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
Applicant’s amendment filed 12 May 2026 is entered. Claims 1 and 7-11 are amended, claims 2-6 are canceled, and claims 12-18 are new. Claims 1 and 7-18 are pending and under examination.
Claim Objections
Claims 15 and 18 are objected to because of the following informalities: The redundant term “the method comprising” on line 2 of both claims 15 and 18 should be deleted.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
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 and 7-18 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.
Claim 1 recites an increased intracellular copy number of a gene encoding the nuo operon. It is unclear what is meant by “a gene” encoding the nuo operon because the nuo operon is a series of genes and is not encoded by a single gene.
Claims 7-14 are dependent on claim 1, so are indefinite for the same reason.
Claim 15 recites a method for enhancing NADH:quinone oxidoreductase activity in a strain, and then recites one method step “inserting a heterologous promoter operably linked to a nuo operon in a downstream region of an endogenous promoter in the strain, thereby producing the recombinant strain of claim 1”. The claim only recites that the method step produces the recombinant strain of claim 1. The claim does not recite any steps that produce a NADH:quinone oxidoreductase activity enhancing effect; thus it is unclear how the recited method step achieves the enhancing effect recited in the preamble.
Claims 16-17 are dependent on claim 15, so are indefinite for the same reason.
Claim 16 recites the activity of phosphoserine phosphatase (SerB) in the recombinant strain is weakened compared to its endogenous activity. It is unclear what is meant by “weakened”, i.e., it is unclear if the gene encoding the enzyme is modified so that the enzyme activity is decreased, or if the gene encoding the enzyme exhibits decreased expression, etc.
Claim 17 recites the activity of O-phosphoserine export protein (YhhS) in the recombinant strain is enhanced compared to its endogenous activity. It is unclear what is meant by “enhanced”, i.e., it is unclear if the individual export proteins are modified so as to export more O-phosphoserine, or if the genes encoding the export proteins are overexpressed leading to increased O-phosphoserine export, etc.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 16-17 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
The limitations “an activity of phosphoserine phosphatase (SerB) in the recombinant strain is weakened compared to its endogenous activity” in claim 16 and “the activity of O-phosphoserine export protein (YhhS) in the recombinant strain is enhanced compared to its endogenous activity” in claim 17 are desired properties of the phosphoserine phosphatase and O-phosphoserine export protein expressed by the recombinant strain, but the claims do not add any new active method steps or structural limitations to the method steps required by claim 15; thus, claims 16 and 17 fail to further limit the subject matter of claim 15.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
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.
Claims 1, 7-9, and 12-13 are rejected under 35 U.S.C. 103 as being unpatentable over Spehr et al., (Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I). Biochemistry. 1999 Dec 7;38(49):16261-7, cited in the IDS filed 12/21/2023) in view of Kim et al., (EP3348567 B1, cited in the IDS filed 02/24/2025).
Regarding claim 1, Spehr teaches a recombinant Escherichia coli microorganism comprising a nuo-operon which was overexpressed by replacing its upstream 5’ promoter region with the phage-T7 RNA polymerase promoter (a modified promoter operably linked to the nuo-operon) (Spehr abstract).
Spher does not teach that the microorganism has O-phosphoserine producing ability.
However, Kim teaches an O-phosphoserine-producing microorganism (para. [0001]).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to engineer the recombinant microorganism of Spehr to produce O-phosphoserine, as taught by Kim. One of ordinary skill in the art would have been motivated to do so because Kim teaches that O-phosphoserine may be used to produce L-cysteine, which is an amino acid with an important role in sulfur metabolism in all living organisms (para. [0002]- [0003]). One of ordinary skill in the art would have had a reasonable expectation of success because Spehr and Kim are in the same field of endeavor of recombinant microorganism development.
Regarding claim 7, Spehr teaches that the recombinant microorganism is Escherichia coli (Spehr Abstract). Kim teaches that the microorganism may be Escherichia coli (Kim [0027]).
