{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T00:49:12Z","timestamp":1793321352318,"version":"4.5.0"},"reference-count":69,"publisher":"Springer Science and Business Media LLC","issue":"19-20","license":[{"start":{"date-parts":[[2022,9,7]],"date-time":"2022-09-07T00:00:00Z","timestamp":1662508800000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2022,9,7]],"date-time":"2022-09-07T00:00:00Z","timestamp":1662508800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100005416","name":"Norges Forskningsr\u00e5d","doi-asserted-by":"publisher","award":["226973\/E40"],"award-info":[{"award-number":["226973\/E40"]}],"id":[{"id":"10.13039\/501100005416","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Appl Microbiol Biotechnol"],"published-print":{"date-parts":[[2022,10]]},"abstract":"<jats:sec>\n                <jats:title>Abstract<\/jats:title>\n                <jats:p><jats:italic>Nannochloropsis oceanica<\/jats:italic> is a unicellular oleaginous microalga of emerging biotechnological interest with a sequenced, annotated genome, available transcriptomic and proteomic data, and well-established basic molecular tools for genetic engineering. To establish <jats:italic>N. oceanica<\/jats:italic> as a eukaryotic host for recombinant protein synthesis and develop molecular technology for vaccine production, we chose the viral surface protein 2 (VP2) of a pathogenic fish virus that causes infectious pancreatic necrosis as a model vaccine. Upon stable nuclear transformation of <jats:italic>N. oceanica<\/jats:italic> strain CCMP1779 with the codon-optimized <jats:italic>VP2<\/jats:italic> gene, a Venus reporter fusion served to evaluate the strength of different endogenous promoters in transformant populations by qPCR and flow cytometry. The highest VP2 yields were achieved for the elongation factor promoter, with enhancer effects by its N-terminal leader sequence. Individual transformants differed in their production capability of reporter-free VP2 by orders of magnitude. When subjecting the best candidates to kinetic analyses of growth and VP2 production in photobioreactors, recombinant protein integrity was maintained until the early stationary growth phase, and a high yield of 4.4% VP2 of total soluble protein was achieved. The maximum yield correlated with multiple integrations of the expression vector into the nuclear genome. The results demonstrate that <jats:italic>N. oceanica<\/jats:italic> was successfully engineered to constitute a robust platform for high-level production of a model subunit vaccine. The molecular methodology established here can likely be adapted in a straightforward manner to the production of further vaccines in the same host, allowing their distribution to fish, vertebrates, or humans via a microalgae-containing diet.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Key points<\/jats:title>\n                <jats:p><jats:italic>\u2022 We engineered N. oceanica for recombinant protein production.<\/jats:italic><\/jats:p>\n                <jats:p><jats:italic>\u2022 The antigenic surface protein 2 of IPN virus could indeed be expressed in the host.<\/jats:italic><\/jats:p>\n                <jats:p><jats:italic>\u2022 A high yield of 4.4% VP2 of total soluble protein was achieved in N. oceanica.<\/jats:italic><\/jats:p>\n              <\/jats:sec>","DOI":"10.1007\/s00253-022-12106-7","type":"journal-article","created":{"date-parts":[[2022,9,7]],"date-time":"2022-09-07T08:03:54Z","timestamp":1662537834000},"page":"6535-6549","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Production of a viral surface protein in Nannochloropsis oceanica for fish vaccination against infectious pancreatic necrosis virus"],"prefix":"10.1007","volume":"106","author":[{"given":"Sweta Suman","family":"Rout","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Imke","family":"de Grahl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaohong","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3856-3880","authenticated-orcid":false,"given":"Sigrun","family":"Reumann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,9,7]]},"reference":[{"issue":"11","key":"12106_CR1","doi-asserted-by":"publisher","first-page":"2783","DOI":"10.1007\/s11274-014-1714-0","volume":"30","author":"F