Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2002-10-16
2004-09-21
Hutson, Richard (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S183000, C435S189000, C435S190000, C435S195000, C536S023100, C536S023200
Reexamination Certificate
active
06794165
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of molecular biology and microbiology. More specifically, adipic acid has been produced from cyclohexanol by micro-biological means. The reaction is mediated by a set of enzymes resident on a 17 kb gene cluster, isolated from Acinetobacter sp.
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 17 kb nucleic acid fragment from Acinetobacter sp. that is responsible for mediating the conversion of cyclohexanol to adipic acid. Recombinant
E. coli
hosts with the DNA containing the 17 kb gene cluster conveys on the host the ability to convert cyclohexanol to adipic acid.
SUMMARY OF THE INVENTION
The invention provides an isolated nucleic acid fragment encoding an adipic acid synthesizing enzyme selected from the group consisting of: an isolated nucleic acid fragment encoding an adipic acid synthesizing enzyme selected from the group consisting of: (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:24, and SEQ ID NO:26, or an enzymatically active fragment thereof; (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:12, SEQ ID NO:20, SEQ ID NO:24, and SEQ ID NO:26, based on the partial sequence of said 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, said transformed host cell comprising a nucleic acid fragment encoding SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:24, and SEQ ID NO:26 under the control of suitable regulatory sequences.
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:12, SEQ ID NO:20, SEQ ID NO:24, and SEQ ID NO:26.
Additionally, the invention provides host cells transformed with all or a substantial portion of the 17 kb gene cluster.
REFERENCES:
patent: 3843466 (1974-10-01), Ajinomoto Co. Inc.
patent: 4400468 (1983-08-01), Faber
patent: 5616496 (1997-04-01), Frost et al.
patent: 5629190 (1997-05-01), Petre et al.
patent: 5635391 (1997-06-01), Petre et al.
patent: 7403156 (1974-11-01), None
patent: 61128890 (1986-06-01), None
patent: 01023894 (1989-01-01), None
patent: 01023895 (1989-01-01), None
Steinbuechel, Alexander CLB Chem. Labor Biotech., Microorganisms for manufacturing polymers. (1995)46(6) 277-8.
Draths et al., ACS Symp. Ser., Microbial Biocatalysis. Synthesis of Adipic Acid from D-Glucose. (1994). 577 (Benign by Design) 32-45.
Cho, Takeshi et al., Bio Ind., Manufacture of adipic acid by biotechnology. (1991).8(10). 671-8.
Hasegawa et al., Biosci., Biotechnol., Biochem., The Metabolism of Cyclohexanol by Exophiala jeanselmei. (1992). 56(8). 1319-20.
Yoshizako et al., J. Ferment. Bioeng., Metabolism of n-Alkylcyclohexanes with an Even Nubmer of Carbon Atoms in the Side Chain by Micrococcus sp. RCO-4M. (1989), 67(5), 335-8.
Kim et al., Sanop Misaengmul Ilakhoechi . Utilization of cyclohexanol and characterization of Acinetobacter calcoaceticus C-15,(1985), 13(1), 71-7.
Donoghue et al., Eur. J. Biochem., The Meta
Cheng Qiong
Nagarajan Vasantha
Thomas Stuart M.
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