crtI Genes Research Articles

Efficient synthesis of functional isoprenoids from acetoacetate through metabolic pathway-engineered Escherichia coli.

We show here an efficient synthesis system of isoprenoids from acetoacetate as the main substrate. We expressed in Escherichia coli a Streptomyces mevalonate pathway gene cluster starting from HMG-CoA synthase and including isopentenyl diphosphate isomerase (idi) type 2 gene and the yeast idi type 1 and rat acetoacetate-CoA ligase (Aacl) genes. When the alpha-humulene synthase (ZSS1) gene of shampoo ginger was expressed in this transformant, the resultant E. coli produced 958 mug/mL culture of alpha-humulene with a lithium acetoacetate (LAA) supplement, which was a 13.6-fold increase compared with a control E. coli strain expressing only ZSS1. Next, we investigated if this E. coli strain engineered to utilize acetoacetate can synthesize carotenoids effectively. When the crtE, crtB, and crtI genes required for lycopene synthesis were expressed in the transformant, lycopene amounts reached 12.5 mg/g dry cell weight with addition of LAA, an 11.8-fold increase compared with a control expressing only the three crt genes. As for astaxanthin production with the E. coli transformant, in which the crtE, crtB, crtI, crtY, crtZ, and crtW genes were expressed, the total amount of carotenoids produced (astaxanthin, lycopene, and phytoene) was significantly increased to 7.5 times that of a control expressing only the six crt genes.

Increased β-Carotene Production in Recombinant Escherichia coli Harboring an Engineered Isoprenoid Precursor Pathway with Mevalonate Addition

When pT-LYCm4 containing lycopene synthetic genes was co-transformed with pSUcrtY or pSHcrtY containing crtY gene of Pantoea ananatis (P. ananatis) or Pantoea agglomerans (P. agglomerans), beta-carotene productions of 36 and 35 mg/L were obtained, respectively. No lycopene was detected in the beta-carotene production culture. pT-HB, constructed by addition of P. ananatis crtY gene into pT-LYCm4, was used for co-transformation with pSdxs and pSSN12Didi, which increased isopentenyl diphosphate and dimethylallyl diphosphate synthesis. beta-Carotene production significantly increased 1.5-fold (51 mg/L) with the amplification of the dxs gene through pSdxs and 4-fold (135 mg/L) with the mevalonate bottom pathway of pSSN12Didi in the presence of 3.3 mM mevalonate. The pT-DHB, constructed by integrating the dxs gene into pT-HB, was used for cotransformation of Escherichia coli (E. coli) harboring pSSN12Didi, resulting in beta-carotene production of 141 mg/L. Recombinant E. coli harboring pT-DHB and pSSN12Didi was used to maximize beta-carotene production by adjusting the available amounts of glycerol, a carbon source, and mevalonate, the precursor of the mevalonate bottom pathway. When recombinant E. coli was given 16.5 mM mevalonate and 2.5% (w/v) glycerol, beta-carotene production of 503 mg/L in concentration and 49.3 mg/g DCW in content was obtained at 144 h, which was the highest level of carotenoid production in E. coli ever reported in the literature.

Knockout of crtB or crtI gene blocks the carotenoid biosynthetic pathway in Deinococcus radiodurans R1 and influences its resistance to oxidative DNA-damaging agents due to change of free radicals scavenging ability

Deinococcus radiodurans R1, a red-pigmented strain of the extremely radioresistant genus Deinococcus, contains a major carotenoid namely deinoxanthin. The high resistance of this organism against the lethal actions of DNA-damaging agents including ionizing radiation and ultraviolet light (UV) has been widely reported. However, the possible antioxidant role of carotenoids in this strain has not been completely elucidated. In this study, we constructed two colorless mutants by knockout of crtB and crtI genes, respectively. Comparative analysis of the two colorless mutants and the wild type showed that the two colorless mutants were more sensitive to ionizing radiation, UV, and hydrogen peroxide, but not to mitomycin-C (MMC). With electron spin resonance (ESR) and spin trapping techniques, we observed that hydroxyl radical signals occurred in the suspensions of UV irradiated Deinococcus radiodurans cells and the intensity of signals was influenced by carotenoids levels. We further showed that the carotenoid extract from the wild type could obviously scavenge superoxide anions generated by the irradiated riboflavin/EDTA system. These results suggest that carotenoids in D. radiodurans R1 function as free radical scavengers to protect this organism against the deleterious effects of oxidative DNA-damaging agents.