Akbari","year":"2014","unstructured":"Akbari F, Eskandani M, Khosroushahi AY (2014) The potential of transgenic green microalgae; a robust photobioreactor to produce recombinant therapeutic proteins. World J Microbiol Biotechnol 30(11):2783\u20132796. https:\/\/doi.org\/10.1007\/s11274-014-1714-0","journal-title":"World J Microbiol Biotechnol"},{"issue":"26","key":"12106_CR2","doi-asserted-by":"publisher","first-page":"4880","DOI":"10.1016\/j.vaccine.2007.04.068","volume":"25","author":"FC Allnutt","year":"2007","unstructured":"Allnutt FC, Bowers RM, Rowe CG, Vakharia VN, LaPatra SE, Dhar AK (2007) Antigenicity of infectious pancreatic necrosis virus VP2 subviral particles expressed in yeast. Vaccine 25(26):4880\u20134888. https:\/\/doi.org\/10.1016\/j.vaccine.2007.04.068","journal-title":"Vaccine"},{"key":"12106_CR3","doi-asserted-by":"publisher","first-page":"237","DOI":"10.3389\/fpls.2020.00237","volume":"11","author":"N Betterle","year":"2020","unstructured":"Betterle N, Hidalgo Martinez D, Melis A (2020) Cyanobacterial production of biopharmaceutical and biotherapeutic proteins. Front Plant Sci 11:237. https:\/\/doi.org\/10.3389\/fpls.2020.00237","journal-title":"Front Plant Sci"},{"issue":"3","key":"12106_CR4","doi-asserted-by":"publisher","first-page":"912","DOI":"10.1021\/acssynbio.7b00431","volume":"7","author":"N Betterle","year":"2018","unstructured":"Betterle N, Melis A (2018) Heterologous leader sequences in fusion constructs enhance expression of geranyl diphosphate synthase and yield of \u03b2-phellandrene production in cyanobacteria (Synechocystis). ACS Synth Biol 7(3):912\u2013921. https:\/\/doi.org\/10.1021\/acssynbio.7b00431","journal-title":"ACS Synth Biol"},{"key":"12106_CR5","unstructured":"Biering E, Villoing S, Sommerset I, Christie KE (2005) Update on viral vaccines for fish. In: PJ M (ed) Progress in fish vaccinology. 121. Karger, Basel, 97\u2013113"},{"issue":"3","key":"12106_CR6","doi-asserted-by":"publisher","first-page":"743","DOI":"10.1007\/s10811-013-9983-9","volume":"25","author":"MA Borowitzka","year":"2013","unstructured":"Borowitzka MA (2013) High-value products from microalgae\u2014their development and commercialisation. J Appl Phycol 25(3):743\u2013756. https:\/\/doi.org\/10.1007\/s10811-013-9983-9","journal-title":"J Appl Phycol"},{"key":"12106_CR7","doi-asserted-by":"publisher","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","volume":"72","author":"MM Bradford","year":"1976","unstructured":"Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248\u2013254","journal-title":"Anal Biochem"},{"key":"12106_CR8","doi-asserted-by":"publisher","first-page":"141","DOI":"10.1159\/000149667","volume":"25","author":"F Brown","year":"1986","unstructured":"Brown F (1986) The classification and nomenclature of viruses: summary of results of meetings of the international committee on taxonomy of viruses in Sendai, September 1984. Intervirology 25:141\u2013143. https:\/\/doi.org\/10.1159\/000149667","journal-title":"Intervirology"},{"issue":"7261","key":"12106_CR9","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1038\/nature08450","volume":"461","author":"BR Cairns","year":"2009","unstructured":"Cairns BR (2009) The logic of chromatin architecture and remodelling at promoters. Nature 461(7261):193\u2013198. https:\/\/doi.org\/10.1038\/nature08450","journal-title":"Nature"},{"issue":"6","key":"12106_CR10","doi-asserted-by":"publisher","first-page":"2429","DOI":"10.1007\/s00253-013-5413-9","volume":"98","author":"J Camacho-Rodr\u00edguez","year":"2014","unstructured":"Camacho-Rodr\u00edguez J, Gonz\u00e1lez-C\u00e9spedes AM, Cer\u00f3n-Garc\u00eda C, Fern\u00e1ndez-Sevilla JM, Aci\u00e9n-Fern\u00e1ndez FG, Molina-Grima E (2014) A quantitative study of eicosapentaenoic acid (EPA) production by Nannochloropsis gaditana for aquaculture as a function of dilution rate, temperature and average irradiance. Appl Microbiol Biotechnol 98(6):2429\u20132440. https:\/\/doi.org\/10.1007\/s00253-013-5413-9","journal-title":"Appl Microbiol Biotechnol"},{"issue":"1","key":"12106_CR11","doi-asserted-by":"publisher","first-page":"118","DOI":"10.1016\/j.bbrc.2012.06.086","volume":"424","author":"S Cao","year":"2012","unstructured":"Cao S, Zhang X, Ye N, Fan X, Mou S, Xu D, Liang C, Wang Y, Wang