Skip to main content

Advertisement

SpringerLink
Log in

Metabolic engineering of Dunaliella salina for production of ketocarotenoids

  • Original Article
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

Dunaliella is a commercially important marine alga producing high amount of β-carotene. The use of Dunaliella as a potential transgenic system for the production of recombinant proteins has been recently recognized. The present study reports for the first time the metabolic engineering of carotenoid biosynthesis in Dunaliella salina for ketocarotenoid production. The pathway modification included the introduction of a bkt gene from H. pluvialis encoding β-carotene ketolase (4,4′β-oxygenase) along with chloroplast targeting for the production of ketocarotenoids. The bkt under the control of Dunaliella Rubisco smaller subunit promoter along with its transit peptide sequence was introduced into the alga through standardized Agrobacterium-mediated transformation procedure. The selected transformants were confirmed using GFP and GUS expression, PCR and southern blot analysis. A notable upregulation of the endogenous hydroxylase level of transformants was observed where the BKT expression was higher in nutrient-limiting conditions. Carotenoid analysis of the transformants through HPLC and MS analysis showed the presence of astaxanthin and canthaxanthin with maximum content of 3.5 and 1.9 µg/g DW, respectively. The present study reports the feasibility of using D. salina for the production of ketocarotenoids including astaxanthin.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Anila N, Chandrashekar A, Ravishankar GA, Sarada R (2011) Establishment of Agrobacterium mediated genetic transformation in D. bardawil. Eur J Phycol 46(1):36–44

    Article  CAS  Google Scholar 

  • Arun N, Singh DP (2013) Differential response of Dunaliella salina and Dunaliella tertiolecta isolated from brines of Sambhar Salt Lake of Rajasthan (India) to salinities: a study on growth, pigment and glycerol synthesis. J Mar Biol Assoc India 55(1):65–70

    Article  Google Scholar 

  • Becker W (2004) Microalgae for aquaculture. The nutritional value of microalgae for aquaculture. In: Richmond A (ed) Handbook of microalgal culture. Blackwell, Oxford, pp 380–391

    Google Scholar 

  • Ben Amotz A, Avron M (1983) On the factors which determine massive beta carotene accumulation in the halotolerant alga Dunaliella bardawil. Plant Physiol 72:593–597

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Coesel SN, Baumgartner AC, Teles LM, Ramos AA, Henriques NM, Cancela L, Varela JCS (2008) Nutrient limitation is the main regulatory factor for carotenoid accumulation and for Psy and Pds steady state transcript levels in Dunaliella salina (Chlorophyta) exposed to high light and salt stress. Mar Biotechnol 10(5):602–611

    Article  CAS  PubMed  Google Scholar 

  • Cottage A, Yang A, Maunders H, de Lacy RC, Ramsay NA (2001) Identification of DNA sequences flanking T-DNA insertions by PCR-walking. Plant Mol Biol 19(4):321–327

    Article  CAS  Google Scholar 

  • Degui G, Yiqin W, Wenbin L, Yongru S (2002) Transient expression of gus gene in Dunaliella salina. High Technol Lett 2:35–39

    Google Scholar 

  • Dipak SP, Lele SS (2005) Carotenoid production from microalga Dunaliella salina. Indian J Biotechnol 4:476–483

    Google Scholar 

  • Fraser PD, Römer S, Shipton CA, Mills PB, Kiano JW, Misawa N, Drake RG, Schuch W, Bramley PM (2002) Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner. Proc Natl Acad Sci USA 99:1092–1097

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Geng D, Wang Y, Wang P, Li W, Sun Y (2003) Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta). J Appl Phycol 15:451–456

    Article  CAS  Google Scholar 

  • Goswami G, Chaudhuri S, Dutta D (2010) The present perspective of astaxanthin with reference to biosynthesis and pharmacological importance. World J Microbiol Biotechnol 26:1925–1939

    Article  CAS  Google Scholar 

  • Hata N, Ogbonna JC, Hasegawa Y, Taroda H, Tanaka H (2001) Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture. J App Phycol 13:395–402

    Article  CAS  Google Scholar 

  • Hirschberg J (2001) Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol 4:210–218

    Article  CAS  PubMed  Google Scholar 

  • Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16(20):9877

    Article  PubMed Central  PubMed  Google Scholar 

  • Huang JC, Zhong YJ, Liu J, Sandmann G, Chen F (2013) Metabolic engineering of tomato for high-yield production of astaxanthin. Metab Eng 17:59–67

    Article  CAS  PubMed  Google Scholar 

  • Jayaraj J, Devlin R, Punja Z (2008) Metabolic engineering of novel keto-carotenoid production in carrot plants. Transgen Res 17:489–501

    Article  CAS  Google Scholar 

  • Jin ES, Polle JE, Melis A (2001) Involvement of zeaxanthin and of the Cbr protein in the repair of photosystem II from photoinhibition in the green alga Dunaliella salina. Biochim et Biophys Acta 1506:244–259

