Induction of cytochrome P-450 and ethanol oxidation in Yarrowia lipolytica

The study of the effect of different ethanol concentrations in the medium on the growth and the activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. But when the initial concentration of ethanol in the medium was 3 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17

The relationship between 17alpha-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51alpha, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17alpha-hydroxyprogesterone and 17alpha,20(alpha,beta)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(alpha,beta)-ol-3-ones were determined. They included cycles of 20-oxidation, 17alpha-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17alpha-hydroxyprogesterone or progesterone 17alpha,20(alpha,beta)-diols, could be obtained from progesterone.     

Range of substrates and steroid bioconversion reactions performed by recombinant microorganisms Saccharomyces cerevisiae and Yarrowia lipolytica expressing cytochrome P450c17

The relationship between 17α-hydroxylation and 20-oxidation-reduction of progesterone and some of its derivatives was studied in yeast strains Saccharomyces cerevisiae YEp51α, Yarrowia lipolytica E129A15, and expressing cytochrome P450c17. The key metabolites were found to be 17α-hydroxyprogester-one and 17α,20(α,β)-dihydroxypregn-4-ene-3-ones. The bioconversion pathways of pregn-4-ene-20(α,β)-ol-3-ones were determined. They included cycles of 20-oxidation, 17α-hydroxylation, and stereospecific 20-reduction. The efficiency and kinetic parameters of steroid bioconversion by the recombinant strains were determined. The role of yeast analogs of mammalian steroid dehydrogenases is discussed. It was found that any of the desired derivatives, 17α-hydroxyprogesterone or progesterone 17α,20(α,β)-diols, could be obtained from progesterone. Cholesterol bioconversion yields important metabolites: steroid hormones, the vitamin-D group, and bile acids [1, 2]. Attention to various cytochrome-P450 species participating in the biosynthesis of mammalian steroid hormones is caused by two circumstances: (1) the necessity of detecting structural-function abnorm alities of some of the enzymes of steroid-synthesis that cause human diseases, and (2) the potential of regio-and stereospecific cytochrome P450 species of mammals in chemoenzymatic synthesis of pharmacologically valuable steroids. Concerning the second line of inquiry, the development of transgenic Saccharomyces cerevisiae yeast for the complete synthesis of cortisol by additional expression and elimination of a total of 13 genes was reported [3]. To increase the yield of the target compound, the genes for enzymes performing undesirable steroid modifications were inactivated. These modifications included esterification of pregnenolone [4] and 20α-reduction of 17α-hydroxyprogesterone [5]. A search for analogs of mammalian 20α-hydroxysteroid dehydrogenase (20α-HSD) in the Saccharomyces cerevisiae genome revealed two candidate proteins: Ypr1p (yeast aldo-keto reductase) and Gcy1p (yeast galactose-inducible crystallin-like protein) [3]. Indeed, it was formerly shown that expression of cytochrome P450 from bovine adrenal cortex, performing 17α-hydroxylation and the C17,20-lyase reaction (P450c17) in S. cerevisiae under the control of the GAL10-promoter with the presence of D-galactose as an inducer, was accompanied by the sequential conversion of progesterone to 17α-hydroxyprogesterone and 17α,20(α,β)-dihydroxypregn-4-ene-3-one with a high yield [5].     

Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.     

Partial purification of human liver cytochrome P 450.

Human liver cytochrome P 450 was partially purified by hydrophobic chromatography on Octyl-Sepharose, followed by ion-exchange chromatography on DEAE-cellulose. Two fractions (A and B) were obtained; cytochrome P 450 of fraction A was purified sixfold, with an overall yield of about 6 %. Its spectral properties were similar to those previously described in animal cytochromes P 450. Moreover, p-nitroanisole-O-demethylase activity could be obtained in a reconstituted system involving cytochrome P 450 of fraction A, human NADPH-cytochrome $\text{c}$ reductase and phospholipids.     

Activity of human P450 2D6 in biphasic solvent systems

Several limitations have restricted the use of P450 enzymes in synthesis, including the narrow substrate specificity of some P450 isoforms, the need for a redox partner and an expensive cofactor, incompatibility with organic solvents, and poor stability. We previously demonstrated that the natural redox partner and cofactor of the promiscuous P450s 3A4 and 2D6 can be efficiently substituted with some cheap hydrogen peroxide donors or organic peroxides. We report here that P450 2D6 maintains as much as 76% of its activity when used in buffer/organic emulsions. Product formation in biphasic solvent systems is comparable whether the natural redox partner and cofactor are used, or a surrogate. As reported for other enzymes, a correlation is observed between the logP and the suitability of a solvent for enzymatic activity. Moreover, the utility of our system was established by demonstrating the transformation of a novel hydrophobic substrate, not modified by P450 2D6 in the absence of organic solvent.     