W (2012) Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp by quantitative real-time RT-PCR. Biochem Biophys Res Commun 424(1):118\u2013123. https:\/\/doi.org\/10.1016\/j.bbrc.2012.06.086","journal-title":"Biochem Biophys Res Commun"},{"issue":"2","key":"12106_CR12","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3390\/biology7020024","volume":"7","author":"P Charoonnart","year":"2018","unstructured":"Charoonnart P, Purton S, Saksmerprome V (2018) Applications of microalgal biotechnology for disease control in aquaculture. Biology (basel) 7(2):1\u201314. https:\/\/doi.org\/10.3390\/biology7020024","journal-title":"Biology (basel)"},{"issue":"3","key":"12106_CR13","doi-asserted-by":"publisher","first-page":"768","DOI":"10.1111\/j.1529-8817.2008.00508.x","volume":"44","author":"HL Chen","year":"2008","unstructured":"Chen HL, Li SS, Huang R, Tsai HJ (2008) Conditional production of a functional fish growth hormone in the transgenic line of Nannochloropsis oculata (Eustigmatophyceae). J Phycol 44(3):768\u2013776. https:\/\/doi.org\/10.1111\/j.1529-8817.2008.00508.x","journal-title":"J Phycol"},{"issue":"10","key":"12106_CR14","doi-asserted-by":"publisher","first-page":"2279","DOI":"10.1016\/j.bpj.2018.03.015","volume":"114","author":"RV Chereji","year":"2018","unstructured":"Chereji RV, Clark DJ (2018) Major determinants of nucleosome positioning. Biophys J 114(10):2279\u20132289. https:\/\/doi.org\/10.1016\/j.bpj.2018.03.015","journal-title":"Biophys J"},{"issue":"4","key":"12106_CR15","doi-asserted-by":"publisher","first-page":"1792","DOI":"10.1128\/JVI.01536-09","volume":"84","author":"F Coulibaly","year":"2010","unstructured":"Coulibaly F, Chevalier C, Delmas B, Rey FA (2010) Crystal structure of an Aquabirnavirus particle: insights into antigenic diversity and virulence determinism. J Virol 84(4):1792\u20131799. https:\/\/doi.org\/10.1128\/JVI.01536-09","journal-title":"J Virol"},{"issue":"11","key":"12106_CR16","doi-asserted-by":"publisher","first-page":"2025","DOI":"10.3390\/v3112025","volume":"3","author":"M Crane","year":"2011","unstructured":"Crane M, Hyatt A (2011) Viruses of fish: an overview of significant pathogens. Viruses 3(11):2025\u20132046. https:\/\/doi.org\/10.3390\/v3112025","journal-title":"Viruses"},{"key":"12106_CR17","doi-asserted-by":"publisher","first-page":"8303648","DOI":"10.1155\/2019\/8303648","volume":"2019","author":"E Criscuolo","year":"2019","unstructured":"Criscuolo E, Caputo V, Diotti RA, Sautto GA, Kirchenbaum GA, Clementi N (2019) Alternative methods of vaccine delivery: an overview of edible and intradermal vaccines. J Immunol Res 2019:8303648. https:\/\/doi.org\/10.1155\/2019\/8303648","journal-title":"J Immunol Res"},{"issue":"20","key":"12106_CR18","doi-asserted-by":"publisher","first-page":"8747","DOI":"10.1007\/s00253-020-10789-4","volume":"104","author":"I de Grahl","year":"2020","unstructured":"de Grahl I, Rout SS, Maple-Gr\u00f8dem J, Reumann S (2020) Development of a constitutive and an auto-inducible high-yield expression system for recombinant protein production in the microalga Nannochloropsis oceanica. Appl Microbiol Biotechnol 104(20):8747\u20138760. https:\/\/doi.org\/10.1007\/s00253-020-10789-4","journal-title":"Appl Microbiol Biotechnol"},{"issue":"4","key":"12106_CR19","doi-asserted-by":"publisher","first-page":"562","DOI":"10.1016\/j.fsi.2009.12.006","volume":"28","author":"AI de Las Heras","year":"2010","unstructured":"de Las Heras AI, Rodriguez Saint-Jean S, Perez-Prieto SI (2010) Immunogenic and protective effects of an oral DNA vaccine against infectious pancreatic necrosis virus in fish. Fish Shellfish Immunol 28(4):562\u201370. https:\/\/doi.org\/10.1016\/j.fsi.2009.12.006","journal-title":"Fish Shellfish Immunol"},{"key":"12106_CR20","doi-asserted-by":"publisher","first-page":"25","DOI":"10.1016\/0959-8030(95)00003-8","volume":"5","author":"P Dobos","year":"1995","unstructured":"Dobos P (1995) The molecular biology of infectious pancreatic necrosis virus (IPNV). Annu Rev Fish Dis 5:25\u201354. https:\/\/doi.org\/10.1016\/0959-8030(95)00003-8","journal-title":"Annu Rev Fish Dis"},{"issue":"198","key":"12106_CR21","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1139\/m83-062","volume":"29","author":"P Dobos","year":"1983","unstructured":"Dobos