Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
This application is a 371 of PCT/EP2021/068282.
The amendment filed on February 20, 2026 has been entered.
Election/Restrictions
Applicant elected without traverse of Group I with a species election of
Bacterial cell: Pseudomonas putida,
Enzymes:
benzoate 1,2-dioxygenase – BenABC of SEQ ID NO:1, 2, and 3,
benzoate 1,2-dihydrodiol dehydrogenase – BenD of SEQ ID NO:7, and
catechol 1,2-dioxygenase – CatA-I of SEQ ID NO:9,
Specific promoters for expression of the enzymes
PTac SEQ ID NO:14 for BenABC and BenD and
P14b (constitutive) SEQ ID NO:49 for Cat A-I
Translation initiation sequences
P. putida native translation initiation for BenABC and BenD and
BCD10 SEQ ID NO:16 for translation initation for CatA-I.
in the reply filed on September 12, 2025 is acknowledged.
Claims 3-4, 12-13, 17-18, 26, 30-31, and 33-36 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species/invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on September 12, 2025.
Status of Claims
Claims 1-4, 6, 10, 12-13, 17-18, 26, and 31-36 are pending.
Claims 3-4, 12-13, 17-18, 26, 31, and 33-36 are withdrawn.
Claims 1-2, 6, 10, and 32 are under examination.
Response to Amendments/Arguments
Claim Rejections - 35 USC § 112(b)
Applicant’s arguments, see page 11 of the Remarks, filed February 20, 2026, with respect to claim 1 and claims 2, 6, 10, 27-29, and 32 depending therefrom have been fully considered and are persuasive. Claim 1 has been amended to clarify expression of the second catechol 1,2-dioxygenase occurs under branch (b). Claims 27-29 have been cancelled. Therefore, the rejection of claim 1 and claims 2, 6, 10, 27-29, and 32 depending therefrom under 35 U.S.C. 112(b) has been withdrawn.
Applicant’s arguments, see page 11 of the Remarks, filed February 20, 2026, with respect to claim 1 and claims 2, 6, 10, 27-29, and 32 depending therefrom have been fully considered and are persuasive. Claim 1 has been amended to delete the indefinite phrase “such as”. Claims 27-29 have been cancelled. Therefore, the rejection of claim 1 and claims 2, 6, 10, 27-29, and 32 depending therefrom under 35 U.S.C. 112(b) has been withdrawn.
Applicant’s arguments, see page 11 of the Remarks, filed February 20, 2026, with respect to claim 2 have been fully considered and are persuasive. Claim 2 has been amended to delete the indefinite phrase “such as”. Therefore, the rejection of claim 2 under 35 U.S.C. 112(b) has been withdrawn.
Applicant’s arguments, see page 11 of the Remarks, filed February 20, 2026, with respect to claim 6 have been fully considered and are persuasive. Claim 6 has been amended to delete the indefinite term “similar”. Therefore, the rejection of claim 6 under 35 U.S.C. 112(b) has been withdrawn.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-2, 6, 10, and 32 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' Therefore, the claims have been broadly interpreted to encompass (A) any Pseudomonas cell capable of producing 2-fluoro-cis-cis-muconate (2-FMA) or at least 500 mg/L of 2-FMA from 3-fluorobenzoate (3-FB), wherein the cell expresses (B) any benzoate 1,2-dioxygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, (C) any benzoate 1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and (D) any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein (E) expression of the benzoate 1,2-dioxygenase is under the control of any medium-strong constitutive promoter, and (F) expression of the catechol 1,2-dioxygeanse is under the control of a first promoter expression module comprising any first promoter or a medium-to -weak constitutive promoter P14b or a variant thereof having at least 85% sequence identity to SEQ ID NO:49 and any first strong translational initiation sequence, any strong translation coupler, any first strong translational initiation sequence that has the