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. 2006 Dec;72(12):7477-84.
doi: 10.1128/AEM.01461-06. Epub 2006 Sep 29.

Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

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Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

Jens Steinbrenner et al. Appl Environ Microbiol. 2006 Dec.

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 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.

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Figures

FIG. 1.
FIG. 1.
(A) Comparison of the CrtP phytoene desaturase from Synechococcus sp. strain PCC 7942, including the four known amino acid changes and their resistance factors (RF) for the bleaching herbicide norflurazon, with the Pds from H. pluvialis. The ChloroP program identified a chloroplast transit peptide (TP) at the N terminus. The modified codon at position 504 of Pds from H. pluvialis, leading to an amino acid change from leucine to arginine, corresponds to codon 436 in CrtP from Synechococcus sp. (B) Intron and exon structure of the pds gene. The promoter sequence is indicated by a gray box, the exon sequences are indicated by black boxes, and the intron sequences are indicated by lines. (C) Map of H. pluvialis transformation vector pPlat-pds-L504R with restriction sites indicated. The engineered pds gene (pds-L504R) is inserted into the NaeI site of the vector pBluescript SK(−). The construct also contains the ampicillin resistance gene, the E. coli origin of replication (ColE1), and a multiple cloning site (MCS). The sequence of this vector has been deposited in the GenBank database under accession number DQ404589.
FIG. 2.
FIG. 2.
Dixon plot of the reciprocal of in vitro phytoene-to-ζ-carotene conversion versus concentration of the inhibitor norflurazon for recombinant WT Pds (▪) and Pds-L504R (•) of H. pluvialis. The ki values for norflurazon inhibition were calculated from the intercepts with the x axis to be 0.14 μM and 5.98 μM for the heterologously expressed WT Pds and Pds-L504R, respectively. RF, resistance factor.
FIG. 3.
FIG. 3.
Southern blot analysis of H. pluvialis transformants P1 to P11 and H. pluvialis WT. The DNA from transformants and WT were digested with XbaI and XhoI and electrophoresed on a 0.8% agarose gel. For detection, a pds-specific cDNA probe (A) or a specific probe for the ampicillin resistance cassette (B) was used. The arrow indicates the position of the 5.78-kb XbaI/XhoI fragment containing the endogenous pds gene. The numbers on the left indicate the sizes of the labeled λ HindIII fragments used as markers (lane M).
FIG. 4.
FIG. 4.
(A) Transcript analysis of the phytoene desaturase gene (pds) and carotenoid hydroxylase gene (bhy) in wild-type H. pluvialis cells after 0, 18, and 36 h of high-light (HL) treatment and in transformants P1 to P11 under low-light (LL) conditions. For comparison, total RNA (rRNA) was stained with ethidium bromide. (B) Western analysis of phytoene desaturase protein (Pds) levels.
FIG. 5.
FIG. 5.
Astaxanthin formation after exposure of H. pluvialis WT (▪) and transformants P3 (•) and P11 (▴) to continuous high light.
FIG. 6.
FIG. 6.
Kinetics of NPQ of dark-adapted H. pluvialis WT (▪) and transformants P3 (•) and P11 (▴).

References

    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. - PMC - PubMed
    1. Baroli, I., and K. K. Niyogi. 2000. Molecular genetics of xanthophyll-dependent photoprotection in green algae and plants. Phil. Trans. R. Soc. Lond. B 355:1385-1394. - PMC - PubMed
    1. Boussiba, S. 2000. Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response. Physiol. Plant. 108:111-117.
    1. Boussiba, S., L. Fan, and A. Vonshak. 1992. Enhancement and determination of astaxanthin accumulation in green alga Haematococcus pluvialis. Methods Enzymol. 213:386-391.
    1. 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.

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