DETAILED CORRESPONDENCE
Application Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Support for the amendments are within the instant application specification.
Examiner acknowledges the amendment of the claim set dated 2/3/2025 to the claim set dated 10/23/2025 which is now directed to the instant application invention. As such, said claims are examined on the merits.
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 02/14/2024 has been entered.
Applicant’s amendment to the claims filed on 11/4/2025 in response to the Nonfinal Rejection mailed on 11/4/2024 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
Claims 1-10, 12, 16-18 are canceled.
Claims 11, 13-15, 19-28 are pending and examined on the merits.
Applicant’s remarks filed on 10/23/2025 in response to the Nonfinal rejection dated 11/4/2025 and Final Rejection dated 4/23/2025 have been fully considered and are deemed persuasive to overcome at least one of the rejections and/or objections as previously applied.
The text of those sections of Title 35 U.S. Code not included in the instant action can be found in the prior Office Action.
Withdrawn Rejections
The rejection of Claim 10 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, is withdrawn in view of cancellation of claim 10.
The rejection of Claims 1-2, 4, 7-8, 10-15 under 35 U.S.C. 103 as being unpatentable over Harada et al. (JP2009207376A, priority to 03-03-2008, previously cited in PTO-892 filed 9/25/2023; machine translation generated in Espacenet attached) {herein Harada}, in view of Masignani et al. (WO 02/094868 A2, Date of Publication: 28 November 2002, previously cited in PTO-892 filed 9/25/2023) {herein Masignani}, Demain et al. (1967, Journal of Bacteriology, https://journals.asm.org/journal/jb on 15 April 2024 by 151.207.250.216, cited on IDS filed 4/8/2022) {herein Demain} and GenBank WP_012730718.1 (26 May 2013, https://www.ncbi.nlm.nih.gov/protein/WP_012730718.1, previously cited in PTO-892 filed 5/7/2024) {herein GenBank WP_012730718.1} and as evidenced by Gronenberg et al. (2013, SciVerse ScienceDirect, http://dx.doi.org/10.1016/j.cbpa.2013.03.037, previously cited in PTO-892 filed 5/7/2024) {herein Gronenberg} is withdrawn in view of cancellation of claims 1-2, 4, 7-8, 10 and amendment of claim 11 to recite ‘comprising the enzymes: acetyl-CoA acetyltransferase (AtoB), hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR),mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2.’
The rejection of Claim 5 under 35 U.S.C. 103 as being unpatentable over Harada et al. (JP2009207376A, priority to 03-03-2008, previously cited in PTO-892 filed 9/25/2023; machine translation generated in Espacenet attached) {herein Harada} in view of Masignani et al. (WO 02/094868 A2, Date of Publication: 28 November 2002, previously cited in PTO-892 filed 9/25/2023) {herein Masinani}, Demain et al. (1967, Journal of Bacteriology, https://journals.asm.org/journal/jb on 15 April 2024 by 151.207.250.216, cited on IDS filed 4/8/2022) {herein Demain} and GenBank WP_012730718.1 (26 May 2013, https://www.ncbi.nlm.nih.gov/protein/WP_012730718.1, previously cited in PTO-892 filed 5/7/2024) {herein GenBank WP_012730718.1} as applied to claims 1-2, 4, 7-8, 10-15 above in further view of Mao et al. (2017, Available online 23 October 2017. Green Chem, 10.1039/c7gc02753b, previously cited in PTO-892 filed 9/25/2023) {herein Mao} as evidenced by Gronenberg et al. (2013, SciVerse ScienceDirect, http://dx.doi.org/10.1016/j.cbpa.2013.03.037, previously cited in PTO-892 filed 5/7/2024) {herein Gronenberg} is withdrawn in view of Applicant’s cancellation of claim 5.
112a Written Description
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 11, 13-15, 19-28 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2163.II.A.2.(a).i) states, “Whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention”.
For claims drawn to a genus, MPEP § 2163 states the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406.
