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 .
Claims 1-4 are pending in this application.
Applicant’s amendment to the claims filed 04/08/2026 is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed on 04/08/2026 in response to the non-final rejection mailed on 12/09/2025 are acknowledged and have 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Claim 4 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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
The objection to claim 1 is withdrawn in view of the instant claim amendment.
Claim Rejections - 35 USC § 112(b)
The rejection of claims 1-3 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point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention is withdrawn in view of the instant claim amendments.
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.
The instant rejection is newly stated and necessitated by claim amendment.
Claim 1 (claims 2-3 dependent therefrom) is rejected for the recitation of “the function of pyruvate kinase is impaired”, “the activity of phosphoenolpyruvate carboxylase is enhanced by … a method in which a polynucleotide encoding phosphoenolpyruvate carboxykinase is introduced from the outside to the inside of a host microorganism”, “the activity of phosphoenolpyruvate carboxylase is enhanced by … a method in which copy number of a polynucleotide encoding phosphoenolpyruvate carboxykinase is increased”, and “the activity of an enzyme … is enhanced by introduction of a polynucleotide encoding the enzyme” without a recitation of a standard or reference with which to compare said function, activity, copy number increase, expression or enzyme activity.
Response to Remarks: beginning on page 5 of Applicant’s response to rejections under 35 USC 112(b); Applicant in summary contends the claims have been amended to overcome the 112(b) rejection of record.
Applicant’s arguments are considered the rejection of record has been withdrawn, however the instant amendments have necessitated new ground of rejection set forth above.
Claim Rejections - 35 USC § 112(a)
The rejection of claims 1-3 under 35 U.S.C. 112(a) as failing to comply with the written description requirement as being drawn to an undescribed genus, and
the rejection of claims 1-3 under 35 U.S.C. 112(a) as not reasonably providing enablement for all genetically modified microorganisms encompassed by the claims
are withdrawn in view of the instant amendments to direct the genetic modifications to specific genes in order to impair pyruvate kinase function, enhance phosphoenolpyruvate carboxykinase, and enhance the activity of an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.
Claim Rejections - 35 USC § 103
Claims 1-2 are rejected under 35 U.S.C. 103 as being unpatentable over Japanese Application No. JP 2011-515111A (cited on the IDS submitted 08/23/2022; reference is made to a machine translation cited on the Form PTO-892 mailed 06/07/2024; herein referred to as 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), Zhao et al. (BMC Biotechnol, 2016, 16:52; cited on the Form PTO-892 mailed 05/09/2025; herein referred to as Zhao2), and Chiba et al. (J Biol Chem, 2015, 290:23960; cited on the Form PTO-892 mailed 12/09/2025; herein Chiba).
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Claim 1 is drawn to a genetically modified microorganism with an ability to produce 3-hydroxyadipic acid, α-hydromuconic acid, and/or adipic acid, in which
the function of pyruvate kinase is impaired by disruption of an endogenous gene encoding pyruvate kinase by artificial genetic modification of the endogenous gene encoding pyruvate kinase,
the activity of phosphoenolpyruvate carboxykinase (PEPCK) is enhanced by artificial genetic modification which is at least one selected from the group consisting of
a method in which a polynucleotide encoding phosphoenolpyruvate carboxykinase is introduced from the outside to the inside of a host microorganism;
a method in which copy number of a polynucleotide encoding phosphoenolpyruvate carboxykinase is increased; and
a method in which a promoter region or a ribosome-binding sequence upstream region of an endogenous gene encoding phosphoenolpyruvate carboxykinase is modified compared to an otherwise identical microorganism without the artificial genetic modifications, and
the activity of an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA is by enhanced introduction of a polynucleotide encoding the enzyme.
The claim 1 limitation “the function of pyruvate kinase is impaired by disruption of an endogenous gene encoding pyruvate kinase by artificial genetic modification of the endogenous gene encoding pyruvate kinase” is considered to be a product-by-process limitation, wherein the claimed product of a genetically modified microorganism is not limited to the manipulations of the steps of “disruption of an endogenous gene encoding pyruvate kinase by artificial genetic modification of the endogenous gene encoding pyruvate kinase” drawn to the method of making the genetically modified microorganism, but only the structure implied by the steps (see MPEP 2113.I). The structure implied by the steps is considered to be a microorganism with pyruvate kinase, as the term “disrupted” can be broadly interpreted to encompass changes in the structure of the encoding gene ranging from a single nucleotide polymorphism causing a silent mutation, to the complete deletion of the gene. Additionally, the recitation of “the function of pyruvate kinase is impaired” is a functional limitation that does not structurally limit the genetically modified cell (see MPEP 2112.01).
