Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-03-01
2001-12-25
Prouty, Rebecca E. (Department: 1652)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S252300, C435S419000, C435S325000, C435S254110
Reexamination Certificate
active
06333401
ABSTRACT:
FIELD OF TIHE INVENTION
This invention is in the field of molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding phenol-induced proteins of the denitriying bacterium Thauera aromatics
BACKGROUND OF THE INVENTION
Phenolic compounds are basic chemicals of high interest to the chemical and pharmaceutical industries. Phenolic compounds are important plant constituents and phenol is formed from a variety of natural and synthetic substrates by the activity of microorganisms. The aerobic metabolism of phenol has been studied extensively; in all aerobic metabolic pathways oxygenases initiate the degradation of phenol by hydroxylation to catechol. Catechol can be oxygenolytically cleaved by dioxygenases, either by ortho- or meta-cleavage.
Anaerobic metabolism of phenol, aniline, o-cresol (2-methylphenol), hydroquinone (1,4-dihydroxybenzene), catechol (1,2-dihydroxybenzene), naphthalene and phenanthrene (Zhang et al.,
App. Environ. Microbiol
. 63:4759-4764 (1997)) by denitrifying and sulfate-reducing bacteria involves carboxylation of the aromatic ring ortho orpara to the hydroxy or amino substituent. Products are 4-hydroxybenzoate, 4-aminobenzoate, 4-hydroxy-3-methylbenzoate, gentisate (2,5-dihydroxybenzoate), and protocatechuate (3,4-dihydroxybenzoate) (Heider et al.,
Eur. J. Biochem
. 243:577-596 (1997)). Consortia of fermenting bacteria convert phenol to benzoate and decarboxylate 4-hydroxybenzoate to phenol (Winter et al.,
Appl. Microbiol. Biotechnol
. 25:384-391 (1987); He et al.,
Eur. J. Biochem
. 229:77-82 (1995); He et al.,
J. Bacteriol
. 178:3539-3543 (1996); Van Schie et al.,
Appl. Environ. Microbiol
. 64:2432-2438 (1998)). They also catalyze an isotope exchange between D
2
O and the proton at C4 of the aromatic ring of 4-hydroxybenzoate. Phenol carboxylation to 4-hydroxybenzoate in the denitritying bacterium
Thauera aromatica
is the best studied of these carboxylation reactions and is a paradigm for this new type of carboxylation reaction (Tschech et al.,
Arch. Microbiol
. 148:213-217 (1987); Lack et al.,
Eur. J. Biochem
. 197:473-479 (1991); Lack et al.,
J. Bacteriol
. 174:3629-3636 (1992); Lack et al.,
Arch. Microbiol
. 161:132-139 (1994)).
Without an isolated gene and corresponding sequence of the coding sequence, there remains a need for a convenient way to produce various intermediates in phenol metabolism with a transformed microorganism.
SUMMARY OF THE INVENTION
Five phenol-induced proteins from
Thauera aromnatica
have been isolated. Three dominant phenol-induced proteins called F1, F2, and F3 were purified and sequenced in an attempt to purif the enzyme(s) that catalyze the
14
CO
2
:4-hydroxybenzoate isotope exchange reaction and the carboxylation of phenylphosphate. The N-terminal amino acid sequences of these proteins as well as the N-terminus of the phenol-induced proteins F4 and F5 were determined. Internal sequences of F2 were obtained by trypsin digest. All of these sequences have application in industrial processes that involve the use of phenol or its intermediates. The instant invention provides a means to manipulate phenol metabolism and to produce various phenol intermediates in recombinant micro-organisms. The approach is based on the observation that anoxic growth with phenol and nitrate induces novel proteins that are lacking in cells grown with 4-hydroxybenzoate and nitrate.
BRIEF DESCRIPTION OF THE SEQUENCE DESCRIPTIONS
The following 44 sequence descriptions and sequence listings attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825 (“Requirements for Patent Applications contaning nucleotide sequences and/or Amino Acid Sequence Disclosure—the Sequence Rules”) and consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 4.95(a-bis) and Section 208 and Annex C of the Administrative Instructions). The Sequence Descriptions contain the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IYUB standards described in
Nucleic Acids Research
13:3021-3030 (1985) and in the
Biochemical Journal
219(2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822. The present invention utilizes Wisconsin Package Version 9.0 software from Genetics Computer Group (GCG), Madison, Wis.
