Cite this paper:
Li GUO, Guanpin YANG. Nannochloropsis artificial chromosomes (NannoACs) loom on the horizon[J]. Journal of Oceanology and Limnology, 2023, 41(6): 2336-2347

Nannochloropsis artificial chromosomes (NannoACs) loom on the horizon

Li GUO1, Guanpin YANG1,2,3
1 College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China;
2 Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China;
3 Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003, China
Species in genus Nannochloropsis, especially N. oceanica and N. gaditana, have been evolving as the model microalgae for both application and theory studies. The position effect of genome integration, the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments. The molecular tools including genetic transformation, homologous recombination, gene edition, gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species. The construction of animal and plant artificial chromosomes with “top down” strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes (NannoACs). It seems that the methods and materials to set the foundation for constructing NannoACs are at hands. In this review, we outlined the current status of transgenes in Nannochloropsis species, summarized the limitations of both integrative and non-integrative vectors, and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first, and then truncating the natural chromosomes. NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds, thus contributing towards its genetic improvement and synthetic biological studies.
Key words:    Nannochloropsis|artificial chromosome|microalgal genetic improvement|gene stacking|concatenate gene|trait transfer   
Received: 2022-08-30   Revised:
PDF (854 KB) Free
Print this page
Add to favorites
Email this article to others
Articles by Li GUO
Articles by Guanpin YANG
Abe S, Honma K, Okada A et al.2021.Construction of stable mouse artificial chromosome from native mouse chromosome 10 for generation of transchromosomic mice.Scientific Reports, 11(1):20050,
Abidin A A Z, Suntarajh M, Yusof Z N B.2020.Transformation of a Malaysian species of Nannochloropsis:gateway to construction of transgenic microalgae as vaccine delivery system to aquatic organisms.Bioengineered, 11(1):1071-1079,
Abremski K, Hoess R, Sternberg N.1983.Studies on the properties of P1 site-specific recombination:evidence for topologically unlinked products following recombination.Cell, 32(4):1301-1311,
Andersen R A, Brett R W, Potter D et al.1998.Phylogeny of the Eustigmatophyceae based upon 18s rDNA, with emphasis on Nannochloropsis.Protist, 149(1):61-74,
Araki K, Imaizumi T, Okuyama K et al.1997.Efficiency of recombination by Cre transient expression in embryonic stem cells:comparison of various promoters.The Journal of Biochemistry, 122(5):977-982,
Arnak R, Bruschi C V, Tosato V.2012.Yeast artificial chromosomes.In:Encyclopedia of Life Sciences (eLS).John Wiley & Sons Ltd, Chichester, UK,
Bailey J C, Freshwater D W.1997.Molecular systematics of the Gelidiales:inferences from separate and combined analyses of plastid rbcL and nuclear SSU gene sequences.European Journal of Phycology, 32(4):343-352,
Boussiba S, Vonshak A, Cohen Z et al.1987.Lipid and biomass production by the halotolerant microalga Nannochloropsis salina.Biomass, 12(1):37-47,
Bruno W J, Knill E, Balding D J et al.1995.Efficient pooling designs for library screening.Genomics, 26(1):21-30,
Burke D T, Carle G F, Olson M V.1987.Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors.Science, 236(4803):806-812,
Carpinelli E C, Telatin A, Vitulo N et al.2014.Chromosome scale genome assembly and transcriptome profiling of Nannochloropsis gaditana in nitrogen depletion.Molecular Plant, 7(2):323-335,