P, Roberts TE (1983) The molecular biology of infectious pancreatic necrosis virus: a review. Can J Microbiol 29(198):377\u2013384. https:\/\/doi.org\/10.1139\/m83-062","journal-title":"Can J Microbiol"},{"issue":"2","key":"12106_CR22","doi-asserted-by":"publisher","first-page":"1","DOI":"10.3390\/pathogens9020094","volume":"9","author":"CP Dopazo","year":"2020","unstructured":"Dopazo CP (2020) The infectious pancreatic necrosis virus (IPNV) and its virulence determinants: what is known and what should be known. Pathogens 9(2):1\u201325. https:\/\/doi.org\/10.3390\/pathogens9020094","journal-title":"Pathogens"},{"issue":"6","key":"12106_CR23","doi-asserted-by":"publisher","first-page":"1349","DOI":"10.1093\/nar\/19.6.1349","volume":"19","author":"K Edwards","year":"1991","unstructured":"Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res 19(6):1349","journal-title":"Nucleic Acids Res"},{"key":"12106_CR24","unstructured":"FAO (2017) Fishery and aquaculture statistics, Food and Aquaculture Organisation of the United Nations."},{"issue":"3","key":"12106_CR25","doi-asserted-by":"publisher","first-page":"519","DOI":"10.1007\/s00705-013-1856-7","volume":"159","author":"S Feng","year":"2014","unstructured":"Feng S, Feng W, Zhao L, Gu H, Li Q, Shi K, Guo S, Zhang N (2014) Preparation of transgenic Dunaliella salina for immunization against white spot syndrome virus in crayfish. Arch Virol 159(3):519\u2013525. https:\/\/doi.org\/10.1007\/s00705-013-1856-7","journal-title":"Arch Virol"},{"key":"12106_CR26","doi-asserted-by":"publisher","first-page":"1165","DOI":"10.1099\/0022-1317-76-5-1165","volume":"76","author":"P Frost","year":"1995","unstructured":"Frost P, Hfivarstein LS, Lygren B, Stfihl S, Endresen C, Christie KE (1995) Mapping of neutralization epitopes on infectious pancreatic necrosis viruses. J Gen Virol 76:1165\u20132117. https:\/\/doi.org\/10.1099\/0022-1317-76-5-1165","journal-title":"J Gen Virol"},{"issue":"4","key":"12106_CR27","doi-asserted-by":"publisher","first-page":"879","DOI":"10.1016\/j.cell.2015.04.010","volume":"161","author":"Y Fu","year":"2015","unstructured":"Fu Y, Luo GZ, Chen K, Deng X, Yu M, Han D, Hao Z, Liu J, Lu X, Dore LC, Weng X, Ji Q, Mets L, He C (2015) N6-methyldeoxyadenosine marks active transcription start sites in Chlamydomonas. Cell 161(4):879\u2013892. https:\/\/doi.org\/10.1016\/j.cell.2015.04.010","journal-title":"Cell"},{"issue":"15","key":"12106_CR28","doi-asserted-by":"publisher","first-page":"2657","DOI":"10.1016\/j.vaccine.2011.01.097","volume":"29","author":"E Gomez-Casado","year":"2011","unstructured":"Gomez-Casado E, Estepa A, Coll JM (2011) A comparative review on European-farmed finfish RNA viruses and their vaccines. Vaccine 29(15):2657\u20132671. https:\/\/doi.org\/10.1016\/j.vaccine.2011.01.097","journal-title":"Vaccine"},{"key":"12106_CR29","doi-asserted-by":"publisher","first-page":"229","DOI":"10.1139\/m62-029","volume":"8","author":"RR Guillard","year":"1962","unstructured":"Guillard RR, Ryther JH (1962) Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can J Microbiol 8:229\u2013239","journal-title":"Can J Microbiol"},{"issue":"5","key":"12106_CR30","doi-asserted-by":"publisher","first-page":"1057","DOI":"10.1111\/j.1529-8817.2012.01222.x","volume":"48","author":"MD Guiry","year":"2012","unstructured":"Guiry MD (2012) How many species of algae are there? J Phycol 48(5):1057\u20131063. https:\/\/doi.org\/10.1111\/j.1529-8817.2012.01222.x","journal-title":"J Phycol"},{"issue":"12","key":"12106_CR31","doi-asserted-by":"publisher","first-page":"e28424","DOI":"10.1371\/journal.pone.0028424","volume":"6","author":"F Hempel","year":"2011","unstructured":"Hempel F, Lau J, Klingl A, Maier UG (2011) Algae as protein factories: expression of a human antibody and the respective antigen in the diatom Phaeodactylum tricornutum. PLoS One 6(12):e28424. https:\/\/doi.org\/10.1371\/journal.pone.0028424","journal-title":"PLoS ONE"},{"issue":"3","key":"12106_CR32","doi-asserted-by":"publisher","first-page":"1112","DOI":"10.1104\/pp.18.00630","volume":"178","author":"LG Heredia-Mart\u00ednez","year":"2018","unstructured":"Heredia-Mart\u00ednez LG, Andr\u00e9s-Garrido A, Mart\u00ednez-Force E, Perez-Perez ME, Crespo JL (2018) Chloroplast damage induced by