Cloning and Expression InEscherichia Coli of the Gene Encoding [Beta]-C-4-Oxygenase, That Converts [Beta]-Carotene to the Ketocarotenoid Canthaxanthin … (original) (raw)
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Applied Microbiology and Biotechnology, 2005
The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark-heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a carotenoid oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was found up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting β-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described and the CRTO is termed as carotenoid 4, 4′-β-ionone ring oxygenase.
Plant & cell physiology, 2008
The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW1...
Microbial Cell Factories, 2008
Astaxanthin is a xanthophyll of great interest in animal nutrition and human health. The market prospect in the nutraceutics industries for this health-protective molecule is very promising. Astaxanthin is synthesized by several bacteria, algae and plants from β-carotene by the sequential action of two enzymes: a β-carotene, 3,3'-hydroxylase that introduces an hydroxyl group at the 3 (and 3') positions of each of the two β-ionone rings of β-carotene, and a β-carotene ketolase that introduces keto groups at carbons 4 and 4' of the β-ionone rings. Astaxanthin is also produced by the yeast-like basidiomycete Xanthophyllomyces dendrorhous. A gene crtS involved in the conversion of β-carotene to astaxanthin has been cloned simultaneously by two research groups. Complementation studies of X. dendrorhous mutants and expression analysis in Mucor circinelloides reveals that the CrtS enzyme is a β-carotene hydroxylase of the P-450 monooxygenase family that converts β-carotene to the hydroxylated derivatives β-cryptoxanthin and zeaxanthin, but it does not form astaxanthin or the ketolated intermediates in this fungus. A bifunctional β-carotene hydroxylase-ketolase activity has been proposed for the CrtS protein. The evidence for and against this hypothesis is analyzed in detail in this review. Review Carotenoids are an important group of natural pigments with specific applications as colorants, feed supplements and nutraceuticals; they are also used for medical, cosmetic and biotechnological purposes. A few of the variety of natural and synthetic carotenoids available have been exploited commercially, including β-carotene, lycopene, astaxanthin, canthaxanthin, lutein and capxanthin [1-4]. Although more than 600 different carotenoids have been described from carotenogenic microorganisms [5], only β-carotene, lycopene and astaxanthin are commercially produced by microbial fermentation. These three compounds have various biological functions such as species-specific coloration, light-harvesting, photo-protection, antioxidant, and hormone precursor [6,7]. Dietary carotenoids have beneficial effects delaying the onset of many diseases such as arteriosclerosis, cataracts, age-related macular degeneration, multiple sclerosis, cardiovascular diseases, and some kinds of cancer [4]. For these reasons the demand and market of carotenoids have grown drastically [8]. Production and use of astaxanthin Astaxanthin (Fig. 1) is a xanthophyll widely used as a pigment in aquaculture. The all-trans isomer is found in nature together with small amounts of 9-cis and 13-cis
Applied and Environmental Microbiology, 2006
Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires -carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from Paracoccus sp. strain N81106 (formerly classified as Agrobacterium aurantiacum) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in Escherichia coli cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing -carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin.
The synthesis of the acidic apo-carotenoid neurosporaxanthin by the fungus Fusarium fujikuroi depends on four enzyme activities: phytoene synthase and carotene cyclase, encoded by the bifunctional gene carRA, a carotene desaturase, encoded by carB, and a postulated cleaving enzyme converting torulene (C40) into neurosporaxanthin (C35). Based on sequence homology to carotenoid oxygenases, we identified the novel fungal enzyme CarT. Sequencing of the carT allele in a torulene-accumulating mutant of F. fujikuroi revealed a mutation affecting a highly conserved amino acid, and introduction of a heterologous carT gene in this mutant restored the ability to produce neurosporaxanthin, pointing to CarT as the enzyme responsible for torulene cleavage. Expression of carT in lycopene-accumulating E. coli cells resulted in the formation of minor amounts of apocarotenoids, but no enzymatic activity was observed in b-carotene-accumulating cells, indicating a preference for acyclic or monocyclic carotenes. The purified CarT enzyme efficiently cleaved torulene in vitro to produce b-apo-4Ј-carotenal, the aldehyde corresponding to the acidic neurosporaxanthin, and was also active on other monocyclic synthetic substrates. In agreement with its role in carotenoid biosynthesis, the carT transcript levels are induced by light and upregulated in carotenoid-overproducing mutants, as already found for other car genes.
Plant molecular biology, 1995
We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcuspluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes r-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, r-carotene ketolase (r-carotene oxygenase), which converted r-carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3' S), which was identified after purification by a variety of spectroscopic methods.
Microbiological Synthesis of Carotenoids: Pathways and Regulation
Progress in Carotenoid Research
Carotenoids are naturally produced by plants, algae, and some bacteria and fungi, fulfilling functions as accessory photosynthetic pigments and antioxidants. Among carotenoids, the xanthophyll astaxanthin stands out for its antioxidant and nutraceutical properties, which are beneficial to human health, and also for its use in the aquaculture industry as nutritional supplement of salmonid fish. Many studies have focused on the search of natural sources of astaxanthin as an alternative production that guarantees the beneficial properties of this compound. In nature, few astaxanthin-producing organisms are known, being the microalgae Haematococcus pluvialis and the yeast Xanthophyllomyces dendrorhous the most promising microbiological systems for the biotechnological production of this carotenoid. In this chapter, we describe the carotenogenic pathways in these microorganisms and the proposed carotenogenesis regulation mechanisms. As an example, the influence of the carbon source, the regulation by catabolic repression and by sterols in the carotenogenesis in the yeast X. dendrorhous is described.
Carotenoid Cleavage Oxygenases from Microbes and Photosynthetic Organisms: Features and Functions
International Journal of Molecular Sciences, 2016
Apocarotenoids are carotenoid-derived compounds widespread in all major taxonomic groups, where they play important roles in different physiological processes. In addition, apocarotenoids include compounds with high economic value in food and cosmetics industries. Apocarotenoid biosynthesis starts with the action of carotenoid cleavage dioxygenases (CCDs), a family of non-heme iron enzymes that catalyze the oxidative cleavage of carbon-carbon double bonds in carotenoid backbones through a similar molecular mechanism, generating aldehyde or ketone groups in the cleaving ends. From the identification of the first CCD enzyme in plants, an increasing number of CCDs have been identified in many other species, including microorganisms, proving to be a ubiquitously distributed and evolutionarily conserved enzymatic family. This review focuses on CCDs from plants, algae, fungi, and bacteria, describing recent progress in their functions and regulatory mechanisms in relation to the different roles played by the apocarotenoids in these organisms.