Carotene Hydroxylase Activity Determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots

The typically intense carotenoid accumulation in cultivated orange-rooted carrots (Daucus carota) is determined by a high protein abundance of the rate-limiting enzyme for carotenoid biosynthesis, phytoene synthase (PSY), as compared with white-rooted cultivars. However, in contrast to other carotenoid accumulating systems, orange carrots are characterized by unusually high levels of α-carotene in addition to β-carotene. We found similarly increased α-carotene levels in leaves of orange carrots compared with white-rooted cultivars. This has also been observed in the Arabidopsis thaliana lut5 mutant carrying a defective carotene hydroxylase CYP97A3 gene. In fact, overexpression of CYP97A3 in orange carrots restored leaf carotenoid patterns almost to those found in white-rooted cultivars and strongly reduced α-carotene levels in the roots. Unexpectedly, this was accompanied by a 30 to 50% reduction in total root carotenoids and correlated with reduced PSY protein levels while PSY expression was unchanged. This suggests a negative feedback emerging from carotenoid metabolites determining PSY protein levels and, thus, total carotenoid flux. Furthermore, we identified a deficient CYP97A3 allele containing a frame-shift insertion in orange carrots. Association mapping analysis using a large carrot population revealed a significant association of this polymorphism with both α-carotene content and the α-/β-carotene ratio and explained a large proportion of the observed variation in carrots.     

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio- and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, Δ⁴-androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16α-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio- and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K_d) values of the binding between CYP154C3 and these steroids were less than 8 μM as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of Δ⁴-androstene-3,17-dione were determined (K_m, 31.9 ± 9.1 μM; k_cat, 181 ± 4.5 s⁻¹). We concluded that CYP154C3 is a steroid D-ring 16α-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16α-specific hydroxylation activity.     

Over-expression of Arabidopsis thaliana carotenoid hydroxylases individually and in combination with a β-carotene ketolase provides insight into in vivo functions

Carotenoids represent a group of widely distributed pigments derived from the general isoprenoid biosynthetic pathway that possess diverse functions in plant primary and secondary metabolism. Modification of α- and β-carotene backbones depends in part on ring hydroxylation. Two ferredoxin-dependent non-heme di-iron monooxygenases (AtB1 and AtB2) that mainly catalyze in vivo β-carotene hydroxylations of β,β-carotenoids, and two heme-containing cytochrome P450 (CYP) monooxygenases (CYP97A3 and CYP97C1) that preferentially hydroxylate the ε-ring of α-carotene or the β-ring of β,ε-carotenoids, have been characterized in Arabidopsis by analysis of loss-of-function mutant phenotypes. We further investigated functional roles of both hydroxylase classes in modification of the β- and ε-rings of α-carotene and β-carotene through over-expression of AtB1, CYP97A3, CYP97C1, and the hydroxylase candidate CYP97B3. Since carotenoid hydroxylation is required for generation of ketocarotenoids by the bkt1(CrtO) β-carotene ketolase, all hydroxylase constructs were also introduced into an Arabidopsis line expressing the Haematococcus pluvalis bkt1 β-carotene ketolase. Analysis of foliar carotenoid profiles in lines overexpressing the individual hydroxylases indicate a role for CYP97B3 in carotenoid biosynthesis, confirm and extend previous findings of hydroxylase activities based on knock-out mutants, and suggest functions of the multifunctional enzymes in carotenoid biosynthesis. Hydroxylase over-expression in combination with bkt1 did not result in ketocarotenoid accumulation, but instead unexpected patterns of α-carotene derivatives, accompanied by a reduction of α-carotene, were observed. These data suggest possible interactions between the β-carotene ketolase bkt1 and the hydroxylases that impact partitioning of carbon flux into different carotenoid branch pathways.     