Regarding claim 8, Kim teaches a method for producing O-phosphoserine comprising culturing the microorganism that produces it (para. [0001]; para. [0009]). Kim teaches that the culture process may be performed according to the appropriate medium and conditions for culture known in the art (para. [0064]).
Regarding claim 9, Kim teaches that the O-phosphoserine may be recovered from the culture (para. [0068]).
Regarding claim 12, Spehr teaches that overexpression of the nuo-operon was achieved by replacing its 5’-promoter region, i.e., the region upstream of the operon, with the phage-T7 RNA polymerase (heterologous) promoter and then expressing the genes with the T7 RNA polymerase coded on an inducible plasmid (Spehr Abstract).
Regarding claim 13, it is interpreted that the claim requires that the nuoA gene is upstream of all other genes of the operon. Because nuoA is canonically the first gene in the operon, it is considered that nuoA of Spehr is upstream of all other genes of the operon.
Claims 10 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al., (EP3348567 B1, cited in the IDS filed 02/24/2025) in view of Spehr et al., (Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I). Biochemistry. 1999 Dec 7;38(49):16261-7, cited in the IDS filed 12/21/2023).
Regarding claim 10, Kim teaches a method for producing cysteine or a derivative thereof, comprising culturing an O-phosphoserine-producing E. coli in a medium to produce O-phosphoserine; and reacting the O-phosphoserine with a sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the OPSS (Kim [0069]).
Kim does not teach the cultured O-phosphoserine-producing E. coli comprises a nuo operon with one or more genetic modifications selected from an increased intracellular copy number of a gene encoding the nuo operon, and a modified promoter operably linked to the nuo operon.
Spehr teaches a recombinant Escherichia coli microorganism comprising a nuo-operon which was overexpressed by replacing its upstream 5’ promoter region with the phage-T7 RNA polymerase promoter (a modified promoter operably linked to the nuo-operon) (Spehr abstract).
It would have been obvious to one of ordinary skill in the art, prior to the effective filing date of the claimed invention, to modify Kim’s recombinant E. coli microorganism by inserting and operably linking a heterologous phage-T7 RNA polymerase promoter to the recombinant strain’s nuo operon as taught by Spehr. One of ordinary skill in the art would have been motivated to do so because overexpressing the genes encoding NADH:ubiquinone oxidoreductase, as taught by Spehr, would increase the intracellular concentration of O-phosphoserine, which Kim teaches may be used to produce L-cysteine, an amino acid with an important role in sulfur metabolism in all living organisms (Kim [0002]- [0003]). Therefore, one of ordinary skill in the art would be motivated to overexpress the nuo operon to generate a recombinant microorganism that can produce higher levels of cysteine. One of ordinary skill in the art would have had a reasonable expectation of success because Spehr and Kim are in the same field of endeavor of recombinant microorganism development.
Regarding claim 11, Kim teaches that the sulfide may be Na2S, NaSH, (NH4)2S, or Na2S2O3 (Kim [0073]).
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Spehr in view of Kim as applied to claims 1, 7-9, and 12-13 above, and further in view of Li et al. (A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli, Microbial Cell Factories 2012, 11:19).
Spehr and Kim do not teach that the endogenous nuo operon promoter is maintained in the recombinant strain, nor that the operably linked heterologous promoter is inserted downstream of the endogenous promoter.
Li teaches inserting multiple tandem promoters upsteam of an operon increased the transcription strength of tandem promoter clusters and increased the overexpression of the linked operon, in their case the phbCAB genes (Li abstract and pg. 6 conclusion). Li teaches that older methods of simply adding a copy of the operon created genetic instability and additional metabloic burden on the E. coli, but their new method of inserting multiple tandem promoters maintains genetic stability while also providing increased overexpression of the linked gene operon (Li abstract and pg. 6 conclusion).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to insert Spehr's heterologous phage-T7 RNA polymerase promoter in tandem with the endogenous E. coli nuo operon promoter because Li teaches that tandem promoters increase transcription strength and gene overexpression while maintaining stability (Li abstract and pg. 6 conclusion). It would have been obvious for one of ordinary skill in the art to try to insert Spehr's heterologous phage-T7 RNA polymerase promoter downstream of endogenous E. coli nuo operon promoter because a finite number of identified, predictable options (upstream or downstream) were recognized, and Li taught that inserting tandem promoters would have a reasonable expectation of success.