Claims 11, 13-15, 19-28 are drawn to a method for a genetically modified bacterial host cell producing tetramethyl pyrazine (TMP), comprising (a) providing a genetically modified Corynebacterium host cell comprising the enzymes: acetyl-CoA acetyltransferase (AtoB), hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR),mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2, (b) culturing or growing the host cell in a suitable culture or medium comprising an ionic liquid (IL), such that the host cell produces equal to or more than 5 g of TMP per L of the culture or medium in 48 hours. The structure of HMGR having at least 80% identity to SEQ ID NO: 3 or 4 is a large number of sequences. The structure of the HMGR (SEQ ID NO: 3 or 4) capable of accepting up to 20% sequence variation is a large number of sequences. In addition, the structure of MK having at least 80% sequences identity to SEQ ID NO: 1 or 2 is a large number of sequences. The structure of the MK (SEQ ID NO: 1 or 2) capable of accepting up to 20% sequence variation is a large number of sequences.
In this case, the specification discloses the following representative species of HMGR and MK are encompassed by the claims (i.e. SEQ ID NO: 3, 4 (HMGR) and SEQ ID NO: 1, 2 (MK)). Other than the above disclosed species, there is no prior-art or disclosed teaching as to the large number of HMGR and MK proteins that would encompass at least 20% variability. The breadth of the claims encompass a large number of sequences of HMGR and MK proteins with 20% variability.
An adequate written description of a chemical invention also requires a precise definition, such as by structure, formula, chemical name, or physical properties, and not merely a wish or plan for obtaining the chemical invention claimed. See, e.g., Univ. of Rochester v. G.D. Searle & Co., 358 F.3d 916, 927, 69 USPQ2d 1886, 1894-95 (Fed. Cir. 2004). Here, the disclosure fails to teach which combinations of amino acids out of the numerous possibilities encompass the 20% variability for the production of TMP in culture or medium from genetically modified Corynebacterium.
Accordingly, one of skill in the art would not accept the disclosure of SEQ ID NO: 3 and 4 as being representative of all HMGR proteins as encompassed by the claims. Furthermore, one of skill in the art would not accept the disclosure of SEQ ID NO: 1 and 2 as being representative of all MK proteins as encompassed by the claims. As such, the specification, taken with the pre-existing knowledge in the art of HMGR and MK, fails to satisfy the written description requirement of 35 U.S.C. 112, first paragraph.
RESPONSE TO REMARKS: Applicants remarks filed on 10/23/2025 have been fully considered; however, they are rendered moot. Examiner contends that the recitation ‘at least 80% sequence identity’ encompasses 20% variability which is a large number of sequences that have not been defined in the instant application claims and specification. One of ordinary skill in the art would not ascertain the structure of the 20% variability of SEQ ID Nos: 1-4. It is recommended that Applicant amend the claim to recite ‘at least 90% sequence identity.’
Scope of Enablement
Claims 11, 13-15, 19-28 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for HMGR comprising a full length amino acid sequence having 100% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4 and MK comprising a full length amino acid sequence having 100% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2, it does not reasonably provide enablement for all HMGR and MK having 20% variation which is the same as sequences having 80% sequence identity as said variation is comprised of a large number of sequences as encompassed by the claims. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
The test of enablement is not whether any experimentation is necessary, but whether, if experimentation is necessary, it is undue.” In re Angstadt, 537 F.2d 498, 504, 190 USPQ 214, 219 (CCPA 1976). Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands (858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988)) as follows: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. See MPEP § 2164.01(a). The Factors considered to be most relevant to the instant rejection are addressed in detail below.
(A)The breadth of the claims:
Claims 11, 13-15, 19-28 are drawn to a method for a genetically modified bacterial host cell producing tetramethyl pyrazine (TMP), comprising (a) providing a genetically modified Corynebacterium host cell comprising the enzymes: acetyl-CoA acetyltransferase (AtoB), hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR),mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2, (b) culturing or growing the host cell in a suitable culture or medium comprising an ionic liquid (IL), such that the host cell produces equal to or more than 5 g of TMP per L of the culture or medium in 48 hours. The structure of HMGR having at least 80% identity to SEQ ID NO: 3 or 4 is a large number of sequences. The structure of the HMGR (SEQ ID NO: 3 or 4) capable of accepting up to 20% sequence variation is a large number of sequences. In addition, the structure of MK having at least 80% sequences identity to SEQ ID NO: 1 or 2 is a large number of sequences. The structure of the MK (SEQ ID NO: 1 or 2) capable of accepting up to 20% sequence variation is a large number of sequences.
