DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-11) and species of poly(3-HIBA), SEQ ID NO: 3, SEQ ID NO: 22, ethanol and ADH in the reply filed on 10/06/2025 is acknowledged.
Claims 12-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/06/2025.
Claims 9-10 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/06/2025. Claims 9 and 10 do not read on the elected species of an engineered pathway being ADH.
Claim Objections
Claims 1-4 and 8 are objected to because of the following informalities:
In claims 3 and 4, the Sequence Identifiers in parenthesis are understood as claim limitations and therefore must be recited outside of parenthesis.
In claims 1, 2, 4, and 8, abbreviations should be defined prior to their first use.
Claim 3 recites “the CoA-ligase [singular] has at least 90% sequence identity to one or more of.” In order for a singular CoA-ligase having sequence identity to more than one (e.g. two) of the listed sequences, the same would have to be a fusion protein. Claim 4 with regards to a PHA synthase presents a similar issues wherein “and/or” directly indicates a fusion protein by identity to more than one sequence.” However, since the specification does not discuss fusion proteins it would be appropriate for the claim to be amended to “the CoA-ligase has at least 90% sequence identity to one of” and to remove “and/or” from both claims 3 and 4.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 3 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. For a broad generic claim, the specification must provide adequate written description to identify the genus of the claim.
“A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) (“In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus.”). Regents of the University of California v. Eli Lilly & Co., 119, F.3d 1559, 1568, 43 USPQ2d 1398, 1405 (Fed. Cir. 1997).
MPEP § 2163 further states that if a biomolecule is described only by a functional characteristic, without any disclosed correlation between function and structure of the biomolecule, it is "not sufficient characteristic for written description purposes, even when accompanied by a method of obtaining the claimed biomolecule.”
“The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice . . ., reduction to drawings . . ., or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus.” MPEP 2163(II)(3)(a).
Furthermore, a “‘representative number of species’ means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure ‘indicates that the patentee has invented species sufficient to constitute the gen[us].’ See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615; Noelle v. Lederman, 355 F.3d 1343, 1350, 69 USPQ2d 1508, 1514 (Fed. Cir. 2004) (Fed. Cir. 2004) (‘[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated.’). ‘A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when … the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed.’ In re Curtis, 354 F.3d 1347, 1358, 69 USPQ2d 1274, 1282 (Fed. Cir. 2004).” MPEP 2163(II)(3)(a).
The specification, provides, the following in para. [00102]:
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The above is understood as setting forth a limiting definition that a CoA ligase must have a ligase activity consistent with EC 6.2.1. For example, a transferase classified under EC 2.x.x.x having activity to convert HIBA to HIBA-CoA would not be a CoA ligase as defined by the specification.
McDonald et al. (Enzyme nomenclature and classification: the state of the art, FEBS J. 290, 2023, 2214-31), Table 2, evidences the meaning of the nomenclature of a ligase enzyme classified in EC 6.x.x.x as follows:
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As such, a Ligase creates a covalent bond between two molecules A and B coupled with the hydrolysis of a nucleotide triphosphate.
Claim 3 asserts that an enzyme with SEQ ID NO: 3 (or 90% identity thereto) is a CoA ligase. Uniprot, Accession No. Q5U921, 2020, www.uniprot.org., evidences that an enzyme 100% identical to SEQ ID NO: 3 is understood to be a 2-hydroxyisocaproate-CoA transferase. As evidenced by Table 2 of McDonald, a transferase transfers a moiety (e.g. CoA) between a donor and acceptor molecule without hydrolysis of ATP and is therefore not a ligase.
The specification asserts that SEQ ID NO: 3 has CoA ligase activity. While an enzyme having SEQ ID NO: 3 may have an ability to form HIBA-CoA as a product, the specification provides no recognizable evidence that would allow one having ordinary skill in the art that the transferase of SEQ ID NO: 3 also has CoA ligase activity that requires NTP hydrolysis activity, which is an activity never attributed in the prior art to an enzyme having SEQ ID NO: 3. Further, claims 3 and the specification describes SEQ ID NO: 3 as an isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase, which is a transferase and not a ligase activity For these reason, an ordinarily skilled artisan cannot recognize a genus of CoA-ligase enzymes having at least 90% identity to SEQ ID NO: 3 as recited such that the same lacks adequate written description in the specification.
