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Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study

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Abstract

Natural carotenoids such as astaxanthin, β,β-carotene and lutein are pigments with a high market value. We studied the effects of nitrogen depletion on the carotenoid metabolism of Chlorella zofingiensis (Chlorophyta) and the subsequent treatment with diphenylamine (DPA), an inhibitor of the biosynthesis of secondary ketocarotenoids. Pigments were identified and quantified based on reversed phase ultra-high performance liquid chromatography photodiode array tandem mass spectrometry (RP-UHPLC-PDA-MSn). Nitrogen depletion (without DPA) resulted in a degradation of chlorophylls and primary carotenoids and an accumulation of astaxanthin, ketolutein, canthaxanthin, adonixanthin and β,β-carotene. The DPA treatment decreased the overall production of β,β-carotene derivatives (sum of astaxanthin, canthaxanthin, echinenone and adonixanthin); however, the production of ketolutein and degradation of primary carotenoids were not modified. This suggests that the regulatory mechanisms controlling the flux towards ketolutein and primary carotenoids were not affected by the decreased levels of β,β-carotene derivatives. In addition, DPA increased production of the individual carotenoids, adonixanthin and echinenone. Insight into the regulation of microalgal carotenoid biosynthesis as demonstrated in this paper is essential when a large-scale carotenoid production process is to be optimised or a recombinant C. zofingiensis strain is to be designed with the intention of excessively producing primary or secondary carotenoids.

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Acknowledgments

We gratefully thank Tiny Franssen-Verheijen of Wageningen Electron Microscopy Centre for her help with the cryo-SEM. This work was supported by the FeyeCon D&I and by grants from Rijksdienst voor Ondernemend Nederland (Project no. FND09014).

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Authors and Affiliations

  1. Bioprocess Engineering, AlgaePARC, Wageningen University, P.O. Box 16, 6700 AA, Wageningen, The Netherlands

    Kim J. M. Mulders, Packo P. Lamers, Dirk E. Martens & René H. Wijffels

  2. Laboratory of Food Chemistry, Wageningen University, P.O. Box 17, 6700 AA, Wageningen, The Netherlands

    Yannick Weesepoel, Jean-Paul Vincken & Harry Gruppen

  3. FeyeCon Development and Implementation, Rijnkade 17a, 1382 GS, Weesp, The Netherlands

    Kim J. M. Mulders & Yannick Weesepoel

  4. Polytech Graduate School of Engineering, Nantes University, Bd. de l’Université, 44600, Saint-Nazaire, France

    Pierre Bodenes

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  1. Kim J. M. Mulders
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  2. Yannick Weesepoel
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  3. Pierre Bodenes
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Correspondence to Kim J. M. Mulders.

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Kim J.M. Mulders and Yannick Weesepoel contributed equally to the manuscript.

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Fig. A1

Time courses of chlorophyll a , chlorophyll b, lutein, 9′cis-neoxanthin, violaxanthin, total astaxanthin (sum of free, mono- and diesters), total ketolutein (sum of free, mono- and diesters), canthaxanthin, adonixanthin, echinenone and β,β-carotene in moles per litre culture volume of nitrogen-depleted C. zofingiensis exposed to no DPA (control) (A) and exposed to repeated additions of DPA resulting each time in a concentration increase of 60 μM (B). Data points represent averages of biological duplicates. For separate data points see Fig. 5 and 6. Solid triangles in B indicate DPA additions. Lines are for visual guidance (GIF 43 kb)

High resolution image (TIFF 6933 kb)

Fig. A2

Contents (in mg/g DW) of chlorophyll a (A), chlorophyll b (B), lutein (C), 9′cis-neoxanthin (D), violaxanthin (E), total ketolutein (sum of free, mono- and diesters) (F), total astaxanthin (sum of free, mono- and diesters) (G), adonixanthin (H), echinenone (I), canthaxanthin (J) and β,β-carotene (K) of nitrogen-depleted C. zofingiensis exposed to no DPA (control) and exposed to repeated additions of DPA resulting each time in a concentration increase of 60 μM. Triangles indicate DPA additions. Lines are for visual guidance (GIF 35 kb)

High resolution image (TIFF 4807 kb)

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Mulders, K.J.M., Weesepoel, Y., Bodenes, P. et al. Nitrogen-depleted Chlorella zofingiensis produces astaxanthin, ketolutein and their fatty acid esters: a carotenoid metabolism study. J Appl Phycol 27, 125–140 (2015). https://doi.org/10.1007/s10811-014-0333-3

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