CDNA for phytoene desaturase in Dunaliella salina and its expressed protein as indicators of phylogenetic position of the β‐carotene biosynthetic pathway

AbstractThe desaturations of phytoene are indispensable reaction phases to carotenogenesis. Full‐length cDNA for phytoene desaturase (PDS) in the chlorophyte Dunaliella salina was cloned by reverse transcriptase–polymerase chain reaction and rapid amplification of cDNA ends. The cDNA was expressed in Escherichia coli to produce an expected protein band of molecular mass 65 kDa, as well being expressed in another color complementary E. coli system in which the original phytoene desaturase coding gene crtI of the modified plasmid pACCAR16 ▵ IX was inactivated/complemented. But the colony of the E. coli system did not display the orange color which in the positive control colony appeared to be due to the accumulation of β‐carotene. It is deduced that the expressed enzyme from the green algae could only partially functionally complement the deleted bacterium CrtI, which means another carotene desaturase (ζ‐carotene desaturase) is required for formation of lycopene in D. salina, and the β‐carotene biosynthesis pathway in this alga is similar with that in cyanobacteria and higher plants. The presence of the cloned cDNA and its expressed phytoene desaturase indicated the phylogenetic position of the β‐carotene biosynthesis pathway of D. salina in the molecular evolution of carotenoid biosynthesis from bacteria to plants. Copyright © 2007 Society of Chemical Industry

Metabolic Engineering of Potato Carotenoid Content through Tuber-Specific Overexpression of a Bacterial Mini-Pathway

BackgroundSince the creation of “Golden Rice”, biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids.MethodologyWe transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumulation of leaf carotenoids. Expression of all three genes, under tuber-specific promoter control, results in tubers with a deep yellow (“golden”) phenotype without any adverse leaf phenotypes. In these tubers, carotenoids increase approx. 20-fold, to 114 mcg/g dry weight and beta-carotene 3600-fold, to 47 mcg/g dry weight.ConclusionsThis is the highest carotenoid and beta-carotene content reported for biofortified potato as well as for any of the four major staple foods (the next best event being “Golden Rice 2”, with 31 mcg/g dry weight beta-carotene). Assuming a beta-carotene to retinol conversion of 6∶1, this is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A with 250 gms (fresh weight) of “golden” potatoes.

Astaxanthin formation in the marine photosynthetic bacterium Rhodovulum sulfidophilum expressing crtI, crtY, crtW and crtZ

This study reports the production of astaxanthin in the photosynthetic bacterium Rhodovulum sulfidophilum, which has adequate precursor pools and storage capabilities for heterologous carotenoid production. Chemical mutagenesis was carried out using ethylmethane sulfonate to produce mutants with a modified carotenoid biosynthesis pathway downstream of phytoene. Stable green- and gray-colored mutants were selected. Green mutants contained neurosporene or chloroxanthin as their major carotenoid (>90%), while the gray mutants accumulated phytoene. We previously demonstrated the production of beta-carotene in Rhodovulum sulfidophilum by cloning the Erythrobacter longus crtI (phytoene dehydrogenase) and crtY (lycopene cyclase) genes. In the present study, an expression vector for astaxanthin production was constructed that contained the Paracoccus crtW (beta-carotene oxygenase) and crtZ (beta-carotene hydroxylase) genes in addition to the E. longus crtI and crtY genes. A transconjugant, which can synthesize astaxanthin, was successfully generated (2.0 microg g(-1) DCW).

A carotenoid synthesis gene cluster from Algoriphagus sp. KK10202C with a novel fusion-type lycopene β-cyclase gene

A carotenoid synthesis gene cluster was isolated from a marine bacterium Algoriphagus sp. strain KK10202C that synthesized flexixanthin. Seven genes were transcribed in the same direction, among which five of them were involved in carotenoid synthesis. This cluster had a unique gene organization, with an isoprenoid gene, ispH (previously named lytB), being present among the carotenoid synthesis genes. The lycopene beta-cyclase encoded by the crtY ( cd ) gene appeared to be a fusion of bacterial heterodimeric lycopene cyclase CrtY(c) and CrtY(d). This was the first time that a fusion-type of lycopene beta-cyclase was reported in eubacteria. Heterologous expression of the Algoriphagus crtY ( cd ) gene in lycopene-accumulating Escherichia coli produced bicyclic beta-carotene. A biosynthesis pathway for monocyclic flexixanthin was proposed in Algoriphagus sp. strain KK10202C, though several of the carotenoid synthesis genes not linked with the cluster have not yet been cloned.