A transferase enzyme having SEQ ID NO: 3 is understood to satisfy the claim limitation of a CoA ligase since the specification and claim 3 directly asserts that the same is a CoA ligase. Further any enzyme catalyzing a ligase reaction, joinder of two molecule coupled with ATP hydrolysis, is a ligase even if alternative terminology is used in the art.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 8 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 8 recites a transitional phrase of “comprises or consists of.” The recited transitional phrase is not considered to be a Markush group but rather a transitional phrase that is a combination of comprises or consists of. However, it is unclear if the recited transitional phrase is open, closed, or something else such that one cannot understand how to avoid infringe the claim. See MPEP 2111.03.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-4 and 11 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Mizuno et al. (Biosynthesis of polyhydroxyalkanoates containing 2-hydroxy-4-methylvalerate and 2-hydroxy-3-phenylpropionate units from a related or unrelated carbon source, J. Biosci. Bioeng. 235, 2018, 295-300) as evidenced by Kim et al. (Characterization of (R)-2-Hydroxyisocaproate Dehydrogenase and a Family III Coenzyme A Transferase Involved in Reduction of L-Leucine to Isocaproate by Clostridium difficile, Appl. Environ. Microbiol. 72, 2006, 6062-69), Taguchi et al. (A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme, PNAS 105, 2008, 17323-27), Matsusaki et al. (Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes in Pseudomonas sp. Strain 61-3, J. Bacteriol. 180, 1998, 6459-67) and GenBank, Accession No. AB014758.1, 2016, www.ncbi.nlm.nih.gov.
Mizuno, abstract, states:
The discovery of the lactate-polymerizing enzyme (LPE) enabled the biosynthesis of a polyhydroxyalkanoate (PHA) containing 2-hydroxyalkanoate (2HA). Amino acids are potential precursors of 2HA with various side chain structures if appropriate enzymes are used to convert amino acids to 2HA-coenzyme A (CoA) as the substrate for LPE. In this study, the suitability and utility of (R)-2-hydroxy-4-methylvalerate (2H4MV) dehydrogenase (LdhA) and 2H4MV-CoA transferase (HadA) from Clostridium difficile as 2HA-CoA–supplying enzymes were investigated. By expressing LPE, LdhA, and HadA in Escherichia coli DH5α, we successfully produced poly(3-hydroxybutyrate-co-2HA) [P(3HB-co-2HA)] from a related or unrelated carbon source. The 2HA units incorporated into PHA from unrelated carbon sources were primarily 2H4MV and 2-hydroxy-3-phenylpropionate (2H3PhP), which were assumed to be derived from endogenous leucine and phenylalanine, respectively. Furthermore, P(3HB-co-22 mol% 2HA) synthesis was demonstrated by means of saccharified sugars, which are an abundant and renewable feedstock for polymer production from hemicellulosic biomass (Japanese cedar) as the carbon source. Our study shows that several types of 2HA units such as 2H4MV and 2H3PhP are endogenous monomers for PHA biosynthesis in E. coli expressing LdhA and HadA.
“E. coli strain DH5α (Takara Bio, Ohtsu, Japan) served as the host for PHA production. Plasmid pTTQldhAhadACd_opt was constructed by inserting the ldhA-hadA-containing DNA fragment derived from C. difficile 630 at the PstI and BamHI sites of the pTTQ19 vector. The ldhA-hadA-containing DNA fragment was chemically synthesized with optimized codons for expression in E. coli. Plasmid pBBR1’’C1Ps(STQK)ABRe harbors the following genes: a PHA synthase gene (phaC1Ps) with the STQK mutation (S325T and Q481K), a 3-ketothiolase gene (phaARe), and a NADPH-dependent acetoacetyl-CoA reductase gene (phaBRe). Genes phaC1Ps and phaABRe were derived from Pseudomonas sp. 61-3 and Ralstonia eutropha H16, respectively.”
Table 1 of Mizuno shows E. coli expressing the described pBBR1’’C1Ps(STQK)ABRe plasmid and additionally expressing a plasmid with hadA.
For identity of C. difficile 630 hadA, Mizuno cites reference 16, which is Kim. Fig. 5 of Kim shows that such hadA is identical to recited SEQ ID NO: 3.
For identity of a PHA synthase gene (phaC1Ps) with the STQK mutation (S325T and Q481K), Mizuno cites reference 10, which is Taguchi. In turn, Taguchi in reference 18 of Taguchi is cited for the identity to this PHA synthase, which is Matsusaki. In turn Matsusaki states that the PHA synthase gene sequence is described in GenBank AB014758.1, which is identical to recited SEQ ID NO: 1 upon embodiment of substitutions S325T and Q481K.
