Login Register Cart 0
Generic placeholder image

Current Genomics

Editor-in-Chief

ISSN (Print): 1389-2029
ISSN (Online): 1875-5488

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
General Research Article

Characterization of the Chloroplast Genome Facilitated the Transformation of Parachlorella kessleri-I, A Potential Marine Alga for Biofuel Production

Author(s): Prachi Nawkarkar*, Sagrika Chugh, Surbhi Sharma, Mukesh Jain, Sachin Kajla and Shashi Kumar*

Volume 21, Issue 8, 2020

Page: [610 - 623] Pages: 14

DOI: 10.2174/1389202921999201102164754

Price: $65

Abstract

Introduction: The microalga Parachlorella kessleri-I produces high biomass and lipid content that could be suitable for producing economically viable biofuel at a commercial scale. Sequencing the complete chloroplast genome is crucial for the construction of a species-specific chloroplast transformation vector.

Methods: In this study, the complete chloroplast genome sequence (cpDNA) of P. kessleri-I was assembled; annotated and genetic transformation of the chloroplast was optimized. For the chloroplast transformation, we have tested two antibiotic resistance makers, aminoglycoside adenine transferase (aadA) gene and Sh-ble gene conferring resistance to spectinomycin and zeocin, respectively. Transgene integration and homoplasty determination were confirmed using PCR, Southern blot and Droplet Digital PCR.

Results: The chloroplast genome (109,642 bp) exhibited a quadripartite structure with two reverse repeat regions (IRA and IRB), a long single copy (LSC), and a small single copy (SSC) region. The genome encodes 116 genes, with 80 protein-coding genes, 32 tRNAs and 4 rRNAs. The cpDNA provided essential information like codons, UTRs and flank sequences for homologous recombination to make a species-specific vector that facilitated the transformation of P. kessleri-I chloroplast. The transgenic algal colonies were retrieved on a TAP medium containing 400 mg. L-1 spectinomycin, but no transgenic was recovered on the zeocin-supplemented medium. PCR and Southern blot analysis ascertained the transgene integration into the chloroplast genome, via homologous recombination. The chloroplast genome copy number in wildtype and transgenic P. kessleri-I was determined using Droplet Digital PCR.

Conclusion: The optimization of stable chloroplast transformation in marine alga P. kessleri-I should open a gateway for directly engineering the strain for carbon concentration mechanisms to fix more CO2, improving the photosynthetic efficiency and reducing the overall biofuels production cost.

Keywords: Chloroplast genome, genetic engineering, homologous recombination, photosynthetic organism, microalgae biofuels, parachlorella.