Screening of Drug Metabolizing Enzymes for the Ginsenoside Compound K In Vitro: An Efficient Anti-Cancer Substance Originating from Panax Ginseng

Ginsenoside compound K (CK), a rare ginsenoside originating from Panax Ginseng, has been found to possess unique pharmacological activities specifically as anti-cancers. However, the role of cytochrome P450s (CYPs) in the metabolism of CK is unclear. In this study, we screened the CYPs for the metabolism of CK in vitro using human liver microsomes (HLMs) or human recombinant CYPs. The results showed that CK inhibited the enzyme activities of CYP2C9 and CYP3A4 in the HLMs. The K_m and V_max values of CK were 84.20±21.92 μM and 0.28±0.04 nmol/mg protein/min, respectively, for the HLMs; 34.63±10.48 μM and 0.45±0.05 nmol/nmol P450/min, respectively, for CYP2C9; and 27.03±5.04 μM and 0.68±0.04 nmol/nmol P450/min, respectively, for CYP3A4. The IC₅₀ values were 16.00 μM and 9.83 μM, and K_i values were 14.92 μM and 11.42μM for CYP2C9 and CYP3A4, respectively. Other human CYP isoforms, including CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on CK metabolism. The results suggested that CK was a substrate and also inhibitors for both CYP2C9 and CYP3A4. Patients using CK in combination with therapeutic drugs that are substrates of CYP2C9 and CYP3A4 for different reasons should be careful, although the inhibiting potency of CK is much poorer than that of enzyme-specific inhibitors.     

