New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species

The role of carotenoid genes crtB and crtE has been functionally assigned. These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens. Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation. In vitro reactions from extracts of E. coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP). These results strongly indicate that the crtE gene encodes GGPP synthase. Introduction of the crtB gene into A. tumefaciens led to the conversion of GGPP into phytoene. This activity was absent in similar transformants with the crtE gene. Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E. coli harboring both the crtB and crtE genes.     

Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids,chlorophyll, and abscisic acid

Phytoene synthase catalyzes the dimerization of two molecules of geranylgeranyl pyrophosphate to phytoene and has been shown to be rate limiting for the synthesis of carotenoids. To elucidate if the capacity to produce phytoene is limiting also in the seed of Arabidopsis (Wassilewskija), a gene coding for an endogenous phytoene synthase was cloned and coupled to a seed-specific promoter, and the effects of the overexpression were examined. The resulting transgenic plants produced darker seeds, and extracts from the seed of five overexpressing plants had a 43-fold average increase of beta-carotene and a total average amount of beta-carotene of approximately 260 microg g-1 fresh weight. Lutein, violaxanthin, and chlorophyll were significantly increased, whereas the levels of zeaxanthin only increased by a factor 1.1. In addition, substantial levels of lycopene and alpha-carotene were produced in the seeds, whereas only trace amounts were found in the control plants. Seeds from the transgenic plants exhibited delayed germination, and the degree of delay was positively correlated with the increased levels of carotenoids. The abscisic acid levels followed the increase of the carotenoids, and plants having the highest carotenoid levels also had the highest abscisic acid content. Addition of gibberellic acid to the growth medium only partly restored germination of the transgenic seeds.     

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.     

Expression and functional analysis of a gene cluster involved in the synthesis of decaprenoxanthin reveals the mechanisms for C50 carotenoid formation.

Corynebacterium glutamicum accumulates the C50 carotenoid decaprenoxanthin. Rescued DNA from transposon color mutants of this Gram-positive bacterium was used to clone the carotenoid biosynthetic gene cluster. By sequence comparison and functional complementation, the genes involved in the synthesis of carotenoids with 50 carbon atoms were identified. The genes crtE, encoding a geranylgeranyl pyrophosphate synthase, crtB, encoding a phytoene synthase, and crtI, encoding a phytoene desaturase, are responsible for the formation of lycopene. The products of three novel genes, crtYe and crtYf, with sequence similarities to heterodimeric lycopene cyclase crtYc and crtYd, together with crtEb which exhibits a prenyl transferase motif, were involved in the conversion of C40 acyclic lycopene to cyclic C50 carotenoids. Using functional complementation in Escherichia coli, it could be shown that the elongation of lycopene to the acyclic C50 carotenoid flavuxanthin by the addition of C5 isoprenoid units at positions C-2 and C-2' is catalyzed by the crtEb gene product. Subsequently, the gene products of crtYe and crtYf in a concerted action convert the acyclic flavuxanthin into the cyclic C50 carotene, decaprenoxanthin, forming two epsilon-ionone groups. The mechanisms, involving two individual steps for the formation of cyclic C50 carotenoids from lycopene, are proposed on the basis of these results.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

Carotenoid synthesis and phytoene synthase activity during mating of Blakeslea trispora

Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (−) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of phytoene synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, phytoene synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The phytoene synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional phytoene synthase and lycopene cyclase.     

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, β-carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for β-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.     

beta-Carotene accumulation induced by the cauliflower Or gene is not due to an increased capacity of biosynthesis

The cauliflower (Brassica oleracea L. var. botrytis) Or gene is a rare carotenoid gene mutation that confers a high level of beta-carotene accumulation in various tissues of the plant, turning them orange. To investigate the biochemical basis of Or-induced carotenogenesis, we examined the carotenoid biosynthesis by evaluating phytoene accumulation in the presence of norflurazon, an effective inhibitor of phytoene desaturase. Calli were generated from young seedlings of wild type and Or mutant plants. While the calli derived from wild type seedlings showed a pale green color, the calli derived from Or seedlings exhibited intense orange color, showing the Or mutant phenotype. Concomitantly, the Or calli accumulated significantly more carotenoids than the wild type controls. Upon treatment with norflurazon, both the wild type and Or calli synthesized significant amounts of phytoene. The phytoene accumulated at comparable levels and no major differences in carotenogenic gene expression were observed between the wild type and Or calli. These results suggest that Or-induced beta-carotene accumulation does not result from an increased capacity of carotenoid biosynthesis.     

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1DeltacrtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H2O2 and singlet oxygen than two carotenes (lycopene and beta-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1' position, compared with other tested carotenoids.     

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.     

