DETAILED CORRESPONDENCE
Status of the Application
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant’s submission filed on 07/30/2025 has been entered.
Claims 1-4 are pending in this application.
Applicant’s amendment to the claims filed 07/09/2025 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed on 07/09/2025 in response to the final rejection mailed on 05/09/2025 is acknowledged and has been fully considered.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election
The elected subject matter is
Group I, claims 1-3, drawn to the technical feature of a genetically modified microorganism with an ability to produce 3-hydroxyadipic acid, alpha-hydromuconic acid, and/or adipic acid, in which the function of pyruvate kinase is impaired and the activities of phosphoenolpyruvate carboxykinase and of an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA are enhanced,
elected with traverse in the reply filed 07/29/2024, and
Species A2) a polypeptide composed of an amino acid sequence represented by SEQ ID NO: 2,
elected with traverse in the reply filed 07/29/2024.
Claim 4 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 with traverse in the reply filed on 07/29/2024.
Claims 1-3 are being examined on the merits only to the extent they read on the elected subject matter.
Claim Objections
Claim 1 is objected to for the phrase “at least one selected from the group consisting of recombinant DNA technologies to disrupt the gene by partial or complete deletion of the nucleotide sequence or by partial or complete substitution of the nucleotide sequence with another nucleotide sequence, site-directed mutagenesis, or via introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene”. In the interest of improving claim form, Applicant should consider an amendment to recite “at least one selected from the group consisting of recombinant DNA technologies to disrupt the gene by: partial or complete deletion of the nucleotide sequence; partial or complete substitution of the nucleotide sequence with another nucleotide sequence; site-directed mutagenesis; and introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene” to separate the members of the Markush group with a semicolon and complete the grouping with the conjunction “and”.
Claim Rejections - 35 USC § 112(b)
Claims 1-3 are newly rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 1 (claims 2-3 dependent therefrom) is rejected for the recitation of “the gene” in lines 4 and 7, “the nucleotide sequence” in lines 5 and 7, “the polypeptides” in lines 12, 13 and 15, “the copy numbers” in line 13, and “the region” in line 14. There is insufficient antecedent basis for these limitations in the claim.
Claim Rejections - 35 USC § 112(a)
Claim Interpretation: Claim 1 as amended is drawn to a genetically modified microorganism with an ability to produce 3-hydroxyadipic acid, alpha-hydromuconic acid, and/or adipic acid, in which the function of pyruvate kinase is impaired by artificial genetic modification
which is at least one selected from the group consisting of recombinant DNA technologies to disrupt the gene by partial or complete deletion of the nucleotide sequence or by partial or complete substitution of the nucleotide sequence with another nucleotide sequence, site-directed mutagenesis, or via introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene
compared to an otherwise identical microorganism without the artificial genetic modification, and the activities of phosphoenolpyruvate carboxykinase and of an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA are enhanced by artificial genetic modification
which is at least one selected from the group consisting of a method in which nucleic acids encoding the polypeptides are introduced from the outside to the inside of a host microorganism; a method in which the copy numbers of nucleic acids encoding the polypeptides are increased; and a method in which a promoter region or a ribosome-binding sequence upstream of the region coding for each of the polypeptides is modified
compared to an otherwise identical microorganism without the artificial genetic modification.
The claims are therefore drawn to a genus of genetic modifications to a microorganism that results in the impairment of pyruvate kinase and the enhancement of activities of phosphoenolpyruvate carboxykinase and an enzyme catalyzing the reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl CoA.
Given a broadest reasonable interpretation, and in view of the indefiniteness of the limitations regarding the modifications to “the gene”, “the nucleotide sequence”, and “the polypeptides” set forth above, the genetic modifications to the microorganism are not limited to the genes encoding the recited enzymes but can be modifications to be carried out on any genes, nucleotide sequences, and polypeptides that may directly or indirectly affect the expression and/or activity of the enzymes recited in the claim. As such, the genetic modifications are considered to be widely variant.
A. Claims 1-3 are rejected under 35 U.S.C. 112(a) 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 at the time the application was filed, had possession of the claimed invention.
The instant rejection is maintained from the previous Office Action and any newly recited portions are necessitated by claim amendment.
