Macular pigment-enriched oil production from genome-edited microalgae

Inhwa Song, Sunbin Kim, Jongrae Kim, Hyeonjun Oh, Junhwan Jang, Su Jin Jeong, Kwangryul Baek, Weon Sun Shin, Sang Jun Sim, Eon Seon Jin

Research output: Contribution to journalArticlepeer-review

15 Citations (Scopus)


Background: The photosynthetic microorganism Chlamydomonas reinhardtii has been approved as generally recognized as safe (GRAS) recently, this can excessively produce carotenoid pigments and fatty acids. Zeaxanthin epoxidase (ZEP), which converts zeaxanthin to violaxanthin, and ADP-glucose pyrophosphorylase (AGP). These are key regulating genes for the xanthophyll and starch pathways in C. reinhardtii respectively. In this study, to produce macular pigment-enriched microalgal oil, we attempted to edit the AGP gene as an additional knock-out target in the zep mutant as a parental strain. Results: Using a sequential CRISPR-Cas9 RNP-mediated knock-out method, we generated double knock-out mutants (dZAs), in which both the ZEP and AGP genes were deleted. In dZA1, lutein (2.93 ± 0.22 mg g−1 DCW: dried cell weight), zeaxanthin (3.12 ± 0.30 mg g−1 DCW), and lipids (450.09 ± 25.48 mg g−1 DCW) were highly accumulated in N-deprivation condition. Optimization of the culture medium and process made it possible to produce pigments and oil via one-step cultivation. This optimization process enabled dZAs to achieve 81% higher oil productivity along with similar macular pigment productivity, than the conventional two-step process. The hexane/isopropanol extraction method was developed for the use of macular pigment-enriched microalgal oil for food. As a result, 196 ± 20.1 mg g−1 DCW of edible microalgal oil containing 8.42 ± 0.92 mg g−1 lutein of oil and 7.69 ± 1.03 mg g−1 zeaxanthin of oil was produced. Conclusion: Our research showed that lipids and pigments are simultaneously induced in the dZA strain. Since dZAs are generated by introducing pre-assembled sgRNA and Cas9-protein into cells, antibiotic resistance genes or selective markers are not inserted into the genome of dZA, which is advantageous for applying dZA mutant to food. Therefore, the enriched macular pigment oil extracted from improved strains (dZAs) can be further applied to various food products and nutraceuticals.

Original languageEnglish
Article number27
JournalMicrobial Cell Factories
Issue number1
Publication statusPublished - 2022 Dec

Bibliographical note

Funding Information:
This research was supported by the Basic Science Research Program and “Carbon to X Project” of the National Research Foundation (NRF) of Korea, funded by the Korean government.

Publisher Copyright:
© 2022, The Author(s).


  • ADP-glucose pyrophosphorylase
  • CRISPR-Cas9
  • Chlamydomonas reinhardtii
  • Macular pigment-enriched oil
  • Zeaxanthin epoxidase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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