Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-04-30
2001-12-18
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S183000, C435S189000, C435S252300, C435S254220, C435S320100, C536S023200
Reexamination Certificate
active
06331420
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention relates to novel genes which encode enzymes of the &ohgr;-hydroxylase complex in yeast
Candida tropicalis
strains. In particular, the invention relates to novel genes encoding the cytochrome P450 and NADPH reductase enzymes of the &ohgr;hydroxylase complex in yeast
Candida tropicalis
, and to a method of quantitating the expression of genes.
2. Description of the Related Art
Aliphatic dioic acids are versatile chemical intermediates useful as raw materials for the preparation of perfumes, polymers, adhesives and macrolid antibiotics. While several chemical routes to the synthesis of long-chain alpha, &ohgr;-dicarboxylic acids are available, the synthesis is not easy and most methods result in mixtures containing shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters thereof, chemical synthesis has remained the most commercially viable route, due to limitations with the current biological approaches.
Several strains of yeast are known to excrete alpha, &ohgr;-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. In particular, yeast belonging to the Genus Candida, such as
C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis
and
C. zeylenoides
are known to produce such dicarboxylic acids (
Agr. Biol. Chem
. 35: 2033-2042 (1971)). Also, various strains of
C. tropicalis
are known to produce dicarboxylic acids ranging in chain lengths from C
11
through C
18
(Okino et al., B M Lawrence, B D Mookheijee and B J Willis (eds), in
Flavors and Fragrances: A World Perspective
. Proceedings of the 10
th
International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Bühler and Schindler, in
Aliphatic Hydrocarbons in Biotechnology
, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: &agr;-oxidation of alkanes to alcohols, &ohgr;-oxidation of fatty acids to alpha, &ohgr;-dicarboxylic acids and the degradative &bgr;-oxidation of fatty acids to CO
2
and water. The first two types of oxidations are catalyzed by microsomal enzymes while the last type takes place in the peroxisomes. In
C. tropicalis
, the first step in the &ohgr;-oxidation pathway is catalyzed by a membrane-bound enzyme complex &ohgr;-hydroxylase complex) including a cytochrome P450 monooxygenase and a NADPH cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids (Gilewicz et al.,
Can. J. Microbiol
. 25:201 (1979)). The genes which encode the cytochrome P450 and NADPH reductase components of the complex have previously been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced (Sanglard et al.,
Gene
76:121-136 (1989)). P450ALK has also been designated P450ALK1. More recently, ALK genes have been designated by the symbol CYP and RED genes have been designated by the symbol CPR. See, e.g., Nelson,
Pharmacogenetics
6(1):1-42 (1996), which is incorporated herein by reference. See also Ohkuma et al.,
DNA and Cell Biology
14:163-173 (1995), Seghezzi et al.,
DNA and Cell Bology
, 11:767-780 (1992) and Kargel et al.,
Yeast
12:333-348 (1996), each incorporated herein by reference. For example, P450ALK is also designated CYP52 according to the nomenclature of Nelson, supra. Fatty acids are ultimately formed from alkanes after two additional oxidation steps, catalyzed by alcohol oxidase (Kemp et al.,
Appl. Microbiol. and Biotechnol
. 28: 370-374 (1988)) and aldehyde dehydrogenase. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The &ohgr;-oxidation of fatty acids proceeds via the &ohgr;-hydroxy fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester through the &bgr;-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of &ohgr;-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal &bgr;-oxidation (
J. Biochem
., 102:225-234 (1987)). In yeast, &bgr;-oxidation takes place solely in the peroxisomes (
Agr.Biol.Chem
. 49:1821-1828 (1985)).
The production of dicarboxylic acids by fermentation of unsaturated C
14
-C
16
monocarboxylic acids using a strain of the species
C. tropicalis
is disclosed in U.S. Pat. No. 4,474,882. The unsaturated dicarboxylic acids correspond to the starting materials in the number and position of the double bonds. Similar processes in which other special microorganisms are used are described in U.S. Pat. Nos. 3,975,234 and 4,339,536, in British Patent Specification 1,405,026 and in German Patent Publications 21 64 626, 28 53 847, 29 37 292, 29 51 177, and 21 40 133.
Cytochromes P450 (P450s) are terminal monooxidases of a multicomponent enzyme system as described above. They comprise a superfamily of proteins which exist widely in nature having been isolated from a variety of organisms as described e.g., in Nelson, supra. These organisms include various mammals, fish, invertebrates, plants, mollusk, crustaceans, lower eukaryotes and bacteria (Nelson, supra). First discovered in rodent liver microsomes as a carbon-monoxide binding pigment as described, e.g., in Garfinkel,
Arch. Biochem. Biophys
. 77:493-509 (1958), which is incorporated herein by reference, P450s were later named based on their absorption at 450 nm in a reduced-CO coupled difference spectrum as described, e.g., in Omura et al.,
J. Biol. Chem
. 239:2370-2378 (1964), which is incorporated herein by reference.
P450s catalyze the metabolism of a variety of endogenous and exogenous compounds (Nelson, supra). Endogenous compounds include steroids, prostanoids, eicosanoids, fat-soluble vitamins, fatty acids, mammalian alkaloids, leukotrines, biogenic amines and phytolexins (Nelson, supra). P450 metabolism involves such reactions as epoxidation, hydroxylation, deakylation, N-hydroxylation, sulfoxidation, desulfuration and reductive dehalogenation. These reactions generally make the compound more water soluble, which is conducive for excretion, and more electrophilic. These electrophilic products can have detrimental effects if they react with DNA or other cellular constituents. However, they can react through conjugation with low molecular weight hydrophilic substances resulting in glucoronidation, sulfation, acetylation, amino acid conjugation or glutathione conjugation typically leading to inactivation and elimination as described, e.g., in Klaassen et al.,
Toxicology
, 3
rd
ed, Macmillan, New York, 1986, incorporated herein by reference.
P450s are heme thiolate proteins consisting of a heme moiety bound to a single polypeptide chain of 45,000 to 55,000 Da. The iron of the heme prosthetic group is located at the center of a protoporphyrin ring. Four ligands of the heme iron can be attributed to the porphyrin ring. The fifth ligand is a thiolate anion from a cysteinyl residue of the polypeptide. The sixth ligand is probably a hydroxyl group from an amino acid residue, or a moiety with a similar field strength such as a water molecule as described, e.g., in Goeptar et al.,
Critical Reviews in Toxicology
25(1):25-65 (1995), incorporated herein by reference.
Monooxygenation reactions catalyzed by cytochromes P450 in a eukaryotic membrane-bound system require the transfer of electrons from NADPH to P450 via NADPH-cytochrome P450 reductase (CPR) as described, e.g
Brenner Alfred A.
Cornett Cathy A.
Craft David L.
Eirich L. Dudley
Eshoo Mark
Achutamurthy Ponnathapu
Drach John E.
Kerr Kathleen
Steen Jeffrey S.
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