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A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance

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Abstract

In the present study, we established a genetic system for manipulating the oleaginous heterotrophic microalgae Aurantiochytrium sp. KRS101, using cycloheximide resistance as the selectable marker. The gene encoding ribosomal protein L44 (RPL44) of Aurantiochytrium sp. KRS101 was first identified and characterized. Proline 56 was replaced with glutamine, affording cycloheximide resistance to strains encoding the mutant protein. This resistance served as a novel selection marker. The gene encoding the Δ12-fatty acid desaturase of Mortierella alpina, used as a reporter, was successfully introduced into chromosomal DNA of Aurantiochytrium sp. KRS101 via 18S rDNA-targeted homologous recombination. Enzymatic conversion of oleic acid (C18:1) to linoleic acid (C18:2) was detected in transformants but not in the wild-type strain.

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Acknowledgments

This work was supported the Advanced Biomass R&D Center of Korea (Grant No. 2010-0029737) funded by the Ministry of Education, Science and Technology and by the Ministry of Food, Agriculture, Forestry, and Fisheries, of the Republic Korea.

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Authors and Affiliations

  1. Applied Microbiology Research Center, Bio-Materials Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup, Jeonbuk, 580-185, Korea

    Won-Kyung Hong, Sun-Yeon Heo, Baek-Rock Oh, Chul Ho Kim & Jeong-Woo Seo

  2. Systems and Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, 305-333, Korea

    Jung-Hoon Sohn

  3. Department of Chemical and Biomolecular Engineering and Advanced Biomass R&D Center, KAIST, 291 Daehakno, Yuseong-gu, Daejeon, 305-701, Korea

    Ji-Won Yang

  4. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, 657-8501, Japan

    Akihiko Kondo

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  1. Won-Kyung Hong
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  2. Sun-Yeon Heo
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  3. Baek-Rock Oh
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  4. Chul Ho Kim
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  7. Akihiko Kondo
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  8. Jeong-Woo Seo
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Corresponding authors

Correspondence to Akihiko Kondo or Jeong-Woo Seo.

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A. Kondo and J.-W. Seo are co-corresponding authors.

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449_2012_846_MOESM1_ESM.ppt

Supplementary Fig. 1. GC–MS of fatty acids (FAMEs) extracted from freeze-dried cells of a transformant of Aurantiochytrium sp. KRS101. The cells were cultured in 0.15-0.2 mM CYH containing medium at 28 °C for 3 days, and then cultured for an additional 1 day after addition of 0.1 mM OA. (A) FAMEs from transformant corresponding to the OA methyl ester; (B) OA methyl ester standard; (C) FAMEs from transformant corresponding to the LA methyl ester; (D) LA methyl ester standard. The standards of OA methyl ester and LA methyl ester, which were also analyzed, were Sigma-Aldrich commercial products (Sigma-Aldrich, MO, USA). (PPT 302 kb)

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Hong, WK., Heo, SY., Oh, BR. et al. A transgene expression system for the marine microalgae Aurantiochytrium sp. KRS101 using a mutant allele of the gene encoding ribosomal protein L44 as a selectable transformation marker for cycloheximide resistance. Bioprocess Biosyst Eng 36, 1191–1197 (2013). https://doi.org/10.1007/s00449-012-0846-6

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  • DOI: https://doi.org/10.1007/s00449-012-0846-6

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