An ORF323 with homology to crtE, specifying prephytoene pyrophosphate dehydrogenase, is encoded by cyanelle DNA in the eukaryotic alga Cyanophora paradoxa.

Carotenoids are essential constituents of the light-harvesting and light-protective systems of photosynthetic organisms. The biochemistry of carotenoid biosynthesis in eukaryotes is known, whereas evidence for the genes specifying this biosynthetic pathway is scant. We report here the nucleotide sequence and expression of a gene likely encoding crtE (prephytoene pyrophosphate dehydrogenase). The reaction product of this enzyme is phytoene, a C40 carotenoid precursor common to all organisms. The gene is found in the cyanelle (plastid) DNA of an eukaryotic alga, Cyanophora paradoxa. The expression into protein of cyanelle crtE has been demonstrated in vitro. The identity and similarity scores of CrtE from cyanelles with the corresponding protein from the photosynthetic bacterium Rhodobacter capsulatus are 28.6 and 68.5%, respectively.     

Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis

Carotenoids are essential photoprotective and antioxidant pigments synthesized by all photosynthetic organisms. Most carotenoid biosynthetic enzymes were thought to have evolved independently in bacteria and plants. For example, in bacteria, a single enzyme (CrtI) catalyzes the four desaturations leading from the colorless compound phytoene to the red compound lycopene, whereas plants require two desaturases (phytoene and zeta-carotene desaturases) that are unrelated to the bacterial enzyme. We have demonstrated that carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase. The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants. After positional cloning, enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids. Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis.     

Interallelic complementation provides genetic evidence for the multimeric organization of the Phycomyces blakesleeanus phytoene dehydrogenase.

The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.     

Structural diversity and functional novelty of new carotenoid biosynthesis genes

Many new carotenoid synthesis genes have recently been identified through genomic sequencing or functional cloning. Some of them exhibit novel structures and/or novel functions. This review describes such examples in the families of lycopene β-cyclases, putative homologues of phytoene dehydrogenases and new carotenoid hydroxylases. Both the functionally novel lycopene β-monocyclases and structurally novel fusion-type of lycopene β-cyclases were described. Another newly discovered sequence of lycopene β-cyclase described might represent a new class of lycopene β-cyclases previously not identified in several cyanobacteria. Three examples of putative homologues of phytoene dehydrogenases were described, however, they were confirmed to encode different and/or new functions such as β-carotene ketolase, 4,4′-diapolycopene oxygenase or prolycopene isomerase. Two new carotenoid hydroxylase genes were described that encoded the new function of 2,2′-β-ionone ring hydroxylase or 3,3′-isorenieratene hydroxylase. Phylogenetic analysis of these genes shed light on their possible evolutionary origins. These new genes also provide tools for synthesis of novel and desirable carotenoids by genetic engineering.     

Cloning and nucleotide sequence of the crtI gene encoding phytoene dehydrogenase from the cyanobacterium Aphanocapsa PCC6714

The membrane-bound phytoene dehydrogenase (PD) is an enzyme in carotenoid biosynthesis which is essential in all microorganisms and plants containing these colored pigments. Despite its key role in the regulation of carotenogenesis, the biochemistry and molecular biology of PD are poorly understood. We have cloned, sequenced and expressed a portion of the PD-encoding gene, crtI, from the blue-green algae Aphanocapsa PCC6714. The gene codes for a 532-amino acids (aa) protein, with a calculated Mr of 52,598 Da. Two regions of the aa sequence share significant homology with PD from the purple bacterium Rhodobacter capsulatus, including a 30-aa region which has been proposed to be specific for dehydrogenases in carotenoid biosynthesis.     

Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

ABSTRACT: BACKGROUND: Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. RESULTS: Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 epsilon-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum DeltacrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum DeltacrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 +/- 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum DeltacrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 +/- 0.3 mg/g CDW were obtained. CONCLUSION: C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.     

Light-Stimulated Carotenoid Biosynthesis during Transformation of Maize Etioplasts Is Regulated by Increased Activity of Isopentenyl Pyrophosphate Isomerase

Light-stimulated carotenoid biosynthesis associated with the transformation of etioplasts to chloroplasts was investigated after dark-grown maize (Zea mays) seedlings were transferred into light. These studies focused on the enzymes of the pathway to detect those enzyme activities that were stimulated in the light and thus that were responsible for increased biosynthesis of carotenoids. In preliminary experiments, norflurazon, an inhibitor of phytoene desaturase, was used to prevent phytoene being further metabolized to carotenoids. Light-dependent stimulation of phytoene accumulation indicated that the light-regulated steps are located in the pathway leading to phytoene synthesis. The use of the 14C- labeled precursors mevalonic acid, isopentenyl pyrophosphate, and farnesyl pyrophosphate pointed to increased activity of an enzyme involved in the biosynthetic steps between isopentenyl pyrophosphate and farnesyl pyrophosphate. Determination of the activities of all five enzymes of the pathway involved in the sequence from mevalonic acid to phytoene revealed that the only enzyme activity stimulated by light was isopentenyl pyrophosphate isomerase. Over a 3-h period of illumination, this enzyme activity, like carotenoid biosynthesis, was stimulated 2.8-fold.     

