scholarly journals Transformation of the Green Alga Haematococcus pluvialis with a Phytoene Desaturase for Accelerated Astaxanthin Biosynthesis

2006 ◽  
Vol 72 (12) ◽  
pp. 7477-7484 ◽  
Author(s):  
Jens Steinbrenner ◽  
Gerhard Sandmann
Keyword(s):  
Green Alga ◽  
Haematococcus Pluvialis ◽  
Carotenoid Biosynthesis ◽  
Phytoene Desaturase ◽  
Site Directed Mutagenesis ◽  
Carotenoid Content ◽  
Desaturase Gene ◽  
Vitro Assay ◽  
Gene And Protein Expression

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.

The discovery and structural requirements of inhibitors of p-hydroxyphenylpyruvate dioxygenase

Weed Science ◽  
1997 ◽  
Vol 45 (5) ◽  
pp. 601-609 ◽  
Author(s):  
David L. Lee ◽  
Michael P. Prisbylla ◽  
Thomas H. Cromartie ◽  
Derek P. Dagarin ◽  
Stott W. Howard ◽  
...  
Keyword(s):  
Mechanism Of Action ◽  
Biosynthetic Pathway ◽  
Carotenoid Biosynthesis ◽  
Phytoene Desaturase ◽  
Rat Urine ◽  
Meristematic Tissue ◽  
Chemically Induced ◽  
Mammalian Enzyme ◽  
Unique Mechanism

The benzoylcyclohexane-1,3-diones, the triketones, are potent bleaching herbicides whose structure-activity relationships and physical properties are substantially different from classical bleaching herbicides, which affect phytoene desaturase. The first clue to their unique mechanism of action was the discovery that rats treated with a triketone were found to be tyrosinemic. Additionally, examination of the rat urine revealed the accumulation of p-hydroxyphenylpyruvate (HPP) and p-hydroxyphenyllactate. These results suggested that this chemically induced tyrosinemia was the result of the inhibition of p-hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27), and this suggestion was confirmed when a triketone was shown to be a potent inhibitor of rat liver HPPD. In plants, HPPD is a component of the biosynthetic pathway to plastoquinone (PQ), which in turn is a key cofactor of phytoene desaturase. The expectation that triketone-treated plants should accumulate tyrosine while having reduced PQ levels was dramatically demonstrated in the meristematic tissue of ivyleaf morningglory. Plant HPPD, like the mammalian enzyme, was inhibited in vitro by triketones. These biochemical effects provide evidence that the triketone herbicidal mechanism of action is HPPD inhibition leading to a deficiency of PQ, a key cofactor for carotenoid biosynthesis. Other chemical classes of bleaching herbicides were also examined for their ability to elevate tyrosine and deplete PQ as a definitive means of establishing their mode of action and for delineating the structural and physical chemical requirements for an HPPD herbicide. Evidence is provided to support the claim that a 2-benzoylethen-1-ol substructure is the minimum substructure required for a potent HPPD inhibitor.

Molecular Characterization of Carotenoid Biosynthesis in Plants: The Phytoene Desaturase Gene in Tomato

1992 ◽  
pp. 11-18 ◽  
Author(s):  
Iris Pecker ◽  
Daniel Chamovitz ◽  
Varda Mann ◽  
Gerhard Sandmann ◽  
Peter Böger ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Carotenoid Biosynthesis ◽  
Phytoene Desaturase ◽  
Desaturase Gene

scholarly journals Cytolethal Distending Toxin (CDT)-Negative Campylobacter jejuni Strains and Anti-CDT Neutralizing Antibodies Are Induced during Human Infection but Not during Colonization in Chickens

2005 ◽  
Vol 73 (5) ◽  
pp. 3053-3062 ◽  
Author(s):  
Manal AbuOun ◽  
Georgina Manning ◽  
Shaun A. Cawthraw ◽  
Anne Ridley ◽  
If H. Ahmed ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Neutralizing Antibodies ◽  
Neutralization Assay ◽  
Human Infection ◽  
Site Directed Mutagenesis ◽  
Cytolethal Distending Toxin ◽  
Vitro Assay ◽  
Reverse Transcription Pcr ◽  
Human Sera