« Previous Next »
Graphical Abstract

[1]
Lewis, L.A.; McCourt, R.M. Green algae and the origin of land plants. Am. J. Bot., 2004, 91(10), 1535-1556.
[http://dx.doi.org/10.3732/ajb.91.10.1535] [PMID: 21652308]
[2]
Friedl, T. Inferring taxonomic positions and testing genus level assignments in coccoid green lichen algae: a phylogenetic analysis of 18S ribosomal RNA sequences from Dictyochloropsis reticulata and from members of the genus Myrmecia (Chlorophyta, Trebouxiophyceae cl. nov.). J. Phycol., 1995, 31(4), 632-639.
[http://dx.doi.org/10.1111/j.1529-8817.1995.tb02559.x]
[3]
Friedl, T. The evolution of the green algae. Origins of Algae and their Plastids; Springer: Vienna, 1997, pp. 87-101.
[http://dx.doi.org/10.1007/978-3-7091-6542-3_4]
[4]
Pombert, J.F.; Otis, C.; Lemieux, C.; Turmel, M. The complete mitochondrial DNA sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) highlights distinctive evolutionary trends in the chlorophyta and suggests a sister-group relationship between the Ulvophyceae and Chlorophyceae. Mol. Biol. Evol., 2004, 21(5), 922-935.
[http://dx.doi.org/10.1093/molbev/msh099] [PMID: 15014170]
[5]
Pombert, J.F.; Otis, C.; Lemieux, C.; Turmel, M. The chloroplast genome sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) reveals unusual structural features and new insights into the branching order of chlorophyte lineages. Mol. Biol. Evol., 2005, 22(9), 1903-1918.
[http://dx.doi.org/10.1093/molbev/msi182] [PMID: 15930151]
[6]
Pombert, J.F.; Lemieux, C.; Turmel, M. The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes. BMC Biol., 2006, 4(1), 3.
[http://dx.doi.org/10.1186/1741-7007-4-3] [PMID: 16472375]
[7]
Martin, W.; Rujan, T.; Richly, E.; Hansen, A.; Cornelsen, S.; Lins, T.; Leister, D.; Stoebe, B.; Hasegawa, M.; Penny, D. Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad. Sci. USA, 2002, 99(19), 12246-12251.
[http://dx.doi.org/10.1073/pnas.182432999] [PMID: 12218172]
[8]
Graham, L.E. Green algae to land plants: an evolutionary transition. J. Plant Res., 1996, 109(3), 241-251.
[http://dx.doi.org/10.1007/BF02344471]
[9]
Henry, R.J. Plant diversity and evolution: genotypic and phenotypic variation in higher plants; Cabi Publishing, 2005.
[http://dx.doi.org/10.1079/9780851999043.0000]
[10]
Verma, D.; Daniell, H. Chloroplast vector systems for biotechnology applications. Plant Physiol., 2007, 145(4), 1129-1143.
[http://dx.doi.org/10.1104/pp.107.106690] [PMID: 18056863]
[11]
Daniell, H.; Lin, C.S.; Yu, M.; Chang, W.J. Chloroplast genomes: diversity, evolution, and applications in genetic engineering. Genome Biol., 2016, 17(1), 134.
[http://dx.doi.org/10.1186/s13059-016-1004-2] [PMID: 27339192]
[12]
Gimpel, J.A.; Specht, E.A.; Georgianna, D.R.; Mayfield, S.P. Advances in microalgae engineering and synthetic biology applications for biofuel production. Curr. Opin. Chem. Biol., 2013, 17(3), 489-495.
[http://dx.doi.org/10.1016/j.cbpa.2013.03.038] [PMID: 23684717]
[13]
Cui, Y.; Qin, S.; Jiang, P. Chloroplast transformation of Platymonas (Tetraselmis) subcordiformis with the bar gene as selectable marker. PLoS One, 2014, 9(6)e98607
[http://dx.doi.org/10.1371/journal.pone.0098607] [PMID: 24911932]
[14]
Rathod, J.P.; Prakash, G.; Pandit, R.; Lali, A.M. Agrobacterium-mediated transformation of promising oil-bearing marine algae Parachlorella kessleri. Photosynth. Res., 2013, 118(1-2), 141-146.
[http://dx.doi.org/10.1007/s11120-013-9930-2] [PMID: 24097049]
[15]
Mayfield, S.P.; Franklin, S.E. Expression of human antibodies in eukaryotic micro-algae. Vaccine, 2005, 23(15), 1828-1832.