    Article  CAS  Google Scholar 

  • Kajiwara S, Kakizono T, Saito T, Kondo K, Ohtani T, Nishio N, Nagai S, Misawa N (1995) Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis, and astaxanthin synthesis in Escherichia coli. Plant Mol Biol 29:343–352

    Article  CAS  PubMed  Google Scholar 

  • Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R (2015) Regulation of astaxanthin and its intermediates through cloning and genetic transformation β-carotene ketolase in Haematococcus pluvialis. J Biotechnol 196–197:33–41

    Article  PubMed  Google Scholar 

  • Lamers PP, Janssen M, De Vos RCH, Bino RJ, Wijffels RH (2008) Exploring and exploiting carotenoid accumulation in Dunaliella salina for cell-factory applications. Trends Biotechnol 26:631–638

    Article  CAS  PubMed  Google Scholar 

  • Lamers PP, van de Laak CCW, Kaasenbrood PS, Lorier J, Janssen M, De Vos RCH, Bino RJ, Wijffels RH (2010) Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina. Biotechnol Bioeng 106:638–648

    Article  CAS  PubMed  Google Scholar 

  • Lamers PP, Janssen M, De Vos RCH, Bino RJ, Wijffels RH (2012) Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga. J Biotechnol 162:21–27

    Article  CAS  PubMed  Google Scholar 

  • León R, Couso I, Fernández E (2007) Metabolic engineering of ketocarotenoids biosynthesis in the unicelullar microalga Chlamydomonas reinhardtii. J Biotechnol 130:143–152

    Article  PubMed  Google Scholar 

  • León-Bañares R, González-Ballester D, Galván A, Fernández E (2004) Transgenic microalgae as green cell-factories. Trend Biotechnol 22:45–52

    Article  Google Scholar 

  • Mann V, Harker M, Pecker I, Hirschberg J (2000) Metabolic engineering of astaxanthin production in tobacco flowers. Nat Biotechnol 18:888–892

    Article  CAS  PubMed  Google Scholar 

  • Misawa N, Yamano S, Linden H, Felipe MR, Lucas M, Ikenaga H, Sandmann G (1993) Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of β-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon. Plant J 4(5):833–840

    Article  CAS  PubMed  Google Scholar 

  • Misawa N, Kajiwara S, Konda K, Yokoyama A, Satomi Y, Saito T, Miki W, Ohtani T (1995a) Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon beta-carotene by a single gene. Biochem Biophys Res Commun 209:867–876

    Article  CAS  PubMed  Google Scholar 

  • Misawa N, Satomi Y, Kondo K, Yokoyama A, Kajiwara S, Saito T, Ohtani T, Miki W (1995b) Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level. J Bacteriol 177:6575–6584

    PubMed Central  CAS  PubMed  Google Scholar 

  • Morris WL, Ducreux LJ, Fraser PD, Millam S, Taylor MA (2006) Engineering ketocarotenoid biosynthesis in potato tubers. Metab Eng 8(3):253–263

    Article  CAS  PubMed  Google Scholar 

  • Mulders KJ, Lamers PP, Martens DE, Wijffels RH (2014) Phototrophic pigment production with microalgae: biological constraints and opportunities. J App Phycol 50(2):229–242

    Article  CAS  Google Scholar 

  • Orset S, Young AJ (1999) Low-temperature induced synthesis of α-carotene in the microalga Dunaliella salina (Chlorophyta). J Phycol 35:527–549

    Google Scholar 

  • Ralley L, Enfissi EMA, Misawa N, Schuch W, Bramley PM, Fraser PD (2004) Metabolic engineering of keto-carotenoid formation in higher plants. Plant J 39:477–486

    Article  CAS  PubMed  Google Scholar 

  • Rosati Carlo, Aquilani Riccardo, Dharmapuri Sridhar, Pallara Patrizia, Marusic Carla, Tavazza Raffaela, Bouvier Florence, Camara Bilal, Giuliano Giovanni (2000) Metabolic engineering of beta-carotene and lycopene content in tomato fruit. Plant J 24(3):413–420

    Article  CAS  PubMed  Google Scholar 

  • Shewmaker CK, Sheehy JA, Daley M, Colburn S, Ke DY (1999) Seed-specific overexpression of phytoene synthase: increase in carotenoids and other metabolic effects. Plant J 20:401–412

    Article  CAS  PubMed  Google Scholar 

  • Stalberg K, Lindgren O, Ek B, Hoglund AS (2003) Synthesis of keto-carotenoids in the seed of Arabidopsis thaliana. Plant J 36:771–779

    Article  CAS  PubMed  Google Scholar 

  • Sui X, Kiser PD, von Lintig J, Palczewski K (2013) Structural basis of carotenoid cleavage: from bacteria to mammals. Arch Biochem Biophys 539(2):203–213