B) The nature of the invention; C)The state of the prior art; (D) The level of one of ordinary skill; and (E) The level of predictability in the art: As noted above, the scope of the claimed 20% variability is a large number of sequences.
It is well-known in the prior art that the amino acid sequence of a polypeptide determines the polypeptide’s functional properties. The positions within a protein's sequence where modifications can be made with a reasonable expectation of success in obtaining a polypeptide having the desired activity/utility are limited in any protein and the result of such modifications is highly unpredictable. In addition, one skilled in the art would expect any tolerance to modification for a given protein to diminish with each further and additional modification, e.g., multiple substitutions. The reference of Singh et al. (Current Protein and Peptide Science, 2017; examiner cited) reviews various protein engineering methods and discloses that despite the availability of an ever-growing database of protein structures and highly sophisticated computational algorithms, protein engineering is still limited by the incomplete understanding of protein functions, folding, flexibility, and conformational changes [see p. 7, column 1, top]. The reference of Zhang et al. (Structure, 2018; examiner cited) discloses that a mutation of a residue that was predicted to be benign caused significant structural changes and unexpected effects on the function of a polypeptide [p. 1475, column 1].
It is well-known in the art that even a single amino acid alteration can alter the folding of a polypeptide. See, e.g., MPEP 2144.08.II.A.4.(c), which states, “[i]n the area of biotechnology, an exemplified species may differ from a claimed species by a conservative substitution (“the replacement in a protein of one amino acid by another, chemically similar, amino acid... [which] is generally expected to lead to either no change or only a small change in the properties of the protein.” Dictionary of Biochemistry and Molecular Biology 97 (John Wiley & Sons, 2d ed. 1989)). The effect of a conservative substitution on protein function depends on the nature of the substitution and its location in the chain. Although at some locations a conservative substitution may be benign, in some proteins only one amino acid is allowed at a given position. For example, the gain or loss of even one methyl group can destabilize the structure if close packing is required in the interior of domains. James Darnell et al., Molecular Cell Biology 51 (2d ed. 1990).”
(F) The amount of direction provided by the inventor and (G) The existence of working examples: The specification discloses the following working examples of full-length HMGR and MK (i.e. HMGR comprising at least 80% sequence identity to SEQ ID NO: 3 and 4 (HMGR) and MK comprising at least 80% sequence identity to SEQ ID NO: 1 and 2). Other than the above disclosed species, there is no prior-art or disclosed teaching as to the number of sequences that comprises the 20% variability of HMGR and MK. Other than the above disclosed species, there is no prior-art or disclosed teaching as to the large number of sequences that comprise the 20% variability of the claimed HMGR and MK.
In view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability, and the state of the prior art, undue experimentation would be necessary for a skilled artisan to make and use the entire scope of the claimed invention. Applicants have not provided sufficient guidance to enable one of ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of having the desired biological characteristics is unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
RESPONSE TO REMARKS: Applicants remarks filed on 10/23/2025 have been fully considered; however, they are rendered moot. Examiner contends that the recitation ‘at least 80% sequence identity’ encompasses 20% variability which is a large number of sequences that have not been defined in the instant application claims and specification. As such, Applicant is not enabled for all sequences that may encompass the 20% variability of SEQ ID Nos: 1-4. It is recommended that Applicant amend to the claim to recite ‘at least 90% sequence identity.’
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 23 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.
Regarding claim 23, the phrase " having at least 80% amino acid identity with SEQ ID NO:4" renders the claim indefinite because it is not further limiting from the claim 22, upon which claim 23 depends. Specifically, claim 23 depends from claim 22 but goes back limiting the sequences to 80% (broadening), thus not further limiting claim 23. Instead of narrowing the claim limitation, claim 23 broadens (90% sequence identity versus 80% sequence identity). It is noted that claim 22 claims 90% sequence identity with SEQ ID NO: 3 OR 4. However, both sequences have equal chances to be selected by those skilled in the art and if SEQ ID NO:4 is picked, then 23 is not further limiting but rather broadening the limitation. See MPEP § 2173.05(d).