MPEP 2163.II.A.2.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states 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. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
According to MPEP 2163.II.A.3.(a).ii), [s]atisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’"
The factors considered in the Written Description requirement are (1) level of skill and knowledge in the art, (2) partial structure, (3) physical and/or chemical properties, (4) functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the (5) method of making the claimed invention. Disclosure of any combination of such identifying characteristics that distinguish the claimed invention from other materials and would lead one of skill in the art to the conclusion that the applicant was in possession of the claimed species is sufficient." MPEP § 2163.
The claims recite (in relevant part) a genus of genetic modifications that result in the impairment of pyruvate kinase functions and the enhancement of activities of phosphoenolpyruvate carboxykinase and an enzyme catalyzing the reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl CoA. As stated above, the genetic modifications are considered to be unlimited as the genetic modifications are not limited to be carried out on the enzymes in the claim. In this case, the genus of genetic modifications encompasses species that are considered to be widely variant.
The specification discloses the following representative species of modifications: inactivating genes encoding pyruvate kinase and a phosphotransferase system enzyme and overexpressing genes encoding phosphoenolpyruvate carboxykinase and an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA comprising the amino acid sequence of any one of SEQ ID NOs: 1-7. Aside from these representative species of modifications, there are no other examples of genetic modifications or conditional modifications in the specification disclosed to result in the claimed activity.
Regarding the level of skill and knowledge in the art of amino acid modification, the reference of Singh et al. (Curr. Protein Pept. Sci. 18:1-11, 2017; cited on the Form PTO-892 mailed 11/05/2024) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes [see p. 7, column 1, top]. Also, the unpredictability associated with residue substitution is exemplified by the reference of Zhang et al. (Structure 26:1474-1485, 2018; cited on the Form PTO-892 mailed 11/05/2024), which discloses that even a substitution of a surface residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide [p. 1475, column 1].
Further regarding the genus of genetic modifications, it is noted that the field of metabolic engineering can be inherently unpredictable and advances in metabolic pathway engineering are often achieved only by empirical experimentation. According to Guo et al. (Comp Struct Biotechnol J, 2017, 15:161; cited on the Form PTO-892 mailed 11/05/2024), “First, a lot of organisms are difficult to be engineered because of unknown regulation patterns and the lack of engineering tools for non-model organisms [16]. Even for model microorganisms like Escherichia coli and Saccharomyces cerevisiae, which are well studied and equipped with a broad spectrum of biomolecular tools to allow metabolic engineering easily, the effects of heterologous expression of pathways are often unpredictable to guarantee a high productivity…Second, a key challenge in metabolic engineering is balancing the tug-of-war that exists between the cell's physiological and evolutionary objectives on one side and the engineer's process objectives on the other [20]. Such conflict of resource allocation sometimes cannot be well addressed and toxic intermediates could be built up in the unbalanced pathway” [p. 162, column 1, middle].
In view of the high level of unpredictability in the art regarding structure and function of polypeptides, and the high level of unpredictability in the art regarding metabolic engineering, because the genus of genetic modifications is widely variant, and the specification discloses the actual reduction to practice of only 7 representative species among a widely variant genus, one of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the genus, and thus, that the applicant was not in possession of the recited genus of genetic modifications. The claimed subject matter is not supported by an adequate written description because a representative number of species has not been described.
B. Claims 1-3 are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for a genetically modified microorganism with inactivated genes encoding pyruvate kinase and a phosphotransferase system enzyme and overexpressing genes encoding a phosphoenolpyruvate carboxykinase and an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA comprising any one of the amino acid sequences of SEQ ID NOs: 1-7, does not reasonably provide enablement for all genetically modified microorganisms as encompassed by the claims. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
The instant rejection is maintained from the previous Office Action and any newly recited portions are necessitated by claim amendment.
“The test of enablement is not whether any experimentation is necessary, but whether, if experimentation is necessary, it is undue.” In re Angstadt, 537 F.2d 498, 504, 190 USPQ 214, 219 (CCPA 1976). Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)) as follows: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. See MPEP § 2164.01(a). The Factors considered to be most relevant to the instant rejection are addressed in detail below.
The nature of the invention: According to the specification at para 0012, “an object of the present invention is to provide a genetically modified microorganism for producing 3-hydroxyadipic acid, alpha-hydromuconic acid, and/or adipic acid in high yield.” The object of the invention is therefore to provide a mechanism for high yield production of the disclosed metabolites through genetic modification of the organism.