The claim 1 limitation “the activity of phosphoenolpyruvate carboxykinase is enhanced by artificial genetic modification which is at least one selected from the group consisting of a method in which a polynucleotide encoding phosphoenolpyruvate carboxykinase is introduced from the outside to the inside of a host microorganism; [and] a method in which copy number of a polynucleotide encoding phosphoenolpyruvate carboxykinase is increased” is considered to be a product-by-process limitation, wherein the claimed product of a genetically modified microorganism is not limited to the manipulations of the steps of the artificial genetic modifications recited above that are drawn to the method of making the genetically modified microorganism, but only the structure implied by the steps (see MPEP 2113.I). The structure implied by the steps is considered to be a microorganism comprising a polynucleotide encoding the PEPCK enzyme. While the claim recites a method wherein the copy number of the polynucleotide is increased, without a reference for comparison there is no way to determine the bounds of said increase as discussed in the rejection under 112(b) above. Additionally, the recitation of “the activity of pyruvate kinase is enhanced” is a functional limitation that does not structurally limit the genetically modified cell (see MPEP 2112.01).
Regarding the limitation “a method in which a promoter region or a ribosome-binding sequence upstream region of an endogenous gene encoding phosphoenolpyruvate carboxykinase is modified compared to an otherwise identical microorganism without the artificial genetic modifications”, the phrase “compared to an otherwise identical microorganism without the artificial genetic modifications” is interpreted as only applying to the Markush alternative of “a method in which a promoter region or a ribosome-binding sequence upstream region of an endogenous gene encoding phosphoenolpyruvate carboxykinase is modified”, and therefore the other enzyme functions or activities resulting from the genetic modifications and Markush alternatives recited by the claim are considered presented without a reference for comparison. In view of this interpretation, with the exception of the Markush alternative described above, “the function of pyruvate kinase”, “the activity of PEPCK”, and “the activity of an enzyme that catalyzes a reaction to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA” recited by the claim encompass any function or activity of the recited enzymes.
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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the teachings of 111A are considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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], which is an activity of PEPCK encompassed by the claim. 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 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 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).
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
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 on page 6 of Applicant’s response to 35 USC 103; Applicant in summary contends the amended claims require defined and coordinated set of modifications to impair PK function and enhance the activities of PEPCK and an enzyme catalyzing the reduction of 3-OA-CoA to 3-HA-CoA; Applicant further contends Chiba does not disclose the effects of increasing PEPCK expression or the impairment of pyruvate kinase, and does not teach or suggest how to produce 3-hydroxyadipic acid, α-hydromuconic acid, and/or adipic acid; Applicant further contends there is no motivation to combine the prior art of record, particularly because of the difference in the focus of Sanchez on the PEPC enzyme compared to the modification of PEPCK by Chiba; Applicant further contends there is no reasonable expectation of success in combining the prior art of record because the art does not teach that enhancing PEPCK decreases production of the claimed compounds, and therefore one of ordinary skill in the art would not expect to have improved yields with the described combination of modifications; Applicant further contends that the instant specification shows unexpected results regarding the claimed modifications to PEPCK and PK resulting in increased yields of desired products in Serratia and E. coli, which represents a non-additive synergistic effect.
Applicants remarks are considered and found not convincing.
Regarding the assertion that the amended claims require defined and coordinated set of modifications to impair PK function and enhance the activities of PEPCK and an enzyme catalyzing the reduction of 3-OA-CoA to 3-HA-CoA; as stated in the rejection above, the functional limitations set forth in the claim are recited without reference comparison, and therefore the results of the recited genetic modifications encompass any function or activity of the recited enzymes.
Regarding the assertion that Applicant further contends Chiba does not disclose the effects of increasing PEPCK expression or the impairment of pyruvate kinase, and does not teach or suggest how to produce 3-hydroxyadipic acid, α-hydromuconic acid, and/or adipic acid; the role of Chiba in the rejection is described above to the role of PEPCK in metabolism, and discloses the effect of PEPCK on increasing OAA production. As stated in the rejection, 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, which is an activity of PEPCK encompassed by the claim. As described in the rejection above, Chiba does not teach about pyruvate kinase, and the references of 111A and Zhao2 are considered for limitations regarding pyruvate kinase.
Regarding the assertion that there is no reasonable expectation of success in combining the prior art of record because the art does not teach that enhancing PEPCK decreases production of the claimed compounds, and therefore one of ordinary skill in the art would not expect to have improved yields with the described combination of modifications; if the prior art does not teach enhancing PEPCK decreases production of the claimed compounds, it is unclear why there would be no reasonable expectation of success that combining the prior art would improve yields of the claimed compounds, particularly considering the claims do not require improving yields of any compound.
