DETAILED ACTION
This application has a granted petition for participation in the Patent Prosecution Highway (PPH).
“Any claims amended or added after the grant of the request for participation in the Global/IP5 PPH pilot program must sufficiently correspond to one or more allowable/patentable claims in the OEE application. The applicant is required to submit, along with the amendment, a statement certifying that the amended or newly added claims sufficiently correspond to the allowable/patentable claims in the OEE application. If the certification statement is omitted, the amendment will not be entered and will be treated as a non-responsive reply.” 1400 OG 172 (March 18, 2014).
All objections and rejections set forth in prior Office Actions are withdrawn unless restated below.
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 .
Continued Examination Under 37 CFR 1.114
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 10/07/2025 has been entered.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1, 2 and 4-22 is/are rejected under 35 U.S.C. 103 as being unpatentable over Schneider et al. (WO 2022/008276 A1) (published 01/13/2022, filed 06/28/2021) further in view of Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22) and Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org. A translation of CN’560 is provided and cited herein.
Schneider, abstract, teaches:
The present invention relates to a microorganism transformed with L-arginine:glycine amidinotransferase and reduced malate synthase and a glyoxylate aminotransferase to be capable of producing guanidino acetic acid (GAA) and to a method for the fermentative production of GAA using such microorganism. The present invention also relates to a method for the fermentative production of creatine.
Schneider, in the claims, states:
1 . A microorganism comprising at least one gene coding for a protein having the function of a L-arginine:glycine amidinotransferase and wherein the activity of a protein having the function of a malate synthase is decreased compared to the respective activity in the wildtype microorganism.
4. The microorganism of any of claims 1 to 3, comprising at least one gene encoding a protein having the enzymic activity of a glyoxylate aminotransferase is overexpressed.
5. The microorganism of any of the preceding claims, wherein the microorganism has an increased ability to produce L-arginine compared with the ability of the wildtype microorganism.
6. The microorganism of claim 5, wherein the microorganism has increased activities of an enzyme having the function of a carbamoylphosphate synthase compared to the respective enzymic activity in the wildtype microorganism.
7. The microorganism of claims 5 or 6, wherein the microorganism further comprises an enzyme having the function of an argininosuccinate lyase with an increased activity compared to the respective enzymic activity in the wildtype microorganism.
8. The microorganism of any of claims 5 to 7, wherein the microorganism further comprises an enzyme having the function of an ornithine carbamoyltransferase with an increased activity compared to the respective enzymic activity in the wildtype microorganism.
9. The microorganism of any of claims 5 to 8, wherein the microorganism further comprises an enzyme having the function of an argininosuccinate synthetase with an increased activity compared to the respective enzymic activity in the wildtype microorganism.
10. The microorganism of any of claims 5 to 9, wherein the increased activities of the enzymes are achieved by overexpressing the genes encoding the respective enzymes.
11 . The microorganism of any of claims 5 to 10, wherein the arginine operon ( argCJBDFR ) is overexpressed.
12. The microorganism of any of claims 5 to 10, wherein the expression of an argR gene coding for the arginine responsive repressor protein ArgR is attenuated compared to the expression of the argR gene in the wildtype microorganism or wherein the argR gene is deleted.
13. The microorganism of any of claims 5 to 10 or 12, wherein at least one or more of the genes coding for an enzyme of the biosynthetic pathway of L-arginine, comprising gdh, argJ, argB, argC and/or argD coding for a glutamate dehydrogenase, an ornithine acetyltransferase, an acetylglutamate kinase, an acetylglutamylphosphate reductase and for an acetylornithine aminotransferase, respectively, is overexpressed.
14. The microorganism of any of the preceding claims, wherein the gene coding for a protein having the function of an L-arginine:glycine amidinotransferase is heterologous.
15. The microorganism of any of the preceding claims, wherein the gene coding for a protein having the function of an L-arginine:glycine amidinotransferase is overexpressed [i.e. increased activity relative to a respective activity in a wildtype microorganism].
18. The microorganism of any of the preceding claims, wherein the microorganism belongs to the genus Corynebacterium, to the genus Enterobacteriaceae or to the genus Pseudomonas.
19. The microorganism of claim 18, wherein the microorganism is Corynebacterium glutamicum.
20. The microorganism of claim 18 wherein the microorganism is Escherichia coli.
21 . The microorganism of claim 18 wherein the microorganism is Pseudomonas putida.
22. A method for the fermentative production of guanidino acetic acid (GAA), comprising the steps of a) cultivating the microorganism as defined in any of the preceding claims in a suitable medium under suitable conditions, and b) accumulating GAA in the medium to form a GAA containing fermentation broth.
