DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Objections
Claims 6, 7, 11 and 12 are objected to because of the following informalities:
Claims 6 and 11 recite ornithine carbamoyltransferase subunit F (ArgF) is further weakened. Claims 7 and 12 recite arginine repressor (ArgR) is further weakened. The claims do not have any prior recitation of ArgF or ArgR. As such, recitation of “further” is understood as a reference to a further modification of the recited microorganism. However, claims should be placed in better form to reflect the same. For example, claim 6 can be amended to recite “wherein the microorganism further has weakened activity of ornithine carbamoyltransferase subunit F (ArgF).”
Appropriate correction is required.
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.
Claim 5 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 5 recites SEQ ID NO: 1 and SEQ ID NO: 2 or amino acid sequences having 90% or more homology thereto. Reeck et al. (“Homology” in Proteins and Nucleic Acids, Cell 50, 1987, 667) states:
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Since “homology” cannot be described as a “percent homology,” the meaning of “90% or more homology thereto” as recited in claim 5 is unclear such that an ordinarily skilled artisan cannot determine how to avoid infringement of claim 5, particularly since percent homology is apparently different and distinct from a percent identity.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claim(s) 1-4, 7-10 and 12-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Enhanced production of L-arginine by improving carbamoyl phosphate supply in metabolically engineered Corynebacterium crenatum, Appl. Microbiol. Biotechnol. 105, 2021, 3265-76) further in view of Zhang et al. (Improvement of l‑ornithine production by attenuation of argF in engineered Corynebacterium glutamicum S9114, AMB Expr. 8, 2018, 26) as evidenced by Xu et al. (Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of L-arginine production, J. Ind. Microbiol. Biotechnol. 39, 2012, 495-502).
Wang, abstract, teaches:
Carbamoyl phosphate is an important precursor for L-arginine and pyrimidines biosynthesis. In view of this importance, the cell factory should enhance carbamoyl phosphate synthesis to improve related compound production. In this work, we verified that carbamoyl phosphate is essential for L-arginine production in Corynebacterium sp., followed by engineering of carbamoyl phosphate synthesis for further strain improvement. First, carAB encoding carbamoyl phosphate synthetase II was overexpressed to improve the synthesis of carbamoyl phosphate. Second, the regulation of glutamine synthetase increases the supply of L-glutamine, providing an effective substrate for carbamoyl phosphate synthetase II. Third, carbamate kinase, which catalyzes inorganic ammonia synthesis carbamoyl phosphate, was screened and selected to assist in carbamoyl phosphate supply. Finally, we disrupted ldh (encoding lactate dehydrogenase) to decrease by-production formation and save NADH to regenerate ATP through the electron transport chain. Subsequently, the resulting strain allowed a dramatically increased L-arginine production of 68.6 ± 1.2 g∙L−1, with an overall productivity of 0.71 ± 0.01 g∙L−1∙h−1 in 5-L bioreactor. Stepwise rational metabolic engineering based on an increase in the supply of carbamoyl phosphate resulted in a gradual increase in L-arginine production. The strategy described here can also be implemented to improve L-arginine and pyrimidine derivatives.
“In our previous work, we obtained an L-arginine high-producing Corynebacterium crenatum SYPA5-5 (Corynebacterium glutamicum var. CCTCC AB 2021051) through screening and mutation breeding. Systematic metabolic engineering strategies, such as improvement of the intracellular environment, upregulation of nitrogen metabolism, and promotion of L-arginine excretion, alleviating feedback inhibition of key pathways, were applied to C. crenatum to increase the production of L-arginine (Man et al. 2016; Xu et al. 2013, 2019, 2020).
Xu, abstract, evidences that Corynebacterium crenatum SYPA5-5 “is an industrialized L-arginine producer, had a lethal mutation occurring in the ArgR repressor encoding gene,” which is a description of ArgR being further weakened.
“CP [carbamoyl phosphate] is an important precursor of L-arginine, and some metabolic engineering strategies have begun to focus on it for constructing a high-yielding L-arginine strain. We first constructed a carB deletion strain 5-5/ΔcarB to confirm the essential role of CP in growth and metabolism. As shown in Fig. 2a, 5-5/ΔcarB did not grow. This is because C. crenatum has a single CPS-II that produces the CP needed for arginine and pyrimidine biosynthesis.” Wang, page 3269, left col. “The growth results showed that 5-5/ΔcarB, which lacks CPS-II, can restore growth similar to the SYPA 5-5 strain, only when 1 g∙L−1 L-arginine and 1 g∙L−1 UMP are simultaneously added into the CGXII minimal medium. This result indicates that 5-5/ΔcarB is arginine and pyrimidine auxotrophic. Furthermore, we did not detect L-arginine production by 5-5/ΔcarB in the fermentation medium, although the medium contained yeast extract and added 1 g∙L−1 UMP sufficient to sustain 5-5/ΔcarB growth.” Wang, page 3269, left col.