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.
Claims 11, 13-15, 19-28 are newly rejected under 35 U.S.C. 103 as being unpatentable over Demain et al. (1967, Journal of Bacteriology, https://journals.asm.org/journal/jb on 15 April 2024 by 151.207.250.216, cited on IDS filed 4/8/2022) {herein Demain} in view of Garsin et al (Date Published: April 2010, NIH Public Access, Examiner cited) {herein Garsin}, Harada et al. (JP2009207376A, priority to 03-03-2008, previously cited in PTO-892 filed 9/25/2023; machine translation generated in Espacenet) {herein Harada} and Masinani et al. (WO 02/094868 A2, Date of Publication: 28 November 2002, previously cited in PTO-892 dated 5/7/2024) {herein Masignani} as evidenced by ChemSpider (2025, Royal Society, of Chemistry, Examiner cited) {herein ChemSpider}, GenBank WP_012730718.1 (26 May 2013, https://www.ncbi.nlm.nih.gov/protein/WP_012730718.1, previously cited in PTO-892 filed 5/7/2024) {herein Genbank WP_012730718.1}, Gronenberg et al. (2013, SciVerse ScienceDirect, http://dx.doi.org/10.1016/j.cbpa.2013.03.037, previously cited in PTO-892 filed 5/7/2024) {herein Gronenberg} and Andressi et al (2009, NIH Public Access, Examiner cited) {herein Andressi}. The new rejection is necessitated by Applicant’s amendment of claim 11 to recite ‘comprising the enzymes: acetyl-CoA acetyltransferase (AtoB), hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR),mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2.’
As amended, claims 11, 13-15, 19-28 are drawn to a method for a genetically modified bacterial host cell producing tetramethyl pyrazine (TMP), comprising (a) providing a genetically modified Corynebacterium host cell comprising the enzymes: acetyl-CoA acetyltransferase (AtoB), hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR),mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2, (b) culturing or growing the host cell in a suitable culture or medium comprising an ionic liquid (IL), such that the host cell produces equal to or more than 5 g of TMP per L of the culture or medium in 48 hours.
With respect to claims 11, 20, Demain teaches a method wherein a mutant of Corynebacterium glutamicum was found to accumulate tetramethylpyrazine in the presence of thiamine (abstract). Furthermore, Examiner is interpreting a mutant of Corynebacterium glutamicum as being genetically modified as said strain is not wildtype. Accumulation of tetramethylepyrazine is dependent upon the addition of thiamine (vitamin B) in growth media (abstract). Evidentiary reference of Takeno is cited to demonstrate that Corynebacterium natively contains acetyl-CoA transferase, which is the same as the claimed acetyl-CoA acetyltransferase (claim 11) (page 6779, fig 3 legend). As such, it would be obvious to one of ordinary skill in that art that the mutant of Corynebacterium glutamicum, taught by Demain, natively contains acetyl-CoA acetyltransferase sequence since Demain does not teach said enzyme is deleted from the mutant Corynebacterium glutamicum. Regarding the limitation “such that the host cell produces equal to or more than 5 g of TMP per L of the culture or medium in 48 hours,” (claim 11) this language does not require steps to be performed or limit the claim to a particular structure and is just the characteristic of the microorganism which characteristic could be inherent. .
With respect to claim 14, Demain teaches a method wherein TMP is separated from culture by filtration and crystallization (page 324, column 1, paragraph 5).
With respect to claim 21, the Examiner takes the position that the strain taught by Demain and the strain within the instant application claim 21 are inherently the same as they belong to the same genus and species. Absent evidence otherwise, it would be obvious to one of ordinary skill in the art that Demain could utilize a genetically modified bacterial strain of ATCC 13032 Corynebacterium glutamicum , as said strain is the same genus and species of the mutant strain Corynebacterium glutamicum taught by Demain.