Chankova S G, Dimova E, Dimitrova M et al.2007.Induction of DNA double-strand breaks by zeocin in Chlamydomonas reinhardtii and the role of increased DNA double-strand breaks rejoining in the formation of an adaptive response.Radiation and Environmental Biophysics, 46(4):409-416,
Chen Y W, Hu H H.2019.High efficiency transformation by electroporation of the freshwater alga Nannochloropsis limnetica.World Journal of Microbiology and Biotechnology, 35(8):119,
Chen J W, Huang Y F, Shu Y X et al.2022.Recent progress on systems and synthetic biology of diatoms for improving algal productivity.Frontiers in Bioengineering and Biotechnology, 10:908804,
Daugbjerg N, Andersen R A.1997.A molecular phylogeny of the heterokont algae based on analyses of chloroplast-encoded rbcL sequence data.Journal of Phycology, 33(6):1031-1041,
Echeverri D, Romo J, Giraldo N et al.2019.Microalgae protoplasts isolation and fusion for biotechnology research.Revista Colombiana de Biotecnología, 21(1):101-112,
Ehrhardt A, Haase R, Schepers A et al.2008.Episomal vectors for gene therapy.Current Gene Therapy, 8(3):147-161,
Elnifro E M, Ashshi A M, Cooper R J et al.2000.Multiplex PCR:optimization and application in diagnostic virology.Clinical Microbiology Reviews, 13(4):559-570,
Fabris M, Abbriano R M, Pernice M et al.2020.Emerging technologies in algal biotechnology:toward the establishment of a sustainable, algae-based bioeconomy.Frontiers in Plant Science, 11:279,
Fawley K P, Fawley M W.2007.Observations on the diversity and ecology of freshwater Nannochloropsis (Eustigmatophyceae), with descriptions of new taxa.Protist, 158(3):325-336,
Fawley M W, Jameson I, Fawley K P.2015.The phylogeny of the genus Nannochloropsis (Monodopsidaceae, Eustigmatophyceae), with descriptions of N.australis sp.nov.and Microchloropsis gen.nov.Phycologia, 54(5):545-552,
Galloway R E.1990.Selective conditions and isolation of mutants in salt-tolerant, lipid-producing microalgae.Journal of Phycology, 26(4):752-760,
Gee C W, Niyogi K K.2017.The carbonic anhydrase CAH1 is an essential component of the carbon-concentrating mechanism in Nannochloropsis oceanica.Proceedings of the National Academy of Sciences of the United States of America, 114(17):4537-4542,
Golic K G, Lindquist S.1989.The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.Cell, 59(3):499-509,
Gong Y H, Kang N K, Kim Y U et al.2020.The NanDeSyn database for Nannochloropsis systems and synthetic biology.The Plant Journal, 104(6):1736-1745,
Gossen M, Bujard H.1992.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.Proceedings of the National Academy of Sciences of the United States of America, 89(12):5547-5551,
Guo C L, Anwar M, Mei R et al.2022.Establishment and optimization of PEG-mediated protoplast transformation in the microalga Haematococcus pluvialis.Journal of Applied Phycology, 34(3):1595-1605,
Guo L, Liang S J, Zhang Z Y et al.2019.Genome assembly of Nannochloropsis oceanica provides evidence of host nucleus overthrow by the symbiont nucleus during speciation.Communications Biology, 2(1):249,
Halpin C.2005.Gene stacking in transgenic plants-the challenge for 21st century plant biotechnology.Plant Biotechnology Journal, 3(2):141-155,
Hibberd D J.1981.Notes on the taxonomy and nomenclature of the algal classes Eustigmatophyceae and Tribophyceae (synonym Xanthophyceae).Botanical Journal of the Linnean Society, 82(2):93-119,
Hicks L, Van Der Graaf C M, Childress J et al.2018.Streamlined preparation of genomic DNA in agarose plugs for pulsed-field gel electrophoresis.Journal of Biological Methods, 5(1):e86,
Ikeno M, Suzuki N.2011.Construction and use of a bottom-up HAC vector for transgene expression.Methods in Molecular Biology, 738:101-110,
Kadkhodaei S, Memari H R, Abbasiliasi S et al.2016.Multiple overlap extension PCR (MOE-PCR):an effective technical shortcut to high throughput synthetic biology.RSC Advances, 6(71):66682-66694,