A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway.

In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway. We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form. The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH. A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant. We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.     

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene （PDS3） encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarfphenotypes ofthepds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in thepds3 mutant. Further analysis showed that high level ofphytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype ofthepds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.     

Carotenoid biosynthetic pathway: molecular phylogenies and evolutionary behavior of crt genes in eubacteria

Phylogenetic analysis of carotenoid biosynthetic pathway genes and their evolutionary rate variations were studied among eubacterial taxa. The gene sequences for the enzymes involved in this pathway were obtained for major phylogenetic groups of eubacteria (green sulfur bacteria, green nonsulphur bacteria, Gram-positive bacteria, proteobacteria, flavobacteria, cyanobacteria) and archeabacteria. These gene datasets were distributed under five major steps of carotenoid biosynthesis in eubacteria; isoprenoid precursor biosynthesis, phytoene synthesis, dehydrogenation of phytoene, lycopene cyclization, formation of acyclic xanthophylls, formation of cyclic xanthophylls and carotenoid biosynthesis regulation. The NJ algorithm was used on protein coding DNA sequences to deduce the evolutionary relationship for the respective crt genes among different eubacterial lineages. The rate of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S)) were calculated for different clades of the respective phylogenetic tree for specific crt genes. The phylogenetic analysis suggests that evolutionary pattern of crt genes in eubacteria is characterized by lateral gene transfer and gene duplication events. The d(N) values indicate that carotenoid biosynthetic genes are more conserved in proteobacteria than in any other eubacterial phyla. Furthermore, of the genes involved in carotenoid biosynthesis pathway, structural genes evolve slowly than the regulatory genes in eubacteria.     

Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases.

The nucleotide sequence of the 1794-bp fragment containing the crtD gene from Rhodobacter sphaeroides 2.4.1 encoding for methoxyneurosporene dehydrogenase has been determined. A 63% sequence identity was found when compared with the nucleotide sequence of the crtD gene from Rhodobacter capsulatus. A putative regulatory palindromic motif present in the crtD gene from R. capsulatus also exists in this gene from R. sphaeroides. The translated open reading frame of the crtD gene of R. sphaeroides has identified a polypeptide of 495 amino acids which shares a 56% sequence identity with the same CrtD protein of R. capsulatus. The N- and C-termini of these CrtD proteins present a high degree of similarity with the N- and C-termini of other carotenoid dehydrogenases including those encoded by crtI genes. This is in good agreement with the previously hypothesized homology between CrtI and CrtD proteins.     

Examination of some morphologically unusual cultures of Phytophthora species using a mitochondrial DNA miniprep technique and a standardised sporangium caducity assessment

Using the mitochondrial DNA miniprep technique, the identity of sixteen morphologically unusual cultures allocated to Phytophthora nicotianae, Phytophthora mexicana or Phytophthora porri was determined by comparison with a library of mtDNA band patterns obtained from reference cultures. Seven cultures were identified as Phytophthora nicotianae (including those assigned to Phytophthora mexicana and Phytophthora porri), six as strains of Phytophthora palmivora with small, ovoid, weakly caducous sporangia, and one as Phytophthora citrophthora. Some cultures of P. nicotianae had a low percentage of caducous sporangia. Percentage sporangium caducity, but not sporangium L : B ratio, is considered a useful taxonomic criterion for separating species morphologically similar to Phytophthora nicotianae. One culture from tobacco in New Zealand had a highly unusual morphology and a unique DNA band pattern, but was not identifiable. One culture from Acacia mearnsii in South Africa had a unique DNA band pattern which was identical to that of an isolate from Annona squamosa from Australia previously identified as Phytophthora palmivora, the precise identity of which is still unclear. The identity of most isolates from diseased durian was found to be Phytophthora palmivora, confirming its role as the main pathogen, but P. nicotianae was also identified from this host. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and gene cloning of a dehydrogenase from Lactobacillus brevis that catalyzes a reaction involved in aflatoxin biosynthesis

When 10 strains of lactic acid bacteria were incubated with 5'-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5'-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.