Production of phytoene, a carotenoid, and induction of connexin 26 in transgenic mice carrying the phytoene synthase gene crtB

Carotenoids have been recognized as chemopreventive agents against human diseases, such as cancer and cardiovascular disease. Mammalians utilize carotenoids supplied from their food since they are unable to perform the de novo synthesis of carotenoids. We previously created mammalian cultured cells producing phytoene, a type of carotenoid, and showed that these cells acquired resistance against oxidative stress and oncogenic transformation. In the present study, we established a transgenic mouse line, carrying the crtB gene encoding phytoene synthase, which could produce phytoene endogenously. It was found that connexin 26 was induced in these phytoene-producing mice. Since it is known that carotenoids enhance gap junctional communication by inducing the expression of connexin genes, the present data suggest that the induction of connexin 26 in phytoene-producing mice may play a role in controlling cell-to-cell communication. Phytoene-producing mice provide a useful system in which to investigate the in vivo function of the carotenoid phytoene.     

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis,chemiluminescence, and DNA damage analyses

Deinococcus radiodurans is highly resistant to reactive oxygen species (ROS). The antioxidant effect of carotenoids in D. radiodurans was investigated by using a targeted mutation of the phytoene synthase gene to block the carotenoid synthesis pathway and by evaluating the survival of cells under environmental stresses. The colorless mutant R1ΔcrtB of D. radiodurans failed to synthesize carotenoids, and was more sensitive to ionizing radiation, hydrogen peroxide, and desiccation than the wild type, suggesting that carotenoids in D. radiodurans help in combating environmental stresses. Chemiluminescence analyses showed that deinoxanthin, a major product in the carotenoid synthesis pathway, had significantly stronger scavenging ability on H₂O₂ and singlet oxygen than two carotenes (lycopene and β-carotene) and two xanthophylls (zeaxanthin and lutein). Deinoxanthin also exhibited protective effect on DNA. Our findings suggest that the stronger antioxidant effect of deinoxanthin contribute to the resistance of D. radiodurans. The higher antioxidant effect of deinoxanthin may be attributed to its distinct chemical structure which has an extended conjugated double bonds and the presence of a hydroxyl group at C-1′ position, compared with other tested carotenoids.     

Progress on molecular breeding and metabolic engineering of biosynthesis pathways of C(30), C(35), C(40), C(45), C(50) carotenoids

At least 700 natural carotenoids have been characterized; they can be classified into C(30), C(40) and C(50) subfamilies. The first step of C(40) pathway is the combination of two molecules of geranylgeranyl pyrophosphate to synthesize phytoene by phytoene synthase (CrtB or PSY). Most natural carotenoids originate from different types and levels of desaturation by phytoene desaturase (CrtI or PDS+ZDS), cyclization by lycopene cyclase (CrtY or LYC) and other modifications by different modifying enzyme (CrtA, CrtU, CrtZ or BCH, CrtX, CrtO, etc.) of this C(40) backbone. The first step of C(30) pathway is the combination of two molecules of FDP to synthesize diapophytoene by diapophytoene synthase (CrtM). But natural C(30) pathway only goes through a few steps of desaturation to form diaponeurosporene by diapophytoene desaturase (CrtN). Natural C(50) carotenoid decaprenoxanthin is synthesized starting from the C(40) carotenoid lycopene by the addition of 2 C(5) units. Concerned the importance of carotenoids, more and more attention has been concentrated on achieving novel carotenoids. The method being used successfully is to construct carotenoids biosynthesis pathways by metabolic engineering. The strategy of metabolic engineering is to engineer a small number of stringent upstream enzymes (CrtB, CrtI, CrtY, CrtM, or CrtN), then use a lot of promiscuous downstream enzymes to obtain large number of novel carotenoids. Two key enzymes phytoene desaturase (CrtI(m)) and lycopene cyclase (CrtY(m)) have been modified and used with a series of downstream modifying enzymes with broad substrate specificity, such as monooxygenase (CrtA), carotene desaturase (CrtU), carotene hydroxylase (CrtZ), zeaxanthin glycosylase (CrtX) and carotene ketolase (CrtO) to extend successfully natural C(30) and C(40) pathways in E. coli. Existing C(30) synthase CrtM to synthesize carotenoids with different chain length have been engineered and a series of novel carotenoids have been achieved using downstream modifying enzymes. C(35) carotenoid biosynthesis pathway has been constructed in E. coli as described. C(45) and C(50) carotenoid biosynthesis pathways have also been constructed in E. coli, but it is still necessary to extend these two pathways. Those novel acyclic or cyclic carotenoids have a potential ability to protect against photooxidation and radical-mediated peroxidation reactions which makes them interesting pharmaceutical candidates.     

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-¹⁴C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [¹⁴C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [¹⁴C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [¹⁴C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [¹⁴C]geranylgeranyl pyrophosphate from [1-¹⁴C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.