The breadth of the claims: The claims recite (in relevant part) genetic modification(s) that result in the impairment of pyruvate kinase and a phosphotransferase system enzyme and the enhancement of activities of phosphoenolpyruvate carboxykinase and an enzyme catalyzing the reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl CoA. As stated above, with the exception of inactivating genes encoding pyruvate kinase and a phosphotransferase system enzyme and overexpressing genes encoding phosphoenolpyruvate carboxykinase and an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA comprising the amino acid sequence set forth in any one of SEQ ID NOs: 1-7, the remaining genetic modifications are considered to be unlimited and are not limited to be carried out on the enzymes in the claim.
The state of the prior art; The level of one of ordinary skill; and The level of predictability in the art: According to MPEP 2164.03, “…what is known in the art provides evidence as to the question of predictability” and “[I]f one skilled in the art cannot readily anticipate the effect of a change within the subject matter to which that claimed invention pertains, then there is lack of predictability in the art.”
As noted above, other than the recited cell comprising the amino acid sequences set forth by SEQ ID NOs: 1-7, the modifications are unlimited. The reference of Singh (supra) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes [see p. 7, column 1, top].
The unpredictability associated with amino acid modification is exemplified by the reference of Zhang (supra) which discloses that even a mutation that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide [p. 1475, column 1].
The unpredictability associated with metabolic engineering is exemplified by the reference of Guo (supra)w which discloses “First, a lot of organisms are difficult to be engineered because of unknown regulation patterns and the lack of engineering tools for non-model organisms [16]. Even for model microorganisms like Escherichia coli and Saccharomyces cerevisiae, which are well studied and equipped with a broad spectrum of biomolecular tools to allow metabolic engineering easily, the effects of heterologous expression of pathways are often unpredictable to guarantee a high productivity… Second, a key challenge in metabolic engineering is balancing the tug-of-war that exists between the cell's physiological and evolutionary objectives on one side and the engineer's process objectives on the other [20]. Such conflict of resource allocation sometimes cannot be well addressed and toxic intermediates could be built up in the unbalanced pathway” [p. 162, column 1, middle].
As such, one of skill in the art would recognize a high level of unpredictability that all genetic modifications as encompassed by the claims would maintain the desired activity of impairing pyruvate kinase and enhancing the of activities of phosphoenolpyruvate carboxykinase and an enzyme catalyzing the reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl CoA.
The amount of direction provided by the inventor and The existence of working examples: The specification discloses the following working examples of the recited modification: inactivating genes encoding pyruvate kinase and a phosphotransferase system enzyme and overexpressing genes encoding phosphoenolpyruvate carboxykinase and an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA comprising the amino acid sequence of any one of SEQ ID NOs: 1-7. Other than these working examples, the specification fails to disclose any other modifications that would result in the desired impairment of the functions of pyruvate kinase and a phosphotransferase system enzyme and the enhancement of activities of phosphoenolpyruvate carboxykinase and an enzyme catalyzing the reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl CoA.
Also, the specification fails to provide guidance for using any cells bearing genetic modifications that are non-functional.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure: While methods of modifying the amino acid sequence of a polypeptide and genetically modifying cells were known at the time of the invention, it was not routine in the art to make and determine a use for genetic modifications recited by the claims.
In view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability, and the state of the prior art, undue experimentation would be necessary for a skilled artisan to make and use the entire scope of the claimed invention. Applicants have not provided sufficient guidance to enable one of ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of having the desired biological characteristics is unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
Response to Remarks: beginning p 4 of Applicant’s response to rejections under 35 USC 112; Applicant in summary contends the claim amendments recite particular artificial genetic modifications that overcome the rejections of record.
Applicant’s remarks are considered and found not convincing. While the claims are amended to recite artificial genetic modifications resulting from particular recombinant DNA techniques and methods for altering nucleic acids, the claims do not limit these techniques or methods to be carried out on the enzymes in the claim. For example, the artificial gene modification which impairs pyruvate kinase function is recited as “at least one selected from the group consisting of recombinant DNA technologies to disrupt the gene by partial or complete deletion of the nucleotide sequence or by partial or complete substitution of the nucleotide sequence with another nucleotide sequence, site-directed mutagenesis, or via introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene”, however the claims do not recite a gene, and the gene being affected by these recombinant DNA technologies is not limited to a gene encoding any enzyme recited in the claims.
