Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids

  • 1164 Accesses

  • 28 Citations


The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3′-hydroxylase (CrtZ) and β,β-carotenoid 4,4′-ketolase (4,4′-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8 %) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2 % of total carotenoids), astaxanthin monoester (18.2 %), and the free forms of astaxanthin (10.0 %) and the other ketocarotenoids (17.5 %), which indicated that artificial ketocarotenoids corresponded to 94.9 % of total carotenoids (230 μg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8 %) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Ando S, Osada K, Hatano M, Saneyoshi M (1989) Comparison of carotenoids in muscle and ovary from four genera of salmonoid fishes. Comp Biochem Physiol 93B:503–508

  2. Britton G, Liaaen-Jensen S, Pfander H (2004) Carotenoids handbook. Birkhäuser Verlag, Basel

  3. Choi SK, Nishida Y, Matsuda S, Adachi K, Kasai H, Peng X, Komemushi S, Miki W, Misawa N (2005) Characterization of β-carotene ketolases, CrtW, from marine bacteria by complementation analysis in Escherichia coli. Mar Biotechnol 7:515–522

  4. Choi SK, Matsuda S, Hoshino T, Peng X, Misawa N (2006) Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli. Appl Microbiol Biotechnol 72:1238–1246

  5. Day A, Goldschmidt-Clermont M (2011) The chloroplast transformation toolbox: selectable markers and marker removal. Plant Biotechnol J 9:540–553

  6. Demmig-Adams B, Adams WW III (1996) The role of xanthophyll cycle carotenoids in the protection of photosynthesis. Trends Plant Sci 1:21–26

  7. Farré G, Sanahuja G, Naqvi S, Bai C, Capell T, Zhu C, Christou P (2010) Travel advice on the road to carotenoids in plants. Plant Sci 179:28–48

  8. Fraser PD, Enfissi EMA, Bramley PM (2009) Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches. Arch Biochem Biophys 483:196–204

  9. Fujisawa M, Takita E, Harada H, Sakurai N, Suzuki H, Ohyama K, Shibata D, Misawa N (2009) Pathway engineering of Brassica napus seeds using multiple key-enzyme genes involved in ketocarotenoid formation. J Exp Bot 60:1319–1332

  10. Gerjets T, Sandmann G (2006) Ketocarotenoid formation in transgenic potato. J Exp Bot 57:3639–3645

  11. Gloor A, Simon W (2007) Astaxanthin esters. United States Patent US 7,253,297 B2

  12. Guerin M, Huntley ME, Olaizola M (2003) Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 21:210–216

  13. Hasunuma T, Miyazawa SI, Yoshimura S, Shinzaki Y, Tomizawa KI, Shindo K, Choi SK, Misawa N, Miyake C (2008) Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J 55:857–868

  14. Hirayama O, Nakamura K, Hamada S, Kobayashi Y (1994) Singlet oxygen quenching ability of naturally occurring carotenoids. Lipids 29:149–150

  15. 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

  16. Jayaraj J, Devlin R, Punja Z (2008) Metabolic engineering of novel ketocarotenoid production in carrot plants. Transgenic Res 17:489–501

  17. Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa KI (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217

  18. Kidd PM (2011) Astaxanthin, cell membrane nutrient with diverse clinical benefits and anti-aging potential. Altern Med Rev 16:355–364

  19. Kobayashi M, Sakamoto Y (1999) Singlet oxygen quenching ability of astaxanthin esters from the green alga Haematococcus pluvialis. Biotechnol Lett 21:265–269

  20. Krinsky NI, Landrum JT, Bone RA (2003) Biological mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu Rev Nutr 23:171–201

  21. Liscombe DK, MacLeod BP, Loukanina N, Nandi OI, Facchini PJ (2005) Evidence for the monophyletic evolution of benzylisoquinoline alkaloid biosynthesis in angiosperms. Phytochemistry 66:1374–1393

  22. Maeda H, Hosokawa M, Sashima T, Funayama K, Miyashita K (2005) Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues. Biochem Biophys Res Commun 332:392–397

  23. Maoka T, Etoh T, Kishimoto S, Sakata S (2011) Carotenoids and their fatty acid esters in the petals of Adonis aestivalis. J Oleo Sci 60:47–52

  24. Maruyama T, Kasai H, Choi SK, Ramasamy AK, Inomata Y, Misawa N (2007) Structure of a complete carotenoid biosynthesis gene cluster of marine bacterium Paracoccus sp. strain N81106. Carotenoid Sci 11:50–55

  25. Matsuno T, Katsuyama M, Nagata S (1980) Comparative biochemical studies of carotenoids in fishes-XIX Carotenoids of chum salmon, coho salmon, biwa trout, red-spotted masu salmon, masu salmon, kokanee. Bull Jpn Soc Sci Fish 46:879–884