Exploring the potential of the bacterial carotene desaturase CrtI to increase the β-carotene content in Golden Rice

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.

Light-induced carotenogenesis in Myxococcus xanthus: functional characterization of the ECF sigma factor CarQ and antisigma factor CarR.

Illumination of dark-grown Myxococcus xanthus with blue light leads to the induction of carotenoid synthesis. Central to this response is the activation of the light-inducible promoter, PcarQRS, and the transcription of three downstream genes, carQ, carR and carS. Sequence analysis predicted that CarQ is a member of the ECF (extracytoplasmic function) subfamily of RNA polymerase sigma factors, and that CarR is an inner membrane protein. Genetic analysis strongly implied that CarR is an antisigma factor that sequesters CarQ in a transcriptionally inactive complex. Using in vitro transcription run-off assays, we present biochemical evidence that CarQ functions as a bacterial sigma factor and is responsible for transcription initiation at PcarQRS. Similar experiments using the crtI promoter failed to implicate CarQ in direct transcription of the crtI gene. Experiments using the yeast two-hybrid system demonstrated a protein-protein interaction between CarQ and CarR, providing evidence of a CarQ-CarR complex. The yeast two-hybrid system data also indicated that CarR is capable of oligomerization. Fractionation of M. xanthus membranes with the detergent sarkosyl showed that CarR was associated with the inner membrane. Furthermore, CarR was found to be unstable in illuminated stationary phase cells, providing a possible mechanism by which the CarR-CarQ complex is disrupted.

Genetic basis of microbial carotenogenesis

The synthesis of carotenoids begins with the formation of a phytoene from geranylgeranyl pyrophosphate, a well conserved step in all carotenogenic organisms and catalyzed by a phytoene synthase, an enzyme encoded by the crtB ( spy) genes. The next step is the dehydrogenation of the phytoene, which is carried out by phytoene dehydrogenase. In organisms with oxygenic photosynthesis, this enzyme, which accomplishes two dehydrogenations, is encoded by the crtP genes. In organisms that lack oxygenic photosynthesis, dehydrogenation is carried out by an enzyme completely unrelated to the former one, which carries out four dehydrogenations and is encoded by the crtI genes. In organisms with oxygenic photosynthesis, dehydrogenation of the phytoene is accomplished by a zeta-carotene dehydrogenase encoded by the crtQ ( zds) genes. In many carotenogenic organisms, the process is completed with the cyclization of lycopene. In organisms exhibiting oxygenic photosynthesis, this step is performed by a lycopene cyclase encoded by the crtL genes. In contrast, anoxygenic photosynthetic and non-photosynthetic organisms use a different lycopene cyclase, encoded by the crtY ( lyc) genes. A third and unrelated type of lycopene beta-cyclase has been described in certain bacteria and archaea. Fungi differ from the rest of non-photosynthetic organisms in that they have a bifunctional enzyme that displays both phytoene synthase and lycopene cyclase activity. Carotenoids can be modified by oxygen-containing functional groups, thus originating xanthophylls. Only two enzymes are necessary for the conversion of beta-carotene into astaxanthin, using several ketocarotenoids as intermediates, in both prokaryotes and eukaryotes. These enzymes are a beta-carotene hydroxylase ( crtZ genes) and a beta-carotene ketolase, encoded by the crtW (bacteria) or bkt (algae) genes.

Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene.

