Recombinant Saccharomyces cerevisiae strain expressing a model cytochrome P450 in the rat digestive environment: viability and bioconversion activity

An innovative "biodrug" concept, based on the oral administration of living recombinant microorganisms, has recently emerged for the prevention or treatment of various diseases. An engineered Saccharomyces cerevisiae strain expressing plant P450 73A1 (cinnamate-4-hydroxylase [CA4H] activity) was used, and its survival and ability to convert trans-cinnamic acid (CIN) into p-coumaric acid (COU) were investigated in vivo. In rats, the recombinant yeast was resistant to gastric and small intestinal secretions but was more sensitive to the conditions found in the large intestine. After oral administration of yeast and CIN, the CA4H activity was shown in vivo, with COU being found throughout the rat's digestive tract and in its urine. The bioconversion reaction occurred very fast, with most of the COU being produced within the first 5 min. The gastrointestinal sac technique demonstrated that the recombinant yeast was able to convert CIN into COU (conversion rate ranging from 2 to 5%) in all the organs of the rat's digestive tract: stomach, duodenum, jejunum, ileum, cecum, and colon. These results promise new opportunities for the development of drug delivery systems based on engineered yeasts catalyzing a bioconversion reaction directly in the digestive tract.     

Recombinant Saccharomyces cerevisiae Strain Expressing a Model Cytochrome P450 in the Rat Digestive Environment: Viability and Bioconversion Activity

An innovative “biodrug” concept, based on the oral administration of living recombinant microorganisms, has recently emerged for the prevention or treatment of various diseases. An engineered Saccharomyces cerevisiae strain expressing plant P450 73A1 (cinnamate-4-hydroxylase [CA4H] activity) was used, and its survival and ability to convert trans-cinnamic acid (CIN) into p-coumaric acid (COU) were investigated in vivo. In rats, the recombinant yeast was resistant to gastric and small intestinal secretions but was more sensitive to the conditions found in the large intestine. After oral administration of yeast and CIN, the CA4H activity was shown in vivo, with COU being found throughout the rat's digestive tract and in its urine. The bioconversion reaction occurred very fast, with most of the COU being produced within the first 5 min. The gastrointestinal sac technique demonstrated that the recombinant yeast was able to convert CIN into COU (conversion rate ranging from 2 to 5%) in all the organs of the rat's digestive tract: stomach, duodenum, jejunum, ileum, cecum, and colon. These results promise new opportunities for the development of drug delivery systems based on engineered yeasts catalyzing a bioconversion reaction directly in the digestive tract.     

Improvement in the expression efficiency of mammalian cytochrome P450 and NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae

Mammalian cytochrome P450 (P450) cDNAs were modified by partial or complete removal of their untranslated regions (UTRs). Expression efficiency of P450s in Saccharomyces cerevisiae was increased by the complete removal of the UTRs from the P450 cDNAs prior to insertion into an expression vector. A similar modification was effective in improving the expression of mammalian NADPH-P450 oxidoreductases in S. cerevisiae. This revised version was published online in November 2006 with corrections to the Cover Date.     

Occurrence of cytochrome P450 in continuous cultures of Saccharomyces cerevisiae

Summary Cytochrome P450 of Saccharomyces cerevisiae is an inducible enzyme system. Hitherto, its induction was related to semi-anaerobic culture conditions and high glucose concentrations in the growth medium respectively. Since glucose and oxygen are main regulatory effectors in this yeast, the relationship between the occurrence of cytochrome P450 and these two effectors was established in continuous culture. At glucose-derepressed conditions it was not possible to induce the formation of cytochrome P450 by oxygen limitation alone. The oxygen supply had to be decreased to a level where glucose repression also became active. At glucose-repressed conditions cytochrome P450 was obtained in good yield (3 to 5 pmol per mg dry cell weight) below a dissolved oxygen tension of appproximately 15%. There was a correlation between the content of mitochondrial cytochromes and that of cytochrome P450. The presence of mitochondrial cytochromes was reciprocal with cytochrome P450 when its content was increased by lowering the dissolved oxygen tension.     

Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

ABSTRACT: BACKGROUND: Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. RESULTS: For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems.Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system.Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. CONCLUSIONS: Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes.     

Toxin-binding properties of cytochrome P450 in Saccharomyces cerevisiae and Kluyveromyces marxianus

Microsomes from Kluyveromyces marxianus GK1005 examined by carbon monoxide difference spectroscopy showed no evidence of cytochrome P450, in contrast to microsomes isolated from a control strain of Saccharomyces cerevisiae. Benzo[a]pyrene produced a typical Type I-binding spectrum with microsomes of both yeasts, with K _s values of 82 M (S. cerevisiae) and 70 M (K. marxianus). While aflatoxin B₁ generated a typical Type I-binding spectrum with microsomes from S. cerevisiae (K _s of 178 M), the toxin did not produce a recognisable binding spectrum with microsomes from K. marxianus.     

