MICROORGANISMS AND METHODS FOR PRODUCING CIS,CIS-MUCONIC ACID

Application No. 18/891,498

• Filed: Sep 20, 2024
• Priority: Sep 21, 2023 — provisional 63/584,362
• Examiner: PAK, YONG D
• Assignee: Wisconsin Alumni Research Foundation
• OA Round: 1 (Non-Final)

Office Action

Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .

DETAILED ACTION

Status of Claims
Claims 1-20 are pending.
Claims 1-20 are under examination.

Claim for Domestic Priority
Applicants' claim for domestic priority under 35 USC 119(e) to US provisional application 63/584,36, filed 09/21/2023, is acknowledged.

Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.

The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.

Claims 2 and 4 and claims 5-16 depending therefrom are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claims 2 and 4 recite the broad recitation “at least 80%” and the claim also recites “at least 99%” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.

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 1-20 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.'' In the instant case, the claims have been broadly interpreted to encompass (A) any microorganism, any phenol-degrading bacterium, or any Novosphigobium comprising one or more modifications of (B) (i) any modification that increases any flavin prenyltransferase activity or the flavin prenyltransferase activity of a flavin prenyltransferase having at least 80% or 95% sequence identity to SEQ ID NO:2, (ii) any modification that increases any protocatechuate decarboxylase activity or the protocatechuate decarboxylase activity of a protocatechuate decarboxylase having at least 80% or 95% sequence identity to SEQ ID NO:4, (iii) any recombinant protocatechuate decarboxylase D gene encoding any protocatechuate decarboxylase D prenyltransferase or a protocatechuate decarboxylase D having at least 80% or 95% sequence identity to SEQ ID NO:6, (iv) any modification that increases any catechol 1,2-dioxygenase activity or the catechol 1,2-dioxygenase activity of a catechol 1,2-dioxygenase having at least 80% or 95% sequence identity to SEQ ID NO:8, (v) any modification that decreases any muconate lactonizing enzyme activity or the muconate lactonizing enzyme activity of a muconate lactonizing enzyme having at least 80% or 95% sequence identity to SEQ ID NO:10, (vi) any modification that decreases any muconolactone isomerase activity or the muconolactone isomerase activity of a muconolactone isomerase having at least 80% or 95% sequence identity to SEQ ID NO:12, (vii) any modification that decreases any catechol 2,3-dioxygenase activity or the catechol 2,3-dioxygenase activity of a catechol 2,3-dioxygenase having at least 80% or 95% sequence identity to SEQ ID NO:14, and/or (viii) any modification that decreases any protocatechuate 4,5-dioxgyenase activity or the protocatechuate 4,5-dioxgyenase activity of a protocatechuate 4,5-dioxgyenase having at least 80% or 95% sequence identity to SEQ ID NO:16, and (C) a method of producing cis-cis-muconic acid with said microorganism.
Thus, the claims are drawn to a genus comprising of (A) any microorganism, any phenol-degrading bacterium, or any Novosphigobium comprising one or more modifications of (B) (i) genus of modifications that increase flavin prenyltransferase activity of flavin prenyltransferase having unknown structure or a flavin prenyltransferase having at least 80% or 95% sequence identity to SEQ ID NO:2, (ii) genus of modifications that increase protocatechuate decarboxylase activity or a protocatechuate decarboxylase having unknown structure or a protocatechuate decarboxylase having at least 80% or 95% sequence identity to SEQ ID NO:4, (iii) genus of recombinant protocatechuate decarboxylase D gene encoding any protocatechuate decarboxylase D prenyltransferase having unknown structure or a protocatechuate decarboxylase D having at least 80% or 95% sequence identity to SEQ ID NO:6, (iv) genus of modifications that increase catechol 1,2-dioxygenase activity of a catechol 1,2-dioxygenase having unknown structure or a catechol 1,2-dioxygenase having at least 80% or 95% sequence identity to SEQ ID NO:8, (v) genus of modifications that decrease muconate lactonizing enzyme activity of a muconate lactonizing enzyme having unknown structure or muconate lactonizing enzyme having at least 80% or 95% sequence identity to SEQ ID NO:10, (vi) genus of modifications that decrease muconolactone isomerase activity of a muconolactone isomerase having unknown structure or a muconolactone isomerase having at least 80% or 95% sequence identity to SEQ ID NO:12, (vii) genus of modifications that decrease any catechol 2,3-dioxygenase activity of a catechol 2,3-dioxygenase having unknown structure or a catechol 2,3-dioxygenase having at least 80% or 95% sequence identity to SEQ ID NO:14, and/or (viii) genus of modifications that decrease protocatechuate 4,5-dioxgyenase activity of a protocatechuate 4,5-dioxgyenase having unknown structure or a protocatechuate 4,5-dioxgyenase having at least 80% or 95% sequence identity to SEQ ID NO:16, and (C) a method of producing cis-cis-muconic acid with said genus of microorganisms.
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 “flavin prenyltransferase activity”, “protocatechuate decarboxylase activity”, “protocatechuate decarboxylase D”, and “catechol 1,2-dioxygenase” fail to provide a sufficient description of the genus of the enzymes comprised in the genus of microorganism 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 specifically define any of the species that fall 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.
Further, genes encoding muconate lactonizing enzyme, muconolactone isomerase, catechol 2,3-dioxygenae, and protocatechuate 4,5-dioxgyenase must be known in order to decreased their activities in any microorganism, any phenol-degrading bacterium, or any Novosphigobium. The recitation of muconate lactonizing enzyme, muconolactone isomerase, catechol 2,3-dioxygenae, and protocatechuate 4,5-dioxgyenase fails to provide a sufficient description of the genus of genes/encoded proteins as it does not provide any definition of the structural or functional features of the species within the genus. The specification does not specifically define any of the species that fall within the genus. The specification does not define any functional or 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.
Production of cis,cis-muconic acid is limited to recombinant P. putida and Novosphingobium aromaticivorans, see Sonoki (US 2021/0371885 – form PTO-892), Johnson (Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity. Metab Eng Commun. 2016 Apr 22;3:111-119 – form PTO-892), and Perez (Expanding the frontiers of plants utilization: The metabolic power of Novosphingobium aromaticivorans for lignin valorization. UNIVERSITY OF WISCONSIN-MADISON. 2020 – form PTO-892). However, the claimed genus of any microorganism, any phenolic-degrading bacterium, any Novosphingobium comprising the inserted/deleted enzymes recited in claim 1 were not known in the art.
The specification is limited to one example, a recombinant Novosphingobium aromaticivorans DSM12444 expressing (i) flavin prenyltransferase having the amino acid sequence of SEQ ID NO:2, (ii) protocatechuate decarboxylase having the amino acid sequence of SEQ ID NO:4, (iii) protocatechuate decarboxylase D having the amino acid sequence of SEQ ID NO:6, and (iv) catechol 1,2-dioxygenase having the amino acid sequence of SEQ ID NO:8, and deletion of genes encoding the muconate lactonizing enzyme having the amino acid sequence of SEQ ID NO:10, muconolactone isomerase having the amino acid sequence of SEQ ID NO:12, catechol 2,3-dioxygenase having the amino acid sequence of SEQ ID NO:14, and protocatechuate 4,5-dioxgyenase having the amino acid sequence of SEQ ID NO:16, wherein said recombinant Novosphingobium aromaticivorans produces cis-cis-muconic acid. 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 one 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 specification does not provide an actual reduction to practice of the genus because the specification fails to disclose the structure of the genes encoding muconate lactonizing enzyme, muconolactone isomerase, catechol 2,3-dioxygenae, and protocatechuate 4,5-dioxgyenase from non-Novosphingobium aromaticivorans, which must be known in order to decrease the activity of the encoded enzymes in any microorganism, any phenol-degrading bacterium, or any Novosphingobium. The specification does not disclose the isolation or cloning of any non- Novosphingobium aromaticivorans genes ecoding muconate lactonizing enzyme, muconolactone isomerase, catechol 2,3-dioxygenae, and protocatechuate 4,5-dioxgyenase. Because an Novosphingobium aromaticivorans comprising decreased activity of genes encoding muconate lactonizing enzyme having the amino acid sequence of SEQ ID NO:10, muconolactone isomerase having the amino acid sequence of SEQ ID NO:12, catechol 2,3-dioxygenase having the amino acid sequence of SEQ ID NO:14, and protocatechuate 4,5-dioxgyenase having the amino acid sequence of SEQ ID NO:16 is not representative of the entire genus any microorganism, any phenol-degrading bacterium, or any Novosphingobium, and the specification does not disclose structural features shared by members of the genus, the description of the above recombinant Novosphingobium aromaticivorans would not have put the application in possession of the common structural attributes or features shared by members of the genus that structurally distinguish the members of the genus from other materials at the time of filing.
The claimed invention requires a defined set of (A) microorganism, any phenol-degrading bacterium, or any Novosphingobium and (B) increased activities of (i) flavin prenyltransferase, (ii) protocatechuate decarboxylase, (iii) protocatechuate decarboxylase and (iv) catechol 1,2-dioxygenase, and decreased activities of (v) muconate lactonizing enzyme, (vi) muconolactone isomerase, (vii) catechol 2,3-dioxygenase, and (viii) protocatechuate 4,5-dioxgyenase. Although the specification discloses exemplary microorganism and enzymes, 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 microorganism and enzymes 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 the microorganism and enzymes to arrive at the claimed invention.
Further, one of skill in the art could identify polypeptides having at least 80-95% sequence identity to SEQ ID NO:2, 4, 6, and 8. However, there is no teaching regarding which 5-20% of the amino acids can vary from SEQ ID NO:2, 4, 6, and 8 and result in a polypeptide having (i) flavin prenyltransferase activity, (ii) protocatechuate decarboxylase activity, (iii) protocatechuate decarboxylase activity and (iv) catechol 1,2-dioxygenase activity, respectively.
Fransceus (J Ind Microbiol Biotechnol. 2017 May;44(4-5):687-695. – 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. – 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.
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 1-20.

Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –

(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.

Claim(s) 1, 17, and 19 is/are rejected under 35 U.S.C. 102(a)(1) and/or as being anticipated by Johnson (Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity. Metab Eng Commun. 2016 Apr 22;3:111-119 – form PTO-892).
Regarding claim 1, Johnson discloses recombinant Pseudomonas putida expressing protocatechuate decarboxylase (PCA decarboxylase) and catechol 1,2-dioxygenase (CatA) driven by promoters, which encompasses increased activities of said PCA decarboxylase and CatA, and deletions of the gene encoding muconate lactonizing enzyme (CatB) and the gene encoding muconolactone isomerase (CatC), which encompasses decreased activities of said CatBC (Abstract, Fig. 1 at page 112, and pages 114-116).
Regarding claims 17 and 19, Johnson discloses a method of producing cis,cis-muconic acid by culturing the recombinant Pseudomonas putida in a medium comprising p-coumarate, a plant derived phenolic compound and depolymerized lignin from plant (3rd full paragraph at page 114).
Therefore, the reference of Johnson anticipates claims 1, 17, and 19.

Claim(s) 1 and 17-20 is/are rejected under 35 U.S.C. 102(a)(1) and/or as being anticipated by Sonoki (US 2021/0371885 – form PTO-892).
Regarding claim 1, Sonoki discloses recombinant Pseudomonas putida overexpressing protocatechuate decarboxylase (PCA decarboxylase) encoded by the aroY gene and overexpressing catechol 1,2-dioxygenase (CatA), which encompasses increased activities of said PCA decarboxylase and CatA, and deletions of the gene encoding muconate lactonizing enzyme (CatB) and the gene encoding muconolactone isomerase (CatC), which encompasses decreased activities of said CatBC (claims 1-13 and [0082]-[0113]).
Regarding claims 17-19, Sonoki discloses a method of producing cis-cis-muconic acid by culturing the recombinant Pseudomonas putida in a medium comprising syringyl lignin, a plant derived phenolic compound and depolymerized lignin from plant (claim 13).
Regarding claim 20, Sonoki discloses obtaining cis-cis-muconic acid, which encompasses isolating the cis-cis-muconic acid (claim 13).
Therefore, the reference of Sonoki anticipates claims 1 and 17-20.

Claim(s) 1 is/are rejected under 35 U.S.C. 102(a)(1) and/or as being anticipated by Perez (Expanding the frontiers of plants utilization: The metabolic power of Novosphingobium aromaticivorans for lignin valorization. UNIVERSITY OF WISCONSIN-MADISON. 2020 – form PTO-892)
Regarding claim 1, Perez discloses a recombinant Novosphingobium aromaticivorans DSM12444 having a deletion of its ligAB, protocatechuate 4,5-dioxygenase (Table 4.3 at page 112 and Figure 4.11 at page 120).
Therefore, the reference of Perez anticipates claim 1.

Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.

The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.

Claims 1-6 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sonoki (US 2021/0371885 – form PTO-892), Johnson (Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity. Metab Eng Commun. 2016 Apr 22;3:111-119 – form PTO-892), Perez (Expanding the frontiers of plants utilization: The metabolic power of Novosphingobium aromaticivorans for lignin valorization. UNIVERSITY OF WISCONSIN-MADISON. 2020 – form PTO-892), A4XDV7 (UnitProtKB/TrEMBL Database. August 3, 2022 – form PTO-892), A4XDW1 (UnitProtKB/TrEMBL Database. June 2, 2021 – form PTO-892), A4XDW2 (UnitProtKB/TrEMBL Database. May 3, 2023 – form PTO-892), and A4XFH8 (UnitProtKB/TrEMBL Database. May 25, 2022 – form PTO-892).
Regarding claims 1 and 4, Sonoki discloses recombinant Pseudomonas putida overexpressing protocatechuate decarboxylase (PCA decarboxylase) encoded by the aroY gene and overexpressing catechol 1,2-dioxygenase (CatA), which encompasses increased activities of said PCA decarboxylase and CatA, and deletions of the gene encoding muconate lactonizing enzyme (CatB) and the gene encoding muconolactone isomerase (CatC), which encompasses decreased activities of said CatBC, wherein said P. putida produces cis,cis-muconic acid (claims 1-13 and [0082]-[0113]).
Regarding claims 5-6, Sonoki discloses that Novosphingobium aromaticivorans can be used as an alternative to P. putida ([0083]).
Regarding claims 1 and 4, Johnson also discloses recombinant Pseudomonas putida expressing protocatechuate decarboxylase (PCA decarboxylase) and catechol 1,2-dioxygenase (CatA) driven by promoters, which encompasses increased activities of said PCA decarboxylase and CatA, and deletions of the gene encoding muconate lactonizing enzyme (CatB) and the gene encoding muconolactone isomerase (CatC), which encompasses decreased activities of said CatBC (Abstract, Fig. 1 at page 112, and pages 114-116).
Sonoki and Johnson do not disclose Novosphingobium aromaticivorans strain 1244 having increased activities or overexpression of (i) Novosphingobium aromaticivorans flavin prenyltransferase having the amino acid sequence of SEQ ID NO:2, (ii) Novosphingobium aromaticivorans protocatechuate (PCA) decarboxylase having the amino acid sequence of SEQ ID NO:4, (iii) Novosphingobium aromaticivorans protocatechuate decarboxylase D having the amino acid sequence of SEQ ID NO:6, (iv) Novosphingobium aromaticivorans catechol 1,2-dioxygenase having the amino acid sequence of SEQ ID NO:8, and deletion of protecatechuate 4,5-dioxygenase.
However, Sonoki discloses that it is desirable that the organism of origin from which the inserted gene is derived is a host organism to be transformed by the insertion of the gene ([00084]). Johnson discloses additional metabolic engineering to increase the flux of carbon to muconate and industrially-relevant levels of production (last full paragraph at page 116).
Regarding claim 1, Perez discloses a recombinant Novosphingobium aromaticivorans DSM12444 having a deletion of its ligAB, protocatechuate 4,5-dioxygenase (Table 4.3 at page 112 and Figure 4.11 at page 120).
Regarding claims 5-6, Perez discloses that Novosphingobium aromaticivorans DSM 12444 has an advantage over P. putida in producing cis,cis-muconic acid because Novosphingobium aromaticivorans has a wider range of aromatic substrate compared to P. putida (Section 1.6 at page 8). Perez discloses genetic modification of Novosphingobium aromaticivorans DSM 12444 for expressing genes (Section 4.5 at pages 127-129).
Regarding the flavin prenyltransferase of claims 1-3, Johnson discloses that flavin prenyltransferase is responsible for the production of a prenylated-FMN co-factor required by UbiD, homologous to the PCA decarboxylase (1st full paragraph at page 117). A4XDV7 discloses Novosphingobium aromaticivorans DSM 12444 flavin prenyltransferase having 100% sequence identity to the flavin prenyltransferase having the amino acid sequence of SEQ ID NO:2 (pages 1-2 and see the sequence alignment below).
Regarding the PCA decarboxylase of claims 1-3, A4XDW1 discloses Novosphingobium aromaticivorans DSM 12444 UbiD family decarboxylase, a PCA decarboxylase, having 100% sequence identity to the flavin prenyltransferase having the amino acid sequence of SEQ ID NO:4 (page 1 and see the sequence alignment below).
Regarding the PCA decarboxylase D gene of claims 1-3, A4XDW2 discloses a gene encoding Novosphingobium aromaticivorans DSM 12444 phenolic acid decarboxylase subunit D, having 100% sequence identity to the PCA decarboxylase D having the amino acid sequence of SEQ ID NO:6 (page 1 and see the sequence alignment below). Johnson discloses that PCA decarboxylase is co-expressed as an operon with two other small genes shown to be important to activity of the PCA decarboxylase, which includes the subunit D (1st full paragraph at page 112).
Regarding the catechol 1,2-dioxygenase of claims 1-4, A4XFH8 discloses Novosphingobium aromaticivorans DSM 12444 catechol 1,2-dioxygenase (CatA) having 100% sequence identity to the catechol 1,2-dioxygenase having the amino acid sequence of SEQ ID NO:8 (pages 1-2 and see the sequence alignment below).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to modify Novosphingobium aromaticivorans DSM 12444 by increasing the activities of (i) Novosphingobium aromaticivorans DSM 12444 flavin prenyltransferase, (ii) Novosphingobium aromaticivorans DSM 12444 PCA decarboxylase, (iii) Novosphingobium aromaticivorans DSM 12444 protocatechuate decarboxylase D, and (iv) Novosphingobium aromaticivorans DSM 12444 catechol 1,2-dioxygenase. One having ordinary skill in the art would have been motivated to do so in order to increase production of cis,cis-muconic acid. One having ordinary skill in the art would have had a reasonable expectation of success because (A) Sonoki and Johnson disclose increasing activities of PCA decarboxylase and catechol 1,2-dioxygenase in a recombinant microorganism, (B) Perez discloses advantages of using Novosphingobium aromaticivorans DSM 12444 as a host microorganism, (C) Sonoki discloses inserting genes native to the host microorganism, (D) Johnson discloses additional metabolic engineering to increase the flux of carbon to meconate and industrially-relevant levels of production, (E) Johnson discloses that flavin prenyltransferase is responsible for the production of a prenylated-FMN co-factor required by UbiD, homologous to the PCA decarboxylase, (F) Johnson discloses that PCA decarboxylase is co-expressed as an operon with two other small genes shown to be important to activity of the PCA decarboxylase, which includes the subunit D, and (G) the enzymes having the amino acid sequences of SEQ ID NO:2, 4, 6, and 8 were known in the art.
Therefore, the above references render claims 1-6 prima facie obvious.

