Isolation and Characterization of the Epoxide Hydrolase-Encoding Gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the α/β hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the α/β hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min⁻¹ (mg protein)⁻¹ towards 1,2-epoxyhexane. Received: 2 August 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

Soluble epoxide hydrolase homologs in Strongylocentrotus purpuratus suggest a gene duplication event and subsequent divergence

The mammalian soluble epoxide hydrolase (sEH) is a multidomain enzyme composed of C- and N-terminal regions that contain active sites for epoxide hydrolase (EH) and phosphatase activities, respectively. We report the cloning of two 60 kDa multidomain enzymes from the purple sea urchin Strongylocentrotus purpuratus displaying significant sequence similarity to both the N- and C-terminal domains of the mammalian sEH. While one urchin enzyme did not exhibit EH activity, the second enzyme hydrolyzed several lipid messenger molecules metabolized by the mammalian sEH, including the epoxyeicosatrienoic acids. Neither of the urchin enzymes displayed phosphatase activity. The urchin EH was inhibited by small molecule inhibitors of the mammalian sEH and is the likely ancestor of the enzyme. Sequence comparisons suggest that the urchin sEH homologs are the result of a gene fusion event between a gene encoding for an EH and a gene for an enzyme of undetermined function. This fusion event was followed by a duplication event to produce the urchin enzymes.     

Production of (S)-styrene oxide by recombinant Pichia pastoris containing epoxide hydrolase from Rhodotorula glutinis

A recombinant yeast Pichia pastoris carrying the gene encoding epoxide hydrolase (EH) of Rhodotorula glutinis was constructed and used for producing (S)-styrene oxide by enantioselective hydrolysis of racemic mixtures of styrene oxides. The EH gene was obtained by PCR amplification of cDNA of R. glutinis and integrated into the chromosomal DNA of P. pastoris to express EH under the control of AOX promoter. The recombinant yeast has a high hydrolytic activity toward (R)-styrene oxide as 358 nmol min⁻¹ (mg cell)⁻¹, which is about 10-fold higher than that of wild type R. glutinis. When kinetic resolution was conducted by the recombinant yeast at a high initial epoxides concentration of 526 mM that constitutes an epoxide–water two-liquid phase, chiral (S)-styrene oxide with an enantiomeric excess (e.e.) higher than 98% was obtained as 36% yield (theoretical, 50%) at 16 h.     

Cloning and characterization of the astaxanthin biosynthetic gene encoding phytoene desaturase of Xanthophyllomyces dendrorhous.

The first carotenoid biosynthetic gene from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated by heterologous complementation in Escherichia coli. The isolated gene, denominated as crtI, was found to encode for phytoene desaturase. The coding region is interrupted by 11 introns. The deduced amino acid sequence showed significant homology with its bacterial and eukaryotic counterparts, especially those of fungal origin. A plasmid containing the geranylgeranyl diphosphate synthase and phytoene synthase encoding genes from Erwinia uredovora was introduced in E. coli together with the phytoene desaturase encoding cDNA from X. dendrorhous. As a result, lycopene accumulation was observed in these transformants. We conclude that in X. dendrorhous the four desaturase steps, by which phytoene is converted into lycopene, are carried out by a single gene product.     

Biogeography, host specificity, and molecular phylogeny of the basidiomycetous yeast Phaffia rhodozyma and its sexual form, Xanthophyllomyces dendrorhous

Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous) is a basidiomycetous yeast that has been found in tree exudates in the Northern Hemisphere at high altitudes and latitudes. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We isolated X. dendrorhous from the Southern Hemisphere (Patagonia, Argentina), where it was associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus trees. We compared internal transcribed spacer (ITS)-based phylogenies of P. rhodozyma and its tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae) and found them to be generally concordant, suggesting that different yeast lineages colonize different trees and providing an explanation for the phylogenetic distance observed between the type strains of P. rhodozyma and X. dendrorhous. We hypothesize that the association of Xanthophyllomyces with Cyttaria derives from a previous association of the yeast with Nothofagus, and the sister relationship between Nothofagaceae and Betulaceae plus Fagaceae correlates with the phylogeny of X. dendrorhous strains originating from these three plant families. The two most basal strains of X. dendrorhous are those isolated from Cornus, an ancestral genus in the phylogenetic analysis of the host trees. Thus, we question previous conclusions that P. rhodozyma and X. dendrorhous represent different species since the polymorphisms detected in the ITS and intergenic spacer sequences can be attributed to intraspecific variation associated with host specificity. Our study provides a deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny. Such knowledge is essential for the comprehension of many aspects of the biology of this organism and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.     