Fig. 1 of Wang shows that carbamoyl phosphate and ornithine are converted to citrulline by the activity of ornithine carbamoyltransferase (OTC) and further shows production of carbamoyl phosphate by
Wang, Table S1, describe strains with the following genotype:
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Fig. 1 of Wang
To summarize, Wang teaches the production of a C. glutamicum strain (Table S1 identifies the SYPA 5-5 strain as C. glutamicum) having a genetic background for overproduction of arginine with a deletion of carB gene that is a weakening of carbamoyl phosphate synthetase. Deletion of carB eliminated arginine production and introduced both arginine and pyrimidine auxotrophy. Wang teaches successful culturing of the carB deletion strain under conditions wherein auxotrophy is complemented by arginine and UMP addition to growth media. The above is also understood as a description that in addition to the strain SYPA 5-5 having an endogenous carB, but also has an endogenous carA gene in the form of a carAB operon. “The CPS-II encoded by the carAB operon converts HCO3-, ATP, and glutamine to CP.” Wang, page 3273, right col.
In reference to Fig. 1 of Wang, arginine production is eliminated in the carB deletion strain since arginine production requires the activity of ornithine carbamoyltransferase (OTC) that converts carbamoyl phosphate and ornithine to citrulline. However, interruption of activity of ornithine carbamoyltransferase (OTC) is known in the prior art to result in converting a C. glutamicum strain from producing arginine to producing ornithine.
Zhang, abstract, teaches:
l-Ornithine, a non-essential amino acid, has enormous industrial applications in food, pharmaceutical, and chemical industries. Currently, l-ornithine production is focused on microorganism fermentation using Escherichia coli or Corynebacterium glutamicum. In C. glutamicum, development of high l-ornithine producing C. glutamicum was achieved by deletion of argF, but was accompanied by growth deficiency and arginine auxotrophy. l-Arginine has been routinely added to solve this problem; however, this increases production cost and causes feedback inhibition of N-acetyl-l-glutamate kinase activity. To avoid the drawbacks of growth disturbance due to disruption of ArgF, strategies were adopted to attenuate its expression. Firstly, ribosome binding site substitution and start codon replacement were introduced to construct recombinant C. glutamiucm strains, which resulted in an undesirable l-ornithine production titer. Then, we inserted a terminator (rrnB) between argD and argF, which significantly improved l-ornithine production and relieved growth disturbance. Transcription analysis confirmed that a terminator can be used to downregulate expression of argF and simultaneously improve the transcriptional level of genes in front of argF. Using disparate terminators to attenuate expression of argF, an optimal strain (CO-9) with a T4 terminator produced 6.1 g/L of l-ornithine, which is 42.8% higher than that produced by strain CO-1, and is 11.2-fold higher than that of the parent CO strain. Insertion of terminators with gradient termination intensity can be a stable and powerful method to exert precise control of the expression level of argF in the development of l-ornithine producing strains, with potential applications in metabolic engineering and synthetic biology.
The argF gene encodes ornithine carbamoyltransferase and promotes conversion of ornithine to citrulline in producing arginine. See Zhang, Fig. 1, legend. “In this study, a C. glutamicum S9114 mutant strain, with deletion of ncgl1221, lysE, putP, and argR, was selected as the parent strain for the attenuation of argF.” Zhang, page 2, left col. “We speculate that deletion of ArgR inactivated the feedback inhibition of the transcription of the argCJBDFR operon in the l-arginine biosynthetic pathway.” Zhang, page 8, left col. That is, argF is attenuated in a strain that is modified to have an increased arginine biosynthetic pathway such that carbon flow is diverted through the arginine biosynthetic pathway and its intermediates including ornithine.
As reviewed above, Wang teaches the production of a C. glutamicum strain (Table S1 identifies the strain as C. glutamicum) having a genetic background for overproduction of arginine with a deletion of carB gene that is a weakening of carbamoyl phosphate synthetase. Wang does not characterize the production of any other amino acid other than L-arginine (e.g. ornithine).
