A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from ␤-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3-hydroxyechinenone, and 3-hydroxyechinenone.During the last 6 years, significant advances have been made in our understanding of the genes coding for the enzymes involved in carotenoid biosynthesis. Many carotenoid biosynthesis genes have been cloned from various organisms, and their functions have been determined (3, 54, 62). Phytoene synthase genes, which mediate the formation of the first colorless carotenoid phytoene from geranylgeranyl PP i (GGPP) (11,46,55), have been isolated from the photosynthetic bacteria Rhodobacter species (5, 31), the nonphotosynthetic bacteria Erwinia species (4,45,60) and Thermus thermophilus (20), the cyanobacterium Synechococcus sp. strain PCC7942 (11), the fungus Neurospora crassa (57), and higher plants (7,50,51,58). Many genes involved in the desaturation (dehydrogenation) steps to convert phytoene to -carotene, neurosporene, or lycopene have also been obtained from the photosynthetic bacteria Rhodobacter species (5, 6, 31), the nonphotosynthetic bacteria Erwinia species (4, 45, 60) and Myxococcus xanthus (16), the cyanobacterium Synechococcus sp. strain PCC7942 (12), the fungi N. crassa (56) and Cercospora nicotianae (15), and higher plants (8,21,48). A second desaturase gene, which mediates the desaturation reaction from -carotene to lycopene, has been obtained from the cyanobacterium Anabaena sp. strain PCC7120 (33). The genes coding for lycopene cyclase, which catalyzes the formation of a cyclic carotenoid ␤-carotene from lycopene, have been isolated from the nonphotosynthetic bacteria Erwinia species (25, 45) and the cyanobacterium Synechococcus sp. strain PCC7942 (14).Among these organisms, the carotenoid biosynthesis genes of the yellow-pigmented nonphotosynthetic soil bacteria Erwinia uredovora (45) and Erwinia herbicola (4, 22, 60) have been used most frequently in the study o...

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes beta-carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, beta-carotene ketolase (beta-carotene oxygenase), which converted beta-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.

A broad-specificity delta 9 desaturase gene was cloned from the cyanobacterium Anacystis nidulans. The enzyme introduces a cis-double bond at the delta 9 position of both 16 and 18 carbon saturated fatty acids linked to many kinds of membrane lipids. The gene was stably introduced into tobacco plants under transcriptional control of the cauliflower mosaic virus 35S promoter, and the enzyme was targeted into plastids by the transit peptide of the pea RuBisCO small subunit. The transgenic plants had a highly reduced level of saturated fatty acid content in most membrane lipids and exhibited a significant increase in chilling resistance.

No abstract