Functional expression of mammalian cytochromes P450IIB in the yeast Saccharomyces cerevisiae

Three mammalian cytochromes P450 from the IIB subfamily, P450IIB11 from canine and P450IIB4 and P450IIB5 from rabbit, have been expressed in the yeast Saccharomyces cerevisiae by use of an autonomously replicating vector containing the galactose-inducible gal10 promoter. Cytochromes P450IIB4 and P450IIB5 are closely related proteins, with only 11 amino acid substitutions between them. P450IIB11 is a homologous protein, likely orthologous with IIB4 or IIB5, with 102 amino acid substitutions compared with the P450IIB4 protein and 106 compared with the P450IIB5 protein. The expressed proteins are functional in yeast microsomes, exhibiting activity toward androstenedione, 7-ethoxycoumarin, and, in some cases, progesterone. Expressed cytochromes P450IIB4 and P450IIB11 hydroxylate androstenedione with regio- and stereoselectivity characteristic of the purified, reconstituted proteins. A striking difference in the androstenedione metabolite profiles of IIB4 and IIB5 was observed, with IIB4 producing almost exclusively the 16 beta-hydroxy metabolite and IIB5 producing the 16 alpha-hydroxy and 15 alpha-hydroxy products. This is the first time that 15 alpha-hydroxylase activity has been associated with IIB4/IIB5. This activity has also been detected in liver microsomes from some, but not all, individual phenobarbital-induced rabbits tested and is largely inhibited by anti-rabbit P450IIB immunoglobulin G. These studies illustrate the utility of the yeast expression system for defining catalytic activities of individual mammalian cytochromes P450 and identifying new marker activities that can be utilized in liver microsomes.     

Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae.

We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.     

Disruption of the Saccharomyces cerevisiae gene for NADPH-cytochrome P450 reductase causes increased sensitivity to ketoconazole

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14 alpha-demethylase. Resistance is restored through complementation by the plasmid-borne wild type gene from either S. cerevisiae or Candida tropicalis. Neither Southern hybridization nor Western immunoblot techniques provided evidence for a second NADPH-cytochrome P450 reductase gene, suggesting that an alternate pathway may provide for the functions of this reductase in S. cerevisiae.     

Isolation of the Candida tropicalis gene for P450 lanosterol demethylase and its expression in Saccharomyces cerevisiae

We have isolated the gene for cytochrome P450 lanosterol 14 alpha-demethylase (14DM) from the yeast Candida tropicalis. This was accomplished by screening genomic libraries of strain ATCC750 in E. coli using a DNA fragment containing the yeast Saccharomyces cerevisiae 14DM gene. Identity of this gene was confirmed by a) observing a heme binding region common to all P450s after sequencing the 3' portion of the gene, and b) based upon tests of its expression in strains of Saccharomyces cerevisiae.     

Effect of mitochondrial cytochromes and haem content on cytochrome P450 in Saccharomyces cerevisiae

It is well established that the mitochondrial and the microsomal cytochromes in Saccharomyces cerevisiae are regulated differently. Mutations affecting the mitochondrial cytochromes aa3 or c had no effect on the concentration of the microsomal cytochrome P450 even during haem limitation. Moreover, a defect in the cytochrome P450 gene did not affect mitochondrial cytochromes. However, a regulatory mutation present in strain SG1 decreased both mitochondrial and microsomal cytochrome contents. This mutation also affected the intracellular haem concentration. The haem precursor 5-aminolaevulinate increased both mitochondrial and microsomal cytochrome contents. Our results indicate that carbon source and haem concentration are involved in the regulation of cytochrome P450.     

Constitutive and inducible expression of human cytochrome P450IA1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies

A cDNA of human cytochrome P450IA1 was expressed in yeast Saccharomyces cerevisiae on a multicopy plasmid under the control of the constitutive GAPFL or the inducible PHO5 promoter. Microsomes of transformed yeast contained substantial amounts of the heterologous enzyme as determined by reduced CO-difference spectra (156-68 pmol/mg). Enzyme kinetics with 7-ethoxyresorufin as substrate resulted in a Km value of 92 nM and a Vmax value of 223 pmol/mg/min, which is comparable to data obtained with human liver microsomes. The antimycotic drug ketoconazole (Ki = 22nM) as well as the isozyme specific P450 inhibitor alpha-naphthoflavone (Ki = 1.2 nM) were shown to be strong inhibitors of human P450IA1. Taken together, these data show that heterologous P450 gene expression in yeast is a potent instrument for the study of enzyme specific parameters and might be used to answer further questions with regard to substrate specificity as well as drug interaction in a background with no interfering activities.     

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].     

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.     