same the translational initiation strength as BCD10 of SEQ ID NO:16, or a BCD10 or variant thereof having at least 80% sequence identity to the SEQ ID NO:16. Therefore, the claims are drawn to (A) genus of Pseudomonas cells capable of producing 2-fluoro-cis-cis-muconate (2-FMA) or at least 500 mg/L of 2-FMA from 3-fluorobenzoate (3-FB), wherein the cell expresses (B) a genus of benzoate 1,2-dioxygenases BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, (C) a genus of benzoate 1,2-dihydrodiol dehydrogenases BenD having at least 85% sequence identity to SEQ ID NO:7, and (D) a genus of catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein (E) expression of the benzoate 1,2-dioxygenase is under the control of a genus of medium-strong constitutive promoters or the pTac promoter of SEQ ID NO:14, and (F) expression of the catechol 1,2-dioxygeanse is under the control of a genus of first promoter expression modules comprising a genus of first promoters or medium-to -weak constitutive promoter P14b or a variant thereof having at least 85% sequence identity to SEQ ID NO:49 and a genus of first strong translational initiation sequences, strong translation couplers, first strong translational initiation sequences that have the same translational initiation strength as BCD10 of SEQ ID NO:16, or a BCD10 or variant thereof having at least 80% sequence identity to the SEQ ID NO:16.
MPEP 2163 I. states that to “satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention.
MPEP 2163. II.A.3.(a) sates that “Possession may be shown in many ways. For example, possession may be shown by describing an actual reduction to practice of the claimed invention. Possession may also be shown by a clear depiction of the invention in detailed drawings or in structural chemical formulas which permit a person skilled in the art to clearly recognize that inventor had possession of the claimed invention. An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention.
According to MPEP 2163.II.A.3.(a).ii), “Satisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’"
The recitations of “benzoate 1,2-dioxygenase”, “benzoate-1,2-dihydrodiol dehydrogenase”, “catechol 1,2-disoxygenase”, “medium-strong constitutive promoter”, “first promoter”, “first strong translation initiation sequence”, and “strong translational coupler” fail to provide a sufficient description of the genus of the genetically modified Pseudomonas cells capable of producing 2-fluoro-cis,cis-muconate from 3-FB as it merely describes the functional features of the genus without providing any definition of the structural features of the species within the genus. The specification does not define any structural features commonly possessed by members of the genus that distinguish them from others. One skilled in the art therefore cannot, as one can do with a fully described genus, visualize or recognize the identity of the members of the genus.
The prior art discloses genetically modified P. putida producing cis-cis-muconate from benzoate, wherein said P. putida expresses a benzoate 1,2-dixoygenase encoded by benABC gene, benzoate-1,2-dihydrodiol dehydrogenase encoded by the benD gene, and catechol 1,2-dioxygenase encoded by the CatA, see Moreno (The target for the Pseudomonas putida Crc global regulator in the benzoate degradation pathway is the BenR transcriptional regulator. J Bacteriol. 2008 Mar;190(5):1539-45 – form PTO-892) and Van Duuren (EP 2 562 249– form PTO-1449). Van Duuren (EP 2 562 249– form PTO-1449) discloses that 3-FB is toxic when converted by the benzoate pathway (encoded by ben and cat) ([0076]). However, any Pseudomonas cell capable of producing 2-FMA from 3-FB or 500 mg/L of 2-FMA by the expression of any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I is modulated via promoters and translation initiation sequences/couplers was not known.