Since the E. coli described by Mizuno et al. has a CoA-ligase with recited SEQ ID NO: 3 and a PHA synthase with SEQ ID NO: 21 as described by the specification and recited in the claims, such E. coli described by Mizuno et al. has a latent ability to produce poly(HIBA), including poly(3-HIBA), when provided with an appropriate feedstock containing HIBA. It is noted that the rejected claims do not recite any step of a method and do not require any active synthesis of poly(HIBA) from a feedstock, but only that embodiments be “capable of producing a poly(HIBA) from a feedstock” as recited.
Claim(s) 1, 2, 5, 6, 7 and 11 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Marx et al. (U.S. 2010/0068773 A1).
The present invention relates to cells which have been genetically modified in comparison with their wild type, to methods of generating a genetically modified cell, to the genetically modified cells obtainable by these methods, to a process for the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, to a process for the preparation of methacrylic acid or methacrylic esters, and to a process for the preparation of polymethacrylic acid or polymethacrylic esters. The present invention furthermore relates to an isolated DNA, to a vector, to the use of this vector for the transformation of a cell, to a transformed cell, and to a polypeptide.” Marx, para. [0001].
“In accordance with a first special embodiment of this first variant of the cell according to the invention, it is preferred that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid preferentially takes place via succinyl-coenzyme A as intermediate, where the cell preferentially is capable of utilizing carbohydrates, glycerol or glutamate as the carbon source.” Marx, para. [0020].
“In accordance with a second special embodiment of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate semialdehyde as precursor, it is preferred that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place via propionyl-coenzyme A as intermediate, where the cell is capable of preferentially utilizing carbohydrates, glycerol, methane or methanol as carbon source. In this context, a variety of pathways exist for arriving at 3-hydroxy-isobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, departing from propionyl-coenzyme A.” Marx, para. [0256].
In Marx, para. [0089]-[0095]:
Starting from the oxaloacetate intermediate stage, there are several possibilities for arriving at succinyl-coenzyme A, which can then be converted into 3-hydroxyisobutyric acid via methylmalonyl-coenzyme A by means of the three variants mentioned at the outset.
A first pathway leads via fumarate as intermediate. In this case it is preferred in accordance with a first special embodiment of the above-described first, second or third alternative of the cell according to the invention, where methylmalonate-semialdehyde is formed as precursor and succinyl-coenzyme A as intermediate, that the cell, if appropriate additionally to an increased activity of the enzyme E10 or E11, features an activity of at least one of the following enzymes E12 to E15 which is increased in comparison with its wild type (see FIG. 7):
of an enzyme E12, which catalyzes the conversion of oxaloacetate into malate;
of an enzyme E13, which catalyzes the conversion of malate into fumarate;
of an enzyme E14, which catalyzes the conversion of fumarate into succinate;
of an enzyme E15, which catalyzes the conversion of succinate into succinyl-coenzyme A.
In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E12, E13, E14, E15, E12E13, E12E14, E12E15, E13E14, E13E15, E14E15, E12E13E14, E12E13E15, E12E14E15, E13E14E15, E12E13E14E15, with E12E13E14E15 being most preferred.
In this context, it is especially preferred that the enzyme
E12 is a malate dehydrogenase (EC 1.1.1.37) or a malate quinone oxidoreductase (1.1.99.16),
E13 is a fumarate hydratase (EC 4.2.1.2),
E14 is a succinate dehydrogenase (EC 1.3.99.1 or EC 1.3.5.1) or a succinate quinone oxidoreductase (1.3.5.1), and
E15 is a succinate coenzyme A ligase (EC 6.2.1.4 or EC 6.2.1.5).
See pathway in Fig. 8 of Marx. Figs. 1 and 2 of Marx and related text show conversion of succinate coenzyme A to 3-hydroxyisobutyrate.