Claim Rejections - 35 USC § 103
The rejection of claims 1-2 under 35 U.S.C. 103 as being unpatentable over 111A in view of Zhao et al. (Metabolic Eng, 2018, 47:254; cited on the Form PTO-892 mailed 05/09/2025; herein referred to as Zhao), Sanchez et al. (Metabolic Eng, 2005, 7:229; cited on the Form PTO-892 mailed 05/09/2025; herein referred to as Sanchez), and Zhao et al. (BMC Biotechnol, 2016, 16:52; cited on the Form PTO-892 mailed 05/09/2025; herein referred to as Zhao2), and
the rejection of claim 3 under 35 U.S.C. 103 as being unpatentable over 111A, Zhao, Sanchez, and Zhao2 as applied to claims 1-2 above, and further in view of UniProt Accession No. A0A2V4GCF0_SERMA (1 page, 09/12/2018; cited on the Form PTO-892 mailed 05/09/2025; herein referred to as UNI1).
are withdrawn in view of Applicant’s persuasive arguments regarding the teachings of Sanchez.
Claims 1-2 are newly rejected under 35 U.S.C. 103 as being unpatentable over 111A in view of Zhao, Sanchez, Zhao2, and Chiba et al. (J Biol Chem, 2015, 290:23960; cited on the attached Form PTO-892; herein Chiba).
Claim 1 is drawn to a genetically modified microorganism with an ability to produce 3-hydroxyadipic acid, alpha-hydromuconic acid, and/or adipic acid,
in which the function of pyruvate kinase is impaired by artificial genetic modification
which is at least one selected from the group consisting of recombinant DNA technologies to disrupt the gene by partial or complete deletion of the nucleotide sequence or by partial or complete substitution of the nucleotide sequence with another nucleotide sequence, site-directed mutagenesis, or via introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene
compared to an otherwise identical microorganism without the artificial genetic modification, and
the activities of phosphoenolpyruvate carboxykinase and of an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA are enhanced by artificial genetic modification
which is at least one selected from the group consisting of a method in which nucleic acids encoding the polypeptides are introduced from the outside to the inside of a host microorganism; a method in which the copy numbers of nucleic acids encoding the polypeptides are increased; and a method in which a promoter region or a ribosome-binding sequence upstream of the region coding for each of the polypeptides is modified
compared to an otherwise identical microorganism without the artificial genetic modification.
111A discusses microorganisms to produce adipic acid and other compounds [title], and discloses non-naturally occurring microorganisms with an adipate pathway and methods to produce adipate [abstract].
Regarding claim 1, 111A discloses a genetically modified E. coli strain for the production of adipate wherein the strain has been engineered by introducing nucleic acids encoding enzymes that include PaaH, a 3-hydroxyacyl-CoA dehydrogenase [p 20, section “Example II”, para 2-3], wherein the microorganism of 111A has the ability to generate 3-OA-CoA and CoA from acetyl-CoA and succinyl-CoA and 3-hydroxyadipic acid from 3-HA-CoA [p 7, para 3], which satisfies the limitation of a genetically modified cell with enhanced activity of an enzyme that catalyzes a reaction to reduce 3-OA-CoA to 3-HA-CoA.
111A does not teach the genetically modified cell has an impaired pyruvate kinase function and an enhanced PEPCK function.
Zhao relates to the metabolic engineering of E. coli for producing adipic acid through the reverse adipate-degradation pathway [title], and discusses that adipic acid is an important molecule used in the production of nylon fibers and resins, and the elimination of pathways for major metabolites competing for carbon flux in an engineered organism aimed at the accumulation of succinyl-CoA resulted in the highest adipate acid titer reported in E. coli [abstract].
Regarding claim 1, Zhao teaches enhancing the carbon flow through the TCA cycle to produce more succinyl-CoA via the genetic modification to remove sucD responsible for cycling succinyl-CoA, wherein accumulated succinyl-CoA can be shunted out of the TCA to react with acetyl-CoA to produce 3-OA-CoA that is further reduced to 3-HA-CoA on the path to adipate production [Figure 1], which is considered to correspond to a genetic modification comprising recombinant DNA technology to disrupt a gene by complete deletion of a nucleotide sequence.
Sanchez relates to engineering the anaerobic central metabolic pathway of E. coli to increase succinate yield [title], and discusses strategies to increase succinate production through metabolic pathway alterations [abstract]. Sanchez teaches overexpression of PEPC is one of the known strategies to increase succinate yield and productivity [p 229, col 2, para 1, to p 230, col 1, para 1], wherein increased PEPC activity is understood to result in the production of the TCA intermediate OAA that results in the production of succinate [Figure 1]. One of skill in the art would recognize that the carbon flow through the TCA cycle that proceeds along the right side of the TCA cycle depicted in [Figure 1] would pass through succinyl-CoA before reaching succinate at the bottom of the cycle.