The specification is limited to a genetically modified Pseudomonas putida expressing (1) a benzoate 1,2-dioxygeanse comprising the subunits BenABC, wherein BenACB has the amino acid sequence of SEQ ID NO:1-3, respectively, (2) a benzoate-1,2-dihydrodiol dehydrogenase BenD having the amino acid sequence of SEQ ID NO:7, and (3) a catechol 1,2-dioxygenase CatA-I having the amino acid sequence of SEQ ID NO:9 and/or CatA-II having the amino acid sequence of SEQ ID NO:11, wherein (4) expression of the benzoate 1,2-dixoygenase and the benzoate-1,2-dihydrodiol dehydrogenase are under control of the promoter PTac of SEQ ID NO:14, (5) the expression of the catechol 1,2-dioxygenase is under control of a inducible promoter p14b of SEQ ID NO:49 and a translation initiation sequence/coupler BCD2 of SEQ ID NO:22 or BCD10 of SEQ ID NO:16, and (6) the genetically modified Pseudomonas putida converts 3-FB into 2-FMA . While MPEP 2163 acknowledges that in certain situations “one species adequately supports a genus,” it also acknowledges that “[f]or inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.” In view of the widely variant species encompassed by the genus, the example described above is not enough and does not constitute a representative number of species to describe the whole genus. Therefore, the specification fails to describe a representative species of the claimed genus.
The claimed invention requires a defined set of Pseudomonas cells, benzoate 1,2-dixoygenases, benzoate-1,2-dihydrodiol dehydrogenases, catechol 1,2-dioxygenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or couplers. Although the specification discloses exemplary Pseudomonas cells, benzoate 1,2-dixoygenases, the benzoate-1,2-dihydrodiol dehydrogenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or coupler, a “laundry list” disclosure of every possible moiety does not necessarily constitute a written description of every species in a genus because it would not “reasonably lead” those skilled in the art to any particular species, see Fujikawa v. Wattanasin, 93 F.3d 1559, 1571, 39 USPQ2d 1895, 1905 (Fed. Cir. 1996) or MPEP 2163. While the exemplary Pseudomonas cells, benzoate 1,2-dixoygenases, the benzoate-1,2-dihydrodiol dehydrogenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or coupler were known in the art, this knowledge alone would not allow one level of skill in the art to immediately envisage the claimed genus. Therefore, the level of skill and knowledge in the art is such that one of ordinary skill would not be able to identify without further testing which combination of Pseudomonas cells, benzoate 1,2-dixoygenases, the benzoate-1,2-dihydrodiol dehydrogenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or coupler to use to arrive at the claimed invention.
One of skill in the art could identify variants having 85% sequence identity to SEQ ID NO:1-3, 7, or 9. However, there is no teaching regarding which amino acids of SEQ ID NO:1-3, 7, or 9 can vary and result in a polypeptide having benzoate 1,2-dixoygenase activity, benzoate-1,2-dihydrodiol dehydrogenase activity, and catechol 1,2-dioxygenase activity, respectively, wherein expression of the benzoate 1,2-dixoygenase, benzoate-1,2-dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase under the control of the above variant promoters and translation initiation sequence/couplers results in any Pseudomonas cell capable of producing 2-FMA from 3-FB or producing 500 mg/L of 2-FMA.
Regarding claim 6 and 32, one of skill in the art could identify variants having
at least 85 % sequence identity to SEQ ID NO:16 or 49. However, there is no teaching regarding which nucleic acids of SEQ ID NO:16 or 49 can vary and result in a nucleotide sequence having the function of a translation initiation sequence/coupler or a medium-to-weak promoter, result in the expression of the benzoate 1,2-dixoygenase, benzoate-1,2-dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase, and result in
any Pseudomonas cell capable of producing 2-FMA from 3-FB.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – cited previously on form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – cited previously on form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
An important consideration is that structure is not necessarily a reliable indicator of function. In the instant case, there is no disclosure relating similarity of structure to conservation of function. Conservation of structure is not necessarily a surrogate for conservation of function.
While general knowledge in the art may have allowed one of skill in the art to identify other variants expected to have the same or similar tertiary structure, there was no general knowledge in the art in the ability of any Pseudomonas cell producing 2-FMA or 500 mg/L of 2-FMA from 3-FB expressing benzoate 1,2-dixoygenase variants of SEQ ID NO:1-3 under the control of medium-strong constitutive promoter, expressing benzoate-1,2-dihydrodiol dehydrogenase variants of SEQ ID NO:7, and expressing catechol 1,2-dioxygenase variants of SEQ ID NO:9 under the control of medium-to-weak constitutive promoter variants of SEQ ID NO:49.