Biosynthetic pathways for carotenoids in the purple photosynthetic bacterium, Rubrivivax gelatinosus, which synthesizes spirilloxanthin in addition to spheroidene and OH-spheroidene, were investigated by means of genetic manipulation. A phytoene desaturase gene (crtI) found in the photosynthesis gene cluster of this bacterium was expressed in an Escherichia coli strain that can produce phytoene. Both neurosporene and lycopene were synthesized in the recombinant, probably by three- and four-step desaturation reactions of CrtI. A mutant of RVI: gelatinosus lacking the crtI gene produced only phytoene, indicating that this organism had no other phytoene desaturases. When the crtI deletion mutant was complemented by the three-step phytoene desaturase of Rhodobacter capsulatus, spirilloxanthin and its precursors were not synthesized, although spheroidene and OH-spheroidene were accumulated. It was concluded that neurosporene and lycopene are produced by a single phytoene desaturase in RVI: gelatinosus resulting in the synthesis of spheroidene and spirilloxanthin, and that there are no pathways for spirilloxanthin synthesis via spheroidene.

Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization

A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 5′ and 3′ ends of the genes and a putative RNase E site was placed between the genes. This construct was transformed into Escherichia coli cells harboring the genes for phytoene production. By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of β-carotene relative to lycopene. In addition, intermediates in the pathway from phytoene to β-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates. These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates.

Photosynthesis gene regulation in Rubrivivax gelatinosus; transcription factor PpsR is involved in both negative and positive control

Induction of the photosystem biosynthesis in anoxygenic photosynthetic bacteria arises in the absence of oxygen. The regulation of this induction occurs at the transcriptional level, with photosynthesis genes expressed preferentially under anaerobic conditions. Here we report analysis of the transcriptional control of three photosynthesis promoters, puc (light-harvesting II apoproteins), crtI (carotenoid biosynthesis) and bchG (bacteriochlorophyll biosynthesis) by the ppsR transcription factor in R. gelatinosus. This was accomplished by analysing the photosytem production in the wild type and in the PpsR null mutant grown under anaerobic and semiaerobic conditions and by assessing the beta galactosidase activity of lacZ transcriptionnal fusion to promoters possessing the putative ppsR binding boxes. It was found that under semi-aerobic conditions, while inactivation of the ppsR gene results in overproduction of carotenoid pigments, the production bacteriochlorophyll was slightely reduced and the production of LHII was completely abolished. These finding suggest that in contrast to what has been found previously for Rhodobacter species, PpsR acts in R. gelatinosus as an aerobe repressor of crtI gene, meanwhile it acts as an aerobe activator for the expression of both pucBA and an unyet identified bch gene. These results were confirmed by the b-galactosidase activities from pucB::lacZ and crtl::lacZ fusions. Inspection of the putative ppsR binding boxes revealed significant differences (sequence and spacing between the boxes) that could explain the different levels of expression of the studied genes and based on these analyses a model addressing the PpsR involvement in positive and negative regulation is proposed.

Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid

The Erwinia uredovora crtE, crtB, and crtI genes, which are responsible for the synthesis of carotenoid lycopene from farnesyl pyrophosphate, were expressed in Candida utilis under the control of the promoters and terminators derived from the C. utilis GAP, PGK, and PMA genes, respectively. The yeast transformant carrying the carotenoid biosynthesis genes produced 758 μg/g dry weight of lycopene along with 407 μg/g dry weight of phytoene in the stationary phase. It was observed in the C. utilis transformant that ergosterol content was decreased to 65% of that in the parent strain that accumulated 6.04 mg/g dry weight of ergosterol. It is therefore possible that the carbon flux for the ergosterol biosynthesis has been branched at farnesyl pyrophosphate to generate a new pathway for the lycopene production in this yeast transformant. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:306–308, 1998.

Production of lycopene by the food yeast,Candida utilis that does not naturally synthesize carotenoid

The Erwinia uredovora crtE, crtB, and crtI genes, which are responsible for the synthesis of carotenoid lycopene from farnesyl pyrophosphate, were expressed in Candida utilis under the control of the promoters and terminators derived from the C. utilis GAP, PGK, and PMA genes, respectively. The yeast transformant carrying the carotenoid biosynthesis genes produced 758 microg/g dry weight of lycopene along with 407 microg/g dry weight of phytoene in the stationary phase. It was observed in the C. utilis transformant that ergosterol content was decreased to 65% of that in the parent strain that accumulated 6.04 mg/g dry weight of ergosterol. It is therefore possible that the carbon flux for the ergosterol biosynthesis has been branched at farnesyl pyrophosphate to generate a new pathway for the lycopene production in this yeast transformant.