However, Demain does not teach the method of claim 11 of host cell comprising enzymes hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR), mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2; a method wherein ‘culturing or growing the host cell in a suitable culture or medium comprising an ionic liquid (IL), such that the host cell produces equal to or more than 5 g of TMP per L of the culture or medium in 48 hours’ (claim 11). The method of claim 13, wherein step (b) further comprises producing isopentenol (claim 13). ( The method of claim 15, further comprises (c) extracting or separating the isopentenol from the rest of the culture or medium, and/or host cell (claim 15). The method of claim 19, wherein the genetically modified bacterial host cell also produces isopentenol (claim 19). The method of claim 22, wherein the HMGR comprises a full length amino acid sequence having at least 90% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4 (claim 22). The method of claim 23, wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:4, and a conserved domain having an amino acid sequence TAVNGILGRGK (SEQ ID NO:7) (claim 23). The method of claim 24, wherein the MK comprises an amino acid sequence RGLGSSAA (SEQ ID NO:5) in a GHMP kinase N domain and/or KLTGXGRGG (SEQ ID NO:6) in a GHMP kinase C domain, wherein X is any naturally occurring amino acid residue (claim 24). The method of claim 25, wherein the culture medium comprises 50 mM to 150 mM of the IL (claim 25). The method of claim 26, wherein the IL is an acetate salt, imidazolium, cholinium, biogenic, or protic IL (claim 26). The method of claim 27, wherein the IL comprises an ethanolamine [ETA],diethanolamine [DEOA], [C2Cim], or [Ch] IL (claim 27). The method of claim 28, wherein the ionic liquid comprises a 1-ethyl-3- methyl imidazolium ([C2C1im]+), cholinium ([Ch]+), ethanolamine [ETA], or and diethanolamine [DEOA] cation (claim 28)
With respect to claims 11, 25-28, Garsin teaches a method wherein Corynebacterium species are able to utilize ionic liquid culture medium comprised of ethanolamine as a sole source of carbon and/or nitrogen (page 1, para 1). Evidentiary reference of ChemSpider is cited to demonstrate ethanolamine is an acetate salt (page 3). As such, Examiner is interpreting the ethanolamine containing ionic liquid, taught by Garsin, as being an acetate salt. Garsin further discloses the claimed ionic culture medium except for the culture medium comprises 50 mM to 150 mM of the ionic liquid. However, Examiner takes the position that it would have been obvious to one having ordinary skill in the art at the time the invention was made to find the most appropriate concentration of ionic liquid for the culture medium, since it has been held that where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. In re Aller, 105 USPQ 233.
However, Garsin does not teach the method of claim 11 of host cell comprising enzymes hydroxymethylqlutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylqlutaryl-CoA reductase (HMGR), mevalonate kinase (MK), and/or phosphomevalonate decarboxylase (PMD); wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4, the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 or SEQ ID NO:2 (claim 11).The method of claim 13, wherein step (b) further comprises producing isopentenol (claim 13). The method of claim 15, further comprises (c) extracting or separating the isopentenol from the rest of the culture or medium, and/or host cell (claim 15). The method of claim 19, wherein the genetically modified bacterial host cell also produces isopentenol (claim 19). The method of claim 22, wherein the HMGR comprises a full length amino acid sequence having at least 90% amino acid identity with SEQ ID NO:3 or SEQ ID NO:4 (claim 22). The method of claim 23, wherein the HMGR comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:4, and a conserved domain having an amino acid sequence TAVNGILGRGK (SEQ ID NO:7) (claim 23). The method of claim 24, wherein the MK comprises an amino acid sequence RGLGSSAA (SEQ ID NO:5) in a GHMP kinase N domain and/or KLTGXGRGG (SEQ ID NO:6) in a GHMP kinase C domain, wherein X is any naturally occurring amino acid residue (claim 24).