Given this lack of description of the representative species encompassed by the genus of the claims, the specification fails to sufficiently describe the claimed invention in such full, clear, concise, and exact terms that a skilled artisan would recognize that applicants were in possession of the inventions of claims 1-2, 6, 10, and 32
Applicant's arguments filed February 20, 2026 have been fully considered but they are not persuasive.
Applicant argues that the specification provides sufficient description of the newly amended claims because claim 1 has been amended to disclose sequences for the recited enzymes in claim 1, there is mapping between specific sequences and functional classes, and a person of ordinary skill in the art would readily recognize that the exemplified species are equally attributable to other species of Pseudomonas beside P. putida.
This is not found persuasive.
The claims are directed to any Pseudomonas expressing a genus of BenABC, BenC, and CatA-I and genus of promoters, strong translation initiation sequences, wherein the Pseudomonas produces 2-FMA from 3-FB or 500 mg/L of 2-FMA. Van Duuren (EP 2 562 249– form PTO-1449) discloses that 3-FB is toxic when converted by the benzoate pathway (encoded by ben and cat) ([0076]). However, any Pseudomonas cell capable of producing 2-FMA from 3-FB or 500 mg/L of 2-FMA by the expression of any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I is modulated via promoters and translation initiation sequences/couplers was not known. One of skill in the art could identify variants having 85% sequence identity to SEQ ID NO:1-3, 7, or 9. However, there is no teaching regarding which amino acids of SEQ ID NO:1-3, 7, or 9 can vary and result in a polypeptide having benzoate 1,2-dixoygenase activity, benzoate-1,2-dihydrodiol dehydrogenase activity, and catechol 1,2-dioxygenase activity, respectively, wherein expression of the benzoate 1,2-dixoygenase, benzoate-1,2-dihydrodiol dehydrogenase, and catechol 1,2-dioxygenase under the control of the above variant promoters and translation initiation sequence/couplers results in any Pseudomonas cell capable of producing 2-FMA from 3-FB or producing 500 mg/L of 2-FMA.
The claimed invention requires a defined set of Pseudomonas cells, benzoate 1,2-dixoygenases, benzoate-1,2-dihydrodiol dehydrogenases, catechol 1,2-dioxygenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or couplers. Although the specification discloses exemplary Pseudomonas cells, benzoate 1,2-dixoygenases, the benzoate-1,2-dihydrodiol dehydrogenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or coupler, a “laundry list” disclosure of every possible moiety does not necessarily constitute a written description of every species in a genus because it would not “reasonably lead” those skilled in the art to any particular species, see Fujikawa v. Wattanasin, 93 F.3d 1559, 1571, 39 USPQ2d 1895, 1905 (Fed. Cir. 1996) or MPEP 2163. While the exemplary Pseudomonas cells, benzoate 1,2-dixoygenases, the benzoate-1,2-dihydrodiol dehydrogenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or coupler were known in the art, this knowledge alone would not allow one level of skill in the art to immediately envisage the claimed genus. Therefore, the level of skill and knowledge in the art is such that one of ordinary skill would not be able to identify without further testing which combination of Pseudomonas cells, benzoate 1,2-dixoygenases, the benzoate-1,2-dihydrodiol dehydrogenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or coupler to use to arrive at the claimed invention.
Hence the rejection has been maintained.