As shown in Fig. 5 of Marx, upon formation of 3-HIBA by any pathway as described by Marx, polyhydroxyalkanoates based on 3-hydroxyisobutyric acid (i.e. poly(3-HIBA)) is formed by conversion of 3-HIBA to 3-HIBA-CoA and to poly(3-HIBA) by action of a PHA synthase (enzyme E9). From paras. [0073]-[0076] of Marx:
Furthermore, it may in accordance with the first special embodiment of the cell according to the invention (and also in accordance with all embodiments which are still to be described hereinbelow) also be preferred that the cell is capable of converting the formed 3-hydroxyisobutyric acid into a polyhydroxy-alkanoate. Such polyhydroxydalkanoates are deposited intracellularly by many microorganisms in the form of highly refractive granula. In this context, it is especially preferred that the cell according to the invention features an activity of at least one of, preferably of the two, the following enzymes E9 and E10 which is increased [i.e. engineered] in comparison with its wild type (see FIG. 5):
of an enzyme E8, which catalyzes the conversion of 3-hydroxyisobutyric acid into 3-hydroxyisobutyryl-coenzyme A [i.e. a CoA-ligase];
of an enzyme E9, which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A to a polyhydroxy-alkanoate based on 3-hydroxyisobutyric acid.
[0076]
In this context, it is especially preferred that the enzyme
E8 is a 3-hydroxyisobutyryl CoA hydrolase (EC 3.1.2.4) and
E9 is a polyhydroxyalkanoate synthase [i.e. a PHA polymerase].
E. coli is a preferred embodiment of a host cell in the examples of Marx.
In view of the above, Marx discloses an engineered microorganism comprising a succinate CoA ligase and a PHA polymerase capable of producing poly(3-HIBA) and further comprising an engineered pathway for production 3-HIBA from a carbohydrate or glycerol feedstock.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1, 2, 5, 6, 7, 8, and 11 is/are rejected under 35 U.S.C. 103 as being unpatentable over Marx et al. (U.S. 2010/0068773 A1) further in view of Lee et al. (WO 2018/159965 A1) and Sofeo et al. (Altering the Substrate Specificity of Acetyl-CoA Synthetase by Rational Mutagenesis of the Carboxylate Binding Pocket, ACS Synth. Biol. 8, 2019, 1325-1336). A machine translation of Lee et al. is provided and cited herein.
“The present invention relates to cells which have been genetically modified in comparison with their wild type, to methods of generating a genetically modified cell, to the genetically modified cells obtainable by these methods, to a process for the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, to a process for the preparation of methacrylic acid or methacrylic esters, and to a process for the preparation of polymethacrylic acid or polymethacrylic esters. The present invention furthermore relates to an isolated DNA, to a vector, to the use of this vector for the transformation of a cell, to a transformed cell, and to a polypeptide.” Marx, para. [0001].
“In accordance with a second special embodiment of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate semialdehyde as precursor, it is preferred that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place via propionyl-coenzyme A as intermediate, where the cell is capable of preferentially utilizing carbohydrates, glycerol, methane or methanol as carbon source. In this context, a variety of pathways exist for arriving at 3-hydroxy-isobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, departing from propionyl-coenzyme A.” Marx, para. [0256].
In Marx, para. [0350]-[0356]:
In the event that 3-hydroxyisobutyric acid is formed from C1-carbon sources such as, for example, methane or methanol, it may be preferred that the cell features an activity of at least one of the enzymes E67 to E71 which is increased [i.e. engineered] in comparison with its wild type:
of an enzyme E67, which catalyzes the conversion of methane into methanol;
of an enzyme E68, which catalyzes the conversion of methanol into formaldehyde;
of an enzyme E69, which catalyzes the conversion of formaldehyde into 5,10-methylenetetrahydrofolate;
of an enzyme E70, which catalyzes the conversion of 5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate;
of an enzyme E71, which catalyzes the conversion of 5-methyltetrahydrofolate into acetyl-coenzyme A.
In this context, it is especially preferred that the enzyme
E67 is a methane monooxygenase (EC 1.14.13.25),
E68 is a methanol dehydrogenase (EC 1.1.1.244),
E69 is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27),
E70 is a methylenetetrahydrofolate reductase (EC 1.5.1.20),
E71 is a carbon monoxide dehydrogenase (EC 1.2.99.2).