Claim(s) 7-16 is/are rejected under 35 U.S.C. 103 as being unpatentable over Sonoki (US 2021/0371885 – form PTO-892), Johnson (Enhancing muconic acid production from glucose and lignin-derived aromatic compounds via increased protocatechuate decarboxylase activity. Metab Eng Commun. 2016 Apr 22;3:111-119 – form PTO-892), Perez (Expanding the frontiers of plants utilization: The metabolic power of Novosphingobium aromaticivorans for lignin valorization. UNIVERSITY OF WISCONSIN-MADISON. 2020 – form PTO-892), A4XDV7 (UnitProtKB/TrEMBL Database. August 3, 2022 – form PTO-892), A4XDW1 (UnitProtKB/TrEMBL Database. June 2, 2021 – form PTO-892), A4XDW2 (UnitProtKB/TrEMBL Database. June 2, 2021 – form PTO-892), and A4XFH8 (UnitProtKB/TrEMBL Database. May 24, 2022 – form PTO-892) as applied to claims 1-6 above, and further in view of A4XDU1 (UniprotKB/TrEMBL Database. May 25, 2022 – form PTO-892), Q2G4H4 (UniprotKB/TrEMBL Database. May 25, 2022 – form PTO-892), A4XFH6 (UniprotKB/TrEMBL Database. August 3, 2022 – form PTO-892), and A4XFH7 (UniprotKB/TrEMBL Database. September 29, 2021 – form PTO-892).
In combined teachings of Sonoki, Johnson, Perez, A4XDV7, A4XDW1, A4XDW2, and A4XFH8, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to modify Novosphingobium aromaticivorans DSM 12444 by increasing the activities of (i) Novosphingobium aromaticivorans DSM 12444 flavin prenyltransferase of SEQ ID NO:2, (ii) Novosphingobium aromaticivorans DSM 12444 PCA decarboxylase of SEQ ID NO:4, (iii) Novosphingobium aromaticivorans DSM 12444 protocatechuate decarboxylase D of SEQ ID NO:6, (iv) Novosphingobium aromaticivorans DSM 12444 catechol 1,2-dioxygenaseNovosphingobium aromaticivorans DSM12444 of SEQ ID NO:4. Sonoki and Johnson disclose deletions of the gene encoding muconate lactonizing enzyme (CatB) and the gene encoding muconolactone isomerase (CatC), see above. Perez discloses a recombinant Novosphingobium aromaticivorans DSM12444 having a deletion of its ligAB, protocatechuate 4,5-dioxygenase, see above.
However, the above references do not disclose deleting (v) Novosphingobium aromaticivorans DSM12444 muconate lactonizing enzyme (CatB) having the amino acid sequence of SEQ ID NO:10, (vi) Novosphingobium aromaticivorans DSM12444 muconolactone isomerase (CatC) having the amino acid sequence of SEQ ID NO:12, (vii) Novosphingobium aromaticivorans DSM12444 catechol 2,3-dioxygenase having the amino acid sequence of SEQ ID NO:14, and (viii) Novosphingobium aromaticivorans DSM12444 protocatechuate 4,5-dioxgyenase (LigA) having the amino acid sequence of SEQ ID NO:16.
Regarding deletion of catechol 2,3-dioxygenase of claims 7 and 13, AXDU1 discloses Novosphingobium aromaticivorans DSM12444 catechol 2,3-dioxygenase having 100% sequence identity to the amino acid sequence of SEQ ID NO:14 (pages 1-2 and see the sequence alignment below). AXDU1 discloses that said catechol 2,3-dioxygenase converts catechol, precursor to cis-cis-muconic acid, to (2Z,4E)-2-hydroxy-6-oxohexa-2,4-dienoate (page 1). Therefore, catechol 2,3-dioxygenase directs the carbon flux away from cis,cis-muconic acid production.
Regarding deletion of protocatechuate 4,5-dioxygenase of claims 7 and 13, Q2G4H4 discloses Novosphingobium aromaticivorans DSM12444 protocatechuate 4,5-dioxygenase having 100% sequence identity to the amino acid sequence of SEQ ID NO:16 (page 1 and see the sequence alignment below). Perez discloses a recombinant Novosphingobium aromaticivorans DSM12444 having a deletion of its ligAB, protocatechuate 4,5-dioxygenase (Table 4.3 at page 112 and Figure 4.11 at page 120). Perez discloses that protocatechuate 4,5-dioxygenase converts protocatechuic acid, precursor to cis-cis-muconic acid, to CHMS (page 1). Therefore, protocatechuate 4,5-dioxygenase directs the carbon flux away from cis,cis-muconic acid production.
Regarding deletion of muconate lactonizing enzyme activity of claims 10, 13, and 16, A4XFH6 discloses Novosphingobium aromaticivorans DSM12444 muconate cycloisomerase, synonymous with muconate lactonizing enzyme (CatB), having 100% sequence identity to the amino acid sequence of SEQ ID NO:10 (pages 1-2 and see the sequence alignment below). Johnson discloses increased cis,cis-muconic acid production due to deletion of CatBC, which metabolizes cis-cis-muconic acid (Fig. 1).
Regarding deletion of muconate lactonizing enzyme activity of claims 10, 13, and 16, A4XFH7 discloses Novosphingobium aromaticivorans DSM12444 muconolactone isomerase (CatC) having 100% sequence identity to the amino acid sequence of SEQ ID NO:10 (pages 1-2 and see the sequence alignment below). Johnson discloses increased cis,cis-muconic acid production due to deletion of CatBC, which metabolizes cis-cis-muconic acid (Fig. 1).
Regarding claims 8-9, 11-12, and 14-15, Perez discloses that Novosphingobium aromaticivorans DSM 12444 has an advantage over P. putida in producing cis-cis-muconic acid because Novosphingobium aromaticivorans has a wider range of aromatic substrate compared to P. putida (Section 1.6 at page 8). Perez discloses genetic modification of Novosphingobium aromaticivorans DSM 12444 for expressing genes (Section 4.5 at pages 127-129).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to further modify Novosphingobium aromaticivorans DSM 12444 by deleting (v) Novosphingobium aromaticivorans DSM12444 muconate lactonizing enzyme (CatB) having the amino acid sequence of SEQ ID NO:10, (vi) Novosphingobium aromaticivorans DSM12444 muconolactone isomerase (CatC) having the amino acid sequence of SEQ ID NO:12, (vii) Novosphingobium aromaticivorans DSM12444 catechol 2,3-dioxygenase having the amino acid sequence of SEQ ID NO:14, and (viii) Novosphingobium aromaticivorans DSM12444 protocatechuate 4,5-dioxgyenase (LigA) having the amino acid sequence of SEQ ID NO:16. One having ordinary skill in the art would have been motivated to do so to increase production of cis,cis-muconic acid by directing the carbon flux towards cis,cis-muconic acid, thus increasing production of cis,cis-muconic acid. One having ordinary skill in the art would have had a reasonable expectation of success because (A) Sonoki and Johnson disclose increasing activities of PCA decarboxylase and catechol 1,2-dioxygenase in a recombinant microorganism, (B) Perez discloses advantages of using Novosphingobium aromaticivorans DSM 12444 as a host microorganism, (C) Sonoki discloses inserting genes native to the host microorganism, (D) Johnson discloses additional metabolic engineering to increase the flux of carbon to muconate and industrially-relevant levels of production, (E) Johnson discloses that flavin prenyltransferase is responsible for the production of a prenylated-FMN co-factor required by UbiD, homologous to the PCA decarboxylase, (F) Johnson discloses that PCA decarboxylase is co-expressed as an operon with two other small genes shown to be important to activity of the PCA decarboxylase, which includes the subunit D, and (G) the enzymes having the amino acid sequences of SEQ ID NO:10, 12, 14, and 16 were known in the art.
Therefore, the above references render claims 1-16 prima facie obvious.