With respect to claims 11, 13, 15, 19, 22-23, Harada teaches a method wherein a recombinant bacterial strain that produces heterologous isoprenoid enzymes such as MK (referred to as MVA kinase by Harada) and HMGR is provided (referred to as HMG-CoA reductase by Harada) (page 4, para 0009, lines 5; pages 11-12, paragraph 0041, lines 456 - 460) in a culture medium comprised of salt as a substrate for the more efficient production of isoprenoids (para 0011). Evidentiary reference of GenBank WP_012730718.1 is cited to demonstrate that full-length HMGR is 100% identical to the claimed SEQ ID NO: 4 also having a conserved domain of TAVNGILGRGK (SEQ ID NO: 7) (Appendix A). As such, absent evidence otherwise, Examiner is interpreting the enzyme HMGR taught by Harada as being the same as (100% identical) to the instant application SEQ ID NO: 4 with a conserved domain of TAVNGILGRGK (SEQ ID NO: 7) since the enzyme taught by Harada is unmutated. Harada further teaches a method of producing isoprenoids in bacteria and obtaining it from the culture (para 0023). Evidentiary reference of Gronenberg is cited to demonstrate that isopentenol is an isoprenoid derivative (page 17, column 2, paragraph 1). As such, absent evidence otherwise, Examiner is interpreting a cell that produces isoprenoids would necessarily also produce isopentenol since isopentenol is a derivative of isoprenoids. Furthermore, Examiner is interpreting the teaching of ‘obtaining isoprenoid from the culture’ to be the separation of isoprenoid from the culture, as recited in the instant application claim 15.
However, Harada does not teach the method of claim 11, wherein the MK comprises a full length amino acid sequence having at least 80% amino acid identity with SEQ ID NO:1 (claim 11). The method of claim 24, wherein the MK comprises an amino acid sequence RGLGSSAA (SEQ ID NO:5) in a GHMP kinase N domain and/or KLTGXGRGG (SEQ ID NO:6) in a GHMP kinase C domain, wherein X is any naturally occurring amino acid residue (claim 24).
With respect to claims 11, 24, Masignani teaches a method wherein MK is 100% identical to full length MK of SEQ ID NO: 1 within the instant application (Masignani: claim 1; appendix B) and consists of an amino acid sequence RGLGSSAA, which has 100% identity to SEQ ID NO: 5 (appendix B, highlighted) of the instant application (appendix B). Evidentiary reference of Andreassi is cited to demonstrate that the mevalonate pathway consists of mevalonate kinase (MK), which is the N-terminal sequence of GHMP (page 4, paragraph 1). As such, Examiner is interpreting MK with amino acid sequence RGLGSSAA, taught by Masignani, as being the same as the MK recited in the instant application claims 11 and 24 (appendix B (highlighted)), which is the N-terminal sequence of GHMP.
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to apply the teachings of Demain of a method of a mutant of Corynebacterium glutamicum to accumulate tetramethylpyrazine in the presence of thiamine (abstract) or combine the teachings of Garsin, Harada and Masignani because Masignani teaches a method wherein MK is 100% identical to full length MK of SEQ ID NO: 1 within the instant application (Masignani: claim 1; appendix B) and consists of an amino acid sequence RGLGSSAA, which has 100% identity to SEQ ID NO: 5 (appendix B, highlighted) of the instant application (appendix B). Harada teaches a method wherein a recombinant bacterial strain that produces heterologous isoprenoid enzymes such as MK (referred to as MVA kinase by Harada) and HMGR (SEQ ID NO: 4) are provided (referred to as HMG-CoA reductase by Harada) (page 4, para 0009, lines 5; pages 11-12, paragraph 0041, lines 456 - 460) in a culture medium comprised of salt as a substrate for the more efficient production of isoprenoids (para 0011). Whereas, Garsin teaches a method wherein Corynebacterium species in an ionic liquid culture medium comprised of ethanolamine are able to use the ethanolamine as a sole source of carbon and/or nitrogen (page 1, para 1).