Claims 1-2, 6, 10, and 32 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a genetically modified Pseudomonas putida expressing (1) a benzoate 1,2-dioxygeanse comprising the subunits BenABC, wherein BenACB has the amino acid sequence of SEQ ID NO:1-3, respectively, (2) a benzoate-1,2-dihydrodiol dehydrogenase BenD having the amino acid sequence of SEQ ID NO:7, and (3) a catechol 1,2-dioxygenase CatA-I having the amino acid sequence of SEQ ID NO:9 and/or CatA-II having the amino acid sequence of SEQ ID NO:11, wherein (4) expression of the benzoate 1,2-dixoygenase and the benzoate-1,2-dihydrodiol dehydrogenase are under control of the promoter PTac of SEQ ID NO:14, (5) the expression of the catechol 1,2-dioxygenase is under control of a inducible promoter p14b of SEQ ID NO:49 and a translation initiation sequence/coupler BCD2 of SEQ ID NO:22 or BCD10 of SEQ ID NO:16, and (6) the genetically modified Pseudomonas putida converts 3-FB into 2-FMA, does not reasonably provide enablement for any Pseudomonas cell capable of producing 2-FMA or 500 mg/L of 2-FMA from 3-FB. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
The breadth of the claims.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' Therefore, the claims have been broadly interpreted to encompass (A) any Pseudomonas cell capable of producing 2-fluoro-cis-cis-muconate (2-FMA) or at least 500 mg/L of 2-FMA from 3-fluorobenzoate (3-FB), wherein the cell expresses (B) any benzoate 1,2-dioxygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, (C) any benzoate 1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7 and (D) any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein (E) expression of the benzoate 1,2-dioxygenase is under the control of any medium-strong constitutive promoter, and (F) expression of the catechol 1,2-dioxygeanse is under the control of a first promoter expression module comprising any first promoter or a medium-to -weak constitutive promoter P14b or a variant thereof having at least 85% sequence identity to SEQ ID NO:49 and any first strong translational initiation sequence, any strong translation coupler, any first strong translational initiation sequence that has the same the translational initiation strength as BCD10 of SEQ ID NO:16, or a BCD10 or variant thereof having at least 80% sequence identity to the SEQ ID NO:16.
The claim is not commensurate with the enablement provided by the disclosure with regard to the extremely large number of Pseudomonas cells, benzoate 1,2-dixoygenases, benzoate-1,2-dihydrodiol dehydrogenases, catechol 1,2-dioxygenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or couplers. The specification is limited to a genetically modified Pseudomonas putida expressing (1) a benzoate 1,2-dioxygeanse comprising the subunits BenABC, wherein BenACB has the amino acid sequence of SEQ ID NO:1-3, respectively, (2) a benzoate-1,2-dihydrodiol dehydrogenase BenD having the amino acid sequence of SEQ ID NO:7, and (3) a catechol 1,2-dioxygenase CatA-I having the amino acid sequence of SEQ ID NO:9 and/or CatA-II having the amino acid sequence of SEQ ID NO:11, wherein (4) expression of the benzoate 1,2-dixoygenase and the benzoate-1,2-dihydrodiol dehydrogenase are under control of the promoter PTac of SEQ ID NO:14, (5) the expression of the catechol 1,2-dioxygenase is under control of a inducible promoter p14b of SEQ ID NO:49 and a translation initiation sequence/coupler BCD2 of SEQ ID NO:22 or BCD10 of SEQ ID NO:16, and (6) the genetically modified Pseudomonas putida converts 3-FB into 2-FMA.
The quantity of experimentation required to practice the claimed invention based on the teachings of the specification.
While enzyme isolation techniques, recombinant and mutagenesis techniques were known in the art at the time of the invention, e.g. mutagenesis, and it is routine in the art to screen for variants comprising multiple substitutions or multiple modifications as encompassed by the instant claims, the specific amino acid positions within the protein's sequence where amino acid modifications can be made with a reasonable expectation of success in obtaining the desired activity/utility are limited in any protein and the result of such modifications is unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g. multiple substitutions.