Conclusion

Claims 1-20 are pending.

Claims 1-20 are rejected.

Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is [phone]. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
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/YONG D PAK/Primary Examiner, Art Unit 1652

Sequence alignment of SEQ ID NO:2 of the instant application (“Qy”) and the flavin prenyltransferase of A4XDV7 (“Db”)

A4XDV7_NOVAD
ID A4XDV7_NOVAD Unreviewed; 204 AA.
AC A4XDV7;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 18-JUN-2025, entry version 88.
DE RecName: Full=Flavin prenyltransferase UbiX {ECO:0000256|HAMAP-Rule:MF_01984};
DE EC=2.5.1.129 {ECO:0000256|HAMAP-Rule:MF_01984};
GN Name=ubiX {ECO:0000256|HAMAP-Rule:MF_01984};
GN OrderedLocusNames=Saro_3873 {ECO:0000313|EMBL:ABP64118.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL1 {ECO:0000313|EMBL:ABP64118.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64118.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64118.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL1 {ECO:0000313|EMBL:ABP64118.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL1 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
CC -!- FUNCTION: Flavin prenyltransferase that catalyzes the synthesis of the
CC prenylated FMN cofactor (prenyl-FMN) for 4-hydroxy-3-polyprenylbenzoic
CC acid decarboxylase UbiD. The prenyltransferase is metal-independent and
CC links a dimethylallyl moiety from dimethylallyl monophosphate (DMAP) to
CC the flavin N5 and C6 atoms of FMN. {ECO:0000256|HAMAP-Rule:MF_01984}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=dimethylallyl phosphate + FMNH2 = prenylated FMNH2 +
CC phosphate; Xref=Rhea:RHEA:37743, ChEBI:CHEBI:43474,
CC ChEBI:CHEBI:57618, ChEBI:CHEBI:87467, ChEBI:CHEBI:88052;
CC EC=2.5.1.129; Evidence={ECO:0000256|ARBA:ARBA00050612,
CC ECO:0000256|HAMAP-Rule:MF_01984};
CC -!- SIMILARITY: Belongs to the UbiX/PAD1 family.
CC {ECO:0000256|ARBA:ARBA00060793, ECO:0000256|HAMAP-Rule:MF_01984}.
CC -!- CAUTION: Lacks conserved residue(s) required for the propagation of
CC feature annotation. {ECO:0000256|HAMAP-Rule:MF_01984}.
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DR EMBL; CP000676; ABP64118.1; -; Genomic_DNA.
DR RefSeq; WP_010891036.1; NC_009426.1.
DR AlphaFoldDB; A4XDV7; -.
DR KEGG; nar:Saro_3873; -.
DR HOGENOM; CLU_074522_0_1_5; -.
DR Proteomes; UP000009134; Plasmid pNL1.
DR GO; GO:0016831; F:carboxy-lyase activity; IEA:TreeGrafter.
DR GO; GO:0106141; F:flavin prenyltransferase activity; IEA:UniProtKB-EC.
DR FunFam; 3.40.50.1950:FF:000001; Flavin prenyltransferase UbiX; 1.
DR Gene3D; 3.40.50.1950; Flavin prenyltransferase-like; 1.
DR HAMAP; MF_01984; ubiX_pad; 1.
DR InterPro; IPR036551; Flavin_trans-like.
DR InterPro; IPR003382; Flavoprotein.
DR InterPro; IPR004507; UbiX-like.
DR NCBIfam; NF004685; PRK06029.1; 1.
DR NCBIfam; TIGR00421; ubiX_pad; 1.
DR PANTHER; PTHR43374; FLAVIN PRENYLTRANSFERASE; 1.
DR PANTHER; PTHR43374:SF1; FLAVIN PRENYLTRANSFERASE PAD1, MITOCHONDRIAL; 1.
DR Pfam; PF02441; Flavoprotein; 1.
DR SUPFAM; SSF52507; Homo-oligomeric flavin-containing Cys decarboxylases, HFCD; 1.
PE 3: Inferred from homology;
KW Flavoprotein {ECO:0000256|ARBA:ARBA00022630, ECO:0000256|HAMAP-
KW Rule:MF_01984};
KW FMN {ECO:0000256|ARBA:ARBA00022643, ECO:0000256|HAMAP-Rule:MF_01984};
KW Lyase {ECO:0000313|EMBL:ABP64118.1}; Plasmid {ECO:0000313|EMBL:ABP64118.1};
KW Prenyltransferase {ECO:0000256|ARBA:ARBA00022602, ECO:0000256|HAMAP-
KW Rule:MF_01984}; Reference proteome {ECO:0000313|Proteomes:UP000009134};
KW Transferase {ECO:0000256|ARBA:ARBA00022679, ECO:0000256|HAMAP-
KW Rule:MF_01984}.
FT DOMAIN 2..165
FT /note="Flavoprotein"
FT /evidence="ECO:0000259|Pfam:PF02441"
FT BINDING 10..12
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_01984"
FT BINDING 37
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_01984"
FT BINDING 88..91
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_01984"
FT BINDING 123
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_01984"
FT BINDING 153
FT /ligand="dimethylallyl phosphate"
FT /ligand_id="ChEBI:CHEBI:88052"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_01984"
FT BINDING 169
FT /ligand="dimethylallyl phosphate"
FT /ligand_id="ChEBI:CHEBI:88052"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_01984"
SQ SEQUENCE 204 AA; 21973 MW; 25E072D3081342AF CRC64;

Query Match 100.0%; Score 1035; Length 204;
Best Local Similarity 100.0%;
Matches 204; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MKRMVVGITGATGSVYGLRLLELLRETGGWETHLVMSPAALLNIREELPEGKARLEALAD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MKRMVVGITGATGSVYGLRLLELLRETGGWETHLVMSPAALLNIREELPEGKARLEALAD 60

Qy 61 VVHNVRNVGASIASGSFVCEGMAIA PCSMRTLGAVAHALSDNLITRAADVMLKERRRLVM 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 VVHNVRNVGASIASGSFVCEGMAIA PCSMRTLGAVAHALSDNLITRAADVMLKERRRLVM 120

Qy 121 ITREAPLNLAHLRNMTACTEMGAVIFPPVPAFYARPTSLADVVDHTCMRVLDLFGLHAKS 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ITREAPLNLAHLRNMTACTEMGAVIFPPVPAFYARPTSLADVVDHTCMRVLDLFGLHAKS 180

Qy 181 EKRWQGLSKEAASLVPGAGQMEGN 204
||||||||||||||||||||||||
Db 181 EKRWQGLSKEAASLVPGAGQMEGN 204

Sequence alignment of SEQ ID NO:4 of the instant application (“Qy”) and the PCA decarboxylase of A4XDW1 (“Db”)

