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 .
Status of Claims
Receipt of Arguments/Remarks filed on 10/10/2025 is acknowledged. Claim 1 was amended. Claims 1-4 are pending.
Withdrawn Claims
The rejections of claims 1-4 on the grounds of non-statutory double patenting are withdrawn.
New and modified rejections necessitated by amendment
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 1-4 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 applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The terms “improved” and “enhanced” in claim 1 are relative terms which render the claim indefinite. The terms “improved” and “enhanced” are not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is not clear from the specification what degree of improvement or enhancement of the L-citrulline productivity or enzyme activities would be considered within the scope of the invention.
Claim 1 recites the limitation "the parent strain". There is insufficient antecedent basis for this limitation in the claim. There is no prior reference to a parent strain in the claim, and therefore it is unclear what “the parent strain” refers to. It is suggested that the claim be amended to recite “a parent strain”.
Claims 2-4 are included in this rejection because they depend on a rejected claim and do not clarify the issue.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1-4 are rejected under 35 U.S.C. 103 as being unpatentable over Stansen et al., Applied and environmental microbiology; 71(10):5920-8 in view of Yamada et al., US 2019/0161780 A1.
Regarding claim 1, Stansen teaches a Corynebacterium glutamicum strain with inactivated lactate permease gene NCgl2816 (Stansen “Abstract”, Table 1, pg. 5921 “Construction of plasmids and strains”). Stansen teaches that NCgl2816 is a lactate permease gene involved in glutamate production by C. glutamicum (Stansen pg. 5920 “Abstract”; pg. 5926 “Discussion” para. 5). Instant claim 1 recites a functional limitation of the C. glutamicum strain, i.e. improved L-citrulline productivity. As Stansen teaches a C. glutamicum strain with the same structure as instantly claimed, NCgl2816 inactivation, it is expected that this strain would have the same functional characteristic of improved L-citrulline productivity.
Regarding claim 2, Stansen teaches that C. glutamicum ATCC 13032 was used to construct the inactivation mutant (Stansen pg. 5921 “Construction of plasmids and strains”). The nucleotide sequence of NCgl2816 from C. glutamicum ATCC 13032 is 100% identical to instant SEQ ID NO: 1 (see Search Results from 6/27/2025, file “20250627_091819_us-18-025-056-1.rge”, Result 1).
Regarding claim 3, Stansen teaches that a portion of the NCgl2816 gene is deleted (Stansen pg. 5921 “Construction of plasmids and strains”).
Stansen does not teach that the NCgl2816 gene is inactivated in the parent strain of Corynebacterium glutamicum which has enhanced activities of ornithine carbamoyl transferase and carbamoyl phosphate synthase as recited in instant claim 1. Stansen does not teach a method of producing citrulline comprising the steps of culturing the mutant strain and recovering L-citrulline from the culture medium as recited in instant claim 4.
Regarding claim 1, Yamada teaches methods for producing L-amino acids by culturing bacteria having modified enzyme activity (Yamada Abstract). Yamada teaches L-citrulline producing bacteria (Yamada p. 14 para. 229). Yamada teaches that L-citrulline and L-ornithine are intermediates of the biosynthetic pathway of L-arginine, and to impart or enhance an ability to produce L-citrulline and/or L-ornithine a bacterium can be modified so that the bacterium has an increased activity or activities of one or more of the L-arginine biosynthesis enzymes (Yamada p. 14 para. 230). Yamada teaches that these enzymes include ornithine carbamoyl transferase and carbamoyl phosphate synthetase (Yamada p. 14 para. 230). Yamada teaches that L-citrulline is an L-amino acid of the glutamate family, which refers to L-amino acids that are biosynthesized via L-glutamic acid as an intermediate (Yamada p. 3 para. 66).
Regarding claim 4, Yamada teaches producing L-amino acids, including L-citrulline, by culturing the modified bacteria (such as a bacterium having increased activity of ornithine carbamoyl transferase and carbamoyl phosphate synthetase) in a culture medium to accumulate the amino acid, and then collecting the amino acid from the medium (Yamada p. 28 para. 367).
It would have been obvious to a skilled artisan, before the effective filing date, create a modified bacterium with a deletion of the NCgl2816 gene in a parent strain having increased activity of ornithine carbamoyl transferase and carbamoyl phosphate synthetase. Stansen teaches that NCgl2816 is a lactate permease involved in glutamate production. As L-citrulline is a glutamate family amino acid that is synthesized with glutamic acid as an intermediate, it would have been obvious to modify NCgl2816 in a strain having modifications to ornithine carbamoyl transferase and carbamoyl phosphate synthetase. It would further be obvious that a modified C. glutamicum strain could be cultured to produce L-citrulline, as Yamada teaches doing so with a strain having increased ornithine carbamoyl transferase and carbamoyl phosphate synthetase activity.
A person of ordinary skill in the art would have been motivated to modify the NCgl2816 gene using a parent strain with increased ornithine carbamoyl transferase and carbamoyl phosphate synthetase activity, because Yamada teaches that increasing the production of these enzymes results in increased L-citrulline production. As NCgl2816 is regulated in a carbon source-dependent manner during glutamate production in C. glutamicum, and glutamate is the precursor of L-citrulline, a skilled artisan would have been motivated to culture an NCgl2816 mutant strain with additional modifications to L-citrulline biosynthetic enzymes to produce L-citrulline (Stansen pg. 5925 “Comparative transcriptome analysis of L-lactate- and pyruvate-grown C. glutamicum ATCC 13032”). As L-citrulline has industrial utility and it is of interest to create bacterial strains to produce amino acids, including citrulline, a skilled artisan would have been motivated to create a strain having all of these modifications and culture the strain for production of L-citrulline as instantly claimed.
A skilled artisan would have a reasonable expectation of success in creating and culturing a strain with the instantly claimed modifications for L-citrulline production, as metabolic engineering of various enzymes in amino acid biosynthetic pathways for amino acid production is a well-established technique in the prior art, as taught by Yamada, and increased expression of both ornithine carbamoyltransferase and carbamoyl phosphate is associated with enhanced L-citrulline production.
Response to Arguments
Applicant’s arguments, filed 10/15/2025 have been fully considered, and in light of amendments to the claims, the rejection of claims 1-3 under 35 U.S.C. § 102 has been withdrawn. However, upon further consideration, new grounds of rejection of claims 1-4 are made under 35 U.S.C. § 103 in view of Stansen and Yamada as set forth above. Given these new grounds of rejection, the arguments presented regarding claims 1-3 rejected under 35 U.S.C. § 102 and claim 4 under 35 U.S.C. § 103 are moot.
Regarding the rejection on the grounds of non-statutory double patenting rejection, this rejection is withdrawn as the instant claims and the claims of copending 18/280,846 recite different genes that are modified.
Conclusion
Claims 1-4 are rejected. No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/EMILY F EIX/Examiner, Art Unit 1653
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653