One of ordinary skill in the art would be motivated to either use the teachings of Demain et al. by itself or combine the teachings of Garsin, Harada and Masignani because Garsin provides the motivation for Demain to utilize an ionic liquid containing ethanolamine since Garsin teaches Corynebacterium species are able to use the ethanolamine as a sole source of carbon and/or nitrogen (page 1, para 1). Additionally, since only a select variety of bacteria are able to use ethanolamine as a carbon source (Garsin: abstract) said ionic compound could serve as a means for limiting the growth of contaminants. Furthermore, Demain would be motivated to modify the genetically modified Corynebacteium by adding heterologous isoprenoid enzymes such as MK (SEQ ID NO: 1) (referred to as MVA kinase by Harada) (Masignani: appendix B) and HMGR (referred to as HMG-CoA reductase by Harada) (Harada: pages 11-12, paragraph 0041, lines 456 – 460, appendix A) to the culture medium taught by Garsin because the expression of said genes in heterologous bacteria have been shown to result in the production of Isoprenoids (Harada: para 0001, 0009). Furthermore, since HMGR is from Corynebacterium (SEQ ID NO: 4) (appendix A) one of ordinary skill in the art would expect limited to no deleterious effects from the recombination of the heterologous genes and the appropriate expression of TMP and isopentenol. One of ordinary skill in the art would have a reasonable expectation of success to make and use a culture medium comprising an ionic liquid and a genetically modified bacterial host cell capable of producing tetramethyl pyrazine comprising the enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MK) because doing so would result in a culture medium wherein the isoprenoids (HMGR and MK) along with the ionic liquid within the culture medium would serve as a continuous carbon source for the production of TMP, thereby bypassing the need for the continuous replenishment of a carbon source in cultures for the production of TMP.
One of skill in the art would have a reasonable expectation of success to make and use the claimed method for producing TMP because Demain provides the basic method for producing TMP in genetically modified Corynebacterium and its uses and methods of making it. Reference of Garsin provides the teaching of a method wherein Corynebacterium species withing an ionic culture medium comprising ethanolamine is able to utilize the ethanolamine as a sole source of carbon and/or nitrogen (Garsin: page 1, para 1). Harada provides a method for the efficient production of isoprenoids (para 0011) from recombinant bacterial strain that produces heterologous isoprenoid enzymes such as MK (referred to as MVA kinase by Harada) and HMGR (SEQ ID NO: 4) is (referred to as HMG-CoA reductase by Harada) (page 4, para 0009, lines 5; pages 11-12, paragraph 0041, lines 456 - 460) in a culture medium comprised of salt. Whereas, Masignani teaches a method wherein MK is 100% identical to full length MK of SEQ ID NO: 1 within the instant application (Masignani: claim 1; appendix B) and consists of an amino acid sequence RGLGSSAA, which has 100% identity to SEQ ID NO: 5 (appendix B, highlighted) of the instant application (appendix B). Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Applicant's arguments filed 10/23/2025 have been fully considered but they are not persuasive. Beginning on page 6 of Applicant’s remarks, Applicant’s in summary contends that the references do not teach ‘a suitable culture or medium comprising an ionic liquid (IL).’ Applicant contends that the specification teaches the unexpected result of increasing the IL resulting in an increase in the production of TMP.
The arguments are found to be not persuasive in view of the new rejection set forth. Examiner contends that the reference Garsin provides the motivation for Demain to utilize an ionic liquid for the production of TMP, as Garsin teaches an ionic liquid culture medium comprised of ethanolamine as a sole source of carbon and/or nitrogen (page 1, para 1), thereby resulting in a culture medium with an abundant supply of carbon/nitrogen for the production of TMP. Additionally, since only a select variety of bacteria are able to use ethanolamine, which comprises the ionic liquid recited by Applicant and taught by Garsin, as a carbon source (Garsin: abstract) said ionic compound could serve as a means for limiting the growth of contaminants thereby providing additional motivation for Demain to utilize said ionic liquid in the production of TMP. In response to Applicant’s assertion that increasing the concentration of IL results in the unexpected increased production of TMP. Examiner acknowledges said assertion. However, Applicant is reminded that the claims are read in light of the specification. Since said assertion is not encompassed within the claims, Examiner contends that it is not a required limitation of the claims.
Conclusion
Status of the Claims
Claims 11, 13-15, 19-28 are pending. Claims 1-10, 12, 16-18 are canceled.
Claims 11, 13-15, 19-28 are rejected.