Chiba relates to phosphoenolpyruvate carboxykinase (PEPCK) genes in eukaryotes and bacteria [title].
Regarding claim 1 and the limitation of enhancing PEPCK activity, Chiba teaches that PEPCK catalyzes the interconversion of PEP-OAA, and works as a major crossroad to connect glycolysis/gluconeogenesis and organic acid metabolism such as the TCA [p 23961, col 1, para 5], and discloses the conditions for OAA production by PEPCK [Reactions 6-9, and p 23961, col 2, para 4]. While Sanchez teaches the overexpression of PEPC increases succinate production, one of skill in the art would reasonably conclude that the overexpression of the PEPCK of Chiba would result in a similar increase in succinate production as evidenced by both of these enzymes catalyzing the conversion of PEP to OAA [see Figure 1 of Sanchez, and Reactions 6-9 of Chiba]. Therefore one of skill in the art would be motivated to genetically modify a cell to enhance the activity of PEPCK by overexpression, as taught by Sanchez, because Sanchez teaches the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao teaches increased carbon flow through succinyl-CoA is a metabolic engineering strategy to increase adipate production.
Zhao2 relates to the optimization of central carbon metabolism for engineering succinate production in E. coli [title], and discusses succinate as an important chemical for the synthesis of high value products, and optimizing central carbon metabolism at the PEP node improves succinate production [abstract].
Regarding claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 teaches the use of small RNA (sRNA) to interfere with pykF translation to pyruvate kinase, an enzyme responsible for converting PEP to pyruvate [Figure 1], in order to fine tune its activity to increase succinate production [p 5, col 2, para 2], and noted an increase in succinate titer compared to a control that did not have impaired pyruvate kinase activity [Figure 4D, and p 6, col 2, para 1]. One of skill in the art would recognize that increased succinate titer through this pathway would involve carbon flow from PEP to OAA and subsequently through the TCA through succinyl-CoA as well [Figure 1].
Furthermore, as Zhao2 teaches reduced pykF translation increases succinate production, and Zhao teaches the removal of sucD impairs the sucD function to increase succinate yield, one of skill in the art would be capable of reducing pykF translation as taught by Zhao2 by removing the gene as taught by Zhao, as one of skill in the art would recognize that both gene deletion and the use of sRNA are techniques for impairing gene translation, which is considered to be encompassed by the limitation of impairing the function of pyruvate kinase.
Therefore one of skill in the art would have been motivated to carry out the genetic modification of Zhao on the gene of Zhao2 to impair activity of pyruvate kinase, because Zhao2 teaches this modification increases succinate titer, and Zhao teaches increased carbon flow through succinyl-CoA is a metabolic engineering strategy to increase adipate production.
In view of 111A, Zhao, Sanchez, Zhao2 and Chiba, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the engineered cell of 111A by enhancing PEPCK activity, as taught by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity, as taught by Zhao and Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the engineered cell of 111A by enhancing PEPCK activity because Sanchez teaches this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba teaches PEPCK is an enzyme that converts PEP to OAA, and because Zhao teaches increased carbon flow through succinyl-CoA is a metabolic engineering strategy to increase adipate production. One of ordinary skill in the art would have been motivated to modify the engineered cell of 111A by impairing pyruvate kinase activity because Zhao2 teaches this modification increases succinate titer, and Zhao teaches increased carbon flow through succinyl-CoA is a metabolic engineering strategy to increase adipate production. One of ordinary skill in the art would have had a reasonable expectation of success because 111A and Zhao relate to strategies for improving adipate production, Zhao, Sanchez and Zhao2 relate to strategies for tuning carbon through succinyl-CoA, and Sanchez and Chiba relate to enzymes that catalyze the conversion of PEP to OAA.
Regarding claim 2, Zhao2 teaches the inactivation and mutation of genes involved in the PTS system is beneficial to succinate production [p 2, col 2, para 3], which is interpreted to encompass the further impairment of a PTS enzyme by genetic modification.