In the absence of: (a) rational and predictable scheme for producing Pseudomonas cell producing 2-FMA or 500 mg/L of 2-FMA from 3-FB by expressing any benzoate 1,2-dixoygenases under the control of any medium-strong constitutive promoter, expressing any benzoate-1,2-dihydrodiol dehydrogenase, and expressing any catechol 1,2-dioxygenases under the control of a medium-to-weak constitutive promoter, translation initiation sequence, and/or couplers, (b) a correlation between structure and the function of a polypeptide having benzoate 1,2-dixoygenase activity, having benzoate-1,2-dihydrodiol dehydrogenase activity, and catechol 1,2-dioxygenase activity, and (c) a correlation between structure and the function of an oligonucleotide having the function of a medium-strong constitutive promoter, medium-to-weak constitutive promoter, translation initiation sequence, and/or couplers, a the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. One of skill in the art would have to test these infinite possible combination of Pseudomonas cells, benzoate 1,2-dixoygenases, benzoate-1,2-dihydrodiol dehydrogenases, catechol 1,2-dioxygenases, medium-strong constitutive promoters, medium-to-weak constitutive promoters, translation initiation sequences, and/or couplers to arrive at a Pseudomonas cell producing 2-FMA or 500 mg/L of 2-FMA from 3-FB. While enablement is not precluded by the necessity for routine screening, if a large amount of screening is required, as is the case herein, the specification must provide a reasonable amount of guidance which respect to the direction in which the experimentation should proceed so that a reasonable number of species can be selected for testing. In view of the fact that such guidance has not been provided in the instant specification, it would require undue experimentation to enable the full scope of the claims.
The state of prior art, the relative skill of those in the art, and predictability or unpredictability of the art.
The prior art discloses genetically modified P. putida producing cis-cis-muconate from benzoate, wherein said P. putida expresses a benzoate 1,2-dixoygenase encoded by benABC gene, benzoate-1,2-dihydrodiol dehydrogenase encoded by the benD gene, and catechol 1,2-dioxygenase encoded by the CatA, see Moreno (The target for the Pseudomonas putida Crc global regulator in the benzoate degradation pathway is the BenR transcriptional regulator. J Bacteriol. 2008 Mar;190(5):1539-45 – form PTO-892) and Van Duuren (EP 2 562 249– form PTO-1449). Van Duuren (EP 2 562 249– form PTO-1449) discloses that 3-FB is toxic when converted by the benzoate pathway (encoded by ben and cat) ([0076]). However, any Pseudomonas cell capable of producing 2-FMA from 3-FB or 500 mg/L of 2-FMA by the expression of any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I is modulated via promoters and translation initiation sequences/couplers was not known.
Since the amino acid sequence of the mutant determines its structural and functional properties, predictability of which changes can be tolerated in a protein's amino acid sequence and obtain the desired activity requires a knowledge of and guidance with regard to which amino acids in the protein's sequence, if any, are tolerant of modification and which are conserved (i.e. expectedly intolerant to modification), and detailed knowledge of the ways in which the proteins' structure relates to its function. In the instant case, neither the specification or the art provide a correlation between structure and activity such that one of skill in the art can envision the structure of any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I, or predict said function of a polypeptide from its primary structure. In addition, the art does not provide any teaching or guidance as to (1) which amino acids within the polypeptide of SEQ ID NO:1-3, 7, and 9 that can be modified and which ones are conserved such that one of skill in the art can make polypeptides having benzoate 1,2-dixoygenase activity, benzoate-1,2-dihydrodiol dehydrogenase activity, and catechol 1,2-dioxygenase activity, (2) which segments of the polypeptide of SEQ ID NO:1-3, 7, and 9 are essential for having the function of having benzoate 1,2-dixoygenase activity, benzoate-1,2-dihydrodiol dehydrogenase activity, and catechol 1,2-dioxygenase activity and (3) the general tolerance of the polypeptide of SEQ ID NO:1-3, 7, and 9 to structural modifications and the extent of such tolerance. The art