[http://dx.doi.org/10.1016/j.vaccine.2004.11.013] [PMID: 15734050]
[16]
Doron, L.; Segal, N.; Shapira, M. Transgene expression in microalgae-from tools to applications. Front. Plant Sci., 2016, 7, 505.
[http://dx.doi.org/10.3389/fpls.2016.00505] [PMID: 27148328]
[17]
Lee, S.B.; Kwon, H.B.; Kwon, S.J.; Park, S.C.; Jeong, M.J.; Han, S.E.; Byun, M.O.; Daniell, H. Accumulation of trehalose within transgenic chloroplasts confers drought tolerance. Mol. Breed., 2003, 11(1), 1-3.
[http://dx.doi.org/10.1023/A:1022100404542]
[18]
Kuroiwa, T.; Kawano, S.; Nishibayashi, S.; Sato, C. Epifluorescent microscopic evidence for maternal inheritance of chloroplast DNA. Nature, 1982, 298(5873), 481-483.
[http://dx.doi.org/10.1038/298481a0] [PMID: 7088193]
[19]
Daniell, H. Transgene containment by maternal inheritance: effective or elusive? Proc. Natl. Acad. Sci. USA, 2007, 104(17), 6879-6880.
[http://dx.doi.org/10.1073/pnas.0702219104] [PMID: 17440039]
[20]
Daniell, H. Molecular strategies for gene containment in transgenic crops. Nat. Biotechnol., 2002, 20(6), 581-586.
[http://dx.doi.org/10.1038/nbt0602-581] [PMID: 12042861]
[21]
Kumar, S. GM algae for biofuel production: biosafety and risk assessment. Collect Biosaf Rev., 2015, 9, 52-75.
[22]
Li, X.; Přibyl, P.; Bišová, K.; Kawano, S.; Cepák, V.; Zachleder, V.; Čížková, M.; Brányiková, I.; Vítová, M. The microalga Parachlorella kessleri-a novel highly efficient lipid producer. Biotechnol. Bioeng., 2013, 110(1), 97-107.
[http://dx.doi.org/10.1002/bit.24595] [PMID: 22766749]
[23]
Ahmad, I.; Fatma, Z.; Yazdani, S.S.; Kumar, S. DNA barcode and lipid analysis of new marine algae potential for biofuel. Algal Res., 2013, 2(1), 10-15.
[http://dx.doi.org/10.1016/j.algal.2012.10.003]
[24]
Guillard, R.R.; Ryther, J.H. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve). Gran. Can. J. Microbiol., 1962, 8(2), 229-239.
[http://dx.doi.org/10.1139/m62-029] [PMID: 13902807]
[25]
Angelova, A.; Park, S.H.; Kyndt, J.; Fitzsimmons, K.; Brown, J.K. Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing. J. Appl. Phycol., 2014, 26(1), 209-218.
[http://dx.doi.org/10.1007/s10811-013-0125-1]
[26]
Clarke, JD Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb. Protoc., 2009, 2009(3) pdb.prot5177.
[27]
Dierckxsens, N.; Mardulyn, P.; Smits, G. NOVOPlasty: de novo assembly of organelle genomes from whole genome data. Nucleic Acids Res., 2017, 45(4)e18
[PMID: 28204566]
[28]
Tillich, M.; Lehwark, P.; Pellizzer, T.; Ulbricht-Jones, E.S.; Fischer, A.; Bock, R.; Greiner, S. GeSeq - versatile and accurate annotation of organelle genomes. Nucleic Acids Res., 2017, 45(W1), W6-W11.
[http://dx.doi.org/10.1093/nar/gkx391] [PMID: 28486635]
[29]
Altschul, S.F.; Gish, W.; Miller, W.; Myers, E.W.; Lipman, D.J. Basic local alignment search tool. J. Mol. Biol., 1990, 215(3), 403-410.
[http://dx.doi.org/10.1016/S0022-2836(05)80360-2] [PMID: 2231712]
[30]
Wyman, S.K.; Jansen, R.K.; Boore, J.L. Automatic annotation of organellar genomes with DOGMA. Bioinformatics, 2004, 20(17), 3252-3255.
[http://dx.doi.org/10.1093/bioinformatics/bth352] [PMID: 15180927]
[31]
Lohse, M.; Drechsel, O.; Bock, R. OrganellarGenomeDRAW (OGDRAW): a tool for the easy generation of high-quality custom graphical maps of plastid and mitochondrial genomes. Curr. Genet., 2007, 52(5-6), 267-274.
[http://dx.doi.org/10.1007/s00294-007-0161-y] [PMID: 17957369]
[32]
Laslett, D.; Canback, B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res., 2004, 32(1), 11-16.
[http://dx.doi.org/10.1093/nar/gkh152] [PMID: 14704338]
[33]