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C₁₃ apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.In plants, the synthesis of carotenoids is plastid localized, with the plastid type determining their function (Ruiz-Sola and Rodríguez-Concepción, 2012; Nisar et al., 2015). In nonphotosynthetic chromoplasts, carotenoids and their volatile derivatives attract pollinating insects or zoochoric animals. Here, carotenoids are sequestered in diverse suborganellar structures, which can be tubulous, globulous, membranous, or crystalline (Sitte et al., 1980; Egea et al., 2010). In chloroplasts, carotenoids are present in light-harvesting complex proteins and photosynthetic reaction centers. They extend the light spectrum used for photosynthetic energy transformation and act photoprotectively because of their ability to quench excitation energy from singlet- or triplet-state chlorophylls, thereby decreasing the risk that singlet oxygen forms (Niyogi, 1999; Demmig-Adams and Adams, 2002). Furthermore, the regulated epoxidation and deepoxidation of zeaxanthin in the xanthophyll cycle contribute to the nonphotochemical quenching of energy (Niyogi, 1999; Ballottari et al., 2014). In contrast to these processes, which maintain carotenoid integrity, carotenoids are also capable of chemically quenching singlet oxygen by their own oxidation, which is accompanied by the release of various carotenoid degradation products (Ramel et al., 2012a, 2013).The various functions of carotenoids require their dynamic qualitative and quantitative tuning in response to environmental conditions to attain and maintain adequate steady-state concentrations. These include both the regulation of their synthesis and the formation, release, or disposal of their breakdown products. The synthesis of carotenoids is initiated by the condensation of two molecules of geranylgeranyl diphosphate to form phytoene catalyzed by the enzyme phytoene synthase (PSY), which is considered as the rate-limiting enzyme (von Lintig et al., 1997; Li et al., 2008; Rodríguez-Villalón et al., 2009; Welsch et al., 2010; Zhou et al., 2015). In plants, two desaturases, phytoene desaturase and ζ-carotene desaturase, and two carotene cis-trans-isomerases convert the colorless phytoene into the red-colored all-trans-lycopene (Isaacson et al., 2002; Park et al., 2002; Chen et al., 2010; Yu et al., 2011). Two lycopene cyclases introduce either β- or ε-ionone rings, yielding α-(ε,β-)-carotene and β-(β,β)-carotene. In Arabidopsis (Arabidopsis thaliana), four enzymes hydroxylate carotenes with partially overlapping substrate specificity (Kim et al., 2009). Two nonheme iron-dependent β-carotene hydroxylases (BCH), BCH1 and BCH2, convert β-carotene into zeaxanthin. The second set of hydroxylases, cytochrome P450 (CYP)97A3 and CYP97C1, prefers α-carotene and produces zeinoxanthin and lutein, respectively. Absence of each cytochrome P450 hydroxylase constitutes a distinct phenotype, named lutein deficient5 (lut5) for CYP97A3 deficiency and lut1 for CYP97C1 deficiency, characterized by altered pigment compositions and the accumulation of monohydroxylated intermediates, whereas deficiency in BCH1 and BCH2 does not affect the pigment composition.In green tissues, photooxidative destruction seemingly predominates and consumes carotenoids (Simkin et al., 2003). Moreover, ¹⁴CO₂ pulse-chase experiments with Arabidopsis leaves identified α- and β-carotene as the main targets for photooxidation, whereas xanthophylls were less affected (Beisel et al., 2010). Oxidation assays with β-carotene showed epoxy- and peroxy-derivatives as the main primary products, which however, undergo additional reactions, yielding more stable degradation products that are, in part, the same apocarotenals/ones as those being produced enzymatically (Ramel et al., 2012a, 2013).In Arabidopsis, genes coding for carotenoid cleaving enzymes (carotenoid cleavage dioxygenases [CCDs]) form a small gene family comprising nine members, five of which are attributed to the synthesis of abscisic acid (ABA; nine-cis-epoxycarotenoid cleavage dioxygenases [NCEDs]; AtNCED2, AtNCED3, AtNCED5, AtNCED6, and AtNCED9; Iuchi et al., 2001; Tan et al., 2003), whereas two are committed to strigolactone biosynthesis (CCD7/MORE AXILLARY GROWTH3 [MAX3] and CCD8/MAX4; Alder et al., 2012; Bruno et al., 2014). Orthologs of CCD1 are involved in the generation of volatile apocarotenoids contributing to flower scents and aroma production (e.g. saffron [Crocus sativus; Rubio et al., 2008; Frusciante et al., 2014] and tomato [Solanum lycopersicum; Simkin et al., 2004]), whereas CCD4 enzymes are involved in citrus peel and chrysanthemum (Chrysanthemum morifolium) petal coloration (Ohmiya et al., 2006; Rodrigo et al., 2013). Recent analysis of Arabidopsis mutants revealed a major function of CCD4 in regulating seed carotenoid content with only a minor contribution of CCD1 (Gonzalez-Jorge et al., 2013). Moreover, CCD4 activity was required for the synthesis of an apocarotenoid-derived signaling molecule involved in leaf development and retrograde gene expression (Avendaño-Vázquez et al., 2014).Elevated carotenoid pathway flux caused by PSY overexpression increases carotenoid accumulation in various nongreen tissues, such as tomato fruits, canola (Brassica napus) seeds, cassava (Manihot esculenta) roots, and rice (Oryza sativa) endosperm (Shewmaker et al., 1999; Ye et al., 2000; Fraser et al., 2002; Welsch et al., 2010). Similarly, the constitutive overexpression of PSY in Arabidopsis results in dramatically increased carotenoid amounts accumulating as crystals in nongreen tissues, such as roots and callus, yielding β-carotene as the main product (Maass et al., 2009). However, leaves of the very same plants do not show altered pigment composition, and phytoene or other pathway intermediates are not detected. Similarly, increased levels of active PSY protein achieved though overexpression of the ORANGE protein exclusively affect carotenoid amounts in roots but not in leaves (Zhou et al., 2015). Leaves from constitutively PSY-overexpressing tomato and tobacco (Nicotiana tabacum) plants are also reported to show only slightly increased carotenoid levels compared with the wild-type control (Fray et al., 1995; Busch et al., 2002). These contrasting responses of leaves versus nongreen tissues to elevated pathway flux suggest fundamental differences in the modes of carotenoid formation and/or degradation.In this work, we identified xanthophyll-derived apocarotenoid glycosides (AGs) in Arabidopsis leaves that increase upon higher pathway flux. This suggests that apocarotenoid glycosylation functions as a valve regulating carotenoid steady-state levels in leaves. The analysis of Arabidopsis mutants enabled us to conclude on potential precursor carotenoids and assess the contribution of carotenoid cleavage enzymes on their formation. Moreover, apocarotenoids but not the identified glycosides were increased in carotenoid-hyperaccumulating roots, indicating tissue-specific different modes of carotenoid turnover regulation.     