As stated in the rejection above, one of ordinary skill in the art would have had a reasonable expectation of success in combining the prior art 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 the assertion that the instant specification shows unexpected results regarding the claimed modifications to PEPCK and PK resulting in increased yields of desired products in Serratia and E. coli, which represents a non-additive synergistic effect:
MPEP 716.02(a) states evidence must show unexpected results, and evidence of a greater than expected result may be shown by demonstrating an effect which is greater than the sum of each of the effects taken separately (i.e., demonstrating “synergism”). Applicant’s assertion that said results are shown in the instant specification does not correspond to a demonstration of said effect as set forth in the MPEP. Therefore Applicant’s response has not satisfied the requirements of MPEP 716.02(a). Applicant is invited to specifically provide the evidence of unexpected results and demonstration of the asserted non-additive synergism.
MPEP 716.02(b).I states the burden is Applicant to establish results are unexpected and significant, and of both statistical and practical significance. Applicant has proffered no data as evidence of the alleged unexpected results, and therefore has not established any statistical or practical significance of said results. Therefore Applicant’s response has not satisfied the requirements of MPEP 716.02(b).I.
MPEP 716.02(b).II states Applicants have the burden of explaining proffered data. As Applicant has proffered no data, Applicant has therefore not explained proffered data. Therefore Applicant’s response has not satisfied the requirements of MPEP 716.02(b).II.
MPEP 716.02(d) states unexpected results must be commensurate in scope with the claimed invention. As Applicant has proffered no data or offered any explanation of said results. Applicants statement of “the combination of enhancing PEPCK and impairing pyruvate kinase increases yields of the desired products across microorganisms such as Serratia and E. coli” is considered not commensurate in scope with the claimed invention that is drawn to all genetically modified microorganisms comprising impairment of pyruvate kinase by all disruptions to the pyruvate kinase gene, the enhancement of PEPCK by all of the genetic modifications set forth in the Markush alternatives such as all modifications to promoter region or RBS of the gene encoding PEPCK and all copy numbers of a polynucleotide encoding PEPCK. Therefore Applicant’s response has not satisfied the requirements of MPEP 716.02(d).
MPEP 716.02(e) states allegations of unexpected results must be compared with the closest prior art. Applicant has not compared the alleged unexpected results to the prior art. Therefore Applicant’s response has not satisfied the requirements of MPEP 716.02(e).
For these reasons, Applicant’s allegation of unexpected results is considered insufficient to rebut a prima facie case of obviousness.
Double Patenting
A. Claims 1-2 are 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.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the patent by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the patent and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
Claim 3 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,078,503, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the patent and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the patent do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the patent, 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.
B. Claims 1-2 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10,858,677 (herein “patent”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 1 of the patent recites a method of producing 3-hydroxyadipic acid comprising a microorganism having the capacity to produce 3-hydoxyadipic 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the patent by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the patent and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
Claim 3 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10,858,677, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the patent and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the patent do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the patent, 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.
C. Claims 1-2 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,275,979 (previously applied as a provisional rejection from claim 12 of co-pending Application 18/766979; cited on the attached Form PTO-892; herein “patent”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 1 of the patent recites a microorganism modified to express a polypeptide to increase 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the patent by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the patent by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the patent and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
Claim 3 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,275,979, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the patent and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the patent do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the patent, 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.
D. Claims 1-2 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 5 of co-pending Application No. 17/609964 (herein “reference application”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 5 of the reference application recites a microorganism comprising a polypeptide having an enzymatic ability to reduce 3-oxoadipyl-CoA to 3-hydroxyadipyl CoA that is further limited to produce 3-hydroxyadipic acid.
The claims of the reference application 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the reference application by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the reference application by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the reference application by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the reference application by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the reference application and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 3 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 5 of co-pending Application No. 17/609964, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the reference application and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the reference application do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the reference application, 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
E. Claims 1-2 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,577,592 (herein “patent”; previously provisionally rejected over co-pending Application No. 17/908059) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 1 of the patent recites a microorganism having an ability to produce 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 PEPC 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the patent by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the reference application by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the patent by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the patent by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the patent and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
Claim 3 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 12,577,592 (previously provisionally rejected over co-pending Application No. 17/908059), 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the patent and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the patent do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the patent, 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.
F. Claims 1-2 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 10 of co-pending Application No. 17/924145 (herein “reference application”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 10 of the reference application recites a microorganism expressing a mutated protein and has the ability to produce the organic acid 3-hydroxyadipic acid modified to express a polypeptide to increase 3-hydroxyadipic acid.