Kandilian R, Lee E, Pilon L.2013.Radiation and optical properties of Nannochloropsis oculata grown under different irradiances and spectra.Bioresource Technology,
137:63-73, B J, Diner R E, Lefebvre S C et al.2015.Designer diatom episomes delivered by bacterial conjugation.Nature Communications, 6(1):6925,
Kilian O, Benemann C S E, Niyogi K K et al.2011.High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.Proceedings of the National Academy of Sciences of the United States of America, 108(52):21265-21269,
Kouprina N, Petrov N, Molina O et al.2018.Human artificial chromosome with regulated centromere:a tool for genome and cancer studies.ACS Synthetic Biology, 7(9):1974-1989,
Kurita T, Iwai M, Moroi K et al.2022.Genome editing with removable TALEN vectors harboring a yeast centromere and autonomous replication sequence in oleaginous microalga.Scientific Reports, 12(1):2480,
Kurita T, Moroi K, Iwai M et al.2020.Efficient and multiplexable genome editing using Platinum TALENs in oleaginous microalga, Nannochloropsis oceanica NIES-2145.Genes to Cells, 25(10):695-702,
Kuroiwa Y, Shinohara T, Notsu T et al.1998.Efficient modification of a human chromosome by telomere-directed truncation in high homologous recombination-proficient chicken DT40 cells.Nucleic Acids Research, 26(14):3447-3448,
Kuroiwa Y, Tomizuka K, Shinohara T et al.2000.Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts.Nature Biotechnology, 18(10):1086-1090,
Le-Feuvre R, Moraga-Suazo P, González-Durán J et al.2021.Chemical induction of polyploidy increases astaxanthin accumulation capacity in the microalgae Haematococcus lacustris (Gir.-Chantr.) Rostaf.Algal Research, 59:102465,
Lee N C O, Kim J H, Petrov N S et al.2018.Method to assemble genomic DNA fragments or genes on human artificial chromosome with regulated kinetochore using a multi-integrase system.ACS Synthetic Biology, 7(1):63-74,
Li F J, Gao D W, Hu H H.2014.High-efficiency nuclear transformation of the oleaginous marine Nannochloropsis species using PCR product.Bioscience, Biotechnology, and Biochemistry, 78(5):812-817,
Liang C W, Cao S N, Zhang X W et al.2013.De novo sequencing and global transcriptome analysis of Nannochloropsis sp.(Eustigmatophyceae) following nitrogen starvation.BioEnergy Research, 6(2):494-505,
Lubián L M, Montero O, Moreno-Garrido I et al.2000.Nannochloropsis (Eustigmatophyceae) as source of commercially valuable pigments.Journal of Applied Phycology, 12(3):249-255,
Ma X N, Chen T P, Yang B et al.2016.Lipid production from Nannochloropsis.Marine Drugs, 14(4):61,
Manning W M, Strain H H.1943.Chlorophyll d, a green pigment of red algae.Journal of Biology Chemistry, 151(1):1-19,
Mansouri H, Nezhad F S.2021.Changes in growth and biochemical parameters in Dunaliella salina (Dunaliellaceae) in response to auxin and gibberellin under colchicine-induced polyploidy.Journal of Phycology, 57(4):1284-1294,
Manzoor A, Ahmad T, Bashir M A et al.2019.Studies on colchicine induced chromosome doubling for enhancement of quality traits in ornamental plants.Plants, 8(7):194,
Miga K H, Koren S, Rhie A et al.2020.Telomere-to-telomere assembly of a complete human X chromosome.Nature, 585(7823):79-84,
Moriwaki T, Abe S, Oshimura M et al.2020.Transchromosomic technology for genomically humanized animals.Experimental Cell Research, 390(2):111914,
Muñoz C F, Südfeld C, Naduthodi M I S et al.2021.Genetic engineering of microalgae for enhanced lipid production.Biotechnology Advances, 52:107836,
Murata M, Shibata F, Hironaka A et al.2013.Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination.The Plant Journal, 74(3):363-371,
Muravenko O, Selyakh I, Kononenko N et al.2001.Chromosome numbers and nuclear DNA contents in the red microalgae Cyanidium caldarium and three Galdieria species.European Journal of Phycology, 36(3):227-232,