Therefore, the invention of claims 1-2 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claim 3 is newly rejected under 35 U.S.C. 103 as being unpatentable over 111A in view of Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
Claim 3 is drawn to the genetically modified microorganism according to claim 1, wherein the enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA is any one of the following polypeptides (a) to (c):
(a) a polypeptide composed of the amino acid sequence of any one of SEQ ID NOs: 1 to 7;
(b) a polypeptide composed of the same amino acid sequence as that of any one of SEQ ID NOs: 1 to 7, except that one or several amino acids are substituted, deleted, inserted, and/or added, and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA;
(c) a polypeptide composed of the amino acid sequence with a sequence identity of not less than 70% to the sequence of any one of SEQ ID NOs:1 to 7 and having an enzymatic activity that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.
The teachings of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. These references do not teach the sequence limitations of the enzyme that catalyzes a reaction to reduce 3-OA-CoA to 3-HA-CoA.
UNI discloses a 3-hydroxybutyryl-CoA dehydrogenase from Serratia marsescens that shares 94.1% sequence identity with SEQ ID NO: 2 and contains several amino acids that are substituted and deleted [see Appendix A], and therefore satisfies the sequence limitations (b) and (c) of the claim. As the polypeptide of UNI is encompassed by the structural requirements of the claims, it is presumed to have the activity of catalyzing the reduction of 3-OA-CoA to 3-HA-CoA, as the activity of the polypeptide is presumed to be inherent its structure (see MPEP 2112.01.I).
In view of UNI, it would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of 111A, Zhao, Sanchez, Zhao2 and Chiba by replacing the enzyme of 111A with the enzyme of UNI to arrive at the claimed invention, because the simple substitution of one known element for another results in a predictable result. One of ordinary skill in the art would have recognized that the enzyme of 111A and the enzyme of UNI are both enzymes capable of catalyzing the reduction of 3-OA-CoA to 3-HA-CoA, and as such both are capable of being incorporated into such genetically modified cells as described by 111A. Thus it would have been obvious to one of ordinary skill in the art to replace the enzyme of 111A with the enzyme of UNI, as one of ordinary skill in the art would have been able to carry out such a substitution with a reasonable expectation of success because both the 111A and UNI relate to enzymes that catalyze the reduction of 3-OA-CoA to 3-HA-CoA.
Therefore, the invention of claim 3 would have been obvious to one of ordinary skill in the art before the effective filing date.
Response to Remarks: beginning p 6 of Applicant’s response to 35 USC 103; Applicant in summary contends the claim amendments to recite particular artificial genetic modifications that are note disclosed by the prior art of record; Applicant further contends the Sanchez reference relates to a PEPC enzyme that is phosphoenolpyruvate carboxylase, which is distinct from the enzyme phosphoenolpyruvate carboxykinase recited in the claims; Applicant further alleges the present invention produces unexpected results shown by Comparative Examples 1 and 2 with Example 3 in the instant specification, and notes the conversion of OAA to PEP through increased PEPCK decreases the products of the desired invention to support the unexpected results of the claimed invention; Applicant further contends the number of references combined would not provide a reasonable expectation of success.
Applicant’s remarks are considered and found not convincing.
Regarding the response that the claim amendments to recite particular artificial genetic modifications that are note disclosed by the prior art of record, as stated in the above rejection, the particular artificial genetic modification techniques recited in the claims do not limit the techniques to be carried out on the enzymes recited in the claim. For example, the artificial gene modification which impairs pyruvate kinase function is recited as “at least one selected from the group consisting of recombinant DNA technologies to disrupt the gene by partial or complete deletion of the nucleotide sequence or by partial or complete substitution of the nucleotide sequence with another nucleotide sequence, site-directed mutagenesis, or via introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene”, however the claims do not recite a gene, and the gene being affected by these recombinant DNA technologies is not limited to a gene encoding any enzyme recited in the claims.
For the sake of compact prosecution, the art presented in the rejection under 35 USC 103 above correspond to the genetic modifications recited in the claim to (1) impair pyruvate kinase function and (2) enhance PEPCK and an enzyme that catalyzes the reduction of 3-OA-CoA to 3-HA-CoA.
Regarding the response that the Sanchez reference relates to a PEPC enzyme that is phosphoenolpyruvate carboxylase, which is distinct from the enzyme phosphoenolpyruvate carboxykinase recited in the claims; Applicant’s response is acknowledged, and the reference of Chiba is cited in the rejection above regarding the PEPCK enzyme recited in the claims, wherein Sanchez and Chiba disclose the enzymes PEPC and PEPCK both catalyze the interconversion of OAA to PEP.