the inhibition of fatty acid synthesis triggers autophagy in Chlamydomonas. Plant Physiol 178(3):1112\u20131129. https:\/\/doi.org\/10.1104\/pp.18.00630","journal-title":"Plant Physiol"},{"issue":"2","key":"12106_CR33","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1105\/tpc.16.00637","volume":"29","author":"M Izumi","year":"2017","unstructured":"Izumi M, Ishida H, Nakamura S, Hidema J (2017) Entire photodamaged chloroplasts are transported to the central vacuole by autophagy. Plant Cell 29(2):377\u2013394. https:\/\/doi.org\/10.1105\/tpc.16.00637","journal-title":"Plant Cell"},{"key":"12106_CR34","doi-asserted-by":"publisher","first-page":"253","DOI":"10.2216\/i0031-8884-35-3-253.1","volume":"35","author":"B Karlson","year":"1996","unstructured":"Karlson B, Potter D, Kuylenstierna M, Andersen R (1996) Ultrastructure, pigment composition, and 18S rRNA gene sequence for Nannochloropsis granulata sp. nov. (Monodopsidaceae, Eustigmatophyceae), a marine ultraplankter isolated from the Skagerrak, northeast Atlantic Ocean. Phycologia 35:253\u2013260. https:\/\/doi.org\/10.2216\/i0031-8884-35-3-253.1","journal-title":"Phycologia"},{"key":"12106_CR35","doi-asserted-by":"publisher","first-page":"430","DOI":"10.1104\/pp.125.1.430","volume":"125","author":"H Kuroda","year":"2001","unstructured":"Kuroda H, Maliga P (2001) Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol 125:430\u2013436. https:\/\/doi.org\/10.1104\/pp.125.1.430","journal-title":"Plant Physiol"},{"issue":"1","key":"12106_CR36","doi-asserted-by":"publisher","first-page":"74","DOI":"10.1016\/j.jsb.2006.02.014","volume":"155","author":"CC Lee","year":"2006","unstructured":"Lee CC, Ko TP, Chou CC, Yoshimura M, Doong SR, Wang MY, Wang AH (2006) Crystal structure of infectious bursal disease virus VP2 subviral particle at 26A resolution: implications in virion assembly and immunogenicity. J Struct Biol 155(1):74\u201386. https:\/\/doi.org\/10.1016\/j.jsb.2006.02.014","journal-title":"J Struct Biol"},{"issue":"2","key":"12106_CR37","doi-asserted-by":"publisher","first-page":"316","DOI":"10.1016\/j.fsi.2008.07.004","volume":"26","author":"SS Li","year":"2009","unstructured":"Li SS, Tsai HJ (2009) Transgenic microalgae as a non-antibiotic bactericide producer to defend against bacterial pathogen infection in the fish digestive tract. Fish Shellfish Immunol 26(2):316\u2013325. https:\/\/doi.org\/10.1016\/j.fsi.2008.07.004","journal-title":"Fish Shellfish Immunol"},{"issue":"Pt 7","key":"12106_CR38","doi-asserted-by":"publisher","first-page":"1721","DOI":"10.1099\/vir.0.030270-0","volume":"92","author":"S Mutoloki","year":"2011","unstructured":"Mutoloki S, Evensen O (2011) Sequence similarities of the capsid gene of Chilean and European isolates of infectious pancreatic necrosis virus point towards a common origin. J Gen Virol 92(Pt 7):1721\u20131726. https:\/\/doi.org\/10.1099\/vir.0.030270-0","journal-title":"J Gen Virol"},{"key":"12106_CR39","doi-asserted-by":"publisher","first-page":"66","DOI":"10.1186\/s13068-019-1401-3","volume":"12","author":"MIS Naduthodi","year":"2019","unstructured":"Naduthodi MIS, Mohanraju P, Sudfeld C, D\u2019Adamo S, Barbosa MJ, van der Oost J (2019) CRISPR-Cas ribonucleoprotein mediated homology-directed repair for efficient targeted genome editing in microalgae Nannochloropsis oceanica IMET1. Biotechnol Biofuels 12:66. https:\/\/doi.org\/10.1186\/s13068-019-1401-3","journal-title":"Biotechnol Biofuels"},{"key":"12106_CR40","doi-asserted-by":"publisher","first-page":"101664","DOI":"10.1016\/j.algal.2019.101664","volume":"45","author":"E Poliner","year":"2020","unstructured":"Poliner E, Clark E, Cummings C, Benning C, Farr\u00e9 EM (2020) A high-capacity gene stacking toolkit for the oleaginous microalga, Nannochloropsis oceanica CCMP1779. Algal Res 45:101664. https:\/\/doi.org\/10.1016\/j.algal.2019.101664","journal-title":"Algal Res"},{"issue":"10","key":"12106_CR41","doi-asserted-by":"publisher","first-page":"1383","DOI":"10.1007\/s00299-018-2270-0","volume":"37","author":"E Poliner","year":"2018","unstructured":"Poliner E, Farr\u00e9 EM, Benning C (2018a) Advanced genetic tools enable synthetic biology in the oleaginous microalgae Nannochloropsis sp. Plant Cell Rep 37(10):1383\u20131399. https:\/\/doi.org\/10.1007\/s00299-018-2270-0","journal-title":"Plant