Expression Characteristics of β-Catenin in Scallop Chlamys farreri Gonads and Its Role as a Potential Upstream Gene of Dax1 through Canonical Wnt Signalling Pathway Regulating the Spermatogenesis

β-catenin is a key signaling molecule in the canonical Wnt pathway, which is involved in animal development. However, little information has been reported for β-catenin in bivalves. In the present study, we cloned a homolog of β-catenin from the scallop Chlamys farreri and determined its expression characteristics. The full-length cDNA of β-catenin was 3,353 bp, including a 2,511 bp open reading frame that encoded a predicted 836 amino acid protein. Level of the β-catenin mRNA increased significantly (P<0.05) with C. farreri gonadal development and presented a sexually dimorphic expression pattern in the gonads, which was significantly high in ovaries detected by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis revealed that the β-catenin was mainly located in germ cells of the gonads, with obvious positive immune signals in the oogonia and oocytes of ovaries as well as in the spermatogonia and spermatocytes of testes, implying β-catenin might be involved in the gametogenesis of C. farreri. Furthermore, when 0.1 µg/mL and 0.2 µg/mL DKK-1 (an inhibitor of the canonical Wnt pathway) were added in vitro to culture medium containing testis cells of C. farreri, the expression of β-catenin decreased significantly detected by qRT-PCR (P<0.05), suggesting the canonical Wnt signal pathway exists in the scallop testis. Similarly, when 50 µM and 100 µM quercetin (an inhibitor of β-catenin) were added in vitro to the culture system, Dax1 expression was significantly down-regulated compared with controls (P<0.05), implying the β-catenin is an upstream gene of Dax1 and is involved in the regulation of C. farreri spermatogenesis.     

kin-19/casein kinase Iα has dual functions in regulating asymmetric division and terminal differentiation in C. elegans epidermal stem cells

Casein Kinase I (CKI) is a conserved component of the Wnt signaling pathway that regulates cell fate determination in metazoans. We show that post-embryonic asymmetric division and fate specification of C. elegans epidermal stem cells are controlled by a non-canonical Wnt/β-catenin signaling pathway, involving the β-catenins WRM-1 and SYS-1, and that C. elegans kin-19/CKIα functions in this pathway. Furthermore, we find that kin-19 is the only member of the Wnt asymmetry pathway that functions with, or in parallel to, the heterochronic temporal patterning pathway to control withdrawal from self-renewal and subsequent terminal differentiation of epidermal stem cells. We show that, except in the case of kin-19, the Wnt asymmetry pathway and the heterochronic pathway function separately and in parallel to control different aspects of epidermal stem cell fate specification. However, given the function of kin-19/CKIα in both pathways, and that CKI, Wnt signaling pathway and heterochronic pathway genes are widely conserved in animals, our findings suggest that CKIα may function as a regulatory hub through which asymmetric division and terminal differentiation are coordinated in adult stem cells of vertebrates.Key words: C. elegans, kin-19, casein kinase Ialpha (CKIα), Wnt, stem cell, asymmetric cell division, heterochronic, temporal identity, terminal differentiation, self-renewal     

Searching the Cytochrome P450 Enzymes for the Metabolism of Meranzin Hydrate: A Prospective Antidepressant Originating from Chaihu-Shugan-San

Meranzin hydrate (MH), an absorbed bioactive compound from the Traditional Chinese Medicine (TCM) Chaihu-Shugan-San (CSS), was first isolated in our laboratory and was found to possess anti-depression activity. However, the role of cytochrome P450s (CYPs) in the metabolism of MH was unclear. In this study, we screened the CYPs for the metabolism of MH in vitro by human liver microsomes (HLMs) or human recombinant CYPs. MH inhibited the enzyme activities of CYP1A2 and CYP2C19 in a concentration-dependent manner in the HLMs. The K_m and V_max values of MH were 10.3±1.3 µM and 99.1±3.3 nmol/mg protein/min, respectively, for the HLMs; 8.0±1.6 µM and 112.4±5.7 nmol/nmol P450/min, respectively, for CYP1A2; and 25.9±6.6 µM and 134.3±12.4 nmol/nmol P450/min, respectively, for CYP2C19. Other human CYP isoforms including CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 showed minimal or no effect on MH metabolism. The results suggested that MH was simultaneously a substrate and an inhibitor of CYP1A2 and CYP2C9, and MH had the potential to perpetrate drug-drug interactions with other CYP1A2 and CYP2C19 substrates.     

