Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2002-08-29
2004-09-14
Slobodyansky, Elizabeth (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C536S023200
Reexamination Certificate
active
06790645
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of molecular biology and microbiology. More specifically, genes have been isolated from Brevibacterium sp HCU and sequences that encode for enzymes useful for production of intermediates in the adipic acid biosynthetic pathway or for the production of related molecules.
BACKGROUND OF THE INVENTION
Production of adipic acid in the U.S. was 1.96 billion pounds in 1997 with an estimated 2.0 billion pounds in 1998. Historically the demand for adipic acid has grown 2% per year and 1.5-2% is expected through the year 2002. Adipic acid consistently ranks as one of the top fifty chemicals produced domestically. Nearly 90% of domestic adipic acid is used to produce nylon-6,6. Other uses of adipic acid include production of lubricants and plasticizers, and as a food acidulant.
The dominant industrial process for synthesizing adipic acid employs initial air oxidation of cyclohexane to yield a mixture of cyclohexanone (ketone) and cyclohexanol (alcohol), which is designated KA (see for example U.S. Pat. No. 5,221,800). Hydrogenation of phenol to yield KA is also used commercially, although this process accounts for just 2% of all adipic acid production. KA produced via both methods is oxidized with nitric acid to produce adipic acid. Reduced nitrogen oxides including NO
2
, NO, and N
2
O are produced as by-products and are recycled back to nitric acid at varying levels.
Research has also focused on synthesis of adipic acid from alternative feedstocks. Significant attention has been directed at carbonylation of butadiene (U.S. Pat. No. 5,166,421). More recently, a method of dimerizing methyl acrylates was reported, opening up the possibility of adipic acid synthesis from C-3 feedstocks.
These processes are not entirely desirable due to their heavy reliance upon environmentally sensitive feedstocks, and their propensity to yield undesirable by-products. Non-synthetic, biological routes to adipic acid would be more advantageous to industry and beneficial to the environment.
A number of microbiological routes are known. Wildtype and mutant organisms have been shown to convert renewable feedstocks such as glucose and other hydrocarbons to adipic acid [Frost, John, Chem. Eng. (Rugby, Engl.) (1996), 611, 32-35; WO 9507996; Steinbuechel, AlexanderCLB
Chem. Labor Biotech
. (1995), 46(6), 277-8; Draths et al., ACS Symp. Ser. (1994), 577 (Benign by Design), 32-45; U.S. Pat. No. 4,400,468; JP 49043156 B4; and DE 2140133]. Similarly, organisms possessing nitrilase activity have been shown to convert nitriles to carboxylic acids including adipic acid [Petre et al., AU 669951; CA 2103616].
Additionally, wildtype organisms have been used to convert cyclohexane and cyclohexanol and other alcohols to adipic acid [JP 01023894 A2; Cho, Takeshi et al.,
Bio Ind
. (1991), 8(10), 671-8; Horiguchi et al., JP 01023895 A2; JP 01023894 A2; JP 61128890 A; Hasegawa et al.,
Biosci., Biotechnol., Biochem
. (1992), 56(8), 1319-20; Yoshizako et al., J. Ferment.
Bioeng
. (1989), 67(5), 335-8; Kim et al., Sanop Misaengmul Hakhoechi (1985), 13(1), 71-7; Donoghue et al.,
Eur. J. Biochem
. (1975), 60(1), 1-7].