Cell Rep"},{"key":"12106_CR42","doi-asserted-by":"publisher","first-page":"298","DOI":"10.1111\/pbi.12772","volume":"16","author":"E Poliner","year":"2017","unstructured":"Poliner E, Pulman JA, Zienkiewicz K, Childs K, Benning C, Farr\u00e9 EM (2017) 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 16:298\u2013309. https:\/\/doi.org\/10.1111\/pbi.12772","journal-title":"Plant Biotechnol J"},{"issue":"4","key":"12106_CR43","doi-asserted-by":"publisher","first-page":"962","DOI":"10.1021\/acssynbio.7b00362","volume":"7","author":"E Poliner","year":"2018","unstructured":"Poliner E, Takeuchi T, Du ZY, Benning C, Farr\u00e9 EM (2018b) Nontransgenic marker-free gene disruption by an episomal CRISPR system in the oleaginous microalga, Nannochloropsis oceanica CCMP1779. ACS Synth Biol 7(4):962\u2013968. https:\/\/doi.org\/10.1021\/acssynbio.7b00362","journal-title":"ACS Synth Biol"},{"key":"12106_CR44","unstructured":"Qiagen (2003) The QIAexpressionist: a handbook for high-level expression and purification of 6xHis6-tagged proteins, 5th edn. Qiagen"},{"issue":"19","key":"12106_CR45","doi-asserted-by":"publisher","first-page":"R865","DOI":"10.1016\/j.cub.2013.08.046","volume":"23","author":"H Qiu","year":"2013","unstructured":"Qiu H, Price DC, Weber AP, Reeb V, Yang EC, Lee JM, Kim SY, Yoon HS, Bhattacharya D (2013) Adaptation through horizontal gene transfer in the cryptoendolithic red alga Galdieria phlegrea. Curr Biol 23(19):R865\u2013R866. https:\/\/doi.org\/10.1016\/j.cub.2013.08.046","journal-title":"Curr Biol"},{"key":"12106_CR46","doi-asserted-by":"publisher","first-page":"101708","DOI":"10.1016\/j.algal.2019.101708","volume":"44","author":"M Ramarajan","year":"2019","unstructured":"Ramarajan M, Fabris M, Abbriano RM, Pernice M, Ralph PJ (2019) Novel endogenous promoters for genetic engineering of the marine microalga Nannochloropsis gaditana CCMP526. Algal Res 44:101708. https:\/\/doi.org\/10.1016\/j.algal.2019.101708","journal-title":"Algal Res"},{"key":"12106_CR47","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1186\/s13068-018-1127-7","volume":"11","author":"LV Richter","year":"2018","unstructured":"Richter LV, Yang H, Yazdani M, Hanson MR, Ahner BA (2018) A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts. Biotechnol Biofuels 11:133. https:\/\/doi.org\/10.1186\/s13068-018-1127-7","journal-title":"Biotechnol Biofuels"},{"key":"12106_CR48","doi-asserted-by":"publisher","DOI":"10.1007\/s10811-021-02416-0","author":"B Roncaglia","year":"2021","unstructured":"Roncaglia B, Papini A, Chini Zittelli G, Rodolfi L, Tredici MR (2021) Cell wall and organelle modifications during nitrogen starvation in Nannochloropsis oceanica F&M-M24. J Appl Phycol. https:\/\/doi.org\/10.1007\/s10811-021-02416-0","journal-title":"J Appl Phycol"},{"issue":"3","key":"12106_CR49","doi-asserted-by":"publisher","first-page":"479","DOI":"10.1007\/s00299-011-1186-8","volume":"31","author":"S Rosales-Mendoza","year":"2012","unstructured":"Rosales-Mendoza S, Paz-Maldonado LM, 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\u2013494. https:\/\/doi.org\/10.1007\/s00299-011-1186-8","journal-title":"Plant Cell Rep"},{"key":"12106_CR50","volume-title":"Molecular cloning: a laboratory manual","author":"JF Sambrook","year":"2001","unstructured":"Sambrook JF, Russell D (2001) Molecular cloning: a laboratory manual, vol 1. Cold Springs Harbour Press Springer, Dordrecht"},{"issue":"9","key":"12106_CR51","doi-asserted-by":"publisher","first-page":"722","DOI":"10.1525\/bio.2010.60.9.9","volume":"60","author":"R Sayre","year":"2010","unstructured":"Sayre R (2010) Microalgae: the potential for carbon capture. Bioscience 60(9):722\u2013727. https:\/\/doi.org\/10.1525\/bio.2010.60.9.9","journal-title":"Bioscience"},{"issue":"11","key":"12106_CR52","doi-asserted-by":"publisher","first-page":"1450","DOI":"10.1128\/EC.00183-14","volume":"13","author":"MJ Scholz","year":"2014","unstructured":"Scholz MJ, Weiss TL, Jinkerson RE, Jing J, Roth R, Goodenough U, Posewitz MC, Gerken HG (2014) Ultrastructure and composition of the Nannochloropsis gaditana cell wall. Eukaryot Cell 13(11):1450\u20131464. https:\/\/doi.org\/10.1128\/EC.00183-14","journal-title":"Eukaryot