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

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Abstract

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.

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Acknowledgments

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.

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Correspondence to Norihiko Misawa.

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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

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Keywords

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