A4XDW1_NOVAD
ID A4XDW1_NOVAD Unreviewed; 486 AA.
AC A4XDW1;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 28-JAN-2026, entry version 79.
DE SubName: Full=UbiD family decarboxylase {ECO:0000313|EMBL:ABP64122.1};
GN OrderedLocusNames=Saro_3877 {ECO:0000313|EMBL:ABP64122.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL1 {ECO:0000313|EMBL:ABP64122.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64122.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64122.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL1 {ECO:0000313|EMBL:ABP64122.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL1 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
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CC ---------------------------------------------------------------------------
DR EMBL; CP000676; ABP64122.1; -; Genomic_DNA.
DR AlphaFoldDB; A4XDW1; -.
DR KEGG; nar:Saro_3877; -.
DR HOGENOM; CLU_023348_3_2_5; -.
DR Proteomes; UP000009134; Plasmid pNL1.
DR GO; GO:0005829; C:cytosol; IEA:TreeGrafter.
DR GO; GO:0008694; F:4-hydroxy-3-polyprenylbenzoate decarboxylase activity; IEA:TreeGrafter.
DR GO; GO:0006744; P:ubiquinone biosynthetic process; IEA:TreeGrafter.
DR Gene3D; 3.40.1670.10; UbiD C-terminal domain-like; 1.
DR InterPro; IPR053417; PAD_UbiD-like.
DR InterPro; IPR002830; UbiD.
DR InterPro; IPR049381; UbiD-like_C.
DR InterPro; IPR049383; UbiD-like_N.
DR InterPro; IPR048304; UbiD_Rift_dom.
DR NCBIfam; TIGR00148; UbiD family decarboxylase; 1.
DR NCBIfam; NF041204; VdcC; 1.
DR PANTHER; PTHR30108; 3-OCTAPRENYL-4-HYDROXYBENZOATE CARBOXY-LYASE-RELATED; 1.
DR PANTHER; PTHR30108:SF17; FERULIC ACID DECARBOXYLASE 1; 1.
DR Pfam; PF01977; UbiD; 1.
DR Pfam; PF20696; UbiD_C; 1.
DR Pfam; PF20695; UbiD_N; 1.
DR SUPFAM; SSF50475; FMN-binding split barrel; 1.
DR SUPFAM; SSF143968; UbiD C-terminal domain-like; 1.
PE 4: Predicted;
KW Plasmid {ECO:0000313|EMBL:ABP64122.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134}.
FT DOMAIN 20..99
FT /note="3-octaprenyl-4-hydroxybenzoate carboxy-lyase-like N-
FT terminal"
FT /evidence="ECO:0000259|Pfam:PF20695"
FT DOMAIN 117..318
FT /note="3-octaprenyl-4-hydroxybenzoate carboxy-lyase-like
FT Rift-related"
FT /evidence="ECO:0000259|Pfam:PF01977"
FT DOMAIN 325..447
FT /note="3-octaprenyl-4-hydroxybenzoate carboxy-lyase-like C-
FT terminal"
FT /evidence="ECO:0000259|Pfam:PF20696"
SQ SEQUENCE 486 AA; 52918 MW; DF4F027230434385 CRC64;

Query Match 100.0%; Score 2519; Length 486;
Best Local Similarity 100.0%;
Matches 486; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MTMNDLPNRARSISSLRDFLELLEDAGQAITWSDAVMPEPGVRNIAVAASRDANGAPAIV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTMNDLPNRARSISSLRDFLELLEDAGQAITWSDAVMPEPGVRNIAVAASRDANGAPAIV 60

Qy 61 FDNITGYPGKRLAVGVHGSWDNIALLLGRPKGTTIRELFFEIAGRWGDQEAQISFVPEAQ 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 FDNITGYPGKRLAVGVHGSWDNIALLLGRPKGTTIRELFFEIAGRWGDQEAQISFVPEAQ 120

Qy 121 APVHECRIEQDINLYDVLPVYRINEYDGGFYIGKASVASRDPLDPDNFGKQNVGIYRLQI 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 APVHECRIEQDINLYDVLPVYRINEYDGGFYIGKASVASRDPLDPDNFGKQNVGIYRLQI 180

Qy 181 QGPDTFTLMTIPSHDMGRQIMAAEREGVPLKIAVMLGNHPGLAVFAATPIGYEESEYSYA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 QGPDTFTLMTIPSHDMGRQIMAAEREGVPLKIAVMLGNHPGLAVFAATPIGYEESEYSYA 240

Qy 241 SAMMGAPIRLTKSGNGIDILADSEIVIEAELQPGGRELEGPFGEFPGSYSGVRKAPIFKV 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 SAMMGAPIRLTKSGNGIDILADSEIVIEAELQPGGRELEGPFGEFPGSYSGVRKAPIFKV 300

Qy 301 TAVSHRRDPIFENIYIGRGWTEHDTLIGLHTSAPIYAQLRQSFPEVTAVNALYQHGLTGI 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TAVSHRRDPIFENIYIGRGWTEHDTLIGLHTSAPIYAQLRQSFPEVTAVNALYQHGLTGI 360

Qy 361 ISVKNRMAGFAKTVALRALSTPHGVMYLKNLIMVDADVDPFDLNQVMWALSTRTRADDII 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 ISVKNRMAGFAKTVALRALSTPHGVMYLKNLIMVDADVDPFDLNQVMWALSTRTRADDII 420

Qy 421 VLPNMPAVPIDPSAVVPGKGHRLIIDATSYLPPDPVGEAHLVTPPTGDEIDALSKRIREM 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 VLPNMPAVPIDPSAVVPGKGHRLIIDATSYLPPDPVGEAHLVTPPTGDEIDALSKRIREM 480

Qy 481 QLGALS 486
||||||
Db 481 QLGALS 486
Sequence alignment of SEQ ID NO:6 of the instant application (“Qy”) and the PCA decarboxylase D of A4XDW2 (“Db”)

A4XDW2_NOVAD
ID A4XDW2_NOVAD Unreviewed; 74 AA.
AC A4XDW2;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 28-JAN-2026, entry version 53.
DE RecName: Full=Phenolic acid decarboxylase subunit D {ECO:0008006|Google:ProtNLM};
GN OrderedLocusNames=Saro_3878 {ECO:0000313|EMBL:ABP64123.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL1 {ECO:0000313|EMBL:ABP64123.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64123.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64123.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL1 {ECO:0000313|EMBL:ABP64123.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL1 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
CC ---------------------------------------------------------------------------
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CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; CP000676; ABP64123.1; -; Genomic_DNA.
DR RefSeq; WP_010891041.1; NC_009426.1.
DR AlphaFoldDB; A4XDW2; -.
DR KEGG; nar:Saro_3878; -.
DR HOGENOM; CLU_180856_2_0_5; -.
DR Proteomes; UP000009134; Plasmid pNL1.
DR InterPro; IPR047707; VdcD-like.
DR NCBIfam; NF041205; VdcD; 1.
DR Pfam; PF26358; EcdD_BsdD_detox; 1.
PE 4: Predicted;
KW Plasmid {ECO:0000313|EMBL:ABP64123.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134}.
SQ SEQUENCE 74 AA; 8154 MW; 401EDA0DF7993A20 CRC64;

Query Match 100.0%; Score 401; Length 74;
Best Local Similarity 100.0%;
Matches 74; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MTTTVCGRCKSSGAVTDHQGRQDGAVVWTILRCPTCNFSWRDSEPARAIDPAVRSADFAV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTTTVCGRCKSSGAVTDHQGRQDGAVVWTILRCPTCNFSWRDSEPARAIDPAVRSADFAV 60

Qy 61 DVGDLQRYPKILQQ 74
||||||||||||||
Db 61 DVGDLQRYPKILQQ 74
Sequence alignment of SEQ ID NO:8 of the instant application (“Qy”) and the catechol 1,2-dioxygenase of A4XFH8 (“Db”)

A4XFH8_NOVAD
ID A4XFH8_NOVAD Unreviewed; 298 AA.
AC A4XFH8;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 02-APR-2025, entry version 76.
DE RecName: Full=catechol 1,2-dioxygenase {ECO:0000256|ARBA:ARBA00013118};
DE EC=1.13.11.1 {ECO:0000256|ARBA:ARBA00013118};
GN OrderedLocusNames=Saro_3830 {ECO:0000313|EMBL:ABP64689.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL2 {ECO:0000313|EMBL:ABP64689.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64689.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64689.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL2 {ECO:0000313|EMBL:ABP64689.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL2 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=catechol + O2 = cis,cis-muconate + 2 H(+);
CC Xref=Rhea:RHEA:23852, ChEBI:CHEBI:15378, ChEBI:CHEBI:15379,
CC ChEBI:CHEBI:18135, ChEBI:CHEBI:32379; EC=1.13.11.1;
CC Evidence={ECO:0000256|ARBA:ARBA00001312};
CC -!- COFACTOR:
CC Name=Fe(3+); Xref=ChEBI:CHEBI:29034;
CC Evidence={ECO:0000256|ARBA:ARBA00001965};
CC -!- PATHWAY: Aromatic compound metabolism; beta-ketoadipate pathway; 5-oxo-
CC 4,5-dihydro-2-furylacetate from catechol: step 1/3.
CC {ECO:0000256|ARBA:ARBA00004957}.
CC -!- SIMILARITY: Belongs to the intradiol ring-cleavage dioxygenase family.
CC {ECO:0000256|ARBA:ARBA00007825}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; CP000677; ABP64689.1; -; Genomic_DNA.
DR RefSeq; WP_011907068.1; NC_009427.1.
DR AlphaFoldDB; A4XFH8; -.
DR KEGG; nar:Saro_3830; -.
DR eggNOG; COG3485; Bacteria.
DR HOGENOM; CLU_046727_0_0_5; -.
DR UniPathway; UPA00157; UER00258.
DR Proteomes; UP000009134; Plasmid pNL2.
DR GO; GO:0018576; F:catechol 1,2-dioxygenase activity; IEA:UniProtKB-EC.
DR GO; GO:0008199; F:ferric iron binding; IEA:InterPro.
DR GO; GO:0042952; P:beta-ketoadipate pathway; IEA:UniProtKB-UniPathway.
DR GO; GO:0019614; P:catechol-containing compound catabolic process; IEA:InterPro.
DR CDD; cd03460; 1_2-CTD; 1.
DR Gene3D; 2.60.130.10; Aromatic compound dioxygenase; 1.
DR InterPro; IPR007535; Catechol_dOase_N.
DR InterPro; IPR012801; Cchol_dOase_prob.
DR InterPro; IPR000627; Intradiol_dOase_C.
DR InterPro; IPR015889; Intradiol_dOase_core.
DR InterPro; IPR050770; Intradiol_RC_Dioxygenase.
DR PANTHER; PTHR33711; DIOXYGENASE, PUTATIVE (AFU_ORTHOLOGUE AFUA_2G02910)-RELATED; 1.
DR PANTHER; PTHR33711:SF7; INTRADIOL RING-CLEAVAGE DIOXYGENASES DOMAIN-CONTAINING PROTEIN-RELATED; 1.
DR Pfam; PF00775; Dioxygenase_C; 1.
DR Pfam; PF04444; Dioxygenase_N; 1.
DR SUPFAM; SSF49482; Aromatic compound dioxygenase; 1.
PE 3: Inferred from homology;
KW Aromatic hydrocarbons catabolism {ECO:0000256|ARBA:ARBA00022797};
KW Dioxygenase {ECO:0000256|ARBA:ARBA00022964, ECO:0000313|EMBL:ABP64689.1};
KW Iron {ECO:0000256|ARBA:ARBA00023004};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002};
KW Plasmid {ECO:0000313|EMBL:ABP64689.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134}.
FT DOMAIN 27..94
FT /note="Catechol dioxygenase N-terminal"
FT /evidence="ECO:0000259|Pfam:PF04444"
FT DOMAIN 103..280
FT /note="Intradiol ring-cleavage dioxygenases"
FT /evidence="ECO:0000259|Pfam:PF00775"
SQ SEQUENCE 298 AA; 32556 MW; 4AB6817EB7CE3CA5 CRC64;