Cloning, sequencing and expressing the carotenoid biosynthesis genes, lycopene cyclase and phytoene desaturase, from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% ( Erwinia herbicola), 37.4% ( Erwinia uredovora) and 22.9% ( Synechococcus sp.), and those between phytoene desaturase genes are 50.3% ( E. herbicola), 54.7% ( E. uredovora) and 39.6% ( Rhodobacter capsulatus).

The role of pigments in the assembly of photosynthetic complexes in Rhodobacter capsulatus

AbstractMutants of Rhodobacter capsulatus, blocked at different steps of bacteriochlorophyll a (BChl) synthesis between protoporphyrin IX and 2‐hydroxyethyl bacteriochlorophyllide a, were induced to synthesize the photosynthetic apparatus by lowering of oxygen tension in dark cultures. The cells were pulse‐labeled with [35S]methionine and the radioactivity chased after dilution of [35S]. The specific radioactivity in the pigment‐binding proteins of light‐harvesting and reaction center proteins of the wild‐type strain was not lowered during the chase period of three hours but in the BChl‐free mutants the label disappeared within five to thirty minutes. The polypeptides were inserted into the membrane but did not remain stably incorporated. In the mutant strain NK9 the synthesis of the carotenoid spheroidenone/spheroidene was inhibited by insertion of Tn5 in the crtI gene (phytoene desaturase), which blocked completely the formation of the light‐harvesting (LH) complex II (B800–850) but not of the LHI (B870) complex. In this mutant the polypeptides of the LH complexes were synthesized in a lower amount than in the wild‐type cells and were inserted into the membrane. The LHIIα poly‐peptide disappeared after 60 min of chase while the LHIIβ was more stable. It was concluded that the pigments are not only necessary for absorption of photons and efficient transfer of excitation energy but have a structural role by stabilizing the oligomeric LH complexes. This is in accordance with the crystal structure data of LHII complexes.

Engineering Cyanobacterial Models Resistant to Bleaching Herbicides

Enzymes catalyzing the dehydrogenation steps of the carotenoid biosynthetic pathway are target sites for bleaching herbicides. We introduced the gene crtI, coding for phytoene desaturase of the nonphotosynthetic bacterium Erwinia uredovora into the cyanobacterium Synechococcus PCC 7942, using it as a convenient experimental model for higher-plant transformation. The heterologous expression of the foreign gene in the transformants was demonstrated by Western blot analysis. The respective gene product CRTI, the enzyme phytoene desaturase, catalyzes the conversion of phytoene to lycopene via phytofluene, ζ-carotene, and neurosporene. It is highly resistant against various herbicides that affect either phytoene or ζ-carotene desaturase activity. Molar I 50 values of carotenoid synthesis caused by various bleaching herbicides and determined at the intact cell revealed that the transformant strains exhibited strong cross-resistance toward the herbicides assayed. Resistance factors were more than 3 orders of magnitude greater than those of the control strain.

The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex in Rhodobacter sphaeroides

Coloured carotenoids play some undefined role in the assembly of a functional light-harvesting 2 (LH2) complex in photosynthetic bacteria. We have used a series of transposon Tn5 insertion mutants disrupted at various stages of the carotenoid-biosynthetic pathway, together with an LH2 deletion/insertion mutant, to investigate this effect in Rhodobacter sphaeroides. Mutants were initially characterized by low-temperature absorbance spectroscopy and ultrastructural analysis: Northern-blot analysis demonstrated normal pucBA transcripts for LH2 polypeptides in all the carotenoid mutants. Analysis of translation of the puc transcript and investigation of the fate of any resulting LH2 polypeptides by SDS/PAGE, Western-blot and pulse-chase experiments clearly demonstrated that, in the absence of coloured carotenoids, the LH2 alpha- and beta-polypeptides are synthesized but are rapidly turned over and do not become stably integrated into the membrane. Complementation of mutants with lesions in the crtB and crtI genes, encoding phytoene synthase and phytoene desaturase respectively, with the cloned R. sphaeroides crtI gene, resulted in restoration of carotenoid biosynthesis and stable assembly of the LH2 complex in the crtI mutant but not in the crtB mutant, despite the presence of the CrtI protein.

Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase.

A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.