No claims are in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERICA NICOLE JONES-FOSTER whose telephone number is (571)270-0360. The examiner can normally be reached mf 7:30a - 4:30p.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656
Appendix A
GenBank WP_012730718.1 alignment with SEQ ID NO: 4 (claims 11, 22, 23) with conserved domain SEQ ID NO: 7 highlighted yellow
Query Match 100.0%; Score 1903; Length 369;
Best Local Similarity 100.0%;
Matches 369; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSDNLYAPIPMSWIGPVHISGNVVSGETAGWNAEDGTQNQESGASYEAVSIPMATYETPL 60
Db 1 MSDNLYAPIPMSWIGPVHISGNVVSGETAGWNAEDGTQNQESGASYEAVSIPMATYETPL 60
Qy 61 WPSVGRGAKVSRYVEGGIRATLVDERMTRSVYFEAPNAGVALRVATELDRRRDELQAVVA 120
Db 61 WPSVGRGAKVSRYVEGGIRATLVDERMTRSVYFEAPNAGVALRVATELDRRRDELQAVVA 120
Qy 121 HASRFAKLIDLHVQYAGNLLFVRFEFTTGDASGHNMVTLASDNLMPWILQQYPELRYGSI 180
Db 121 HASRFAKLIDLHVQYAGNLLFVRFEFTTGDASGHNMVTLASDNLMPWILQQYPELRYGSI 180
Qy 181 SGNYCSDKKATAVNGILGRGKNVVTEMLIPRNVVEERLKTTPEQIADLNVRKNLVGTTLA 240
Db 181 SGNYCSDKKATAVNGILGRGKNVVTEMLIPRNVVEERLKTTPEQIADLNVRKNLVGTTLA 240
Qy 241 GGLRTANAHYANMLLGFYLATGQDAANIVEGSQGITHAEVRDGDLYFSCNLPNLIVGTVG 300
Db 241 GGLRTANAHYANMLLGFYLATGQDAANIVEGSQGITHAEVRDGDLYFSCNLPNLIVGTVG 300
Qy 301 NGKGQGLEVVEENLRRLGCREDRPAGDNARRLAVLCAASVFCGELSLLAAQTNPGELMAA 360
Db 301 NGKGQGLEVVEENLRRLGCREDRPAGDNARRLAVLCAASVFCGELSLLAAQTNPGELMAA 360
Qy 361 HVKIERKGE 369
Db 361 HVKIERKGE 369
Appendix B
Alignment of SEQ ID NO: 1 with Masignani et al SEQ ID NO: 3294 (claim 1) with SEQ ID NO: 5 highlighted in yellow below.
Query Match 100.0%; Score 1566; Length 306;
Best Local Similarity 100.0%;
Matches 306; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MTRKGYGESTGKIILIGEHAVTFGEPAIA VPFNAGKIKVLIEALESGNYSSIKSDVYDGM 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTRKGYGESTGKIILIGEHAVTFGEPAIA VPFNAGKIKVLIEALESGNYSSIKSDVYDGM 60
Qy 61 LYDAPDHLKSLVNRFVELNNITEPLAVTIQTNLPPSRGLGSSAAVAVAFVRASYDFLGKS 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LYDAPDHLKSLVNRFVELNNITEPLAVTIQTNLPPSRGLGSSAAVAVAFVRASYDFLGKS 120
Qy 121 LTKEELIEKANWAEQIAHGKPSGIDTQTIVSGKPVWFQKGHAETLKTLSLDGYMVVIDTG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 LTKEELIEKANWAEQIAHGKPSGIDTQTIVSGKPVWFQKGHAETLKTLSLDGYMVVIDTG 180
Qy 181 VKGSTRQAVEDVHKLCEDPQYMSHVKHIGKLVLRASDVIEHHNFEALADIFNECHADLKA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VKGSTRQAVEDVHKLCEDPQYMSHVKHIGKLVLRASDVIEHHNFEALADIFNECHADLKA 240
Qy 241 LTVSHDKIEQLMKIGKENGAIA GKLTGAGRGGSMLLLAKDLPTAKNIVKAVEKAGAAHTW 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LTVSHDKIEQLMKIGKENGAIA GKLTGAGRGGSMLLLAKDLPTAKNIVKAVEKAGAAHTW 300
Qy 301 IENLGG 306
||||||
Db 301 IENLGG 306