Query Match 100.0%; Score 1588; Length 298;
Best Local Similarity 100.0%;
Matches 298; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MPATFASSDSVQKLFDRACGLDCAGGNPRLKAIMRDLLQATADIIVKHDVSESEFWQATR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MPATFASSDSVQKLFDRACGLDCAGGNPRLKAIMRDLLQATADIIVKHDVSESEFWQATR 60

Qy 61 YLADGAGEIGLIVPGIGLEHFLDLYMDAKDAEAGLTGGTPRTIEGPLYVAGAPLVDGSDE 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YLADGAGEIGLIVPGIGLEHFLDLYMDAKDAEAGLTGGTPRTIEGPLYVAGAPLVDGSDE 120

Qy 121 VDLTSDPDDTDTLHMTGTITGPDGEPVKDAILHVWHANSKGWYSHFDPTSEQTPFNNRRR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 VDLTSDPDDTDTLHMTGTITGPDGEPVKDAILHVWHANSKGWYSHFDPTSEQTPFNNRRR 180

Qy 181 IRVPADGRYAFRSKMPHGYSVPPGGATDVLMQALGRHGNRPAHVHFFVEAPGYRTLTTQI 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 IRVPADGRYAFRSKMPHGYSVPPGGATDVLMQALGRHGNRPAHVHFFVEAPGYRTLTTQI 240

Qy 241 NFGDDPFAADDFAFGTREGLLPVPSRQGDTAHIAFDFQLQRARSEDEQRFSERTRAQA 298
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 NFGDDPFAADDFAFGTREGLLPVPSRQGDTAHIAFDFQLQRARSEDEQRFSERTRAQA 298

Sequence alignment of SEQ ID NO:10 of the instant application (“Qy”) and the muconate lactonizing enzyme of A4XFH6 (“Db”)

A4XFH6_NOVAD
ID A4XFH6_NOVAD Unreviewed; 387 AA.
AC A4XFH6;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 28-JAN-2026, entry version 92.
DE SubName: Full=Muconate and chloromuconate cycloisomerase {ECO:0000313|EMBL:ABP64687.1};
GN OrderedLocusNames=Saro_3828 {ECO:0000313|EMBL:ABP64687.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL2 {ECO:0000313|EMBL:ABP64687.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64687.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64687.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL2 {ECO:0000313|EMBL:ABP64687.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL2 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
CC -!- COFACTOR:
CC Name=Mn(2+); Xref=ChEBI:CHEBI:29035;
CC Evidence={ECO:0000256|ARBA:ARBA00001936};
CC -!- PATHWAY: Aromatic compound metabolism. {ECO:0000256|ARBA:ARBA00005211}.
CC -!- SIMILARITY: Belongs to the mandelate racemase/muconate lactonizing
CC enzyme family. {ECO:0000256|ARBA:ARBA00008031}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; CP000677; ABP64687.1; -; Genomic_DNA.
DR RefSeq; WP_011907066.1; NC_009427.1.
DR AlphaFoldDB; A4XFH6; -.
DR KEGG; nar:Saro_3828; -.
DR eggNOG; COG4948; Bacteria.
DR HOGENOM; CLU_030273_4_5_5; -.
DR Proteomes; UP000009134; Plasmid pNL2.
DR GO; GO:0018850; F:chloromuconate cycloisomerase activity; IEA:InterPro.
DR GO; GO:0000287; F:magnesium ion binding; IEA:UniProtKB-ARBA.
DR GO; GO:0030145; F:manganese ion binding; IEA:InterPro.
DR GO; GO:0018849; F:muconate cycloisomerase activity; IEA:InterPro.
DR GO; GO:0016854; F:racemase and epimerase activity; IEA:UniProtKB-ARBA.
DR GO; GO:0009063; P:amino acid catabolic process; IEA:InterPro.
DR GO; GO:0006518; P:peptide metabolic process; IEA:UniProtKB-ARBA.
DR CDD; cd03318; MLE; 1.
DR Gene3D; 3.20.20.120; Enolase-like C-terminal domain; 1.
DR Gene3D; 3.30.390.10; Enolase-like, N-terminal domain; 1.
DR InterPro; IPR013370; Chloromuconate_cycloisomerase.
DR InterPro; IPR036849; Enolase-like_C_sf.
DR InterPro; IPR029017; Enolase-like_N.
DR InterPro; IPR029065; Enolase_C-like.
DR InterPro; IPR018110; Mandel_Rmase/mucon_lact_enz_CS.
DR InterPro; IPR013342; Mandelate_racemase_C.
DR InterPro; IPR013341; Mandelate_racemase_N_dom.
DR NCBIfam; TIGR02534; mucon_cyclo; 1.
DR PANTHER; PTHR48073:SF2; O-SUCCINYLBENZOATE SYNTHASE; 1.
DR PANTHER; PTHR48073; O-SUCCINYLBENZOATE SYNTHASE-RELATED; 1.
DR Pfam; PF13378; MR_MLE_C; 1.
DR Pfam; PF02746; MR_MLE_N; 1.
DR SFLD; SFLDG01258; (chloro)muconate_cycloisomeras; 1.
DR SFLD; SFLDS00001; Enolase; 1.
DR SFLD; SFLDG00180; muconate_cycloisomerase; 1.
DR SMART; SM00922; MR_MLE; 1.
DR SUPFAM; SSF51604; Enolase C-terminal domain-like; 1.
DR SUPFAM; SSF54826; Enolase N-terminal domain-like; 1.
DR PROSITE; PS00908; MR_MLE_1; 1.
DR PROSITE; PS00909; MR_MLE_2; 1.
PE 3: Inferred from homology;
KW Aromatic hydrocarbons catabolism {ECO:0000256|ARBA:ARBA00022797};
KW Isomerase {ECO:0000256|ARBA:ARBA00023235, ECO:0000313|EMBL:ABP64687.1};
KW Manganese {ECO:0000256|ARBA:ARBA00023211};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723};
KW Plasmid {ECO:0000313|EMBL:ABP64687.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134}.
FT DOMAIN 151..248
FT /note="Mandelate racemase/muconate lactonizing enzyme C-
FT terminal"
FT /evidence="ECO:0000259|SMART:SM00922"
FT ACT_SITE 172
FT /note="Proton acceptor"
FT /evidence="ECO:0000256|PIRSR:PIRSR613370-1"
FT ACT_SITE 330
FT /note="Proton donor"
FT /evidence="ECO:0000256|PIRSR:PIRSR613370-1"
SQ SEQUENCE 387 AA; 40776 MW; 754DF0DF2F5B144A CRC64;

Query Match 100.0%; Score 1956; Length 387;
Best Local Similarity 100.0%;
Matches 387; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MTALANPQILGIETILLDLPTIRPHVLAMATMHAQTICLVRLTCSDGIVGLGEATTIGGL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTALANPQILGIETILLDLPTIRPHVLAMATMHAQTICLVRLTCSDGIVGLGEATTIGGL 60