23. The method of claim 22, further comprising isolating GAA from the GAA containing fermentation broth.
27. A microorganism as claimed in any of claims 1 to 21 , further comprising a gene coding for an enzyme having the activity of a guanidinoacetate N-methyltransferase.
28. The microorganism of claim 27, wherein the gene coding for an enzyme having the activity of a guanidinoacetate N-methyltransferase is overexpressed.
29. A method for the fermentative production of creatine, comprising the steps of a) cultivating the microorganism as defined in claims 27 or 28 in a suitable medium under suitable conditions, and b) accumulating creatine in the medium to form a creatine containing fermentation broth.
30. The method of claim 29, further comprising isolating creatine from the creatine containing fermentation broth.
“The protein having the function of an L-arginine:glycine amidinotransferase in the microorganism of the present invention may comprise an amino acid sequence which is at least 80 % homologous, preferably at least 90 % homologous to the amino acid sequence according to SEQ ID NO: 11. Schneider, page 10, ln. 5-10. SEQ ID NO: 11 of Schneider is identical to recited SEQ ID NO: 2.
The above (particularly in reference to underlined terms above and features of claims of Schneider) is a teaching of all of the features of claim 1, 2 and 4-22 except that Schneider does not teach a heterologous gene encoding a glycine dehydrogenase having recited SEQ ID NO: 6 as recited in claims 1 and 14.
Schneider teaches the following:
“Glyoxylate may be used as starting material for the formation of glycine in the presence of an amino donor, such as amino acids, and a glyoxylate transaminase.” “Glyoxylate transaminases catalyze the transfer of an amino group from an amino acid to glyoxylate. The products of this transfer are glycine and the corresponding a-keto acid.” “Glycine transaminase (EC 2.6.1.4) catalyzes the reaction:
L-glutamate + glyoxylate <=> alpha-ketoglutarate + glycine.” “Serine:glyoxylate transaminase (EC 2.6.1.45) catalyzes the reaction: L-serine + glyoxylate <=> 3-hydroxy-pyruvate + glycine.”
As such, Schneider teaches the production of glycine from glyoxylate by transfer of an amino group to the same as to produce glycine.
However, other methods of producing glycine from glyoxylate in an engineered microorganism (including E. coli) are known in the prior art.
Koch, abstract, teaches: The present invention provides biochemical pathways, glyoxylate producing recombinant microorganisms, and methods for the production and yield improvement of glycolic acid and/or glycine via a reverse glyoxylate shunt.
The reverse glyoxylate shunt described by Koch differs from the glyoxylate shunt taught by Schneider, page 2. Regardless, the prior art teaches that glyoxylate produced from an appropriate pathway (reverse glyoxylate shunt or glyoxylate shunt) and beneficially converted to glycine. As discussed, Schneider teach conversion of glyoxylate to glycine with a heterologous glycine transaminase (or other transaminases).
“To increase the production of glycine, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more genes encoding enzymes that catalyze the glyoxylate to glycine conversion. For example, the recombinant microorganism of any one of the embodiments disclosed herein may comprise a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine transaminase, a gene encoding serine-glyoxylate transaminase, and/or a gene encoding glycine oxidase. In one embodiment, the recombinant microorganism of any one of the embodiments disclosed herein may over-express one or more of these genes to increase the yield of glycine.” Koch, para. [0113].
“The overall stoichiometry is glyoxylate+NH3+1 NAD(P)H->glycine. Other enzymes that can facilitate the conversion of glyoxylate to glycine include glycine dehydrogenase (E.C. 1.4.1.10), glycine transaminase (E.C. 2.6.1.4), serine-glyoxylate transaminase (E.C. 2.6.1.45) and glycine oxidase (E.C. 1.4.3.19). Accordingly, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more gene selected from the group consisting of: a gene encoding alanine-glyoxylate aminotransferase (EC 2.6.1.44), a gene encoding glycine dehydrogenase (E.C. 1.4.1.10), a gene encoding glycine transaminase (E.C. 2.6.1.4), a gene encoding serine-glyoxylate transaminase (E.C. 2.6.1.45) and/or a gene encoding glycine oxidase (E.C. 1.4.3.19).” Koch, para. [0156].