The claims of the reference application 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 PEPC 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the reference application by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the reference application by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the reference application by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the reference application by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the reference application and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 3 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 10 of co-pending Application No. 17/924145, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the reference application and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the reference application do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the reference application, 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
G. Claims 1-2 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 18/034102 (herein “reference application”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 1 of the reference application recites a microorganism modified to produce 3-hydroxyadipic acid.
The claims of the reference application 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 PEPC 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the reference application by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the reference application by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the reference application by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the reference application by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the reference application and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 3 is newly rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 18/034102, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the reference application and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the reference application do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the reference application, 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
H. Claims 1-2 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 18/848,396 (herein “reference application”) in view of 111A, Zhao, Sanchez, Zhao2 and Chiba.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
Regarding instant claim 1, claim 1 of the reference application recites a microorganism modified to produce 3-hydroxyadipic acid.
The claims of the reference application 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 PEPC 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.
Regarding the limitation of a function of pyruvate kinase, 111A describes the engineering of an organism for the reverse adipate degradation pathway [p 20, section “Example II”, para 2, pathway shown in Figure 2], wherein the initial reaction involves the consumption of acetyl-CoA and ultimately produces adipic acid in addition to oxidizing NADH to NAD+ along the way. One of skill in the art would be expected to reason that acetyl-CoA is produced from pyruvate, and pyruvate is produced from PEP via pyruvate kinase activity as evidenced by Zhao2 [Figure 1 depicting central carbon metabolism]. Therefore in view of the interpretation set forth above regarding the limitation of “a function of pyruvate kinase is impaired”, the disclosure of 111A is considered to correspond to a cell comprising a genetic modification and having a function of pyruvate kinase encompassed by the claim.
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 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 instant claim 1 and the limitation of enhancing PEPCK activity, Chiba discloses 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 discloses 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 disclosed by Sanchez, because Sanchez discloses the genetic modification of an enzyme that converts PEP to OAA increases succinate production, and because Zhao discloses 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 instant claim 1 and the limitation of impaired pyruvate kinase function, Zhao2 discloses 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 discloses reduced pykF translation increases succinate production, and Zhao discloses 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 disclosed 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 discloses this modification increases succinate titer, and Zhao discloses 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the claims of the reference application by using the PaaH enzyme of 111A, enhancing PEPCK activity as disclosed by Zhao, Sanchez and Chiba, and impairing pyruvate kinase activity as disclosed by Zhao Zhao2, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to modify the claims of the reference application by using the PaaH enzyme because 111A discloses an enzyme that produces 3-HA-CoA from 3-OA-CoA for adipate production.
One of ordinary skill in the art would have been motivated to modify the claims of the reference application by enhancing PEPCK activity because Sanchez discloses this genetic modification to an enzyme that converts PEP to OAA increases succinate production, because Chiba discloses PEPCK is an enzyme that converts PEP to OAA, and because Zhao discloses 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 claims of the reference application by impairing pyruvate kinase activity because Zhao2 discloses this modification increases succinate titer, and Zhao discloses 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 the reference application and 111A relate to organisms capable of producing intermediates in the adipate pathway, 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 instant claim 2, Zhao2 discloses 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 3 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of co-pending Application No. 18/848,396, 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 above, and further in view of UNI1.
The instant rejection is maintained from the previous Office action, and any newly recited portion is necessitated by claim amendment.
The claims of the reference application and disclosures of 111A, Zhao, Sanchez, Zhao2 and Chiba as applied to claims 1-2 are discussed above. The claims of the reference application do not recite 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 instant claim. As the polypeptide of UNI is encompassed by the structural requirements of the instant 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 obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to modify the combined cell of the reference application, 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.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Response to Remarks: beginning on page 9 of Applicant’s response to rejections on the ground of non-statutory double patenting; Applicant requests the rejections be held in abeyance until allowable subject matter is found.
Applicant’s request is acknowledged and the rejections have been updated to reflect the amendments to the claims.
Conclusion
Status of the Application:
Claims 1-4 are pending.
Claim 4 is withdrawn.
Claims 1-3 are rejected.
No claim is in condition for allowance.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JOSEPH SPANGLER whose telephone number is (571)270-0314. The examiner can normally be reached M-F 7:30 am - 4:30 pm.
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/JOSEPH R SPANGLER/
Examiner
Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656
APPENDIX A
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355
574
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Greyscale
Alignment of SEQ ID NO: 2 with UniProt Accession No. A0A2V4GCF0_SERMA (reference UNI1).
Prosecution Insights