As shown in Fig. 5 of Marx, upon formation of 3-HIBA by any pathway as described by Marx, polyhydroxyalkanoates based on 3-hydroxyisobutyric acid (i.e. poly(3-HIBA)) is formed by conversion of 3-HIBA to 3-HIBA-CoA and to poly(3-HIBA) by action of a PHA synthase (enzyme E9). From paras. [0073]-[0076] of Marx:
Furthermore, it may in accordance with the first special embodiment of the cell according to the invention (and also in accordance with all embodiments which are still to be described hereinbelow) also be preferred that the cell is capable of converting the formed 3-hydroxyisobutyric acid into a polyhydroxy-alkanoate. Such polyhydroxyalkanoates are deposited intracellularly by many microorganisms in the form of highly refractive granula. In this context, it is especially preferred that the cell according to the invention features an activity of at least one of, preferably of the two, the following enzymes E9 and E10 which is increased [i.e. engineered] in comparison with its wild type (see FIG. 5):
of an enzyme E8, which catalyzes the conversion of 3-hydroxyisobutyric acid into 3-hydroxyisobutyryl-coenzyme A [i.e. a CoA-ligase];
of an enzyme E9, which catalyzes the conversion of 3-hydroxyisobutyryl-coenzyme A to a polyhydroxy-alkanoate based on 3-hydroxyisobutyric acid.
[0076]
In this context, it is especially preferred that the enzyme
E8 is a 3-hydroxyisobutyryl CoA hydrolase (EC 3.1.2.4) and
E9 is a polyhydroxyalkanoate synthase.
However, Marx does not teach that an enzyme for converting 3-hydroxyisobutyryl CoA to poly(3-HIBA) is a CoA ligase.
The use of a CoA ligase to convert a hydroxyacid to a the CoA derivative thereof for the purpose of producing polyhydroxyalkanoates is known in the prior art. For example, Lee, page 2, provides:
Biosynthesis of PHA undergoes a process in which hydroxy acids are converted to hydroxyacyl-CoA by CoA-transferase or CoA-ligase, and the converted hydroxyacyl-CoA is polymerized by PHA synthase.
An ordinarily skilled artisan at the time of filing would have recognized that a 3-hydroxyisobutyryl CoA hydrolase (EC 3.1.2.4) as taught by Marx catalyzes the hydrolysis reaction in both directions. Synthases and Ligases “catalyz[e] the formation of a thioester bond between Coenzyme A and a carboxylic acid, while hydrolyzing ATP to AMP and pyrophosphate.” Sofeo, abstract. A synthetase forming a bond between two molecule coupled with ATP hydrolysis is within the broadest reasonable definition of ligase and would be recognized as a CoA ligase activity by an ordinarily skilled artisan at the time of filing. “The initial half reaction catalyzed by this superfamily of enzymes activates a carboxylate substrate by converting the hydroxyl leaving group of the substrate to an adenosine monophosphate (adenylate intermediate) using the hydrolysis of PPi to drive the reaction in the forward direction.” Sofeo, page 1325, right col. That is, a ligase/synthase reaction is driven in the forward direction by hydrolysis of ATP wherein a hydrolase reaction is not driven in the forward direction in this manner.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to replace a hydrolase for converting 3-hydroxyisobutyrate to 3-hydroxyisobutyrate-CoA (3-HIBA-CoA) with a suitable CoA ligase/synthase for catalyzing the same reaction since the ligase would have been expected to drive the forward reaction for formation of 3-HIBA-CoA better than a hydrolase, since ligase couple the reaction to hydrolysis of ATP to drive the reaction in the forward direction, which will result in greater amounts of 3-HIBA-CoA for poly(3-HIBA) synthesis.
Sofeo discusses that “We explored the structural basis of the specificity of ACS for only activating acetate, whereas other members of this superfamily utilize a broad range of other carboxylate substrates. By computationally modeling the structure of the Arabidopsis ACS and the Pseudomonas chlororaphis isobutyryl-CoA synthetase using the experimentally determined tertiary structures of homologous ACS enzymes as templates, we identified residues that potentially comprise the carboxylate binding pocket. These predictions were systematically tested by mutagenesis of four specific residues. The resulting rationally redesigned carboxylate binding pocket modified the size and chemo-physical properties of the carboxylate binding pocket. This redesign successfully switched a highly specific enzyme from using only acetate, to be equally specific for using longer linear (up to hexanoate) or branched chain (methylvalerate) carboxylate substrates.” That is, Sofeo teaches many CoA ligases/synthataes that have broad substrate specificity such that an ordinarily skilled artisan would have had an expectation of success in identifying a suitable ligase with activity to convert 3-HIBA to 3-HIBA-CoA.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TODD M EPSTEIN whose telephone number is (571)272-5141. The examiner can normally be reached Mon-Fri 9:00a-5:30p.
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652