All of an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19), are all enzymes that catalyze the conversion of glyoxylate to glycine but differ in the source of an amino group (NH4+ for glycine dehydrogenase and glycine oxidase; another amino acid for transaminases) and/or a source of reducing equivalents (NAD(P)H for dehydrogenases, H2O2 for glycine oxidase, for transaminases the amino acid reactant is the source of both an amino group and reducing equivalents). Regardless, Koch teaches that the enzymes an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19) can interchangeably function to catalyze the desired conversion of glyoxylate to glycine, wherein Koch, Fig. 1, show he operation of a glycine dehydrogenase employing NAD(P)H and NH4+:
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“In an exemplary embodiment, a gene encoding glycine dehydrogenase (EC 1.4.1.10) can be from Mycobacterium sp. (e.g. Mycobacterium tuberculosis, Mycobacterium smegmatis), Pseudomonas sp., Xanthobacter sp., or Bacillus sp.” Koch, para. [0157]. The sequence of glycine dehydrogenase from M. tuberculosis is known in the prior art as taught by Uniprot A5U6D3 that is identical to recited SEQ ID NO: 6.
The teachings of Koch are further reinforced by CN’560. CN’560, abstract:
“The invention claims a production method of glycine. The invention claims a method for constructing engineering strain capable of producing glycine, comprising the following steps: the receptor bacteria expression is derived from Mycobacterium smegmatis, Bacillus sphaericus, Mycobacteriumtuberculosis, bacillus, Bolivemonas, Aeromonas or conglomerate of the glyoxylic acid ammonia enzyme, the obtained strain is named as engineering bacteria 1; the engineering bacteria 1 is engineering strain capable of producing glycine. The invention opens up the first river for synthesizing glycine by biological method.”
“Specifically, the prokaryotic cell can be bacteria; the low eukaryotic cell can be yeast cell. More specifically, the bacteria can be Escherichia coli. the fourth aspect, the invention claims a method for producing glycine. The method for producing glycine claimed by the invention can be any one of the following:
(C1) A method for producing glycine by fermentation, comprising the following steps: performing fermentation culture to the Escherichia coli engineering strain in the second aspect of the previous text, obtaining glycine from the fermentation product.” CN’560, page 9.
“The amino acid sequence of the glyoxylic acid ammonia enzyme of mycobacterium tuberculosis Mycobacterium tuberculosis is shown as SEQ ID No. 7, the corresponding coding gene sequence is shown as SEQ ID No. 14.” CN’560, page 12. SEQ ID NO: 7 of CN’560 is identical to recited SEQ ID NO: 6 and the sequence shown in Uniprot A5U6D3, as such SEQ ID NO: 7 of CN’560 is a NADH-dependent glycine dehydrogenase. That is, CN’560 is describing enzymes that convert glyoxylate and ammonia to glycine, which is glycine dehydrogenase activity wherein Uniprot A5U6D3 evidences that SEQ ID NO: 7 of CN’560 (recited SEQ ID NO: 6) is a NADH-dependent glycine dehydrogenase from M. tuberculosis that is also expressly taught by Koch, and that the same enzyme can be expressed in E. coli to support production of glycine.
CN’560, Fig. 2, shows working embodiments of E. coli expressing Mycobacterium tuberculosis NADH-dependent glycine dehydrogenase having recited SEQ ID NO: 6 (glyoxylic acid ammonia enzyme, “Ms-Ald) as follows:
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“FIG. 2 is a different source of glyoxylic acid ammonia enzyme fermentation producing glycine analysis.Source: Av-ald: Aeromonas veronii, Ba-ald, Bacillus aquimaris, Bc-ald, bacillus cereus Bacillus cereus,Bf-ald: Bacillus flexus, Bl-ald. Bacillus licheniformis, Bv-ald: Bacillus velezensis, Bs-ald: Bacillussubtilis substilis, Gs-ald: Bacillus stearothermophilus Geobacillusstearothermophilus, Hb-ald:Halomonas boliviensis Halomonas boliviensis, La-ald Reagglomerating Laburenz Labrenziaaggregata, Lf-ald: Lysinibacillus fusiformis Lysinibacillus fusiformis, Pa-ald: Pseudomonas aeruginosa,Pg-ald Bacillus leiformis Paucisalibacillus globuius, Ms-ald: Mycobacterium smegmatis Mycobacterium smegmatis, Mt-ald: Mycobacterium tuberculosis. Escherichia coli ATCC 8739 is a control strain. Theordinate represents the yield of glycine in each liter of fermentation broth.” CN’560.
Schneider does not disclose a heterologous gene encoding a glycine dehydrogenase. However, MPEP 2143(I)(B) provides the following:
Substitution of known elements is obvious upon a finding of:
(1) a finding that the prior art contained a device (method, product, etc.) which differed from the claimed device by the substitution of some components (step, element, etc.) with other components;
(2) a finding that the substituted components and their functions were known in the art;
(3) a finding that one of ordinary skill in the art could have substituted one known element for another, and the results of the substitution would have been predictable; and
(4) whatever additional findings based on the Graham factual inquiries may be necessary, in view of the facts of the case under consideration, to explain a conclusion of obviousness.