Claims 15-18 are rejected under 35 U.S.C. 103 as being unpatentable over Spehr et al., (Overexpression of the Escherichia coli nuo-operon and isolation of the overproduced NADH:ubiquinone oxidoreductase (complex I). Biochemistry. 1999 Dec 7;38(49):16261-7, cited in the IDS filed 12/21/2023) in view of Li et al. (A strategy of gene overexpression based on tandem repetitive promoters in Escherichia coli, Microbial Cell Factories 2012, 11:19).
Regarding claims 15 and 18, Spehr teaches a method of overexpressing NADH:ubiquinone oxidoreductase and the nuo operon in recombinant Escherichia coli, and that the overexpression was achieved by inserting a heterologous phage-T7 RNA polymerase promoter upstream of the nuo operon, then expressing the genes with a T7 RNA polymerase (Spehr Abstract).
Spehr does not teach inserting the heterologous phage-T7 RNA polymerase promoter downstream of an endogenous promoter in the E. coli recombinant strain.
Li teaches inserting multiple tandem promoters upsteam of an operon increased the transcription strength of tandem promoter clusters and increased the overexpression of the linked operon, in their case the phbCAB genes (Li abstract and pg. 6 conclusion). Li teaches that older methods of simply adding a copy of the operon created genetic instability and additional metabloic burden on the E. coli, but their new method of inserting multiple tandem promoters maintains genetic stability while also providing increased overexpression of the linked gene operon (Li abstract and pg. 6 conclusion).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to insert Spehr's heterologous phage-T7 RNA polymerase promoter in tandem with the endogenous E. coli nuo operon promoter because Li teaches that tandem promoters increase transcription strength and gene overexpression while maintaining stability (Li abstract and pg. 6 conclusion). It would have been obvious for one of ordinary skill in the art to try to insert Spehr's heterologous phage-T7 RNA polymerase promoter downstream of endogenous E. coli nuo operon promoter because a finite number of identified, predictable options (upstream or downstream) were recognized, and Li taught that inserting tandem promoters would have a reasonable expectation of success.
Regarding claims 16 and 17, the limitations “an activity of phosphoserine phosphatase (SerB) in the recombinant strain is weakened compared to its endogenous activity” in claim 16 and “the activity of O-phosphoserine export protein (YhhS) in the recombinant strain is enhanced compared to its endogenous activity” in claim 17 are interpreted as intended properties of the phosphoserine phosphatase and O-phosphoserine export protein expressed by the recombinant strain, but the claims do not add any new active method steps to claim 15’s method; thus, if the prior art teaches all of the active method steps recited in claim 15, claims 16 and 17 will be considered to be rendered obvious.
Response to Arguments
Applicant's arguments filed 12 May 2026 have been fully considered but they are not persuasive.
Regarding Applicant’s argument that Spehr does not teach “an increased intracellular copy number of a gene encoding the nuo operon” and Kim fails to cure this deficiency (Remarks pg. 4), the limitation “an increased intracellular copy number of a gene encoding the nuo operon” in claim 1 is an alternative limitation recited in a Markush group. Spehr does teach a modified promoter operably linked to the nuo operon.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Alexander M Duryee whose telephone number is (571)272-9377. The examiner can normally be reached Monday - Friday 9:00 am - 5:00 pm.
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/LOUISE W HUMPHREY/ Supervisory Patent Examiner, Art Unit 1657
/Alexander M Duryee/Examiner, Art Unit 1657
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