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Applied and Environmental Microbiology, December 2006, p. 7477-7484, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01461-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis{triangledown}

Jens Steinbrenner1* and Gerhard Sandmann2

Department of Plant Physiology and Biochemistry, Universität Konstanz, D-78434 Konstanz, Germany,1 Institute for Molecular Biosciences 213, J. W. Goethe Universität, P.O. Box 111932, D-60054 Frankfurt/Main, Germany2

Received 26 June 2006/ Accepted 18 September 2006

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to {zeta}-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.

* Corresponding author. Mailing address: Department of Plant Physiology and Biochemistry, Universität Konstanz, D-78434 Konstanz, Germany. Phone: 49-7531-883668. Fax: 49-7531-883042. E-mail: Jens.Steinbrenner{at}uni-konstanz.de.

{triangledown} Published ahead of print on 29 September 2006.

Applied and Environmental Microbiology, December 2006, p. 7477-7484, Vol. 72, No. 12
0099-2240/06/$08.00+0     doi:10.1128/AEM.01461-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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