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...

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The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPAR␣ and -␥, respectively. In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPAR␣ and -␥ in transient co-transfection studies. Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPAR␣ and -␥. Diabetic KK-A y mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone. Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group). C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance. Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes. A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0). These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes.

Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of beta-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating beta-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.

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.

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Nitric oxide (NO) plays an important role in many inflammatory responses and is also involved in carcinogenesis. In the present study, we investigated the inhibitory effect of extracts from Humulus lupulus L. on both the production of NO and the expression of inducible NO synthase (iNOS) in mouse macrophage RAW 264.7 cells. The production of NO was induced by a combination of lipopolysaccharide (LPS) and IFN-g g, and determined by Griess assay. The expression of iNOS was detected by Western blotting. The LPS/IFN-g g-induced production of NO and expression of iNOS were significantly inhibited by the ethyl acetate soluble fraction of Humulus lupulus L. Through bioactivity guided fractionation, humulene, five chalcones, 2,2-di-(3-methyl-2-butyleyl)-4,5-dihydoxy-cyclopent-4-en-1,3-dione, lupulone and three of its derivatives were isolated from the ethyl acetate soluble fraction. The chalcones, including xanthohumol, significantly inhibited the production of NO by suppressing the expression of iNOS.

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