References
-
Kumar, G. et al. Bioengineering of microalgae: recent advances, perspectives, and regulatory challenges for industrial application. Front. Bioeng. Biotechnol. 8, 914 (2020).
-
Rasala, B. A. & Mayfield, S. P. The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng. Bugs 2, 50–54 (2011).
-
Harris, E. H. Chlamydomonas as a model organism. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 363–406 (2001).
-
Fabris, M. et al. Emerging technologies in algal biotechnology: toward the establishment of a sustainable, algae-based bioeconomy. Front. Plant Sci. 11, 279 (2020).
-
Taunt, H. N., Stoffels, L. & Purton, S. Green biologics: the algal chloroplast as a platform for making biopharmaceuticals. Bioengineered 9, 48–54 (2018).
-
Neupert, J. et al. An epigenetic gene silencing pathway selectively acting on transgenic DNA in the green alga Chlamydomonas. Nat. Commun. 11, 6269 (2020).
-
Purton, S. Tools and techniques for chloroplast transformation of Chlamydomonas. Adv. Exp. Med Biol. 616, 34–45 (2007).
-
Ramesh, V. M., Bingham, S. E. & Webber, A. N. A simple method for chloroplast transformation in Chlamydomonas reinhardtii. Methods Mol. Biol. 684, 313–320 (2011).
-
Mordaka, P. M. et al. Regulation of nucleus-encoded trans-acting factors allows orthogonal fine-tuning of multiple transgenes in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J n/a (2024). https://doi.org/10.1111/pbi.14557
-
Larrea-Alvarez, M. & Purton, S. Multigenic engineering of the chloroplast genome in the green alga Chlamydomonas reinhardtii. Microbiology 166, 510–515 (2020).
-
Yeon, J., Miller, S. M. & Dejtisakdi, W. New synthetic operon vectors for expressing multiple proteins in the chlamydomonas reinhardtii chloroplast. Genes 14, 368 (2023).
-
Macedo-Osorio, K. S. et al. Intercistronic expression elements (IEE) from the chloroplast of Chlamydomonas reinhardtii can be used for the expression of foreign genes in synthetic operons. Plant Mol. Biol. 98, 303–317 (2018).
-
Guo, Y., Xiong, H., Fan, Q. & Duanmu, D. Heterologous gene expression in chlamydomonas reinhardtii chloroplast by heterologous promoters and terminators, intercistronic expression elements and minichromosome. Micro. Biotechnol. 17, e70069 (2024).
-
Melero-Cobo, X. et al. MoCloro: an extension of the Chlamydomonas reinhardtii modular cloning toolkit for microalgal chloroplast engineering. Physiol. Plant 177, e70088 (2025).
-
Inckemann, R. M. et al. A modular high-throughput approach for advancing synthetic biology in the chloroplast of Chlamydomonas. Nat, Plants (2025). https://doi.org/10.1038/s41477-025-02126-2
-
Zhang, P. et al. Deep flanking sequence engineering for efficient promoter design using DeepSEED. Nat. Commun. 14, 6309 (2023).
-
Altendorfer, E., Mundlos, S. & Mayer, A. A transcription coupling model for how enhancers communicate with their target genes. Nat. Struct. Mol. Biol. 32, 598–606 (2025).
-
Johnson, C. H. & Schmidt, G. W. The psbB gene cluster of the Chlamydomonas reinhardtii chloroplast: sequence and transcriptional analyses of psbN and psbH. Plant Mol. Biol. 22, 645–658 (1993).
-
Stern, D. S., Higgs, D. C. & Yang, J. Transcription and translation in chloroplasts. Trends Plant Sci. 2, 308–315 (1997).
-
Klein, U., De Camp, J. D. & Bogorad, L. Two types of chloroplast gene promoters in Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. USA 89, 3453–3457 (1992).
-
Hatano-Iwasaki, A., Minagawa, J., Inoue, Y. & Takahashi, Y. Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein, D1. Plant Mol. Biol. 42, 353–363 (2000).
-
Rasala, B. A., Muto, M., Sullivan, J. & Mayfield, S. P. Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5’ untranslated region optimization. Plant Biotechnol. J. 9, 674–683 (2011).
-
Drapier, D. et al. The chloroplast atpA gene cluster in Chlamydomonas reinhardtii. Functional analysis of a polycistronic transcription unit. Plant Physiol. 117, 629–641 (1998).
-
Shimmura, S. et al. Comparative analysis of chloroplast psbD promoters in terrestrial plants. Front Plant Sci. 8, 1186 (2017).