Regarding the allegations that the present invention produces unexpected results shown by Comparative Examples 1 and 2 with Example 3 in the instant specification; Comparative Example 1 of the specification [para 0163] corresponds to a Serratia organism with disrupted intact pyruvate kinase function and enhanced PEPCK activity, Comparative Example 2 [para 0164] corresponds to a Serratia organism with impaired pyruvate kinase function and unenhanced PEPCK activity, and Example 3 [para 0155] utilized the organism from Example 2 [beginning para 0151] corresponding to a Serratia organism with impaired pyruvate kinase function and carrying a plasmid expressing a PEPCK and enzymes catalyzing the reactions A, B, E and F [Scheme 1, para 0027]. These Comparative Examples 1 and 2 to Example 3 show yields of 3-HA-CoA are increased only by both impairing pyruvate kinase function and enhancing PEPCK in the microorganism of the genus Serratia [para 0165].
According to MPEP 716.02(e), unexpected results must be compared with the closest prior art. As the results proffered by Applicant are a comparison to internal control cells disclosed by the specification and are not compared with the closest prior art, Applicant’s allegations of unexpected results do not satisfy the requirements of MPEP 716.02(e).
According to MPEP 716.02(d), unexpected results must be commensurate in scope with the claimed invention. As the results proffered by Applicant correspond specific mutations carried out in a single microorganism, the results are not commensurate with the scope of the claims that are drawn to all microorganisms, and all genetic modifications consisting of recombinant DNA technologies to disrupt the gene by partial or complete deletion of the nucleotide sequence, recombinant DNA technologies to disrupt the gene by partial or complete substitution of the nucleotide sequence with another nucleotide sequence, site-directed mutagenesis, introduction of a frame-shift mutation or a stop codon into the nucleotide sequence of the gene nucleic acids encoding the polypeptides are introduced from the outside to the inside of a host microorganism, a method in which the copy numbers of nucleic acids encoding the polypeptides are increased, and a method in which a promoter region or a ribosome-binding sequence upstream of the region coding for each of the polypeptides is modified carried out on all genes, nucleotide sequences and polypeptides. Therefore Applicant’s allegations of unexpected results do not satisfy the requirements of MPEP 716.02(d).
According to MPEP 716.02(b).I, the burden is on applicant to establish results are unexpected and significant. As Applicant states increased PEPCK caused the conversion of OAA to PEP and resulting decrease in the products of the desired invention, the disclosure of Sanchez indicates the increased flow in the opposite direction from PEP to OAA would produce the results alleged to be unexpected, and Chiba teaches the PEPCK enzyme is involved in the interconversion of OAA to PEP, which is understood to mean that the enzyme catalyzes the reaction in both directions. As Chiba further indicates the reaction conditions required to produce OAA both in [Schemes 6-9] and on [p 23961, col 2, para 4] comprising an excess of PEP in the reaction, the disclosure of Chiba indicates the equilibrium requirements for OAA formation of keeping PEP in high amounts, which one of skill in the art would recognize can be achieved by reducing the activity of the PEP-consuming enzyme pyruvate kinase [see Figure 1 of Sanchez], and therefore one of skill in the art therefore would not recognize that the overexpression of PEPCK resulting in producing more OAA would be unexpected as alleged by Applicant.
For these reasons, Applicant’s allegations of unexpected results are not considered insufficient to rebut a prima facie case of obviousness.
Regarding the response that the number of references combined would not provide a reasonable expectation of success, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991). While Applicant references an excerpt from the rejections under 35 USC 112(a) regarding the unpredictability in the field of genetic modifications [p 10, top, of Applicant’s response], the excerpt quoted is being used to support Applicant’s argument of a lack of a reasonable expectation of success in combining references under 35 USC 103, which is not an argument commensurate with what is stated in the respective rejections. As described in detail in the section regarding 35 USC 112(a), the claims are drawn to unlimited genetic modifications to a cell that would result in specific functional outcomes, and therefore the scope of the claims are not enabled in view of the recognition in the field that genetic modifications can have unpredictable outcomes, amongst other reasons outlined in the 112(a) rejections above. The reasonable expectation of success in combining prior art references for a rejection under 35 USC 103 regards teaching of specific mutations to specific genes, with specific noted outcomes, and specific rationales that would provide one of skill in the art with a reasonable expectation of success that combining said specific mutations to specific genes would result in a modified cell.