The bacterial P450 BM3: a prototype for a biocatalyst with human P450 activities

The use of cytochrome P450 (P450 or CYP) enzymes as biocatalysts for the production of fine chemicals, including pharmaceuticals, has been of increasing interest, primarily owing to their catalytic diversity and broad substrate range. CYP102A1 (P450 BM3) from Bacillus megaterium integrates an entire monooxygenase system into one polypeptide and represents an appropriate prokaryotic model for industrial applications of mammalian P450 activities. CYP102A1 not only exhibits the highest catalytic activity ever detected in a P450 monooxygenase but also provides a potentially versatile biocatalyst for the production of human P450 metabolites. CYP102A1 can be further engineered to be a drug-metabolizing enzyme, making it a promising candidate to use as a biocatalyst in drug discovery and synthesis.     

Saccharomyces cerevisiae as a model organism: a comparative study

Background

Model organisms are used for research because they provide a framework on which to develop and optimize methods that facilitate and standardize analysis. Such organisms should be representative of the living beings for which they are to serve as proxy. However, in practice, a model organism is often selected ad hoc, and without considering its representativeness, because a systematic and rational method to include this consideration in the selection process is still lacking.

Methodology/Principal Findings

In this work we propose such a method and apply it in a pilot study of strengths and limitations of Saccharomyces cerevisiae as a model organism. The method relies on the functional classification of proteins into different biological pathways and processes and on full proteome comparisons between the putative model organism and other organisms for which we would like to extrapolate results. Here we compare S. cerevisiae to 704 other organisms from various phyla. For each organism, our results identify the pathways and processes for which S. cerevisiae is predicted to be a good model to extrapolate from. We find that animals in general and Homo sapiens in particular are some of the non-fungal organisms for which S. cerevisiae is likely to be a good model in which to study a significant fraction of common biological processes. We validate our approach by correctly predicting which organisms are phenotypically more distant from S. cerevisiae with respect to several different biological processes.

Conclusions/Significance

The method we propose could be used to choose appropriate substitute model organisms for the study of biological processes in other species that are harder to study. For example, one could identify appropriate models to study either pathologies in humans or specific biological processes in species with a long development time, such as plants.     

Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant Production of an Inulinase in a Saccharomyces cerevisiae gal80 Strain

The inulinase gene (INU1) from Kluyveromyces marxianus NCYC2887 strain was overexpressed by using GAL10 promotor in a △gal80 strain of Saccharomyces cerevisiae. The inulinase gene lacking the original signal sequence was fused in-frame to mating factor alpha signal sequence for secretory expression. Use of the △gal80 strain allowed the galactose-free induction of inulinase expression using a glucose-only medium. Shake flask cultivation in YPD medium produced 34.6 U/ml of the recombinant inulinase, which was approximately 13-fold higher than that produced by K. marxianus NCYC2887. It was found that the use of the △gal80 strain improved the expression of inulinase in the recombinant S. cerevisiae in both the aerobic and the anaerobic condition by about 2.9- and 1.7-fold, respectively. 5 L fed-batch fermentation using YPD medium was performed under aerobic condition with glucose feeding, which resulted in the inulinase production of 31.7 U/ml at OD600 of 67. Ethanol fermentation of dried powder of Jerusalem artichoke, an inulin-rich biomass, was also performed using the recombinant S. cerevisiae expressing INU1 and K. marxianus NCYC2887. Fermentation in a 5L scale fermentor was carried out at an aeration rate of 0.2 vvm, an agitation rate of 300 rpm, and the pH was controlled at 5.0. The temperature was maintained at 30degrees C and 37degrees C, respectively, for the recombinant S. cerevisiae and K. marxianus. The maximum productivities of ethanol were 59.0 and 53.5 g/L, respectively.     

Generation of the amyloid-beta peptide N terminus in Saccharomyces cerevisiae expressing human Alzheimer's amyloid-beta precursor protein

The Alzheimer's amyloid-beta precursor protein (betaAPP) is a type 1 membrane-spanning protein from which the Alzheimer's disease amyloid-beta peptide (Abeta) is proteolytically derived. To date, attempts to identify the enzymes responsible for Abeta generation have failed. Here we report the accumulation of Abeta-immunoreactive peptides in yeast expressing human betaAPP. Characterization of these peptides by metabolic labeling, immunoprecipitation with Abeta-specific antibodies, and N-terminal radiosequencing indicates that these peptides include the Abeta peptide at their N termini. The Abeta-like peptides generated in yeast were recovered predominantly as 8- and 12-14-kDa species. A 4-kDa species was recovered either when a protease-deficient strain was used to prevent breakdown or when the 8- and 12-14-kDa species were treated with disaggregating agents. The likely existence in yeast of enzymes generating the Abeta N terminus indicates that the molecular identification of yeast beta-secretase-like enzymes may be accomplished using genetic screens or empirical approaches based upon the sequenced genome of Saccharomyces cerevisiae.