Darling, A.C.; Mau, B.; Blattner, F.R.; Perna, N.T. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res., 2004, 14(7), 1394-1403.
[http://dx.doi.org/10.1101/gr.2289704] [PMID: 15231754]
[34]
Marchler-Bauer, A.; Bo, Y.; Han, L.; He, J.; Lanczycki, C.J.; Lu, S.; Chitsaz, F.; Derbyshire, M.K.; Geer, R.C.; Gonzales, N.R.; Gwadz, M.; Hurwitz, D.I.; Lu, F.; Marchler, G.H.; Song, J.S.; Thanki, N.; Wang, Z.; Yamashita, R.A.; Zhang, D.; Zheng, C.; Geer, L.Y.; Bryant, S.H. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res., 2017, 45(D1), D200-D203.
[http://dx.doi.org/10.1093/nar/gkw1129] [PMID: 27899674]
[35]
Almagro Armenteros, J.J.; Sønderby, C.K.; Sønderby, S.K.; Nielsen, H.; Winther, O. DeepLoc: prediction of protein subcellular localization using deep learning. Bioinformatics, 2017, 33(21), 3387-3395.
[http://dx.doi.org/10.1093/bioinformatics/btx431] [PMID: 29036616]
[36]
Gorman, D.S.; Levine, R.P. Photosynthetic electron transport chain of Chlamydomonas reinhardi. V. Purification and properties of cytochrome 553 and ferredoxin. Plant Physiol., 1966, 41(10), 1643-1647.
[http://dx.doi.org/10.1104/pp.41.10.1643] [PMID: 16656452]
[37]
Anila, N.; Chandrashekar, A.; Ravishankar, G.A.; Sarada, R. Establishment of Agrobacterium tumefaciens-mediated genetic transformation in Dunaliella bardawil. Eur. J. Phycol., 2011, 46(1), 36-44.
[http://dx.doi.org/10.1080/09670262.2010.550386]
[38]
Tran, M.; Mayfield, S.P. Expression of full length monoclonal antibodies (mAb) in algal chloroplast. Antibody engineering; Springer: Berlin, Heidelberg, 2010, pp. 503-516.
[http://dx.doi.org/10.1007/978-3-642-01144-3_32]
[39]
Sun, Y.; Joyce, P.A. Application of droplet digital PCR to determine copy number of endogenous genes and transgenes in sugarcane. Plant Cell Rep., 2017, 36(11), 1775-1783.
[http://dx.doi.org/10.1007/s00299-017-2193-1] [PMID: 28849385]
[40]
Boudreau, E.; Takahashi, Y.; Lemieux, C.; Turmel, M.; Rochaix, J.D. The chloroplast ycf3 and ycf4 open reading frames of Chlamydomonas reinhardtii are required for the accumulation of the photosystem I complex. EMBO J., 1997, 16(20), 6095-6104.
[http://dx.doi.org/10.1093/emboj/16.20.6095] [PMID: 9321389]
[41]
Turmel, M.; Otis, C.; Lemieux, C. The chloroplast genomes of the green algae Pedinomonas minor, Parachlorella kessleri, and Oocystis solitaria reveal a shared ancestry between the Pedinomonadales and Chlorellales. Mol. Biol. Evol., 2009, 26(10), 2317-2331.
[http://dx.doi.org/10.1093/molbev/msp138] [PMID: 19578159]
[42]
Alikhan, N.F.; Petty, N.K.; Ben Zakour, N.L.; Beatson, S.A. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics, 2011, 12(1), 402.
[http://dx.doi.org/10.1186/1471-2164-12-402] [PMID: 21824423]
[43]
Green, B.R. Chloroplast genomes of photosynthetic eukaryotes. Plant J., 2011, 66(1), 34-44.
[http://dx.doi.org/10.1111/j.1365-313X.2011.04541.x] [PMID: 21443621]
[44]
Dyo, Y.M.; Purton, S. The algal chloroplast as a synthetic biology platform for production of therapeutic proteins. Microbiology (Reading), 2018, 164(2), 113-121.
[http://dx.doi.org/10.1099/mic.0.000599] [PMID: 29297850]
[45]
Adem, M.; Beyene, D.; Feyissa, T. Recent achievements obtained by chloroplast transformation. Plant Methods, 2017, 13(1), 30.
[http://dx.doi.org/10.1186/s13007-017-0179-1] [PMID: 28428810]
[46]
Yagi, Y.; Shiina, T. Recent advances in the study of chloroplast gene expression and its evolution. Front. Plant Sci., 2014, 5, 61.
[http://dx.doi.org/10.3389/fpls.2014.00061] [PMID: 24611069]
[47]