Naduthodi M I S, Claassens N J, D'Adamo S et al.2021a.Synthetic biology approaches to enhance microalgal productivity.Trends in Biotechnology, 39(10):1019-1036,
Naduthodi M I S, Südfeld C, Avitzigiannis E K et al.2021b.Comprehensive genome engineering toolbox for microalgae Nannochloropsis oceanica based on CRISPR-Cas systems.ACS Synthetic Biology, 10(12):3369-3378,
Naduthodi M I S, Mohanraju P, Südfeld C et al.2019.CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1.Biotechnology for Biofuels, 12(1):66,
Nair S, Karim R, Cardosa M J et al.1999.Convenient and versatile DNA extraction using agarose plugs for ribotyping of problematic bacterial species.Journal of Microbiological Methods, 38(1-2):63-67,
Nezhad F S, Mansouri H.2019.Induction of polyploidy by colchicine on the green algae Dunaliella salina.Russian Journal of Marine Biology, 45(2):106-112,
Oshimura M, Uno N, Kazuki Y et al.2015.A pathway from chromosome transfer to engineering resulting in human and mouse artificial chromosomes for a variety of applications to bio-medical challenges.Chromosome Research, 23(1):111-133,
Pan K H, Qin J J, Li S et al.2011.Nuclear monoploidy and asexual propagation of Nannochloropsis oceanica (Eustigmatophyceae) as revealed by its genome sequence.Journal of Phycology, 47(6):1425-1432,
Park S B, Yun J H, Ryu A J et al.2021.Development of a novel nannochloropsis strain with enhanced violaxanthin yield for large-scale production.Microbial Cell Factories, 20(1):43,
Poliner E, Clark E, Cummings C et al.2020.A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779.Algal Research, 45:101664,
Poliner E, Farré E M, Benning C.2018a.Advanced genetic tools enable synthetic biology in the oleaginous microalgae Nannochloropsis sp.Plant Cell Reports, 37(10):1383-1399,
Poliner E, Pulman J A, Zienkiewicz K et al.2018b.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 Biotechnology Journal, 16(1):298-309,
Poliner E, Takeuchi T, Du Z Y et al.2018c.Nontransgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779.ACS Synthetic Biology, 7(4):962-968,
Ponomartsev S V, Sinenko S A, Skvortsova E V et al.2020.Human alphoidtetO artificial chromosome as a gene therapy vector for the developing hemophilia a model in mice.Cells, 9(4):879,
Radakovits R, Jinkerson R E, Fuerstenberg S I et al.2012.Draft genome sequence and genetic transformation of the oleaginous alga Nannochloropsis gaditana.Nature Communications, 3(1):686,
Reece-Hoyes J S, Walhout A J M.2018.Gateway recombinational cloning.Cold Spring Harbor Protocols, 2018(1):pdb.top094912,
Ren G P, Zhang X, Li Y et al.2021.Large-scale whole-genome resequencing unravels the domestication history of Cannabis sativa.Science Advances, 7(29):eabg2286,
Riaz S, Xiao M, Chen P Y et al.2021.The genome copy number of the thermophilic cyanobacterium Thermosynechococcus elongatus E542 is controlled by growth phase and nutrient availability.Applied and Environmental Microbiology, 87(9):e02993-20,
Ryu A J, Jeong B R, Kang N K et al.2021.Safe-harboring based novel genetic toolkit for Nannochloropsis salina CCMP1776:efficient overexpression of transgene via CRISPR/Cas9-mediated knock-in at the transcriptional hotspot.Bioresource Technology, 340:125676,
Sánchez-Gárate J D, Cira-Chavez L A, Rout N P.2020.Visualization of smaller chromosomes from unicellular microalgae.Brazilian Journal of Botany, 43(3):633-641,
Satofuka H, Abe S, Moriwaki T et al.2022.Efficient human-like antibody repertoire and hybridoma production in trans-chromosomic mice carrying megabase-sized human immunoglobulin loci.Nature Communications, 13(1):1841,
Schweizer H P.2003.Applications of the Saccharomyces cerevisiae Flp-FRT system in bacterial genetics.Journal of Molecular Microbiology and Biotechnology, 5(2):67-77,
Sharma-Kuinkel B K, Rude T H, Fowler V G Jr.2016.Pulse field gel electrophoresis.Methods in Molecular Biology, 1373:117-130,