SEQ ID NO:1 is the deduced amino acid sequence of protein F1 and is coded by orf6.
SEQ ID NO:2 is the nucleotide sequence of orf6 that codes for protein F1.
SEQ ID NO:3 is the deduced amino acid sequence of protein F2 and is coded by orf4.
SEQ ID NO:4 is the nucleotide sequence of orf4 that codes for protein F2.
SEQ ID NO:5 is the deduced amino acid sequence of protein F3 and is coded by orf1.
SEQ ID NO:6 is the nucleotide sequence of orf1 that codes for protein F3.
SEQ ID NO:7 is the deduced amino acid sequence of protein F4 and is coded by orf5.
SEQ ID NO:8 is the nucleotide sequence of orf5 that codes for protein F4.
SEQ ID NO:9 is the deduced amino acid sequence of protein F5 and is coded by orf8.
SEQ ID NO:10 is the nucleotide sequence of orf8 that codes for protein F5.
SEQ ID NO:11 is the deduced amino acid sequence of orf2.
SEQ ID NO:12 is the nucleotide sequence of orf2 that codes for an unknown protein.
SEQ ID NO:13 is the deduced amino acid sequence of orf3.
SEQ ID NO:14 is the nucleotide sequence of orf3 that codes for an unknown protein.
SEQ ID NO:15 is the deduced amino acid sequence of orf7.
SEQ ID NO:16 is the nucleotide sequence of orf7 that codes for an unknown protein.
SEQ ID NO:17 is the deduced amino acid sequence of orf9.
SEQ ID NO:18 is the nucleotide sequence of orf9 that codes for an unknown protein.
SEQ ID NO:19 is the deduced amino acid sequence of orf10.
SEQ ID NO:20 is the nucleotide sequence of orf10 that codes for an unknown protein.
SEQ ID NO:21 is the deduced amino acid sequence of orf-1.
SEQ ID NO:22 is the nucleotide sequence of orf-1 that codes for an unknown protein.
SEQ ID NO:23 is the nucleotide sequence containing two gene clusters that are involved in phenol metabolism.
SEQ ID NO:24 is the N-terminal amino acid sequence of F1 (experimentally determined).
SEQ ID NO:25 is the N-terminal amino acid sequence of F1 (deduced from the genes).
SEQ ID NO:26 is the N-terminal amino acid sequence of F2 (experimentally determined).
SEQ ID NO:27 is the N-terminal amino acid sequence of F2 (deduced from the genes).
SEQ ID NO:28 is the N-terminal amino acid sequence of F3 (experimentally determined).
SEQ ID NO:29 is the N-terminal amino acid sequence of F3 (deduced from the genes).
SEQ ID NO:30 is the amino acid sequence of an internal fragment of F2 that was obtained by trypsin-digest.
SEQ ID NO:31 is the amino acid sequence of an internal fragment of F2 that was obtained by trypsin-digest.
SEQ ID NO:32 is the primer of F2-forward (N-terminus).
SEQ ID NO:33 is the primer of F2T6-reverse.
SEQ ID NO:34 is the primer of F2T43-reverse.
SEQ ID NO:35 is the primer T7.
SEQ ID NO:36 is the primer T3.
SEQ ID NO:37 is the primer designated breib31.
SEQ ID NO:38 is the primer designated breib07r3.
SEQ ID NO:39 is the primer of &lgr;15-forward.
SEQ ID NO:40 is the primer of &lgr;15-reverse.
SEQ ID NO:41 is the N-terminal amino acid sequence of F4 (experimentally determined).
SEQ ID NO:42 is the N-terminal amino acid sequence of F4 (deduced from the genes).
SEQ ID NO:43 is the N-terminal amino acid sequence of F5 (experimentally determined).
SEQ ID NO:44 is the N-terminal amino acid sequence of F5 (deduced from the genes).
REFERENCES:
Brackmann et al., Enzymes of anaerobic metabolism of phenolic compounds: 4-hydroxybenzoyl-CoA reductase (dehydroxylating) from a denitrifying Pseudomonas species,European Journal of Biochemistry, vol. 213, No. 1, pp. 563-571, 1993 XP000921140.
Heider et al., Differential inducti
Breinig Sabine
Fuchs Georg
E. I. Du Pont de Nemours and Company
Prouty Rebecca E.
Steadman David J.
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