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Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene

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Abstract

Aurantiochytrium limacinum is a heterotrophic eukaryotic microorganism that can accumulate high levels of commercial products such as astaxanthin and docosahexaenoic acid. Due to its rapid growth and relatively simple extraction method, A. limacinum is considered a promising astaxanthin resource to replace the conventional microalgal production. However, the astaxanthin biosynthetic process in A. limacinum remains incompletely understood, especially in those catalysed by β-carotene hydroxylase (CrtZ) and ketolase. In this study, we overexpressed a crtZ candidate gene to increase astaxanthin production and expand our understanding of the conversion from beta-carotene to astaxanthin. The resultant transformant AlcrtZ#10 cultivated for 5 days showed a significant increase in astaxanthin production per culture (2.8-fold) and per cell (4.5-fold) compared with that of the wild-type strain. Strikingly, longer light exposure increased astaxanthin production and decreased the beta-carotene content in the wild-type strain, suggesting that light exposure duration is important for astaxanthin production in A. limacinum. Among several predicted intermediates, furthermore, the cantaxanthin produced from β-carotene by ketolase activity were enhanced in the transformant AlcrtZ#10. Although the further investigation is needed, this result suggested that the main route of astaxanthin was via cantaxanthin. Thus, our findings will be valuable not only for its application, but also for understanding the astaxanthin biosynthetic process in A. limacinum.

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Funding

This work was supported by Grants-in Aids for Scientific Research (B) (No. 22H02237) from the Japan Society for the Promotion of Science (JSPS).

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Authors and Affiliations

  1. Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan

    Toru Yoshimi, Sakiko Hashimoto, Yuki Kubo, Masato Takeuchi, Daichi Morimoto, Satoshi Nakagawa & Shigeki Sawayama

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  1. Toru Yoshimi
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  2. Sakiko Hashimoto
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  3. Yuki Kubo
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  4. Masato Takeuchi
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  6. Satoshi Nakagawa
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  7. Shigeki Sawayama
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Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analyses were performed by Toru Yoshimi and Sakiko Hashimoto. The first draft of the manuscript was written by Toru Yoshimi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Daichi Morimoto.

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Fig. S1

Agarose gel electrophoresis of PCR products amplified from transformed A. limacinum colonies. Genomic integration of pAlcrtZ was verified by colony PCR of G418-resistant A. limacinum colonies transformed with pAlcrtZ (AlcrtZ#4, 7, 10, 13, 14). Arrow indicates PCR products amplified from pAlcrtZ using the PCR primers pAl-Neo-F and pAl-Ter-R.

High Resolution Image (TIF 356 KB)

Fig. S2

HPLC chromatogram of carotenoids extracted from transformant AlcrtZ#10 on day 5 (using 6-fold concentrated extract). 1, Astaxanthin; 2, Canthaxanthin; 3, β-carotene

High Resolution Image (TIF 125 KB)

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Yoshimi, T., Hashimoto, S., Kubo, Y. et al. Improvement of Astaxanthin Production in Aurantiochytrium limacinum by Overexpression of the Beta-Carotene Hydroxylase Gene. Appl Biochem Biotechnol 195, 1255–1267 (2023). https://doi.org/10.1007/s12010-022-04172-4

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