Cell"},{"key":"12106_CR53","doi-asserted-by":"publisher","unstructured":"Schroda M (2019) Good news for nuclear transgene expression in Chlamydomonas. Cells 8(12). https:\/\/doi.org\/10.3390\/cells8121534","DOI":"10.3390\/cells8121534"},{"key":"12106_CR54","doi-asserted-by":"publisher","first-page":"121","DOI":"10.1046\/j.1365-313x.2000.00652.x","volume":"21","author":"M Schroda","year":"2000","unstructured":"Schroda M, Blocker D, Beck C (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J 21:121\u201313","journal-title":"Plant J"},{"issue":"1","key":"12106_CR55","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1007\/s10811-017-1234-z","volume":"30","author":"MR Shah","year":"2017","unstructured":"Shah MR, Lutzu GA, Alam A, Sarker P, Kabir Chowdhury MA, Parsaeimehr A, Liang Y, Daroch M (2017) Microalgae in aquafeeds for a sustainable aquaculture industry. J Appl Phycol 30(1):197\u2013213. https:\/\/doi.org\/10.1007\/s10811-017-1234-z","journal-title":"J Appl Phycol"},{"key":"12106_CR56","doi-asserted-by":"publisher","first-page":"1784","DOI":"10.3389\/fpls.2019.01784","volume":"10","author":"N Shahar","year":"2019","unstructured":"Shahar N, Landman S, Weiner I, Elman T, Dafni E, Feldman Y, Tuller T, Yacoby I (2019) The integration of multiple nuclear-encoded transgenes in the green alga Chlamydomonas reinhardtii results in higher transcription levels. Front Plant Sci 10:1784. https:\/\/doi.org\/10.3389\/fpls.2019.01784","journal-title":"Front Plant Sci"},{"key":"12106_CR57","doi-asserted-by":"publisher","first-page":"223","DOI":"10.1016\/s0145-305x(99)00074-9","volume":"24","author":"JM Sharma","year":"2000","unstructured":"Sharma JM, Kim I-J, Rautenschlein S, Yeh H-Y (2000) Infectious bursal disease virus of chickens: pathogenesis and immunosuppression. Dev Comp Immunol 24:223\u2013235. https:\/\/doi.org\/10.1016\/s0145-305x(99)00074-9","journal-title":"Dev Comp Immunol"},{"issue":"6","key":"12106_CR58","doi-asserted-by":"publisher","first-page":"2856","DOI":"10.1038\/nprot.2006.468","volume":"1","author":"A Shevchenko","year":"2006","unstructured":"Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1(6):2856\u20132860. https:\/\/doi.org\/10.1038\/nprot.2006.468","journal-title":"Nat Protoc"},{"key":"12106_CR59","unstructured":"Siripornadulsil S, Dabrowski K, Sayre R (2007) Microalgal vaccines. In: Leon R, Galvan A, Fernandez E (eds) Transgenic microalgae as green cell factories. Landes Bioscience and Springer Science+Business Media, New York"},{"key":"12106_CR60","doi-asserted-by":"publisher","first-page":"60","DOI":"10.3389\/fmicb.2014.00060","volume":"5","author":"EA Specht","year":"2014","unstructured":"Specht EA, Mayfield SP (2014) Algae-Based Oral Recombinant Vaccines Front Microbiol 5:60. https:\/\/doi.org\/10.3389\/fmicb.2014.00060","journal-title":"Algae-Based Oral Recombinant Vaccines Front Microbiol"},{"issue":"3","key":"12106_CR61","doi-asserted-by":"publisher","first-page":"665","DOI":"10.1002\/j.1460-2075.1996.tb00399.x","volume":"15","author":"ML Sprengart","year":"1996","unstructured":"Sprengart ML, Fuchs E, Porter AG (1996) The downstream box: an efficient and independent translation initiation signal in Escherichia coli. EMBO J 15(3):665\u2013674","journal-title":"EMBO J"},{"key":"12106_CR62","doi-asserted-by":"publisher","unstructured":"Sproles AE, Fields FJ, Smalley TN, Le CH, Badary A, Mayfield SP (2021) Recent advancements in the genetic engineering of microalgae. Algal Research 53https:\/\/doi.org\/10.1016\/j.algal.2020.102158","DOI":"10.1016\/j.algal.2020.102158"},{"key":"12106_CR63","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1016\/S0022-5320(69)90033-1","volume":"26","author":"AR Spurr","year":"1969","unstructured":"Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct 26:31\u201343. https:\/\/doi.org\/10.1016\/S0022-5320(69)90033-1","journal-title":"J Ultrastruct"},{"issue":"3","key":"12106_CR64","doi-asserted-by":"publisher","first-page":"741","DOI":"10.1111\/j.1529-8817.2006.00218.x","volume":"42","author":"E Varela-\u00c1lvarez","year":"2006","unstructured":"Varela-\u00c1lvarez E, Andreakis N, Lago-Lest\u00f3n A, Pearson GA, Serrao EA, Procaccini G, Duarte CM, Marb\u00e1 N (2006) Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction. J Phycol 