-
Nickelsen, J., Fleischmann, M., Boudreau, E., Rahire, M. & Rochaix, J.-D. Identification of cis-Acting RNA leader elements required for chloroplast psbD gene expression in chlamydomonas. Plant Cell 11, 957–970 (1999).
-
Klein, U., Salvador, M. L. & Bogorad, L. Activity of the Chlamydomonas chloroplast RBCL gene promoter is enhanced by a remote sequence element. Proc. Natl. Acad. Sci. 91, 10819–10823 (1994).
-
Scranton, M. A., Ostrand, J. T., Fields, F. J. & Mayfield, S. P. Chlamydomonas as a model for biofuels and bio-products production. Plant J. 82, 523–531 (2015).
-
Anthonisen, I. L., Salvador, M. L. & Klein, U. Specific sequence elements in the 5’ untranslated regions of rbcL and atpB gene mRNas stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii. RNA 7, 1024–1033 (2001).
-
Cavaiuolo, M., Kuras, R., Wollman, F. A., Choquet, Y. Vallon, O. Small RNA profiling in Chlamydomonas: insights into chloroplast RNA metabolism. Nucleic Acids Res. 45, 10783–10799 (2017).
-
Loiselay, C. et al. Molecular identification and function of cis- and trans-acting determinants for petA transcript stability in Chlamydomonas reinhardtii chloroplasts. Mol. Cell Biol. 28, 5529–5542 (2008).
-
Johnson, X. et al. MRL1, a conserved Pentatricopeptide repeat protein, is required for stabilization of rbcL mRNA in chlamydomonas and arabidopsis. Plant Cell 22, 234–248 (2010).
-
Boudreau, E., Nickelsen, J., Lemaire, S. D., Ossenbühl, F. & Rochaix, J. D. The Nac2 gene of Chlamydomonas encodes a chloroplast TPR-like protein involved in psbD mRNA stability. Embo J. 19, 3366–3376 (2000).
-
Commault, A. S. et al. Methyl jasmonate treatment affects the regulation of the 2-C-methyl-D-erythritol 4-phosphate pathway and early steps of the triterpenoid biosynthesis in Chlamydomonas reinhardtii. Algal Res. 39, 101462 (2019).
-
Wang, R. et al. Isolation and functional characterization of bidirectional promoters in rice. Front Plant Sci. 7, 766 (2016).
-
In, S., Lee, H. A., Woo, J., Park, E. & Choi, D. Molecular characterization of a pathogen-inducible bidirectional promoter from hot pepper (Capsicum annuum). Mol. Plant Microbe Interact. 33, 1330–1339 (2020).
-
Liu, X. et al. The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter. J. Exp. Bot. 67, 4403–4413 (2016).
-
Thieffry, A. et al. Characterization of arabidopsis thaliana promoter bidirectionality and antisense RNAs by inactivation of nuclear RNA decay pathways. Plant Cell 32, 1845–1867 (2020).
-
Vogl, T. et al. Engineered bidirectional promoters enable rapid multi-gene co-expression optimization. Nat. Commun. 9, 3589 (2018).
-
Yang, S., Sleight, S. C. & Sauro, H. M. Rationally designed bidirectional promoter improves the evolutionary stability of synthetic genetic circuits. Nucleic Acids Res. 41, e33 (2013).
-
Kumar, S. et al. A combinatorial bidirectional and bicistronic approach for coordinated multi-gene expression in corn. Plant Mol. Biol. 87, 341–353 (2015).
-
Poliner, E., Clark, E., Cummings, C., Benning, C. & Farre, E. M. A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779. Algal Res. 45, 101664 (2020).
-
Büschlen, S., Choquet, Y., Kuras, R. & Wollman, F.-A. Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii. FEBS Lett. 284, 257–262 (1991).
-
Sakamoto, W., Chen, X., Kindle, K. L. & Stern, D. B. Function of the Chlamydomonas reinhardtii petd 5’ untranslated region in regulating the accumulation of subunit IV of the cytochrome b6/f complex. Plant J. 6, 503–512 (1994).
-
Loizeau, K. et al. Small RNAs reveal two target sites of the RNA-maturation factor Mbb1 in the chloroplast of Chlamydomonas. Nucleic Acids Res. 42, 3286–3297 (2013).
-
Fong, S. E. & Surzycki, S. J. Organization and structure of plastome psbF, psbL, petG and ORF712 genes in Chlamydomonas reinhardtii. Curr. Genet. 21, 527–530 (1992).