Idoine, A.D.; Boulouis, A.; Rupprecht, J.; Bock, R. The diurnal logic of the expression of the chloroplast genome in Chlamydomonas reinhardtii. PLoS One, 2014, 9(10)e108760
[http://dx.doi.org/10.1371/journal.pone.0108760] [PMID: 25272288]
[48]
Surpin, M.; Larkin, R.M.; Chory, J. Signal transduction between the chloroplast and the nucleus. Plant Cell, 2002, 14(Suppl.), S327-S338.
[http://dx.doi.org/10.1105/tpc.010446] [PMID: 12045286]
[49]
Eberhard, S.; Drapier, D.; Wollman, F.A. Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii. Plant J., 2002, 31(2), 149-160.
[http://dx.doi.org/10.1046/j.1365-313X.2002.01340.x] [PMID: 12121445]
[50]
Michelet, L.; Lefebvre-Legendre, L.; Burr, S.E.; Rochaix, J.D.; Goldschmidt-Clermont, M. Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas. Plant Biotechnol. J., 2011, 9(5), 565-574.
[http://dx.doi.org/10.1111/j.1467-7652.2010.00564.x] [PMID: 20809927]
[51]
Ramundo, S.; Rahire, M.; Schaad, O.; Rochaix, J.D. Repression of essential chloroplast genes reveals new signaling pathways and regulatory feedback loops in chlamydomonas. Plant Cell, 2013, 25(1), 167-186.
[http://dx.doi.org/10.1105/tpc.112.103051] [PMID: 23292734]
[52]
Sun, Y.; Zerges, W. Translational regulation in chloroplasts for development and homeostasis. Biochim. Biophys. Acta, 2015, 1847(9), 809-820.
[http://dx.doi.org/10.1016/j.bbabio.2015.05.008] [PMID: 25988717]
[53]
Weis, B.L.; Schleiff, E.; Zerges, W. Protein targeting to subcellular organelles via MRNA localization. Biochim. Biophys. Acta, 2013, 1833(2), 260-273.
[http://dx.doi.org/10.1016/j.bbamcr.2012.04.004] [PMID: 23457718]
[54]
Uniacke, J.; Zerges, W. Photosystem II assembly and repair are differentially localized in Chlamydomonas. Plant Cell, 2007, 19(11), 3640-3654.
[http://dx.doi.org/10.1105/tpc.107.054882] [PMID: 18055604]
[55]
Odahara, M.; Inouye, T.; Nishimura, Y.; Sekine, Y. RECA plays a dual role in the maintenance of chloroplast genome stability in Physcomitrella patens. Plant J., 2015, 84(3), 516-526.
[http://dx.doi.org/10.1111/tpj.13017] [PMID: 26340426]
[56]
Takahashi, S.; Furusawa, H.; Ueda, T.; Okahata, Y. Translation enhancer improves the ribosome liberation from translation initiation. J. Am. Chem. Soc., 2013, 135(35), 13096-13106.
[http://dx.doi.org/10.1021/ja405967h] [PMID: 23927491]
[57]
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., 2011, 9(6), 674-683.
[http://dx.doi.org/10.1111/j.1467-7652.2011.00620.x] [PMID: 21535358]
[58]
Deprez, L.; Corbisier, P.; Kortekaas, A.M.; Mazoua, S.; Beaz Hidalgo, R.; Trapmann, S.; Emons, H. Validation of a digital PCR method for quantification of DNA copy number concentrations by using a certified reference material. Biomol Detect. Quantif., 2016, 9, 29-39.
[http://dx.doi.org/10.1016/j.bdq.2016.08.002] [PMID: 27617230]
[59]
Hirono, M.; Uryu, S.; Ohara, A.; Kato-Minoura, T.; Kamiya, R. Expression of conventional and unconventional actins in Chlamydomonas reinhardtii upon deflagellation and sexual adhesion. Eukaryot. Cell, 2003, 2(3), 486-493.
[http://dx.doi.org/10.1128/EC.2.3.486-493.2003] [PMID: 12796293]
[60]
Staub, J.M.; Maliga, P. Long regions of homologous DNA are incorporated into the tobacco plastid genome by transformation. Plant Cell, 1992, 4(1), 39-45.
[PMID: 1356049]
[61]
Muñoz, C.F.; Sturme, M.H.; D’Adamo, S.; Weusthuis, R.A.; Wijffels, R.H. Stable transformation of the green algae Acutodesmus obliquus and Neochloris oleoabundans based on E. coli conjugation. Algal Res., 2019, 39101453
[http://dx.doi.org/10.1016/j.algal.2019.101453]

Rights & Permissions Print Cite
Article Metrics
30
© 2024 Bentham Science Publishers | Privacy Policy