One enzymatic pathway for the conversion of cyclohexanol to adipic acid has been suggested as including the intermediates cyclohexanol, cyclohexanone, 2-hydroxycyclohexanone, &egr;-caprolactone, 6-hydroxycaproic acid, and adipic acid. Some specific enzyme activities in this pathway have been demonstrated, including cyclohexanol dehydrogenase, NADPH-linked cyclohexanone oxygenase, &egr;-caprolactone hydrolase, and NAD (NADP)-linked 6-hydroxycaproic acid dehydrogenase (Tanaka et al.,
Hakko Kogaku Kaishi
(1977), 55(2), 62-7). An alternate enzymatic pathway has been postulated to comprise cyclohexanol→cyclohexanone→1-oxa-2-oxocycloheptane→6-hydroxyhexanoate→6-oxohexanoate→adipate [Donoghue et al.,
Eur. J. Biochem
. (1975), 60(1), 1-7]. The literature is silent on the specific gene sequences encoding the cyclohexanol to adipic acid pathway, with the exception of the monoxygenase, responsible for the conversion of cyclohexanone to caprolactone, [Chen,et al., J. Bacteriol., 170, 781-789 (1988)].
The problem to be solved, therefore is to provide a synthesis route for adipic acid which not only avoids reliance on environmentally sensitive starting materials but also makes efficient use of inexpensive, renewable resources. It would further be desirable to provide a synthesis route for adipic acid which avoids the need for significant energy inputs and which minimizes the formation of toxic by-products.
Applicants have solved the stated problem by identifying, isolating and cloning a two unique monooxygenase genes, a hydrolase gene, a hydroxycaproate dehydrogenase gene, a cyclohexanol dehydrogenase gene and a gene encoding an acyl-CoA dehydrogenase, all implicated in the adipic acid biosynthetic pathway.
SUMMARY OF THE INVENTION
The invention provides an isolated nucleic acid fragment encoding an adipic acid synthesizing protein selected from the group consisting of: (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24; (b) an isolated nucleic acid molecule that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS at 65° C.; and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; (c) an isolated nucleic acid molecule that is completely complementary to (a) or (b).
In another embodiment the invention provides methods for the isolation of nucleic acid fragments substantially similar to those encoding the polypeptides as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, and SEQ ID NO:24 based on the partial sequence of the nucleic acid fragments.
The invention further provides a method for the production of adipic acid comprising: contacting a transformed host cell under suitable growth conditions with an effective amount of cyclohexanol whereby adipic acid is produced, the transformed host cell containing the nucleic acid fragments as set forth in SEQ ID NO:15 and SEQ ID NO:16.
The invention additionally provides methods for the production of intermediates in the pathway for the synthesis of adipic acid from cyclohexanol comprising transformed organisms transformed with any one of the open reading frames encoding SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, and SEQ ID NO:22.
Additionally the invention provides for recombinant cells transformed with any gene encoding the polypeptides selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22, and SEQ ID NO:24.
The invention further provides an isolated Brevibacterium sp HCU containing the genes required for the production of adipic acid intermediates as identified by its 16s rDNA profile.
REFERENCES:
patent: 4400468 (1983-08-01), Faber
patent: 669951 (1995-07-01), None
patent: 2103616 (1994-02-01), None
patent: 2140133 (1973-02-01), None
patent: 49043156 (1974-11-01), None
patent: 61128890 (1986-06-01), None
patent: 01023894 (1989-01-01), None
patent: 01023895 (1989-01-01), None
patent: WO9507996 (1995-03-01), None
Brzostowicz et al. (May 2002) Identification of two gene clusters involved in cyclohexanone oxidation inBrevibacterium epidermidisstrain HCU. Applied microbiology and biotechnology, 58 (6), 781-9.*
Brzostowicz et al. (Aug. 2000) Simultaneous identification of two cyclohexanone oxidation genes from an environmental Brevibacterium isolate using mRNA differential display. Journal of bacteriology, 182 (15) 4241-8.*
John Frost, Chem. Engg. (Rugby, Engl.) Renewable feedstocks; 611, 32-35, 1996.
Alexander Steinbuechel, CLB Chem. Labor Biotech., Microorganisms for Manufacturing polymers; 56(6), 277-8, 1995.
Draths et al., ACS Sy
Brzostowicz Patricia C.
Rouviere Pierre E.
E. I. du Pont de Nemours and Company
Slobodyansky Elizabeth
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