clearly teaches that changes in a protein's amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are required for that activity is highly unpredictable. At the time of the invention there was a high level of unpredictability associated with altering a polypeptide sequence with an expectation that the polypeptide will maintain the desired activity. For example, Studer (Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes. Biochem. J. (2013) 449, 581–594. – cited previously on form PTO-892) teach that (1) protein engineers are frequently surprised by the range of effects caused by single mutations that they hoped would change only one specific and simple property in enzymes, (2) the often surprising results obtained by experiments where single mutations are made reveal how little is known about the rules of protein stability, and (3) the difficulties in designing de novo stable proteins with specific functions.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – cited previously on form PTO-892) reviews protein engineering techniques, such as random mutagenesis and recombination, directed evolution and iterative or combinatory saturation “hotspots”. Fransceus states that “a recurring problem, however, is choosing which amino acid positions should be targeted. Answering this question is not an easy feat and requires substantial insight in the relationship between an enzyme’s sequence or structure and its properties.” Sanavia (Computational and Structural Biotechnology Journal, Volume 18, 2020, Pages 1968-1979. – cited previously on form PTO-892) discloses challenges in the prediction of protein stability in the occurrence of multiple mutations. “Multiple-point mutations are common variations of the protein sequence that may be needed in protein engineering when a single-point mutation is not enough to yield the desired stability change. Dealing with multiple-site variations adds another level of complexity beyond the prediction of the effect of a single variant on protein stability, since it requires the learning of many types of combinatorial effects”.
The amount of direction or guidance presented and the existence of working examples.
The specification is limited to a genetically modified Pseudomonas putida expressing (1) a benzoate 1,2-dioxygeanse comprising the subunits BenABC, wherein BenACB has the amino acid sequence of SEQ ID NO:1-3, respectively, (2) a benzoate-1,2-dihydrodiol dehydrogenase BenD having the amino acid sequence of SEQ ID NO:7, and (3) a catechol 1,2-dioxygenase CatA-I having the amino acid sequence of SEQ ID NO:9 and/or CatA-II having the amino acid sequence of SEQ ID NO:11, wherein (4) expression of the benzoate 1,2-dixoygenase and the benzoate-1,2-dihydrodiol dehydrogenase are under control of the promoter PTac of SEQ ID NO:14, (5) the expression of the catechol 1,2-dioxygenase is under control of a inducible promoter p14b of SEQ ID NO:49 and a translation initiation sequence/coupler BCD2 of SEQ ID NO:22 or BCD10 of SEQ ID NO:16, and (6) the genetically modified Pseudomonas putida converts 3-FB into 2-FMA. However, the specification fails to provide any information as to the structural elements required in polypeptide having any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I nor structural elements required in oligonucleotides having the function of a medium-strong constitutive promoter, medium-to-weak constitutive promoter, translation initiation sequence, and/or couplers.
Thus, in view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability of the prior art in regard to structural changes and their effect on function and the lack of knowledge about a correlation between structure and function, an undue experimentation would be necessary one having ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of polypeptides having the desired biological characteristics recited in the claims are unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
Applicant's arguments filed February 20, 2026 have been fully considered but they are not persuasive.
Applicant argues that the specification (pages 23-26) provides multiple working examples, quantitative promoters/BCD strength data, clear guidance on expression balancing to avoid 3-fluorocatechol buildup, and explicit construct that achieved the desired conversion of 3-flurobenzoate to 2-fluoro-cis,cis-muconate, including titer thresholds, and guidance for balancing ben/cat flux to avoid toxic intermediates.
This is not found persuasive.