Double Patenting
The rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,078,503 (cited on the Form PTO-892 mailed 11/05/2024) in view of 111A, Zhao, Sanchez and Zhao2,
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,078,503, 111A, Zhao, Sanchez, and Zhao2, and further in view of UNI1,
the rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10,858,677 (cited on the Form PTO-892 mailed 11/05/2024) in view of 111A, Zhao, Sanchez, and Zhao2,
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10,858,677, 111A, Zhao, Sanchez, and Zhao2 as applied to claims 1-2 above, and further in view of UNI1,
the rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 12 of co-pending Application No. 16/766979 (issued on 04/15/2025 as U.S. Patent No. 12,275,979) in view of 111A, Zhao, Sanchez, and Zhao2,
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 12 of co-pending Application No. 16/766979 (issued on 04/15/2025 as U.S. Patent No. 12,275,979), 111A, Zhao, Sanchez, and Zhao2, and further in view of UNI1,
the rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 5 of co-pending Application No. 17/609964 in view of 111A, Zhao, Sanchez, and Zhao2,
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 5 of co-pending Application No. 17/609964, 111A, Zhao, Sanchez, and Zhao2, and further in view of UNI1,
the rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 17/908059 in view of 111A, Zhao, Sanchez, and Zhao2.
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 17/908059, 111A, Zhao, Sanchez, and Zhao2, and further in view of UNI1,
the rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 10 of co-pending Application No. 17/924145 in view of 111A, Zhao, Sanchez, and Zhao2,
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 10 of co-pending Application No. 17/924145, 111A, Zhao, Sanchez, and Zhao2, and further in view of UNI1,
the rejection of claims 1-2 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 18/034102 in view of 111A, Zhao, Sanchez, and Zhao2, and
the rejection of claim 3 on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 18/034102, 111A, Zhao, Sanchez, and Zhao2, and further in view of UNI1
are withdrawn in view of Applicant’s persuasive arguments regarding the teachings of Sanchez.
A. Claims 1-2 are newly rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,078,503 (herein “patent”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
Regarding instant claim 1, claim 1 of the patent recites a microorganism producing 3-hydroxyadipic acid.
The claims of the patent do not recite the function of pyruvate kinase is impaired by artificial genetic modification compared to an otherwise identical microorganism without the artificial genetic modification, and the activities of PEPCK and of an enzyme that catalyzes a reaction to reduce 3-OA-CoA to 3-HA-CoA are enhanced by artificial genetic modification compared to an otherwise identical microorganism without the artificial genetic modification.
111A discusses microorganisms to produce adipic acid and other compounds [title], and discloses non-naturally occurring microorganisms with an adipate pathway and methods to produce adipate [abstract].
Regarding instant claim 1, 111A discloses a genetically modified E. coli strain for the production of adipate wherein the strain has been engineered by introducing nucleic acids encoding enzymes that include PaaH, a 3-hydroxyacyl-CoA dehydrogenase [p 20, section “Example II”, para 2-3], wherein the microorganism of 111A has the ability to generate 3-OA-CoA and CoA from acetyl-CoA and succinyl-CoA and 3-hydroxyadipic acid from 3-HA-CoA [p 7, para 3], which satisfies the limitation of a genetically modified cell with enhanced activity of an enzyme that catalyzes a reaction to reduce 3-OA-CoA to 3-HA-CoA.
Zhao relates to the metabolic engineering of E. coli for producing adipic acid through the reverse adipate-degradation pathway [title], and discusses that adipic acid is an important molecule used in the production of nylon fibers and resins, and the elimination of pathways for major metabolites competing for carbon flux in an engineered organism aimed at the accumulation of succinyl-CoA resulted in the highest adipate acid titer reported in E. coli [abstract].
Regarding instant claim 1, Zhao discloses enhancing the carbon flow through the TCA cycle to produce more succinyl-CoA via the genetic modification to remove sucD responsible for cycling succinyl-CoA, wherein accumulated succinyl-CoA can be shunted out of the TCA to react with acetyl-CoA to produce 3-OA-CoA that is further reduced to 3-HA-CoA on the path to adipate production [Figure 1], which is considered to correspond to a genetic modification which recombinant DNA technology to disrupt a gene by complete deletion of a nucleotide sequence.
Sanchez relates to engineering the anaerobic central metabolic pathway of E. coli to increase succinate yield [title], and discusses strategies to increase succinate production through metabolic pathway alterations [abstract]. Sanchez discloses overexpression of PEPC is one of