-
Fong, S. E. & Surzycki, S. J. Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement. Curr. Genet 21, 485–497 (1992).
-
Solovyev, V. V. & Salamov, A. A. Recognition of 3′-processing sites of human mRNA precursors. Comput. Appl. Biosci. 13, 23–28 (1997).
-
Ma, K., Deng, L., Wu, H. & Fan, J. Towards green biomanufacturing of high-value recombinant proteins using promising cell factory: chlamydomonas reinhardtii chloroplast. Bioresour. Bioprocess 9, 83 (2022).
-
Miro-Vinyals, B. et al. Chloroplast engineering of the green microalgae Chlamydomonas reinhardtii for the production of HAA, the lipid moiety of rhamnolipid biosurfactants. N. Biotechnol. 76, 1–12 (2023).
-
Di Rocco, G. et al. A PETase enzyme synthesised in the chloroplast of the microalga Chlamydomonas reinhardtii is active against post-consumer plastics. Sci. Rep. 13, 10028 (2023).
-
Barnes, D. et al. Contribution of 5’- and 3’-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol. Genet Genomics 274, 625–636 (2005).
-
Economou, C., Wannathong, T., Szaub, J. & Purton, S. A simple, low-cost method for chloroplast transformation of the green alga Chlamydomonas reinhardtii. Methods Mol. Biol. 1132, 401–411 (2014).
-
Nishimura, Y. & Stern, D. B. Differential replication of two chloroplast genome forms in heteroplasmic Chlamydomonas reinhardtii gametes contributes to alternative inheritance patterns. Genetics 185, 1167–1181 (2010).
-
Khan, A. et al. Plant synthetic promoters: advancement and prospective. Agriculture 13, 298 (2023).
-
Anwar, M., Wang, J., Li, J., Altaf, M. M. & Hu, Z. MYB transcriptional factors affects upstream and downstream MEP pathway and triterpenoid biosynthesis in chlamydomonas reinhardtii. Processes 12, 487 (2024).
-
Wasternack, C. & Hause, B. Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. Ann. Bot. 111, 1021–1058 (2013).
-
Goossens, J., Fernandez-Calvo, P., Schweizer, F. & Goossens, A. Jasmonates: signal transduction components and their roles in environmental stress responses. Plant Mol. Biol. 91, 673–689 (2016).
-
Al Hoqani, U. H. A. Metabolic engineering of the algal chloroplast for terpenoid production. Doctoral thesis, UCL (University College London) (2017).
-
Wichmann, J. et al. Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-alpha-bisabolene production. Micro. Cell Fact. 21, 190 (2022).
-
Pérez-Martín, J. & de Lorenzo, V. Clues and consequences of DNA bending in transcription. Annu. Rev. Microbiol. 51, 593–628 (1997).
-
Summer, E. J., Schmid, V. H., Bruns, B. U. & Schmidt, G. W. Requirement for the H phosphoprotein in photosystem II of Chlamydomonas reinhardtii. Plant Physiol. 113, 1359–1368 (1997).
-
Torabi, S. et al. PsbN is required for assembly of the photosystem II reaction center in Nicotiana tabacum. Plant Cell 26, 1183–1199 (2014).
-
Rosales-Mendoza, S., Paz-Maldonado, L. M. T. & Soria-Guerra, R. E. Chlamydomonas reinhardtii as a viable platform for the production of recombinant proteins: current status and perspectives. Plant Cell Rep. 31, 479–494 (2012).
-
Surzycki, R. et al. Factors effecting expression of vaccines in microalgae. Biologicals 37, 133–138 (2009).
-
Barnes, D. et al. Contribution of 5′- and 3′-untranslated regions of plastid mRNAs to the expression of Chlamydomonas reinhardtii chloroplast genes. Mol. Genet. Genomics 274, 625–636 (2005).
-
Wannathong, T., Waterhouse, J. C., Young, R. E., Economou, C. K. & Purton, S. New tools for chloroplast genetic engineering allow the synthesis of human growth hormone in the green alga Chlamydomonas reinhardtii. Appl Microbiol Biotechnol. 100, 5467–5477 (2016).
-
Odom, O. W., Kang, S., Ferguson, C., Chen, C. & Herrin, D. L. Overcoming poor transgene expression in the wild-type chlamydomonas chloroplast: creation of highly mosquitocidal strains of chlamydomonas reinhardtii. Microorganisms 10, 1087 (2022).
-
Coragliotti, A. T., Beligni, M. V., Franklin, S. E. & Mayfield, S. P. Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast. Mol. Biotechnol. 48, 60–75 (2011).
