Affiliation:
1. Department of Plant Physiology and Biochemistry, Universität Konstanz, D-78434 Konstanz, Germany
2. Institute for Molecular Biosciences 213, J. W. Goethe Universität, P.O. Box 111932, D-60054 Frankfurt/Main, Germany
Abstract
ABSTRACT
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 ζ-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
.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Reference41 articles.
1. Babinger, P., I. Kobl, W. Mages, and R. Schmitt. 2001. A link between DNA methylation and epigenetic silencing in transgenic Volvox carteri. Nucleic Acids Res.29:1261-1271.
2. Baroli, I., and K. K. Niyogi. 2000. Molecular genetics of xanthophyll-dependent photoprotection in green algae and plants. Phil. Trans. R. Soc. Lond. B355:1385-1394.
3. Boussiba, S. 2000. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol. Plant.108:111-117.
4. Boussiba, S., L. Fan, and A. Vonshak. 1992. Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. Methods Enzymol.213:386-391.
5. Böger, P., and G. Sandmann. 1990. Modern herbicides affecting typical plant processes, p. 174-216. In W. S. Bowers, W. Ebing, D. Martin, and R. Wegler (ed.), Chemistry of plant protection,vol. 6. Springer Publ., Berlin, Germany.
Cited by
200 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献