 290(5494):1159–1162
Zetsche B, Gootenberg Jonathan S, Abudayyeh Omar O, Slaymaker Ian M, Makarova Kira S, Essletzbichler P, Volz Sara E, Joung J, van der Oost J, Regev A, Koonin Eugene V, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759–771. doi:10.1016/j.cell.2015.09.038
Acknowledgements
This work was supported jointly by grants from International Center for Genetic Engineering and Biotechnology (ICGEB) Collaborative Research Program (CRP) (#CRP/CHN15-01 to Fan ZC), Natural Science Foundation of Tianjin City (#13JCYBJC41900 to Fan ZC), Tianjin University of Science & Technology (#20130420 and #20140789 to Fan ZC), National Natural Science Foundation of China (#31503544 to Meng DM), National Key Research and Development Program of China (#2016YFD0401202 to Wang J), and Obesita & Algaegen LLC, USA.
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Dong, B., Hu, HH., Li, ZF. et al. A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii . Appl Microbiol Biotechnol 101, 4227–4245 (2017). https://doi.org/10.1007/s00253-017-8177-9
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DOI: https://doi.org/10.1007/s00253-017-8177-9