ABSTRACT The cytolethal distending toxin (CDT) of Campylobacter jejuni was detectable, using an in vitro assay, in most but not all of 24 strains tested. The reason for the absence of toxin activity in these naturally occurring CDT-negative C. jejuni strains was then investigated at the genetic level. CDT is encoded by three highly conserved genes, cdtA, -B, and -C. In the CDT-negative strains, two types of mutation were identified. The CDT activities of C. jejuni strains possessing both types of mutation were successfully complemented with the functional genes of C. jejuni 11168. The first type of mutation comprised a 667-bp deletion across cdtA and cdtB and considerable degeneration in the remainder of the cdt locus. Using a PCR technique to screen for this deletion, this mutation occurred in fewer than 3% of 147 human, veterinary, and environmental strains tested. The second type of mutation involved at least four nonsynonymous nucleotide changes, but only the replacement of proline with serine at CdtB position 95 was considered important for CDT activity. This was confirmed by site-directed mutagenesis. This type of mutation also occurred in fewer than 3% of strains as determined using a LightCycler biprobe assay. The detection of two CDT-negative clinical isolates raised questions about the role of CDT in some cases of human campylobacteriosis. To determine if anti-CDT antibodies are produced in human infection, a toxin neutralization assay was developed and validated using rabbit antisera. Pooled human sera from infected patients neutralized the toxin, indicating expression and immunogenicity during infection. However, no neutralizing antibodies were detected in colonized chickens despite the expression of CDT in the avian gut as indicated by reverse transcription-PCR.

Transformation of Tobacco with a Mutated Cyanobacterial Phytoene Desaturase Gene Confers Resistance to Bleaching Herbicides

2002 ◽  
Vol 57 (7-8) ◽  
pp. 671-679 ◽  
Author(s):  
Tobias Wagner ◽  
Ute Windhövel ◽  
Susanne Römer
Keyword(s):  
Genetic Manipulation ◽  
Enzymatic Reaction ◽  
Photosynthetic Apparatus ◽  
D1 Protein ◽  
Carotenoid Biosynthesis ◽  
Phytoene Desaturase ◽  
Desaturase Gene ◽  
Synechococcus Pcc 7942 ◽  
Pcc 7942 ◽  
Transformed Tobacco

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in ζ-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against ζ-carotene desaturase inhibitors.

The Effect of the Herbicide Glufosinate (BASTA) on Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis

1999 ◽  
Vol 54 (1-2) ◽  
pp. 49-54 ◽  
Author(s):  
Claude Aflalo ◽  
Wang Bing ◽  
Aliza Zarka ◽  
Sammy Boussiba
Keyword(s):  
Cell Growth ◽  
Glutamine Synthetase ◽  
Green Alga ◽  
Nitrogen Starvation ◽  
Haematococcus Pluvialis ◽  
Harmful Effect ◽  
Glufosinate Ammonium ◽  
High Concentrations

The addition of 2.5 mᴍ glufosinate ammonium (BASTA), a well known plant killer, to Haematococcus pluvialis culture efficiently inhibits cell growth, blocks the activity of glutamine synthetase (GS) and induces astaxanthin accumulation. Conversely, methionine-S-sulfoximine (MSX), a well known GS inhibitor, had no effect on neither these parameters. When GS activity was tested in vitro, MSX inhibited the activity at high concentrations (mᴍ), while glufosinate was effective in the μm range. We have found that in the presence of glufosinate, ammonia is excreted from the cells. Therefore, we suggest that this process enables Haematococcus cells to escape the potentially harmful effect of glufosinate. As a consequence of the inability to assimilate nitrogen, astaxanthin is accumulated. This situation resembles the response of Haematococcus cells to nitrogen starvation

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