Molecular Basis for Involvement of CYP1B1 in MYOC Upregulation and Its Potential Implication in Glaucoma Pathogenesis

CYP1B1 has been implicated in primary congenital glaucoma with autosomal recessive mode of inheritance. Mutations in CYP1B1 have also been reported in primary open angle glaucoma (POAG) cases and suggested to act as a modifier of the disease along with Myocilin (MYOC). Earlier reports suggest that over-expression of myocilin leads to POAG pathogenesis. Taken together, we propose a functional interaction between CYP1B1 and myocilin where 17β estradiol acts as a mediator. Therefore, we hypothesize that 17β estradiol can induce MYOC expression through the putative estrogen responsive elements (EREs) located in its promoter and CYP1B1 could manipulate MYOC expression by metabolizing 17β estradiol to 4-hydroxy estradiol, thus preventing it from binding to MYOC promoter. Hence any mutation in CYP1B1 that reduces its 17β estradiol metabolizing activity might lead to MYOC upregulation, which in turn might play a role in glaucoma pathogenesis. It was observed that 17β estradiol is present in Human Trabecular Meshwork cells (HTM) and Retinal Pigment Epithelial cells (RPE) by immunoflouresence and ELISA. Also, the expression of enzymes related to estrogen biosynthesis pathway was observed in both cell lines by RT-PCR. Subsequent evaluation of the EREs in the MYOC promoter by luciferase assay, with dose and time dependent treatment of 17β estradiol, showed that the EREs are indeed active. This observation was further validated by direct binding of estrogen receptors (ER) on EREs in MYOC promoter and subsequent upregulation in MYOC level in HTM cells on 17β estradiol treatment. Interestingly, CYP1B1 mutants with less than 10% enzymatic activity were found to increase the level of endogenous myocilin in HTM cells. Thus the experimental observations are consistent with our proposed hypothesis that mutant CYP1B1, lacking the 17β estradiol metabolizing activity, can cause MYOC upregulation, which might have a potential implication in glaucoma pathogenesis.     

The Biosynthetic pathway for synechoxanthin, an aromatic carotenoid synthesized by the euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002

The euryhaline, unicellular cyanobacterium Synechococcus sp. strain PCC 7002 produces the dicyclic aromatic carotenoid synechoxanthin (χ,χ-caroten-18,18′-dioic acid) as a major pigment (>15% of total carotenoid) and when grown to stationary phase also accumulates small amounts of renierapurpurin (χ,χ-carotene) (J. E. Graham, J. T. J. Lecomte, and D. A. Bryant, J. Nat. Prod. 71:1647-1650, 2008). Two genes that were predicted to encode enzymes involved in the biosynthesis of synechoxanthin were identified by comparative genomics, and these genes were insertionally inactivated in Synechococcus sp. strain PCC 7002 to verify their function. The cruE gene (SYNPCC7002_A1248) encodes β-carotene desaturase/methyltransferase, which converts β-carotene to renierapurpurin. The cruH gene (SYNPCC7002_A2246) encodes an enzyme that is minimally responsible for the hydroxylation/oxidation of the C-18 and C-18′ methyl groups of renierapurpurin. Based on observed and biochemically characterized intermediates, a complete pathway for synechoxanthin biosynthesis is proposed.     

Highly Mutagenic Exocyclic DNA Adducts Are Substrates for the Human Nucleotide Incision Repair Pathway

Background

Oxygen free radicals induce lipid peroxidation (LPO) that damages and breaks polyunsaturated fatty acids in cell membranes. LPO-derived aldehydes and hydroxyalkenals react with DNA leading to the formation of etheno(ε)-bases including 1,N ⁶-ethenoadenine (εA) and 3,N ⁴-ethenocytosine (εC). The εA and εC residues are highly mutagenic in mammalian cells and eliminated in the base excision repair (BER) pathway and/or by AlkB family proteins in the direct damage reversal process. BER initiated by DNA glycosylases is thought to be the major pathway for the removal of non-bulky endogenous base damage. Alternatively, in the nucleotide incision repair (NIR) pathway, the apurinic/apyrimidinic (AP) endonucleases can directly incise DNA duplex 5′ to a damaged base in a DNA glycosylase-independent manner.

