A modular synthetic biology toolkit unlocks metabolic engineering of the industrially relevant alga Nannochloropsis

Abstract

Nannochloropsis is an industrially relevant marine microalga with exceptional potential as a chassis for sunlight-driven CO₂ valorization. However, its broad application in synthetic biology has been constrained by the lack of a standardized and modular genetic toolbox. Here, we report the development of a comprehensive Modular Cloning (MoClo) toolkit for Nannochloropsis, based on Golden Gate assembly and a standard syntax. The toolkit comprises 91 domesticated genetic parts spanning promoters, signal peptides, selectable markers, reporter genes, tags and terminators. A large subset of these parts, including several not previously evaluated in Nannochloropsis, was functionally validated, enabling convenient and reliable transformant selection, immunodetection, and subcellular localization. To demonstrate the utility of the toolkit for multi-gene pathway engineering, modularly assembled keto-carotenoid biosynthetic pathways were introduced into Nannochloropsis, leading to substantial accumulation of canthaxanthin (4.5 mg g⁻¹) or astaxanthin (2.8 mg g⁻¹). Collectively, this flexible and expandable MoClo toolkit establishes a standardized foundation for synthetic biology in Nannochloropsis, enables rapid design-build-test cycles for multi-gene constructs, and advances the use of industrial microalga for sustainable, CO₂-based production of value-added biochemicals.

Keywords

Algal biotechnology / Carotenoid biosynthesis / Green production / Metabolic engineering / Modular assembly / Synthetic biology

References

[1] Canini D, Martini F, Cazzaniga S, Miotti T, Pacenza B, D’Adamo S, Ballottari M. Genetic engineering of Nannochloropsis oceanica to produce canthaxanthin and ketocarotenoids. Microb Cell Fact, 2024, 23: 322

[2] Crozet P, Navarro FJ, Willmund F, Mehrshahi P, Bakowski K, Lauersen KJ, Pérez-Pérez M-E, Auroy P, Gorchs Rovira A, Sauret-Gueto S, Niemeyer Jet al.. Birth of a photosynthetic chassis: a MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii. ACS Synth Biol, 2018, 7: 2074-2086

[3] Daneshvar E, Wicker RJ, Show PL, Bhatnagar A. Biologically-mediated carbon capture and utilization by microalgae towards sustainable CO₂ biofixation and biomass valorization – a review. Chem Eng J, 2022, 427: 130884

[4] Engler C, Youles M, Gruetzner R, Ehnert T-M, Werner S, Jones JDG, Patron NJ, Marillonnet S. A golden gate modular cloning toolbox for plants. ACS Synth Biol, 2014, 3: 839-843

[5] Fayyaz M, Chew KW, Show PL, Ling TC, Ng IS, Chang J-S. Genetic engineering of microalgae for enhanced biorefinery capabilities. Biotechnol Adv, 2020, 43: 107554

[6] Galasso C, Orefice I, Pellone P, Cirino P, Miele R, Ianora A, Brunet C, Sansone C. On the neuroprotective role of astaxanthin: new perspectives?. Mar Drugs, 2018, 16: 247

[7] Guo Y, Dong J, Zhou T, Auxillos J, Li T, Zhang W, Wang L, Shen Y, Luo Y, Zheng Y, Lin Jet al.. Yeastfab: the design and construction of standard biological parts for metabolic engineering in Saccharomyces cerevisiae. Nucleic Acids Res, 2015, 43: e88

[8] Hirano M, Ando R, Shimozono S, Sugiyama M, Takeda N, Kurokawa H, Deguchi R, Endo K, Haga K, Takai-Todaka R, Inaura Set al.. A highly photostable and bright green fluorescent protein. Nat Biotechnol, 2022, 40: 1132-1142

[9] Jeong BR, Jang J, Jin E. Genome engineering via gene editing technologies in microalgae. Bioresour Technol, 2023, 373: 128701

[10] Karas BJ, Diner RE, Lefebvre SC, McQuaid J, Phillips APR, Noddings CM, Brunson JK, Valas RE, Deerinck TJ, Jablanovic J, Gillard JTFet al.. Designer diatom episomes delivered by bacterial conjugation. Nat Commun, 2015, 6: 6925

[11] Kassaw TK, Paton AJ, Peers G. Episome-based gene expression modulation platform in the model diatom Phaeodactylum tricornutum. ACS Synth Biol, 2022, 11: 191-204

[12] Kilian O, Benemann CSE, Niyogi KK, Vick B. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc Natl Acad Sci U S A, 2011, 108: 21265-21269

[13] Koh HG, Kang NK, Jeon S, Shin S-E, Jeong B-R, Chang YK. Heterologous synthesis of chlorophyll b in Nannochloropsis salina enhances growth and lipid production by increasing photosynthetic efficiency. Biotechnol Biofuels, 2019, 12: 122-122