Cloning and sequencing of an epoxide hydrolase gene from Rhodosporidium paludigenum.

Epoxide hydrolase (EH) activity was recently described in yeasts and highly selective hydrolysis of epoxides was observed during whole cell biotransformations. To expand the available molecular data regarding yeast EHs, the EH encoding gene from Rhodosporidium paludigenum (CBS 6565) was isolated, cloned and sequenced. The genomic EH sequence revealed a 1600 bp sequence interrupted by six introns. cDNA sequence analysis revealed an open reading frame of 1236 bp with a deduced polypeptide length of 411 amino acids. The deduced amino acid sequence revealed a relative high degree of sequence homology compared to the amino acid sequence of the EH from Rhodotorula glutinis.     

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.     

Construction and Characterisation of a Genetically Engineered Escherichia coli Strain for the Epoxide Hydrolase-catalysed Kinetic Resolution of Epoxides

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.     

Construction and Characterisation of a Genetically Engineered Escherichia coli Strain for the Epoxide Hydrolase-catalysed Kinetic Resolution of Epoxides

The Rhodotorula glutinis epoxide hydrolase, Eph1, was produced in the heterologous host Escherichia coli BL21(DE3) in order to develop a highly effective epoxide hydrolysis system. A 138-fold increase in Eph1 activity was found in cell extracts of the recombinant E. coli when compared to cell extracts of Rhodotorula glutinis, despite the formation of Eph1 inclusion bodies. Optimization of cultivation conditions and co-expression of molecular chaperones resulted in a further increase in activity and a reduction of the inclusion bodies formation, respectively. Compared to Rhodotorula glutinis cells and cell extracts, a total increase in Eph1 activity of over 200 times was found for both Escherichia coli cells and crude enzyme preparations of these cells. The improved conditions for recombinant Eph1 production were used to demonstrate the Eph1-catalysed kinetic resolution of a new Eph1 substrate, 1-oxaspiro[2.5]octane-2-carbonitrile.     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Heterologous expression of pentalenene synthase (PSS) from Streptomyces UC5319 in Xanthophyllomyces dendrorhous

For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used for the transformation of the wild-type strain of X. dendrorhous. The transformations resulted in white colonies, showing a complete shutdown of the carotenoid production. Furthermore, an additional vector was constructed for the insertion of the PSS gene in the rDNA of the yeast. All the mutant strains produce the sesquiterpene pentalenene and show no difference in growth when compared to the wild-type strain. In this report, we demonstrate that X. dendrorhous is a suitable host for the expression of heterologous terpene cyclases and for the production of foreign terpene compounds.     

Effect of carbon and nitrogen sources on the production of a highly enantioselective epoxide hydrolase from Aspergillus niger

The highly enantioselective epoxide hydrolase from Aspergillus niger is well utilized as biocatalysts for the preparation of enantiopure chiral epoxides and diols. Both growth of the fungus and EH activity production were found greatly affected by changing the carbon or the nitrogen source with fructose and corn steep liquor being the best. Their concentrations were optimized (10 g.l–1 of fructose and 15 g.l–1 of corn steep) which resulted in an increase of both the biomass produced (31%) and the epoxide hydrolase specific activity (38%). The results obtained suggested a complex regulation of the EH production. On the whole, a two times increase of the total EH activity was obtained. © Rapid Science Ltd. 1998     

Clues for the existence of two different epoxide hydrolase activities in the fungus Beauveria bassiana

Epoxide hydrolase activity was produced during the exponential and stationary growth phases of the fungus Beauveria bassiana ATCC 7159. It was completely cell-associated. After cell disruption epoxide hydrolase activity was recovered in both the cell debris (EH "A") and the soluble fraction (EH "B"), but not in the membrane fraction. Activity assays of these fractions with two different substrates indicated that their substrate specificity, as well as the corresponding E value and, to a lesser extent, their regioselectivity, were different. Also, we could observe that the absolute configuration of the residual epoxide was opposite. This indicates that these two epoxide hydrolase activities are substantially different and are, therefore, interestingly complementary biocatalysts for the preparation of the corresponding epoxides and/or vicinal diols in nearly enantiopure form.     