Qy 61 AYGPEAPETIKTAIDTYFAPLLAGQDATRPAAAMALVARHVVGNHFAKCAIETALLDAQG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 AYGPEAPETIKTAIDTYFAPLLAGQDATRPAAAMALVARHVVGNHFAKCAIETALLDAQG 120

Qy 121 KRLGLPVSELLGGRRVDSLPVLWTLASGDTARDIAEAEQMLDTRRHDAFKLKIGKRPIEQ 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 KRLGLPVSELLGGRRVDSLPVLWTLASGDTARDIAEAEQMLDTRRHDAFKLKIGKRPIEQ 180

Qy 181 DVAHVGAIKAALGDRASVRVDVNMAWDEPTARRGLAMLADAGCDLVEQPIIRHNRDGMAR 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 DVAHVGAIKAALGDRASVRVDVNMAWDEPTARRGLAMLADAGCDLVEQPIIRHNRDGMAR 240

Qy 241 LVALGLVPVMADESLTGPASAMDFARAAAADVFAVKIEQSGGLDAARAVAQIGDAACIGL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LVALGLVPVMADESLTGPASAMDFARAAAADVFAVKIEQSGGLDAARAVAQIGDAACIGL 300

Qy 301 YGGTMLEGAIGTIASAHAFATFPALKWGTELFGPLLLTEEILERPLTYADFSLEVPAGPG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 YGGTMLEGAIGTIASAHAFATFPALKWGTELFGPLLLTEEILERPLTYADFSLEVPAGPG 360

Qy 361 LGIALDEDRVEHFRRDRTATQFALQGA 387
|||||||||||||||||||||||||||
Db 361 LGIALDEDRVEHFRRDRTATQFALQGA 387
Sequence alignment of SEQ ID NO:12 of the instant application (“Qy”) and the muconolactone isomerase of A4XFH7 (“Db”)

A4XFH7_NOVAD
ID A4XFH7_NOVAD Unreviewed; 96 AA.
AC A4XFH7;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 28-JAN-2026, entry version 79.
DE RecName: Full=Muconolactone Delta-isomerase {ECO:0000256|ARBA:ARBA00012070, ECO:0000256|NCBIfam:TIGR03221};
DE Short=MIase {ECO:0000256|PIRNR:PIRNR001486};
DE EC=5.3.3.4 {ECO:0000256|ARBA:ARBA00012070, ECO:0000256|NCBIfam:TIGR03221};
GN OrderedLocusNames=Saro_3829 {ECO:0000313|EMBL:ABP64688.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL2 {ECO:0000313|EMBL:ABP64688.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64688.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64688.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL2 {ECO:0000313|EMBL:ABP64688.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL2 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(S)-muconolactone = (4,5-dihydro-5-oxofuran-2-yl)-acetate;
CC Xref=Rhea:RHEA:12348, ChEBI:CHEBI:58425, ChEBI:CHEBI:58736;
CC EC=5.3.3.4; Evidence={ECO:0000256|ARBA:ARBA00001739,
CC ECO:0000256|PIRNR:PIRNR001486};
CC -!- PATHWAY: Aromatic compound metabolism; beta-ketoadipate pathway; 5-oxo-
CC 4,5-dihydro-2-furylacetate from catechol: step 3/3.
CC {ECO:0000256|ARBA:ARBA00005193, ECO:0000256|PIRNR:PIRNR001486}.
CC -!- SUBUNIT: Homodecamer. {ECO:0000256|ARBA:ARBA00011365}.
CC -!- SIMILARITY: Belongs to the muconolactone Delta-isomerase family.
CC {ECO:0000256|ARBA:ARBA00010882, ECO:0000256|PIRNR:PIRNR001486}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; CP000677; ABP64688.1; -; Genomic_DNA.
DR RefSeq; WP_011907067.1; NC_009427.1.
DR AlphaFoldDB; A4XFH7; -.
DR KEGG; nar:Saro_3829; -.
DR eggNOG; COG4829; Bacteria.
DR HOGENOM; CLU_080702_2_0_5; -.
DR UniPathway; UPA00157; UER00260.
DR Proteomes; UP000009134; Plasmid pNL2.
DR GO; GO:0016159; F:muconolactone delta-isomerase activity; IEA:UniProtKB-UniRule.
DR GO; GO:0042952; P:beta-ketoadipate pathway; IEA:UniProtKB-UniRule.
DR Gene3D; 3.30.70.1060; Dimeric alpha+beta barrel; 1.
DR InterPro; IPR011008; Dimeric_a/b-barrel.
DR InterPro; IPR026029; MLI_dom.
DR InterPro; IPR003464; Muconolactone_d_Isoase.
DR NCBIfam; TIGR03221; muco_delta; 1.
DR Pfam; PF02426; MIase; 1.
DR PIRSF; PIRSF001486; CatC; 1.
DR SUPFAM; SSF54909; Dimeric alpha+beta barrel; 1.
PE 3: Inferred from homology;
KW Aromatic hydrocarbons catabolism {ECO:0000256|ARBA:ARBA00022797,
KW ECO:0000256|PIRNR:PIRNR001486};
KW Isomerase {ECO:0000256|ARBA:ARBA00023235, ECO:0000256|PIRNR:PIRNR001486};
KW Plasmid {ECO:0000313|EMBL:ABP64688.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134}.
FT DOMAIN 1..89
FT /note="Muconolactone isomerase"
FT /evidence="ECO:0000259|Pfam:PF02426"
SQ SEQUENCE 96 AA; 11244 MW; EFDE7310399533FD CRC64;

Query Match 100.0%; Score 505; Length 96;
Best Local Similarity 100.0%;
Matches 96; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MLFQVEMTVDIPLDFDKAEAERIKAVEKAYSHELQRSGEWRHIWRVAGQYANLSIFDVAS 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MLFQVEMTVDIPLDFDKAEAERIKAVEKAYSHELQRSGEWRHIWRVAGQYANLSIFDVAS 60

Qy 61 NERLHEILMGLPLYPFMTIKVTPLCRHPSSIRDDDR 96
||||||||||||||||||||||||||||||||||||
Db 61 NERLHEILMGLPLYPFMTIKVTPLCRHPSSIRDDDR 96

Sequence alignment of SEQ ID NO:14 of the instant application (“Qy”) and the catechol 2,3-dioxygenase of A4XDU1 (“Db”)

A4XDU1_NOVAD
ID A4XDU1_NOVAD Unreviewed; 307 AA.
AC A4XDU1;
DT 29-MAY-2007, integrated into UniProtKB/TrEMBL.
DT 29-MAY-2007, sequence version 1.
DT 08-OCT-2025, entry version 90.
DE RecName: Full=Metapyrocatechase {ECO:0000256|ARBA:ARBA00022190};
DE EC=1.13.11.2 {ECO:0000256|ARBA:ARBA00013117};
DE AltName: Full=CatO2ase {ECO:0000256|ARBA:ARBA00031146};
DE AltName: Full=Catechol 2,3-dioxygenase {ECO:0000256|ARBA:ARBA00030369};
GN OrderedLocusNames=Saro_3857 {ECO:0000313|EMBL:ABP64102.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OG Plasmid pNL1 {ECO:0000313|EMBL:ABP64102.1,
OG ECO:0000313|Proteomes:UP000009134}.
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABP64102.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|EMBL:ABP64102.1, ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RC PLASMID=pNL1 {ECO:0000313|EMBL:ABP64102.1,
RC ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C.,
RA Glavina del Rio T., Hammon N., Israni S., Dalin E., Tice H., Pitluck S.,
RA Chertkov O., Han C., Thomson S., Schmutz J., Larimer F., Land M.,
RA Kyrpides N., Ivanova N., Fredrickson J., Romine M.F., Richardson P.;
RT "Complete sequence of plasmid pNL1 of Novosphingobium aromaticivorans DSM
RT 12444.";
RL Submitted (APR-2007) to the EMBL/GenBank/DDBJ databases.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=catechol + O2 = (2Z,4E)-2-hydroxy-6-oxohexa-2,4-dienoate +
CC H(+); Xref=Rhea:RHEA:17337, ChEBI:CHEBI:15378, ChEBI:CHEBI:15379,
CC ChEBI:CHEBI:18135, ChEBI:CHEBI:71198; EC=1.13.11.2;
CC Evidence={ECO:0000256|ARBA:ARBA00000163};
CC -!- COFACTOR:
CC Name=Fe(2+); Xref=ChEBI:CHEBI:29033;
CC Evidence={ECO:0000256|ARBA:ARBA00001954,
CC ECO:0000256|RuleBase:RU000683};
CC -!- SUBUNIT: Homotetramer. {ECO:0000256|ARBA:ARBA00011881}.
CC -!- SIMILARITY: Belongs to the extradiol ring-cleavage dioxygenase family.
CC {ECO:0000256|ARBA:ARBA00008784, ECO:0000256|RuleBase:RU000683}.
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DR EMBL; CP000676; ABP64102.1; -; Genomic_DNA.
DR RefSeq; WP_010891020.1; NC_009426.1.
DR AlphaFoldDB; A4XDU1; -.
DR KEGG; nar:Saro_3857; -.
DR HOGENOM; CLU_052361_3_0_5; -.
DR Proteomes; UP000009134; Plasmid pNL1.
DR GO; GO:0018577; F:catechol 2,3-dioxygenase activity; IEA:UniProtKB-EC.
DR GO; GO:0008198; F:ferrous iron binding; IEA:InterPro.
DR CDD; cd07243; 2_3_CTD_C; 1.
DR Gene3D; 3.10.180.10; 2,3-Dihydroxybiphenyl 1,2-Dioxygenase, domain 1; 2.
DR InterPro; IPR017624; Catechol_2-3_dOase.
DR InterPro; IPR029068; Glyas_Bleomycin-R_OHBP_Dase.
DR InterPro; IPR004360; Glyas_Fos-R_dOase_dom.
DR InterPro; IPR037523; VOC_core.
DR InterPro; IPR000486; Xdiol_ring_cleave_dOase_1/2.
DR InterPro; IPR054560; XylE-like_N.
DR NCBIfam; TIGR03211; catechol_2_3; 1.
DR Pfam; PF22247; Diox-like_N; 1.
DR Pfam; PF00903; Glyoxalase; 1.
DR SUPFAM; SSF54593; Glyoxalase/Bleomycin resistance protein/Dihydroxybiphenyl dioxygenase; 1.
DR PROSITE; PS00082; EXTRADIOL_DIOXYGENAS; 1.
DR PROSITE; PS51819; VOC; 2.
PE 3: Inferred from homology;
KW Aromatic hydrocarbons catabolism {ECO:0000256|ARBA:ARBA00022797,
KW ECO:0000256|RuleBase:RU000683};
KW Dioxygenase {ECO:0000256|ARBA:ARBA00022964, ECO:0000256|RuleBase:RU000683};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|RuleBase:RU000683};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|RuleBase:RU000683}; Plasmid {ECO:0000313|EMBL:ABP64102.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134};
KW Repeat {ECO:0000256|ARBA:ARBA00022737}.
FT DOMAIN 8..122
FT /note="VOC"
FT /evidence="ECO:0000259|PROSITE:PS51819"
FT DOMAIN 149..269
FT /note="VOC"
FT /evidence="ECO:0000259|PROSITE:PS51819"
SQ SEQUENCE 307 AA; 34402 MW; DEE2003AE5F4C3DE CRC64;