“There is no requirement that a person of ordinary skill in the art would have recognized the inherent disclosure at the relevant time, but only that the subject matter is in fact inherent in the prior art reference.” MPEP 2112(II). “In relying upon the theory of inherency, the examiner must provide a basis in fact and/or technical reasoning to reasonably support the determination that the allegedly inherent characteristic necessarily flows from the teachings of the applied prior art." MPEP 2112(IV). One a reference teaching a product appearing to be substantially identical is made the basis of a rejection, and the examiner presents evidence or reasoning to show inherency, the burden of production shifts to the applicant: "[T]he PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his [or her] claimed product. Whether the rejection is based on ‘inherency’ under 35 U.S.C. 102, on ‘prima facie obviousness’ under 35 U.S.C. 103, jointly or alternatively, the burden of proof is the same." MPEP 2112(V).
Here, Wang teaches a product that is substantially identical to the product claimed. That is, Wang teach a strain of Croynebacterium with arginine L-amino acid production ability with a weakening/deletion of carbamoyl phosphate synthetase (carB, large subunit). However, Wang does not expressly describe that such a carB deletion strain retains or manifests ornithine L-amino acid producing ability. However, Zhang teaches that when a C. glutamicum strain otherwise modified for arginine production by deletion/inactivation of at least argR has a further modification to prevent conversion of carbamoyl phosphate and ornithine to citrulline then the C. glutamicum strain will accumulate and produce L-ornithine.
Zhang blocks the conversion of carbamoyl phosphate and ornithine to citrulline by deletion of argF encoding the enzyme that catalyzes such reaction;
Wang blocks the conversion of carbamoyl phosphate and ornithine to citrulline by deletion of carB that eliminates activity of carbamoyl phosphate synthetase that produces carbamoyl phosphate such that the strain becomes auxotrophic for arginine due to lack of conversion of ornithine to citrulline that requires carbamoyl phosphate.
Since Zhang demonstrates that blocking the conversion of carbamoyl phosphate and ornithine to citrulline results in L-ornithine production in a C. glutamiucm strain that would otherwise produce arginine, the same is a basis in fact and/or technical reasoning to reasonably support that the carB deletion strain reported by Wang has ability to and does produce ornithine even if the same is not addressed by the authors of Wang. That is, in view of Zhang teaching that a C. glutamicum having a modification that results in blocking the conversion of carbamoyl phosphate and ornithine to citrulline produces ornithine, at the time of filing an ordinarily skilled artisan would have been motivated to culture and test the carB deletion C. glutamicum strain taught by Wang to produce ornithine as an L-amino acid. For this reason, the features of at least claims 1-4, 7-10 and 12-13 are suggested to an ordinarily skilled artisan at time of filing by Wang in view of the disclosure of Zhang. It is noted that a culture of the carB deletion strain of Wang is a composition.
Claim(s) 1-5, 7-10 and 12-13 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang et al. (Enhanced production of L-arginine by improving carbamoyl phosphate supply in metabolically engineered Corynebacterium crenatum, Appl. Microbiol. Biotechnol. 105, 2021, 3265-76) and Zhang et al. (Improvement of l‑ornithine production by attenuation of argF in engineered Corynebacterium glutamicum S9114, AMB Expr. 8, 2018, 26) as evidenced by Xu et al. (Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of L-arginine production, J. Ind. Microbiol. Biotechnol. 39, 2012, 495-502) as applied to claims 1-4, 7-10 and 12-13 above, and further in view of Uniprot, Accession No. P58893, 2020, www.uniprot.org, and Uniprot, Accession No. P58939, 2020, www.uniprot.org.
Regarding claim 5, we studied the corresponding genes for L-arginine biosynthesis from glutamate in C. crenatum SYPA5-5. Because the similarity of the 16SrDNA sequence and the homology of the functional DNA sequence between C. crenatum and C. glutamicum are highly homogeneous, we investigated the evolution of the genes involved in the L-arginine biosynthesis of C. crenatum with the genomic data of C. glutamicum.” Xu, page 496, left col. As discussed, Wang identifies the carB deletion strain described therein as being a variant of C. glutamicum. Regardless, Xu evidences that the gene sequences between the SYPA5-5 strain and C. glutamicum are expected to be very similar.
Uniprot P58893 evidences that the carA gene in C. glutamicum encodes a protein that is at least 99% identical to recited SEQ ID NO: 1 such that the evidence of record supports that that a protein encoded by the carAB operon of the SYPA5-5 strain of Wang has at least 90% homology to SEQ ID NO: 1.