EH3 (ABHD9): the first member of a new epoxide hydrolase family with high activity for fatty acid epoxides

Epoxide hydrolases are a small superfamily of enzymes important for the detoxification of chemically reactive xenobiotic epoxides and for the processing of endogenous epoxides that act as signaling molecules. Here, we report the identification of two human epoxide hydrolases: EH3 and EH4. They share 45% sequence identity, thus representing a new family of mammalian epoxide hydrolases. Quantitative RT-PCR from mouse tissue indicates strongest EH3 expression in lung, skin, and upper gastrointestinal tract. The recombinant enzyme shows a high turnover number with 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET), as well as 9,10-epoxyoctadec-11-enoic acid (leukotoxin). It is inhibited by a subclass of N,N'-disubstituted urea derivatives, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, 1-cyclohexyl-3-dodecylurea, and 1-(1-acetylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, compounds so far believed to be selective inhibitors of mammalian soluble epoxide hydrolase (sEH). Its sensitivity to this subset of sEH inhibitors may have implications on the pharmacologic profile of these compounds. This is particularly relevant because sEH is a potential drug target, and clinical trials are under way exploring the value of sEH inhibitors in the treatment of hypertension and diabetes type II.     

Cyclopropyl oxiranes: reversible inhibitors of cytosolic and microsomal epoxide hydrolases

A series of aryl- and alkyl-substituted cyclopropyl oxiranes were synthesized as potential suicide inhibitors of mouse liver epoxide hydrolase (EH). The inhibitory potency of each compound and its corresponding alkene precursor was determined with mouse liver EHs using [3H]-cis-stilbene oxide as substrate for microsomal EH (mEH) and for glutathione-S-transferase, and using [3H]-trans-stilbene oxide for cytosolic EH (cEH). The cyclopropyl oxiranes all showed low (26-60% at 5 X 10(-4) M) inhibition of glutathione transferase and moderate inhibition (I50 = 5 X 10(-4) to 6 X 10(-6) M) for cEH and mEH. cis-Phenylcyclopropyl oxirane had an I50 for mEH near that for a commonly used inhibitor, 1,1,1-trichloropropene oxide. Inhibition appeared competitive and reversible, and the cyclopropyl oxiranes appeared to function as alternate substrates. Absence of irreversible inhibition is evidence against a strongly electrophilic epoxide-opening mechanism involving a cyclopropyl carbinyl-homoallyl cation rearrangement. Instead, a concerted mechanism is favored, in which electrophilic opening and hydroxide attack occur in a concerted fashion.     

Pseudomonas aeruginosa Cif defines a distinct class of α/β epoxide hydrolases utilizing a His/Tyr ring-opening pair

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that secretes a multitude of virulence factors during the course of infection. Among these is Cif, an epoxide hydrolase (EH) that reduces the functional localization of the cystic fibrosis transmembrane conductance regulator in epithelial cells. In addition to being the first reported EH virulence factor, Cif possesses unique sequence deviations from canonical EH motifs. Foremost among these is the substitution of a histidine for the first epoxide ring-opening tyrosine in the active site. To test the functional equivalence of Tyr and His side chains at this position, we have generated the mutant Cif-H177Y. Structural analysis confirms that both the WT His and mutant Tyr side chains can be accommodated without large-scale conformational changes. However, the Tyr mutant is functionally inactive. Based on a detailed analysis of the structure of the Tyr mutant, it appears that Cif's main-chain conformation imposes a functional requirement for a His at this position. Comparison with canonical EH structures reveals additional conformational differences, which are coupled to divergent sequence characteristics. When used to probe the genomes of other opportunistic pathogens, these sequence-structure criteria uncover candidate sequences that appear to form a distinct subfamily of Cif-like epoxide hydrolases characterized by a conserved His/Tyr ring-opening pair.     

Production of the UVB-absorbing compound mycosporine-glutaminol-glucoside by Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The ability of the basidiomycetous yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to accumulate astaxanthin is responsible for the industrial use of this yeast as a microbial source of pigments for aquaculture. It is also hypothesized that astaxanthin accounts for its ability to thrive in highly oxidative and UV-exposed habitats. Here, we assessed the ability of this species to synthesize UV-absorbing compounds generally known as mycosporines, evaluated the effect of culture media in the production of these compounds and compared its UV growth resistance and tolerance with other yeasts. The 48 wild and collection strains screened were positive for mycosporines and a unique compound identified as mycosporine-glutaminol-glucoside (MGG) was detected. Thus, the ability of X. dendrorhous to produce MGG, as described here for the first time, is so far unique among the Cystofilobasidiales. The compound was synthesized constitutively, although growth under visible light and, to a greater extent, UVA radiation stimulated its production. Strains from UV-exposed habitats produced larger quantities and oligotrophic complex media seemed to favor MGG accumulation. UV tolerance and survival of X. dendrorhous was high and comparable to that of the polyextremophilic Rhodotorula mucilaginosa. The taxonomical and ecological implications of the production of MGG by X. dendrorhous are discussed.