Query Match 100.0%; Score 1628; Length 307;
Best Local Similarity 100.0%;
Matches 307; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MALTGVIRPGYVQLRVLDLDEAIGHYRDRIGLNFVNRDGDRAFFQAFDEFDRHSIILREA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MALTGVIRPGYVQLRVLDLDEAIGHYRDRIGLNFVNRDGDRAFFQAFDEFDRHSIILREA 60

Qy 61 DQAGMDVMGFKVVKDADLDHFAERLLDIGVHVDVIPAGSDPGVGRKIRFNTPTQHVFELY 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 DQAGMDVMGFKVVKDADLDHFAERLLDIGVHVDVIPAGSDPGVGRKIRFNTPTQHVFELY 120

Qy 121 AEMELSATGPAVKNPDVWVVEPRGMRATRFDHCALNGVDISASAKIFVDALDFSVTEELV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 AEMELSATGPAVKNPDVWVVEPRGMRATRFDHCALNGVDISASAKIFVDALDFSVTEELV 180

Qy 181 DEGSGTRLGIFLSCSNKAHDVAFLGYPEDGKIHHTSFNLESWHDVGHAADIISRYDISLD 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 DEGSGTRLGIFLSCSNKAHDVAFLGYPEDGKIHHTSFNLESWHDVGHAADIISRYDISLD 240

Qy 241 IGPTRHGITRGQTIYFFDPSGNRNETFSGGYIYYPDNPQRMWQAESAGKAIFYYEKALND 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 IGPTRHGITRGQTIYFFDPSGNRNETFSGGYIYYPDNPQRMWQAESAGKAIFYYEKALND 300

Qy 301 RFMTVNT 307
|||||||
Db 301 RFMTVNT 307

Sequence alignment of SEQ ID NO:16 of the instant application (“Qy”) and the protocatechuate 4,5-dioxygenase of Q2G4H4 (“Db”)

Q2G4H4_NOVAD
ID Q2G4H4_NOVAD Unreviewed; 145 AA.
AC Q2G4H4;
DT 21-MAR-2006, integrated into UniProtKB/TrEMBL.
DT 21-MAR-2006, sequence version 1.
DT 18-JUN-2025, entry version 88.
DE SubName: Full=Protocatechuate 4,5-dioxygenase alpha subunit {ECO:0000313|EMBL:ABD27249.1};
DE EC=1.13.11.8 {ECO:0000313|EMBL:ABD27249.1};
GN OrderedLocusNames=Saro_2813 {ECO:0000313|EMBL:ABD27249.1};
OS Novosphingobium aromaticivorans (strain ATCC 700278 / DSM 12444 / CCUG
OS 56034 / CIP 105152 / NBRC 16084 / F199).
OC Bacteria; Pseudomonadati; Pseudomonadota; Alphaproteobacteria;
OC Sphingomonadales; Sphingomonadaceae; Novosphingobium.
OX NCBI_TaxID=279238 {ECO:0000313|EMBL:ABD27249.1, ECO:0000313|Proteomes:UP000009134};
RN [1] {ECO:0000313|Proteomes:UP000009134}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 700278 / DSM 12444 / CCUG 56034 / CIP 105152 / NBRC 16084
RC / F199 {ECO:0000313|Proteomes:UP000009134};
RG US DOE Joint Genome Institute;
RA Copeland A., Lucas S., Lapidus A., Barry K., Detter J.C., Glavina T.,
RA Hammon N., Israni S., Pitluck S., Chain P., Malfatti S., Shin M.,
RA Vergez L., Schmutz J., Larimer F., Land M., Kyrpides N., Ivanova N.,
RA Fredrickson J., Balkwill D., Romine M.F., Richardson P.;
RT "Complete sequence of Novosphingobium aromaticivorans DSM 12444.";
RL Submitted (JAN-2006) to the EMBL/GenBank/DDBJ databases.
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DR EMBL; CP000248; ABD27249.1; -; Genomic_DNA.
DR RefSeq; WP_011446453.1; NC_007794.1.
DR AlphaFoldDB; Q2G4H4; -.
DR STRING; 279238.Saro_2813; -.
DR KEGG; nar:Saro_2813; -.
DR eggNOG; COG3384; Bacteria.
DR HOGENOM; CLU_157261_0_0_5; -.
DR Proteomes; UP000009134; Chromosome.
DR GO; GO:0018579; F:protocatechuate 4,5-dioxygenase activity; IEA:UniProtKB-EC.
DR Gene3D; 1.10.700.10; Dioxygenase LigAB, LigA subunit; 1.
DR InterPro; IPR036622; LigA_sf.
DR InterPro; IPR014159; PCA_LigA.
DR InterPro; IPR011986; Xdiol_dOase_LigA.
DR NCBIfam; TIGR02792; PCA_ligA; 1.
DR NCBIfam; NF009917; PRK13377.1; 1.
DR Pfam; PF07746; LigA; 1.
DR SUPFAM; SSF48076; LigA subunit of an aromatic-ring-opening dioxygenase LigAB; 1.
PE 4: Predicted;
KW Dioxygenase {ECO:0000313|EMBL:ABD27249.1};
KW Oxidoreductase {ECO:0000313|EMBL:ABD27249.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000009134}.
FT DOMAIN 43..128
FT /note="Extradiol ring-cleavage dioxygenase LigAB LigA
FT subunit"
FT /evidence="ECO:0000259|Pfam:PF07746"
SQ SEQUENCE 145 AA; 16761 MW; AE5277CD6BF3AA1C CRC64;

Query Match 100.0%; Score 767; Length 145;
Best Local Similarity 100.0%;
Matches 145; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MTDNSSTDKPKPVQNIHEYLAEFEDIPGTRIYTAARARKGYWINQFAMSLMKPENRERFK 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTDNSSTDKPKPVQNIHEYLAEFEDIPGTRIYTAARARKGYWINQFAMSLMKPENRERFK 60

Qy 61 ANERAYLDEWKISEEAKEALLARDYNRLLDLGGNVYFLSKLFSSDGLPFAEAVSTMTDMT 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 ANERAYLDEWKISEEAKEALLARDYNRLLDLGGNVYFLSKLFSSDGLPFAEAVSTMTDMT 120

Qy 121 WPEYRQMMLDGGRKPEGNRSIKGGY 145
|||||||||||||||||||||||||
Db 121 WPEYRQMMLDGGRKPEGNRSIKGGY 145

Prosecution Timeline

• Sep 20, 2024: Application Filed
• Jun 23, 2026: Non-Final Rejection mailed — §102, §103, §112 (current)