Transgenic sweet potato expressing mammalian cytochrome P450

Sweet potato [Ipomoea batatas (L.) Lam] is considered to be recalcitrant to transformation and regeneration because of its genotype-dependent in vitro responses. The lack of a genotype-independent transformation and regeneration system limits biotechnological applications in this plant species. To establish a transformation system for a diverse group of sweet potato genotypes, we examined sweet potato regeneration after transformation in five cultivars. An Agrobacterium tumefaciens transformation system was used for the introduction of mammalian cytochrome P450 genes, which are capable of conferring herbicide tolerance. Among the different factors studied, including explant type, plasmid vectors, and auxin type in the initiation media, auxin type had the greatest effect on the regeneration response. Of the auxins tested, only 4-fluorophenoxyacetic acid (4FA) induced regeneration from all cultivars. In terms of the quality of calli, 4FA promoted the induction of type I calli, which were capable of somatic embryo formation, whereas type II calli fail to produce somatic embryos. The frequency of somatic embryo formation was also affected by the composition of the embryo-induction media. Transgenic plants were regenerated from all cultivars. The stable integration and expression of transgenes was confirmed by several approaches. This Agrobacterium-mediated transformation system should be applicable to a wide range of sweet potato cultivars.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum.

Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized. Purified human liver microsomal cytochromes P450 (P450)-P450 1A2 and P450 2E1-were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline. The activity was confirmed using recombinant and mutated human P450s expressed in bacteria. In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1. Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties.     

Kinetics of bromodichloromethane metabolism by cytochrome P450 isoenzymes in human liver microsomes

The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have been measured in human liver microsomes. The three CYP isoenzymes, CYP2E1, CYP1A2 and CYP3A4, have been identified previously as important in the metabolism of this compound. To measure the constants for each isoenzyme, enzyme-specific inhibitory antibodies were used to block the activities for two of the three isoenzymes. CYP2E1 was found to have the lowest K(m), 2.9 microM, and the highest catalytic activity, k(cat). The K(m) for the other isoenzymes, CYP1A2 and CYP3A4, were about 60 microM with lower values of k(cat). Apparent kinetic constants obtained from two microsomal samples that were not inhibited were consistent with these results. In addition, 11 human microsome samples characterized for 10 CYP activities were correlated with the metabolism of 9.7 microM BDCM by each sample; statistical analysis showed a correlation with CYP2E1 activity only. This result is consistent with the finding that CYP2E1 is the only isoenzyme with a K(m) lower than the BDCM concentration used. The kinetic constants obtained from the inhibited microsomes were compared to similar results from recombinant human isoenzyme preparations containing only one CYP isoenzyme. The results for CYP2E1 were very similar, while the results for CYP1A2 were somewhat less similar and there was a substantial divergence for CYP3A4 in the two systems. Possible reasons for these differences are differing levels of CYP reductase and/or differing makeup of the membrane lipid environment for the CYPs. Because of the low levels of BDCM exposure from drinking water, it appears likely that CYP2E1 will dominate hepatic CYP-mediated BDCM metabolism in humans.     

High diversity and complex evolution of fungal cytochrome P450 reductase: cytochrome P450 systems.

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450-CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450-CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.     

Single nucleotide polymorphisms in cytochrome P450 genes from barley

Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.     

Steroid interactions with cytochrome P-450 from testis microsomes

The cytochrome P-450 of gonadal microsomes is an integral component of the steroid converting enzymes, 17 alpha-hydroxylase and 17,20-lyase. Interaction of the steroid substrates with this cytochrome results in a shift in the Soret band as measured by difference spectroscopy. In these studies it is shown that in contrast to placental microsomal cytochrome P-450 which binds C19 steroids, testis microsomal cytochrome P-450 primarily binds C21 steroids. However, addition of a 17 alpha- methyl, 17 beta-acetate or a 17 beta-benzoate group to testosterone permits interaction. The addition of hydroxyl or methyl groups to other positions does not affect binding. The presence of multiple oxygen functions on C21 steroids, as in cortisol and corticosterone, precludes interaction. At least one oxygen function seems necessary for binding as 5 alpha- and 5 beta-pregnane do not bind whereas 20-deoxypregnenolone (5-pregnen-3 beta-ol) does bind. These findings indicate that factors in addition to hydrophobic interactions dictate the binding of steroid substrates to testis microsomal cytochrome P-450.     

Phosphate-uptake systems in Yarrowia lipolytica cells grown under alkaline conditions

In this study we used a newly isolated Yarrowia lipolytica strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying phosphate transport systems in cells grown under alkaline conditions. Phosphate uptake by Y. lipolytica yeast cells grown at pH 9.5-10 was shown to be mediated by several kinetically discrete Na+-dependent systems. One of these, a low-affinity transporter, operates at high Pi concentrations and is, to our knowledge, here kinetically characterized for the first time. The other two high-affinity systems are derepressible, come into play under conditions of Pi-starvation, and appear to be controlled by the availability of extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor the bacterial kingdoms.