[14] Kumar M, Sun Y, Rathour R, Pandey A, Thakur IS, Tsang DCW. Algae as potential feedstock for the production of biofuels and value-added products: opportunities and challenges. Sci Total Environ, 2020, 716: 137116

[15] Lai HE, Moore S, Polizzi K, Freemont P. EcoFlex: a multifunctional MoClo kit for E. coli synthetic biology. Methods Mol Biol, 2018, 1772: 429-444

[16] Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular, multipart assembly. ACS Synth Biol, 2015, 4: 975-986

[17] Li F, Gao D, Hu H. High-efficiency nuclear transformation of the oleaginous marine Nannochloropsis species using PCR product. Biosci Biotechnol Biochem, 2014, 78: 812-817

[18] Li DW, Balamurugan S, Yang YF, Zheng JW, Huang D, Zou LG, Yang WD, Liu JS, Guan Y, Li HY. Transcriptional regulation of microalgae for concurrent lipid overproduction and secretion. Sci Adv, 2019, 5: eaau3795

[19] Liu J, Liu M, Pan Y, Shi Y, Hu H. Metabolic engineering of the oleaginous alga Nannochloropsis for enriching eicosapentaenoic acid in triacylglycerol by combined pulling and pushing strategies. Metab Eng, 2022, 69: 163-174

[20] Liu M, Ding W, Yu L, Shi Y, Liu J. Functional characterization of carotenogenic genes provides implications into carotenoid biosynthesis and engineering in the marine alga Nannochloropsis oceanica. Algal Res, 2022, 67: 102853

[21] Liu M, Ding W, Pan Y, Hu H, Liu J. Zeaxanthin epoxidase is involved in the carotenoid biosynthesis and light-dependent growth of the marine alga Nannochloropsis oceanica. Biotechnol Biofuels Bioprod, 2023, 16: 74

[22] Liu M, Yu L, Zheng J, Shao S, Pan Y, Hu H, Shen L, Wang W, Zhou W, Liu J. Turning the industrially relevant marine alga Nannochloropsis red: one move for multifaceted benefits. New Phytol, 2024, 244: 1467-1481

[23] Liu M, Zheng J, Yu L, Shao S, Zhou W, Liu J. Engineering Nannochloropsis oceanica for concurrent production of canthaxanthin and eicosapentaenoic acid. Bioresour Technol, 2024, 413 131525

[24] Marillonnet S, Grützner R. Synthetic DNA assembly using golden gate cloning and the hierarchical modular cloning pipeline. Curr Protoc Mol Biol, 2020, 130: e115

[25] Moog D, Stork S, Reislöhner S, Grosche C, Maier U-G. In vivo localization studies in the stramenopile alga Nannochloropsis oceanica. Protist, 2015, 166: 161-171

[26] Naduthodi MIS, Südfeld C, Avitzigiannis EK, Trevisan N, van Lith E, Alcaide Sancho J, D’Adamo S, Barbosa M, van der Oost J. Comprehensive genome engineering toolbox for microalgae Nannochloropsis oceanica based on CRISPR-Cas systems. ACS Synth Biol, 2021, 10: 3369-3378

[27] Nielsen JR, Lewis MJ, Huang WE. Construction and characterization of MoClo-compatible vectors for modular protein expression in E. coli. ACS Synth Biol, 2025, 14: 398-406

[28] Patron NJ, Orzaez D, Marillonnet S, Warzecha H, Matthewman C, Youles M, Raitskin O, Leveau A, Farré G, Rogers C, Smith Aet al.. Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytol, 2015, 208: 13-19

[29] Poliner E, Pulman JA, Zienkiewicz K, Childs K, Benning C, Farré EM. A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long-chain polyunsaturated fatty acid production. Plant Biotechnol J, 2018, 16: 298-309

[30] Poliner E, Takeuchi T, Du Z-Y, Benning C, Farré EM. Nontransgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779. ACS Synth Biol, 2018, 7: 962-968

[31] Poliner E, Clark E, Cummings C, Benning C, Farre EM. A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779. Algal Res, 2020, 45: 101664

[32] Radakovits R, Jinkerson RE, Fuerstenberg SI, Tae H, Settlage RE, Boore JL, Posewitz MC. Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropis gaditana. Nat Commun, 2012, 3: 686

[33] Rebelo BA, Farrona S, Ventura MR, Abranches R. Canthaxanthin, a red-hot carotenoid: applications, synthesis, and biosynthetic evolution. Plants, 2020, 9: 1039

[34] Shah MMR, Liang Y, Cheng JJ, Daroch M. Astaxanthin-producing green microalga Haematococcus pluvialis: from single cell to high value commercial products. Front Plant Sci, 2016, 7: 531