Pages 23-26 of the specification discloses a general list of promoters and translation initiation sequences. The specification does not provide any information as to the structural elements required in oligonucleotides having the function of a medium-strong constitutive promoter, medium-to-weak constitutive promoter, translation initiation sequence, and/or couplers, wherein the usage of the promoters, translation initiation sequence, and couplers in expressing the enzymes recited in claim 1 avoids 3-fluorocatechol buildup, and explicit construct that achieved in the desired conversion of 3-flurobenzoate to 2-fluoro-cis,cis-muconate, including titer thresholds, and guidance for balancing ben/cat flux to avoid toxic intermediates. Further, the specification fails to provide guidance on a rational and predictable scheme for producing Pseudomonas cell producing 2-FMA or 500 mg/L of 2-FMA from 3-FB by expressing any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I under the control of a medium-to-weak constitutive promoter, translation initiation sequence, and/or couplers. The specification fails to provide any information as to the structural elements required in polypeptide having any benzoate 1,2-dixoygenase BenABC having at least 85% sequence identity to SEQ ID NO:1-3, respectively, any benzoate-1,2-dihydrodiol dehydrogenase BenD having at least 85% sequence identity to SEQ ID NO:7, and any catechol 1,2-dioxygenase CatA-I having at least 85% sequence identity to SEQ ID NO:9, wherein expression of the benzoate 1,2-dixoygenase BenABC, benzoate-1,2-dihydrodiol dehydrogenase BenD, and catechol 1,2-dioxygenase CatA-I nor structural elements required in oligonucleotides having the function of a medium-strong constitutive promoter, medium-to-weak constitutive promoter, translation initiation sequence, and/or couplers.
Hence the rejection has been maintained.
Claim Rejections - 35 USC § 103
Applicant’s arguments, see pages 13-15 of the Remarks, filed February 20, 2026, with respect to claims 1-2, 6, 29, and 32 have been fully considered and are persuasive. The rejection of claims 1-2, 6, and 32 under 35 U.S.C. 103 as being unpatentable over Van Duuren (EP 2 562 249– form PTO-1449) and Zobel (Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. ACS Synthetic Biology 2015 4 (12), 1341-1351 – form PTO-1449) has been withdrawn.
Applicant’s arguments, see page 15 of the Remarks, filed February 20, 2026, with respect to claims 1-2, 6, 10, 27-29, and 32 have been fully considered and are persuasive. The rejection of claims 1-2, 6, 10, 27-29, and 32 under 35 U.S.C. 103 as being unpatentable over Van Duuren (EP 2 562 249– form PTO-1449), Zobel (Tn7-Based Device for Calibrated Heterologous Gene Expression in Pseudomonas putida. ACS Synthetic Biology 2015 4 (12), 1341-1351 – form PTO-1449), Q88I40_PSEPK (UniProtKB/TrEMBL Database. May 8, 2019 – form PTO-892), Q88I39_PSEPK (UniProtKB/TrEMBL Database. May 8, 2019 – form PTO-892), Q88I38_PSEPK (UniProtKB/TrEMBL Database. May 8, 2019 – form PTO-892), and Q88I37_PSEPK (UniProtKB/TrEMBL Database. June 5, 2019 – form PTO-892) has been withdrawn.
Other Relevant Art
Wirth (Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida. Chem Catal. 2021 Nov 18;1(6):1234-1259 – form PTO-1449) discloses a genetically modified Pseudomonas putida producing 2-FMA from 3-FB, wherein the Pseudomonas putida expresses benzoate 1,2-dixoygenase encoded by benABC under the control of the promoter pTac, benzoate-1,2-dihydrodiol dehydrogenase encoded by benD, and catechol 1,2-dioxygenase encoded by catA under the control of the constitutive promoter p14b and BCD10 (bottom of page 1240 through page 1241), but is not available as prior art because the reference was published or made known to the public after the instant invention was effectively filed.
Conclusion
Claims 1-4, 6, 10, 12-13, 17-18, 26, and 31-36 are pending.
Claims 3-4, 12-13, 17-18, 26, 31, and 33-36 are withdrawn.
Claims 1-2, 6, 10, and 32 are rejected.
THIS ACTION IS MADE FINAL. 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.
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/YONG D PAK/Primary Examiner, Art Unit 1652
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