-
Barkan, A. Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold. Plant Physiol. 155, 1520–1532 (2011).
-
Kasai, S. et al. Effect of coding regions on chloroplast gene expression in Chlamydomonas reinhardtii. J. Biosci. Bioeng. 95, 276–282 (2003).
-
Kato, K., Ishikura, K., Kasai, S. & Shinmyo, A. Efficient translation destabilizes transcripts in chloroplasts of Chlamydomonas reinhardtii. J. Biosci. Bioeng. 101, 471–477 (2006).
-
Viola, S. et al. MDA1, a nucleus-encoded factor involved in the stabilization and processing of the atpA transcript in the chloroplast of Chlamydomonas. Plant J. 98, 1033–1047 (2019).
-
Eberhard, S. et al. Dual functions of the nucleus-encoded factor TDA1 in trapping and translation activation of atpA transcripts in Chlamydomonas reinhardtii chloroplasts. Plant J. 67, 1055–1066 (2011).
-
Chaux, F. et al. Chloroplast ATP synthase biogenesis requires peripheral stalk subunits AtpF and ATPG and stabilization of atpE mRNA by OPR protein MDE1. Plant J. 116, 1582–1599 (2023).
-
Anthonisen, I. L., Salvador, M. L. & Klein, U. W. E. Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii. RNA 7, 1024–1033 (2001).
-
Hauser, C. R., Gillham, N. W. & Boynton, J. E. Translational regulation of chloroplast genes: proteins binding to the 5′-untranslated regions of chloroplast mrnas in chlamydomonas reinhardtii(∗). J. Biol. Chem. 271, 1486–1497 (1996).
-
Odom, O. W., Kang, S., Ferguson, C., Chen, C. & Herrin, D. L. Overcoming poor transgene expression in the wild-type chlamydomonas chloroplast: creation of highly mosquitocidal strains of chlamydomonas reinhardtii. Microorganisms 10 (2022). https://doi.org/10.3390/microorganisms10061087
-
Miró-Vinyals, B. et al. Chloroplast engineering of the green microalgae Chlamydomonas reinhardtii for the production of HAA, the lipid moiety of rhamnolipid biosurfactants. N. Biotechnol. 76, 1–12 (2023).
-
Mordaka, P. M. et al. Regulation of nucleus-encoded trans-acting factors allows orthogonal fine-tuning of multiple transgenes in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol. J. 23, 1005–1018 (2025).
-
Kurowska, M. M. et al. Methyl Jasmonate Affects Photosynthesis Efficiency, Expression of HvTIP Genes and Nitrogen Homeostasis in Barley. Int J Mol Sci 21 (2020). https://doi.org/10.3390/ijms21124335
-
Xie, D. X., Feys, B. F., James, S., Nieto-Rostro, M. & Turner, J. G. COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280, 1091–1094 (1998).
-
Devoto, A. et al. COI1 links jasmonate signalling and fertility to the SCF ubiquitin-ligase complex in Arabidopsis. Plant J. 32, 457–466 (2002).
-
Chini, A., Boter, M. & Solano, R. Plant oxylipins: COI1/JAZs/MYC2 as the core jasmonic acid-signalling module. FEBS J. 276, 4682–4692 (2009).
-
Staswick, P. E. JAZing up jasmonate signaling. Trends Plant Sci. 13, 66–71 (2008).
-
Fonseca, S., Chico, J. M. & Solano, R. The jasmonate pathway: the ligand, the receptor and the core signalling module. Curr. Opin. Plant Biol. 12, 539–547 (2009).
-
Lohr, M., Schwender, J. & Polle, J. E. Isoprenoid biosynthesis in eukaryotic phototrophs: a spotlight on algae. Plant Sci. 185-186, 9–22 (2012).
-
Higo, K., Ugawa, Y., Iwamoto, M. & Higo, H. PLACE: a database of plant cis-acting regulatory DNA elements. Nucleic Acids Res. 26, 358–359 (1998).
-
Jansen, R. K. et al. Methods for obtaining and analyzing whole chloroplast genome sequences. Methods Enzymol. 395, 348–384 (2005).
-
Barrera, D., Gimpel, J. & Mayfield, S. in Chloroplast Biotechnology: Methods and Protocols (ed Pal Maliga) 391-399 (Humana Press, 2014).
-
Nouemssi, S. B. et al. Rapid and efficient colony-pcr for high throughput screening of genetically transformed chlamydomonas reinhardtii. Life 10, 186 (2020).
-
Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).