Shizuya H, Kouros-Mehr H.2001.The development and applications of the bacterial artificial chromosome cloning system.The Keio Journal of Medicine, 50(1):26-30,
Südfeld C, Pozo-Rodríguez A, Díez S A M et al.2022.The nucleolus as a genomic safe harbor for strong gene expression in Nannochloropsis oceanica.Molecular Plant, 15(2):340-353,
Sukenik A, Carmeli Y, Berner T.1989.Regulation of fatty acid composition by irradiance level in the eustigmatophyte Nannochloropsis sp.Journal of Phycology, 25(4):686-692,
Suzuki Y, Morishita S.2021.The time is ripe to investigate human centromeres by long-read sequencing.DNA Research, 28(6):dsab021,
Szpyrka E, Broda D, Oklejewicz B et al.2020.A non-vector approach to increase lipid levels in the microalga Planktochlorella nurekis.Molecules, 25(2):270,
Takiguchi M, Kazuki Y, Hiramatsu K et al.2014.A novel and stable mouse artificial chromosome vector.ACS Synthetic Biology, 3(12):903-914,
Tan J T, Zhao Y C, Wang B et al.2020.Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining.Plant Biotechnology Journal, 18(11):2161-2163,
Thorpe H M, Smith M C M.1998.In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family.Proceedings of the National Academy of Sciences of the United States of America, 95(10):5505-5510,
Tomizuka K, Yoshida H, Uejima H et al.1997.Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice.Nature Genetics, 16(2):133-143,
Torella J P, Boehm C R, Lienert F et al.2014.Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly.Nucleic Acids Research, 42(1):681-689,
Verruto J, Francis K, Wang Y J et al.2018.Unrestrained markerless trait stacking in Nannochloropsis gaditana through combined genome editing and marker recycling technologies.Proceedings of the National Academy of Sciences of the United States of America, 115(30):E7015-E7022,
Vieler A, Wu G X, Tsai C H et al.2012.Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.PLoS Genetics, 8(11):e1003064,
Wang D M, Ning K, Li J et al.2014.Nannochloropsis genomes reveal evolution of microalgal oleaginous traits.PLoS Genetics, 10(1):e1004094,
Wang Q T, Gong Y H, He Y H et al.2021.Genome engineering of Nannochloropsis with hundred-kilobase fragment deletions by Cas9 cleavages.The Plant Journal, 106(4):1148-1162,
Wang Q T, Lu Y D, Xin Y et al.2016.Genome editing of model oleaginous microalgae Nannochloropsis CRISPR/Cas9.The Plant Journal, 88(6):1071-1081,
Yang G P, Zhang Z Y, Liu H et al.2019.An investigation of the possible methods and potential benefits of de novo cloning of Nannochloropsis oceanica genes.Marine Life Science & Technology, 1(1):22-27,
Yu W C, Lamb J C, Han F P et al.2006.Telomere-mediated chromosomal truncation in maize.Proceedings of the National Academy of Sciences of the United States of America, 103(46):17331-17336,
Yu W C, Yau Y Y, Birchler J A.2016.Plant artificial chromosome technology and its potential application in genetic engineering.Plant Biotechnology Journal, 14(5):1175-1182,
Zerulla K, Ludt K, Soppa J.2016.The ploidy level of Synechocystis sp.PCC 6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters.Microbiology, 162(5):730-739,
Zhang P, Xin Y, He Y H et al.2022.Exploring a blue-light-sensing transcription factor to double the peak productivity of oil in Nannochloropsis oceanica.Nature Communications, 13(1):1664,
Zhang Y, Song W H, Chen S Y et al.2021.A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2.Antiviral Research, 185:104974,
Zhang Y T, Jiang J Y, Shi T Q et al.2019.Application of the CRISPR/Cas system for genome editing in microalgae.Applied Microbiology and Biotechnology, 103(8):3239-3248,
Zulaiha S.2021.Genetic engineering of microalgae lipid biosynthesis for sustainable biodiesel production.World Journal of Advanced Research and Reviews, 11(3):72-77,
Copyright © Haiyang Xuebao