  26. Miki W (1991) Biological functions and activities of animal carotenoids. Pure Appl Chem 63:141–146

  27. Milborrow BV (2001) The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis. J Exp Bot 52:1145–1164

  28. Misawa N (2009) Pathway engineering of plants toward astaxanthin production. Plant Biotechnol 26:93–99

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

  30. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497

  31. Nishida Y, Adachi K, Kasai H, Shizuri Y, Shindo K, Sawabe A, Komemushi S, Miki W, Misawa N (2005) Elucidation of a carotenoid biosynthesis gene cluster encoding a novel enzyme, 2,2′-β-hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls. Appl Environ Microbiol 71:4286–4296

  32. Nishino H, Murakoshi M, Ii T, Takemura M, Kuchide M, Kanazawa M, Mou XY, Wada S, Masuda M, Ohsaka Y, Yogosawa S, Satomi Y, Jinno K (2002) Carotenoids in cancer chemoprevention. Cancer Metastasis Rev 21:257–264

  33. Okada Y, Ishikura M, Maoka T (2009) Bioavailability of astaxanthin in Haematococcus algal extract: the effects of timing of diet and smoking habits. Biosci Biotechnol Biochem 73:1928–1932

  34. Rogalski M, Carrer H (2011) Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants. Plant Biotechnol J 9:554–564

  35. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

  36. Schiedt K, Liaaen-Jensen S (1995) Isolation and analysis. In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1A. Birkhäuser, Basel, pp 81–108

  37. Schiedt K, Bischof S, Glinz E (1995) Example 5: fish isolation of astaxanthin and its metabolites from skin of Atlantic salmon (Salmo salar). In: Britton G, Liaaen-Jensen S, Pfander H (eds) Carotenoids, vol 1A. Birkhäuser, Basel, pp 243–252

  38. Shindo K, Hasunuma T, Asagi E, Sano A, Hotta E, Minemura N, Miyake C, Maoka T, Misawa N (2008) 4-Ketoantheraxanthin, a novel carotenoid produced by the combination of the bacterial enzyme β-carotene ketolase CrtW and endogeneous carotenoid biosynthetic enzymes in higher plants. Tetrahedron Lett 49:3294–3296

  39. Sugiura M, Nakamura M, Ogawa K, Ikoma Y, Yano M (2012) High serum carotenoids associated with lower risk for bone loss and osteoporosis in post-menopausal Japanese female subjects: prospective cohort study. PLoS ONE 7:e52643

  40. Talegawkar SA, Johnson EJ, Carithers TC, Taylor HA Jr, Bogle ML, Tucker KL (2008) Carotenoid intakes, assessed by food-frequency questionnaires (FFQs), are associated with serum carotenoid concentrations in the Jackson Heart Study: validation of the Jackson Heart Study Delta NIRI Adult FFQs. Public Health Nutr 11:989–997

  41. Tatsuzawa H, Maruyama T, Misawa N, Fujimori K, Nakano M (2000) Quenching of singlet oxygen by carotenoids produced in Escherichia coli-attenuation of singlet oxygen-mediated bacterial killing by carotenoids. FEBS Lett 484:280–284

  42. Wolf AM, Asoh S, Hiranuma H, Ohsawa I, Iio K, Satou A, Ishikura M, Ohta S (2010) Astaxanthin protects mitochondrial redox state and functional integrity against oxidative stress. J Nutr Biochem 21:381–389

  43. Yamashita E (2006) The effects of a dietary supplement containing astaxanthin on skin condition. Carotenoid Sci 10:91–95

  44. Zhu C, Naqvi S, Breitenbach J, Sandamnn G, Christou P, Capell T (2008) Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proc Natl Acad Sci USA 105:18232–18237

Download references


The authors are grateful to Central Laboratories for Frontier Technology, Kirin Holdings Co., Ltd., since this work was initially performed there under support from the New Energy and Industrial Technology Development Organization (NEDO). We thank Dr. Tomohisa Hasunuma for the gift of the pLD7–rrnP–crtZ–crtW plasmid. The authors also thank Mss Miyuki Murakami, Megumi Hashida, and Kazuko Arai for their assistance in experiments on plant transformation and cultivation. We also thank Mss Kumiko Ito and Nami Fukuo, Nihon Women’s University, for their in vitro antioxidative experiments.

Author information

Correspondence to Norihiko Misawa.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 271 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Harada, H., Maoka, T., Osawa, A. et al. Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids. Transgenic Res 23, 303–315 (2014). https://doi.org/10.1007/s11248-013-9750-3

Download citation


  • Astaxanthin
  • Carotenoid
  • Lettuce
  • Lactuca sativa
  • Chloroplast transformation
  • Pathway engineering