Methodology/Principal Findings

Here we have characterized the substrate specificity of human major AP endonuclease 1, APE1, towards εA, εC, thymine glycol (Tg) and 7,8-dihydro-8-oxoguanine (8oxoG) residues when present in duplex DNA. APE1 cleaves oligonucleotide duplexes containing εA, εC and Tg, but not those containing 8oxoG. Activity depends strongly on sequence context. The apparent kinetic parameters of the reactions suggest that APE1 has a high affinity for DNA containing ε-bases but cleaves DNA duplexes at an extremely slow rate. Consistent with this observation, oligonucleotide duplexes containing an ε-base strongly inhibit AP site nicking activity of APE1 with IC₅₀ values in the range of 5–10 nM. MALDI-TOF MS analysis of the reaction products demonstrated that APE1-catalyzed cleavage of εA•T and εC•G duplexes generates, as expected, DNA fragments containing 5′-terminal ε-base residue.

Conclusions/Significance

The fact that ε-bases and Tg in duplex DNA are recognized and cleaved by APE1 in vitro, suggests that NIR may act as a backup pathway to BER to remove a large variety of genotoxic base lesions in human cells.     

Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

The inhibition of the human cholesterol 7alpha-hydroxylase gene (CYP7A1) promoter by fibrates in cultured cells is mediated via the liver x receptor alpha and peroxisome proliferator-activated receptor alpha heterodimer

In previous work, we showed that the binding of the liver x receptor α:peroxisome proliferator-activated receptor α (LXRα:PPARα) heterodimer to the murine Cyp7a1 gene promoter antagonizes the stimulatory effect of their respective ligands. In this study, we determined if LXRα:PPARα can also regulate human CYP7A1 gene promoter activity. Co-expression of LXRα and PPARα in McArdle RH7777 hepatoma cells decreased the activity of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol. In vitro, the human CYP7A1 Site I bound LXRα:PPARα, although with substantially less affinity compared with the murine Cyp7a1 Site I. The binding of LXRα:PPARα to human CYP7A1 Site I was increased in the presence of either LXRα or PPARα ligands. In HepG2 hepatoblastoma cells, fibrates and 25-hydroxycholesterol inhibited the expression of the endogenous CYP7A1 gene as well as the human CYP7A1 gene promoter when co-transfected with plasmids encoding LXRα and PPARα. However, a derivative of the human CYP7A1 gene promoter that contains a mutant form of Site I that does not bind LXRα:PPARα was not inhibited by WY 14,643 or 25-hydroxycholesterol in both McArdle RH7777 and HepG2 cells. The ligand-dependent recruitment of LXRα:PPARα heterodimer onto the human CYP7A1 Site I can explain the inhibition of the human CYP7A1 gene promoter in response to fibrates and 25-hydroxycholesterol.     

Effects of Etomidate on the Steroidogenesis of Rat Immature Leydig Cells

Background

Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established.

Methodology

Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3–30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity.

Results and Conclusions

In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC₅₀) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells.     

Novel Bread Wheat Lines Enriched in Carotenoids Carrying Hordeum chilense Chromosome Arms in the ph1b Background

The use of crop wild relative species to improve major crops performance is well established. Hordeum chilense has a high potential as a genetic donor to increase the carotenoid content of wheat. Crosses between the 7H^ch H. chilense substitution lines in wheat and the wheat pairing homoeologous1b (ph1b) mutant allowed the development of wheat-H. chilense translocation lines for both 7H^chα and 7H^chβ chromosome arms in the wheat background. These translocation lines were characterized by in situ hybridization and using molecular markers. In addition, reverse phase chromatography (HPLC) analysis was carried out to evaluate the carotenoid content and both 7H^chα∙7AL and 7AS∙7H^chβ disomic translocation lines. The carotenoid content in 7H^chα∙7AL and 7AS∙7H^chβ disomic translocation lines was higher than the wheat-7H^ch addition line and double amount of carotenoids than the wheat itself. A proteomic analysis confirmed that the presence of chromosome 7H^ch introgressions in wheat scarcely altered the proteomic profile of the wheat flour. The Psy1 (Phytoene Synthase1) gene, which is the first committed step in the carotenoid biosynthetic pathway, was also cytogenetically mapped on the 7H^chα chromosome arm. These new wheat-H. chilense translocation lines can be used as a powerful tool in wheat breeding programs to enrich the diet in bioactive compounds.     

