Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-07-24
2003-01-07
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023200, C435S254110, C435S255400, C435S320100
Reexamination Certificate
active
06503734
ABSTRACT:
BACKGROUND
1. Field of the Invention
This invention relates to processes and compositions involved in dicarboxylic acid production in yeast. More particularly, the invention relates to a novel gene which encodes a cytochrome b5 protein in
Candida tropicalis.
2. Description of 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 &agr;, &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 &agr;, &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 Mookherjee 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 &agr;, &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 as described, e.g., in Gilewicz et al.,
Can. J. Microbiol.
25:201 (1979), incorporated herein by reference. 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 as described, e.g., in Sanglard et al.,
Gene
76:121-136 (1989), incorporated herein by reference. 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 Biology,
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 as described, e.g., in Kemp et al.,
Appl. Microbiol. and Biotechnol.
28: 370-374 (1988), incorporated herein by reference, 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.
Cytochrome P450 monooxygenases (P450s) are terminal monooxidases of a multicomponent enzyme system including P450 and CPR. In some instances, a second electron carrier, cytochrome b5(CYTb5) and its associated reductase are involved as described below and in Morgan, et al.,
Drug Metab. Disp.
12:358-364, 1984. The P450s 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 as described, e.g., in Nelson, supra, and Nebert et al.,
DNA Cell. Biol.
10:1-14 (1991), which is incorporated herein by reference. Endogenous compounds include steroids, prostanoids, eicosanoids, fat-soluble vitamins, fatty acids, mammalian alkaloids, leukotrines, biogenic amines and phytolexins (Nelson, supra, and Nebert et al., supra). P450 metabolism involves such reactions as epoxidation, hydroxylation, dealkylation, 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 simi
Craft David L.
Loper John C.
Madduri Krishna M.
Cognis Corporation
Drach John E.
Ketter James
Sullivan Daniel
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