    Article  CAS  PubMed  Google Scholar 

  • Sun Y, Yang ZY, Gao XS, Li QY, Zhang QQ, Xu ZK (2005) Expression of foreign genes in Dunaliella by electroporation. Mol Biotechnol 30:185–192

    Article  CAS  PubMed  Google Scholar 

  • Suzuki S, Nishihara M, Nakatsuka T, Misawa N, Ogiwara I, Yamamura S (2007) Flower color alteration in Lotus japonicus by modification of the carotenoid biosynthetic pathway. Plant Cell Rep 26:951–959

    Article  CAS  PubMed  Google Scholar 

  • Tan CP, Qin S, Zhang Q, Jiang P, Zhao FQ (2005) Establishment of a micro-particle bombardment transformation system for Dunaliella salina. J Microbiol 43:361–365

    CAS  PubMed  Google Scholar 

  • Vonshak A (1986) Laboratory techniques for cultivation of microalgae. In: Richmond A (ed) CRC Handbook of microalgal mass culture. CRC, Boca Raton, pp 345–349

    Google Scholar 

  • Walker TL, Becker DK, Collet C (2005a) Characterisation of the Dunaliella tertiolecta RbcS genes and their promoter activity in Chlamydomonas reinhardtii. Plant Cell Rep 23:727–735

    Article  CAS  PubMed  Google Scholar 

  • Walker TL, Becker DK, Dale JL, Collet C (2005b) Towards the development of a nuclear transformation system for Dunaliella tertiolecta. J Appl Phycol 17:363–368

    Article  CAS  Google Scholar 

  • Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (β-carotene) biosynthetic pathway into (carotenoid free) rice endosperm. Science 287:303–305

    Article  CAS  PubMed  Google Scholar 

  • Zhong Y-J, Huang J-C, Liu J, Li Y, Jiang Y, Xu Z-F, Sandmann G, Chen F (2011) 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 62(10):3659–3669

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhu C, Gerjets T, Sandmann G (2007) Nicotiana glauca engineered for the production of keto-carotenoids in flowers and leaves by expressing the cyanobacterial crtO ketolase gene. Transgen Res 16:813–821

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank DST for financial support and NA and DPS are grateful to CSIR, Government of India for a Senior Research Fellowship. The authors thank Director CFTRI for providing facilities. The authors also thank CAS in Botany, Madras University, Chennai for providing the D. salina strain.

Author information

Author notes

  1. Arun Chandrashekar

    Present address: Bhat Bio-Tech India (P) Ltd., 11-A, 4th Cross Veerasandra Industrial Area Electronics City, Bangalore, 561221, India

  2. G. A. Ravishankar

    Present address: Dr. C. D. Sagar Center for Life Sciences, Dayanada Sagar Institutions, Bangalore, 560008, India

Authors and Affiliations

  1. Department of Botany, St. Xavier’s College For Women (Affiliated to Mahatma Gandhi University), Aluva, 683101, India

    N. Anila

  2. Plant Cell Biotechnology Department, Central Food Technological Research Institute (A Constituent Laboratory of the Council of Scientific and Industrial Research, CSIR), Mysore, Karnataka, 570 020, India

    Daris P. Simon & R. Sarada

Authors
  1. N. Anila
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daris P. Simon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Arun Chandrashekar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. A. Ravishankar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. R. Sarada
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to R. Sarada.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Fig. S1

Diagrammatic representation of RBB-BKT and RBS-BKT region in the constructs. Filled box represents 136 bp long transit peptide region and unfilled region represents bkt gene (Scale 500 bp = 1″ in promoter region). Supplementary material 1 (TIFF 42 kb)

Fig. S2

PDA spectrum of peaks 1 and 4: (A) Astaxanthin (AB) standard. (B) Canthaxanthin (CD) standard. (C) Peak 1(AB) from transformant. (D) Peak 4 (CD) from transformant. Supplementary material 2 (TIFF 2452 kb)

Fig. S3

Sequence analysis of RBCS2 promoter region along with transit peptide amplified from D. salina. Pink (TR1), yellow (TR2), blue (TR3) and green(TR4) highlighted portions are used as primers for walking. Sequence in blue font in box is TF1 and in red font in box is TF2. TATA box position is underlined with bold font (-52). Grey highlighted portion indicated start of the RBCS2 gene (position-1286 bp) and subsequent transit peptide sequence. ^ indicates the cleavage region of transit peptide. Supplementary material 3 (TIFF 14951 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Anila, N., Simon, D.P., Chandrashekar, A. et al. Metabolic engineering of Dunaliella salina for production of ketocarotenoids. Photosynth Res 127, 321–333 (2016). https://doi.org/10.1007/s11120-015-0188-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11120-015-0188-8

Keywords

Search

Navigation