Production of a Highly Protease-Resistant Fungal α-Galactosidase in Transgenic Maize Seeds for Simplified Feed Processing

Raffinose-family oligosaccharide (RFO) in soybeans is one of the major anti-nutritional factors for poultry and livestocks. α-Galactosidase is commonly supplemented into the animal feed to hydrolyze α-1,6-galactosidic bonds on the RFOs. To simplify the feed processing, a protease-resistant α-galactosidase encoding gene from Gibberella sp. strain F75, aga-F75, was modified by codon optimization and heterologously expressed in the embryos of transgentic maize driven by the embryo-specific promoter ZM-leg1A. The progenies were produced by backcrossing with the commercial inbred variety Zheng58. PCR, southern blot and western blot analysis confirmed the stable integration and tissue specific expression of the modified gene, aga-F75m, in seeds over four generations. The expression level of Aga-F75M reached up to 10,000 units per kilogram of maize seeds. In comparison with its counterpart produced in Pichia pastoris strain GS115, maize seed-derived Aga-F75M showed a lower temperature optimum (50°C) and lower stability over alkaline pH range, but better thermal stability at 60°C to 70°C and resistance to feed pelleting inactivation (80°C). This is the first report of producing α-galactosidase in transgenic plant. The study offers an effective and economic approach for direct utilization of α-galactosidase-producing maize without any purification or supplementation procedures in the feed processing.     

Production of the Carotenoids Lycopene, β-Carotene,and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.Carotenoids are yellow, orange, and red pigments which are widely distributed in nature (3). Industrially, carotenoid pigments such as β-carotene are utilized as food or feed supplements. β-Carotene is also a precursor of vitamin A in mammals (11). Recently, carotenoids have attracted greater attention, due to their beneficial effect on human health: e.g., the functions of lycopene and astaxanthin include strong quenching of singlet oxygen (12), involvement in cancer prevention (2), and enhancement of immune responses (6). Astaxanthin has also been exploited for industrial use, principally as an agent for pigmenting cultured fish and shellfish.The genes responsible for the synthesis of carotenoids such as lycopene, β-carotene, and astaxanthin have been isolated from the epiphytic Erwinia species or the marine bacteria Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1, and their functions have been elucidated (13, 14). The first substrate of the encoded enzymes for carotenoid synthesis is farnesyl pyrophosphate (diphosphate) (FPP), which is the common precursor for the biosynthesis of numerous isoprenoid compounds such as sterols, hopanols, dolicols, and quinones. The ubiquitous nature of FPP among yeasts has been utilized in the microbial production of lycopene and β-carotene by the yeast Saccharomyces cerevisiae carrying the Erwinia uredovora carotenogenic genes (19). However, the amount of carotenoids produced in these hosts was only 0.1 mg of lycopene and 0.1 mg of β-carotene per g (dry weight) of cells, respectively.The edible yeast Candida utilis is generally recognized as a safe substance by the Food and Drug Administration. Large-scale production of the yeast cells has been developed with cheap biomass-derived sugars as the carbon source for the production of single-cell protein and several chemicals such as glutathione and RNA (1, 4). This yeast was also found to accumulate a large amount of ergosterol in the cell during stationary phase (6 to 13 mg/g [dry weight] of cells) (17). Thus, C. utilis has the potential to produce a large amount of carotenoids by redirecting the carbon flux for the ergosterol biosynthesis into the nonendogenous pathway for carotenoid synthesis via FPP. Previously, a C. utilis strain was made to produce lycopene (0.8 mg/g [dry weight]) by expressing the three nonmodified genes crtE, crtB, and crtI derived from E. uredovora (15).In this paper, the de novo biosynthesis of lycopene, β-carotene, and astaxanthin has been performed in C. utilis by using six carotenogenic genes, which were synthesized according to the codon usage of the C. utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) gene, which is expressed at high levels. By this approach, increased carotenoid production in C. utilis was achieved.