42(3):741\u2013745. https:\/\/doi.org\/10.1111\/j.1529-8817.2006.00218.x","journal-title":"J Phycol"},{"issue":"11","key":"12106_CR65","doi-asserted-by":"publisher","first-page":"e1003064","DOI":"10.1371\/journal.pgen.1003064","volume":"8","author":"A Vieler","year":"2012","unstructured":"Vieler A, Wu G, Tsai CH, Bullard B, Cornish AJ, Harvey C, Reca IB, Thornburg C, Achawanantakun R, Buehl CJ, Campbell MS, Cavalier D, Childs KL, Clark TJ, Deshpande R, Erickson E, Armenia Ferguson A, Handee W, Kong Q, Li X, Liu B, Lundback S, Peng C, Roston RL, Sanjaya SJP, Terbush A, Warakanont J, Zauner S, Farre EM, Hegg EL, Jiang N, Kuo MH, Lu Y, Niyogi KK, Ohlrogge J, Osteryoung KW, Shachar-Hill Y, Sears BB, Sun Y, Takahashi H, Yandell M, Shiu SH, Benning C (2012) Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779. PLoS Genet 8(11):e1003064. https:\/\/doi.org\/10.1371\/journal.pgen.1003064","journal-title":"PLoS Genet"},{"issue":"6","key":"12106_CR66","doi-asserted-by":"publisher","first-page":"962","DOI":"10.3390\/ijms17060962","volume":"17","author":"N Yan","year":"2016","unstructured":"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","journal-title":"Int J Mol Sci"},{"issue":"3","key":"12106_CR67","doi-asserted-by":"publisher","first-page":"261","DOI":"10.1046\/j.1365-313x.2001.00958.x","volume":"25","author":"G-N Ye","year":"2001","unstructured":"Ye G-N, Hajdukiewicz PTJ, Broyles D, Rodriguez D, Xu CW, Nehra N, Staub JM (2001) Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco. Plant J 25(3):261\u2013270. https:\/\/doi.org\/10.1046\/j.1365-313x.2001.00958.x","journal-title":"Plant J"},{"issue":"4","key":"12106_CR68","doi-asserted-by":"publisher","first-page":"810","DOI":"10.1021\/acssynbio.0c00610","volume":"10","author":"X Zhang","year":"2021","unstructured":"Zhang X, Betterle N, Hidalgo Martinez D, Melis A (2021) Recombinant protein stability in cyanobacteria. ACS Synth Biol 10(4):810\u2013825. https:\/\/doi.org\/10.1021\/acssynbio.0c00610","journal-title":"ACS Synth Biol"},{"issue":"8","key":"12106_CR69","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1186\/s13068-016-0686-8","volume":"10","author":"K Zienkiewicz","year":"2017","unstructured":"Zienkiewicz K, Zienkiewicz A, Poliner E, Du Z-Y, Vollheyde K, Herrfurth C, Marmon S, Farr\u00e9 EM, Feussner I, Benning C (2017) Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts. Biotechnol Biofuels 10(8):1\u201320. https:\/\/doi.org\/10.1186\/s13068-016-0686-8","journal-title":"Biotechnol Biofuels"}],"container-title":["Applied Microbiology and Biotechnology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00253-022-12106-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/article\/10.1007\/s00253-022-12106-7\/fulltext.html","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1007\/s00253-022-12106-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,10,3]],"date-time":"2022-10-03T09:04:12Z","timestamp":1664787852000},"score":1,"resource":{"primary":{"URL":"https:\/\/link.springer.com\/10.1007\/s00253-022-12106-7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,7]]},"references-count":69,"journal-issue":{"issue":"19-20","published-print":{"date-parts":[[2022,10]]}},"alternative-id":["12106"],"URL":"https:\/\/doi.org\/10.1007\/s00253-022-12106-7","relation":{},"ISSN":["0175-7598","1432-0614"],"issn-type":[{"value":"0175-7598","type":"print"},{"value":"1432-0614","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,7]]},"assertion":[{"value":"23 May 2022","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"24 July 2022","order":2,"name":"revised","label":"Revised","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"27 July 2022","order":3,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"7 September 2022","order":4,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Declarations"}},{"value":"The authors approved ethical standards.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval"}},{"value":"Not applicable.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent to participate"}},{"value":"All authors agreed to manuscript publication.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Consent for publication"}},{"value":"The authors declare no competing interests.","order":5,"name":"Ethics","group":{"name":"EthicsHeading","label":"Conflict of interest"}}]}}