[35] Shi Y, Liu M, Pan Y, Hu H, Liu J. Δ6 fatty acid elongase is involved in eicosapentaenoic acid biosynthesis via the ω6 pathway in the marine alga Nannochloropsis oceanica. J Agric Food Chem, 2021, 69: 9837-9848

[36] Sproles AE, Fields FJ, Smalley TN, Le CH, Badary A, Mayfield SP. Recent advancements in the genetic engineering of microalgae. Algal Res, 2021, 53: 102158

[37] Vasudevan R, Gale GAR, Schiavon AA, Puzorjov A, Malin J, Gillespie MD, Vavitsas K, Zulkower V, Wang B, Howe CJ, Lea-Smith DJet al.. Cyanogate: a modular cloning suite for engineering Cyanobacteria based on the plant MoClo syntax. Plant Physiol, 2019, 180: 39

[38] Vieler A, Wu G, Tsai C-H, Bullard B, Cornish AJ, Harvey C, Reca I-B, Thornburg C, Achawanantakun R, Buehl CJ, Campbell MSet al.. Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous Alga Nannochloropsis oceanica CCMP1779. PLoS Genet, 2012, 8: e1003064

[39] Wang D, Ning K, Li J, Hu J, Han D, Wang H, Zeng X, Jing X, Zhou Q, Su X, Chang Xet al.. Nannochloropsis genomes reveal evolution of microalgal oleaginous traits. PLoS Genet, 2014, 10: e1004094

[40] Wang Q, Gong Y, He Y, Xin Y, Lv N, Du X, Li Y, Jeong B-r, Xu J. Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages. Plant J, 2021, 106: 1148-1162

[41] Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS ONE, 2011, 6: e16765

[42] Wei H, Shi Y, Ma X, Pan Y, Hu H, Li Y, Luo M, Gerken H, Liu J. A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica. Biotechnol Biofuels, 2017, 10 174

[43] Wei L, Xin Y, Wang Q, Yang J, Hu H, Xu J. RNAi-based targeted gene-knockdown in the model oleaginous microalgae Nannochloropsis oceanica. Plant J, 2017, 89: 1236-1250

[44] Xin Y, Lu Y, Lee Y-Y, Wei L, Jia J, Wang Q, Wang D, Bai F, Hu H, Hu Q, Liu Jet al.. Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases. Mol Plant, 2017, 10: 1523-1539

[45] Xu Y. Biochemistry and biotechnology of lipid accumulation in the microalga Nannochloropsis oceanica. J Agric Food Chem, 2022, 70: 11500-11509

[46] Xu P, Shao S, Qian J, Li J, Xu R, Liu J, Zhou W. Scale-up of microalgal systems for decarbonization and bioproducts: challenges and opportunities. Bioresour Technol, 2024, 398: 130528

[47] Ye Y, Liu M, Yu L, Sun H, Liu J. Nannochloropsis as an emerging algal chassis for light-driven synthesis of lipids and high-value products. Mar Drugs, 2024, 22: 54

[48] Yu Z, Geisler K, Leontidou T, Young REB, Vonlanthen SE, Purton S, Abell C, Smith AG. Droplet-based microfluidic screening and sorting of microalgal populations for strain engineering applications. Algal Res, 2021, 56: 102293

[49] Zhang Y, Ye Y, Bai F, Liu J. The oleaginous astaxanthin-producing alga Chromochloris zofingiensis: potential from production to an emerging model for studying lipid metabolism and carotenogenesis. Biotechnol Biofuels, 2021, 14: 119

[50] Zheng J, Shi Y, Yu L, Sun H, Zhou W, Liu J. Modulation of an omega-3 fatty acid desaturase for eicosapentaenoic acid biosynthesis in the alga Nannochloropsis oceanica. Algal Res, 2025, 85: 103877

[51] Zhong Y-J, Huang J-C, Liu J, Li Y, Jiang Y, Xu Z-F, Sandmann G, Chen F. Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis. J Exp Bot, 2011, 62: 3659-3669

[52] Zhou W, Wang J, Chen P, Ji C, Kang Q, Lu B, Li K, Liu J, Ruan R. Bio-mitigation of carbon dioxide using microalgal systems: advances and perspectives. Renew Sustain Energy Rev, 2017, 76: 1163-1175

[53] Zienkiewicz K, Zienkiewicz A, Poliner E, Du Z-Y, Vollheyde K, Herrfurth C, Marmon S, Farré EM, Feussner I, Benning C. Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts. Biotechnol Biofuels, 2017, 10: 8

Funding

National Natural Science Foundation of China(32472487)

RIGHTS & PERMISSIONS

The Author(s)