Uniprot P58939 evidences that the carB gene in C. glutamicum encodes a protein that is at least 99% identical to recited SEQ ID NO: 2 such that the evidence of record supports that a protein encoded by the carAB operon of the SYPA5-5 strain of Wang has at least 90% homology to SEQ ID NO: 2.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1-13 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-12 of copending Application No. 18/847,617 in view of Kuwabara et al. (U.S. 2002/0090702 A1.
Copending claim 1 recites:
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SEQ ID NO: 1 of the copending claims is identical to recited SEQ ID NO: 2.
The specification of ‘617, pages 1-3, describes:
“Carbamoyl phosphate is an intermediate metabolite used in the pyrimidine biosynthetic pathway and the arginine biosynthetic pathway. Carbamoyl phosphate synthetase is an enzyme that converts bicarbonate to carbamoyl phosphate, and utilizes two molecules of ATP and one molecule of glutamine as a nitrogen donor when producing carbamoyl phosphate.”
“However, it has been reported that excessive intracellular formation of carbamoyl phosphate causes cytotoxicity . . . . Carbamoyl phosphate is a very unstable substance, and cyanate, which is naturally converted from carbamoyl phosphate, may cause cytotoxicity.”
“Accordingly, if the activity of carbamoyl phosphate synthetase in microorganisms is controlled to maintain an appropriate intracellular level of carbamoyl phosphate, to prevent unnecessary waste of ATP, and to control energy balance, it is possible to improve L-amino acid production.”
“An object of the present disclosure is to provide a carbamoyl phosphate synthetase large subunit (CarB) variant derived from the genus Corynebacterium, the variant comprising a substitution of an amino acid corresponding to position 799 with isoleucine, leucine, or valine; a substitution of an amino acid corresponding to position 918 with leucine or valine; a substitution of an amino acid corresponding to position 1079 with isoleucine, leucine, or valine in an amino acid sequence of SEQ ID NO: 1; or a combination thereof.”
The above is understood as a description that “activity of carbamoyl phosphate synthetase in microorganisms is controlled” in a manner that the activity of carbamoyl phosphate synthetase is lowered (i.e. reduced ATP consumption that is a substrate of carbamoyl phosphate synthetase in carbamoyl phosphate production) as would otherwise be present by introducing an amino acid substitution into SEQ ID NO: 2 (SEQ ID NO: 1 of the copending claims). The same is within the broadest reasonable meaning of “activity of carbamoyl phosphate synthetase is weakened,” wherein page 10 of the specification states that weakening includes modification of the polypeptide involved.
It is understood in the prior art that Coryneform bacterium contain a carbamoyl phosphate synthase as encoded by two endogenous genes coding for the small (carA) and large (carB) subunits thereof. Kuwabara, abstract, report polypeptides present in Coryneform bacteria being SEQ ID NOS: 2 and 3 of Kuwabara having at least 99% sequence identity with recited SEQ ID NOS: 1 and 2, respectively, with carbamoyl phosphate synthetase activity. Kuwabara, paras. [0077]-[0096], reports several Coryneform bacteria having corresponding genes encoding the subunits of carbamoyl phosphate synthetase including C. glutamicum. Kuwabara states that expression of genes encoding the carbamoyl phosphate synthetase can be modified by manipulating the endogenous promoters of encoding genes: “Specifically, an expression regulation sequence such as a promoter for a gene coding for carbamoyl-phosphate synthetase on the chromosomal DNA or a plasmid can be replaced with a stronger one.” Kuwabara, para. [0105].
At the time of filing, an ordinarily skilled artisan would have been motivated to implement embodiments of copending claims 1 and 4 by modifying a gene encoding SEQ ID NO: 1 of the copending claims (recited SEQ ID NO: 2), to encode the substitution at positions 799, 918 and/or 1079 as recited in the copending claims. An ordinarily skilled artisan at the time of filing would have been motivated to do this since the copending claims directly instruct that a carbamoyl phosphate synthetase large subunit (CarB) having such mutation should be expressed. Since the CarB is otherwise endogenous in Coryneform bacterium, an ordinarily skilled artisan at time of filing would have been motivated to produce embodiments of the copending claims by modifying the endogenous chromosomal gene encoding CarB to have the substitution(s) as recited in the copending claims. Upon making such a modification to a Corynebacterium and utilizing the same in a method of copending claim 9 and/or composition of copending claim 12, the features of claims 1-5 and 9-12 are met. Copending claim 8 states that the Corynebacterium is C. glutamicum, and copending claims 10 and 11 further state ArgF and ArgR are further weakened as to meet claims 6-8 and 11-12.
This is a provisional nonstatutory double patenting rejection.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652
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