Here, (1) Schneider teaches all of the elements of the rejected claims except for a glyoxylate transaminase (e.g. serine-glyoxylate transaminase) as taught by Schneider substituted for a NADH-dependent glycine dehydrogenase. (2) Koch teaches that either a transaminase or a glycine dehydrogenase can be employed to convert glyoxylate to glycine in a recombinant cell as taught above. CN’560 further teaches that expression of a glycine dehydrogenase having recited SEQ ID NO: 6 supports production of glycine by reductive amination of glyoxylate in E. coli. (3) Based upon the explicit teachings of Koch and CN’560 teaching that a glyoxylate transaminase (e.g. serine-glyoxylate transaminase) can be replaced with a glycine dehydrogenase with predictable results of maintaining production of glycine and ordinarily skilled artisan could have substituted a transaminase as taught by Schneider with a glycine dehydrogenase having recited SEQ ID NO: 6 with predictable results of maintaining increased glycine production. It is noted that there is nothing in the present record supporting that expression of a dehydrogenase having recited SEQ ID NO: 6 has any superior performance relative to expression of a serine-glyoxylate transaminase as shown in Schneider. (4) No other findings are necessary to explain a conclusion of obvious.
For these reasons, an ordinarily skilled artisan at the time of filing would have been motivated to replace a serine-glyoxylate transaminase as taught in embodiments of Schneider with a glycine dehydrogenase as taught by Koch and CN’560 having recited SEQ ID NO: 6.
Claim(s) 1, 2 and 4-23 (all pending claims) is/are rejected under 35 U.S.C. 103 as being unpatentable over Schneider et al. (WO 2022/008276 A1) (published 01/13/2022, filed 06/28/2021), Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22) and Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org as applied to claims 1, 2 and 4-22 above, and further in view of Phogosee et al. (Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties, Archives Microbiol. 200, 2018, 719-27) and GenBank, Accession No. MG430510.1, 2018, www.ncib.nlm.nih.gov.
Regarding claim 23, as discussed above, Koch and CN’560 teach that glycine dehydrogenases enzymes including recited SEQ ID NO: 6 can substitute for transaminase enzymes as taught by Schneider for production of glycine in E. coli. Additional enzymes teaching this reaction, i.e. conversion of glyoxylate and ammonia to glycine by reductive animation, are known and taught in the prior art.
“Functional analysis revealed that ApalaDH encodes a bifunctional protein catalyzing the reversible reaction of pyruvate to l-alanine via its pyruvate reductive aminase (PvRA) activity, the reaction of l-alanine to pyruvate via its alanine oxidative dehydrogenase activity, and the non-reversible reaction of glyoxylate to glycine via its glyoxylate reductive aminase (GxRA) activity.” Phogosee, abstract. Such, reaction of glyoxylate to glycine is NADH-dependent glycine dehydrogenase activity as shown in Fig. 1(b) of Phogosee. That is ApalaDH has NADH-dependent glycine dehydrogenase activity.
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It is noted that, Phogosee, Table 5 compares the activity of dehydrogenase from M. tuberculosis with A. halophytica.
As discussed above, Koch and CN’560 teach that an enzyme having the NADH-dependent glycine dehydrogenase activity as shown in Fig. 1(b) of Phogosee can support glycine production in vivo and substitute for a transaminase as taught by Schneider. As far as Phogosee teach an additional enzyme species having such activity, an ordinarily skilled artisan at time of filing would have further been motivated to substitute a transaminase as taught by Schneider for either a dehydrogenase having recited SEQ ID NO: 6 as discussed or the ApalaDH enzyme of Phogosee that catalyzes the same reductive amination of glyoxylate to glycine with an expectation of success.
“Nucleotide sequence data of ApAlaDH was deposited in GenBank under accession number MG430510,” which encodes an enzyme identical to recited SEQ ID NO: 18. Phogosee, page 721, left col.
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.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 2 and 4-23 (all pending claims) are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-28 of U.S. Patent No. 11,999,982 B2 in view of Schneider et al. (WO 2022/008276 A1), Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22), Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org, Phogosee et al. (Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties, Archives Microbiol. 200, 2018, 719-27) and GenBank, Accession No. MG430510.1, 2018, www.ncib.nlm.nih.gov.
U.S. Patent No. 11,999,982 B2 is previously published as Schneider et al. (WO 2022/008276 A1).
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference.
Patented claim 1 recites:
1. A microorganism, comprising:
at least one gene coding for a protein having a function of a L-arginine:glycine amidinotransferase, and wherein an activity of a protein having a function of a malate synthase is decreased compared to the respective activity in a corresponding wildtype microorganism,
wherein the protein having the function of the L-arginine:glycine amidinotransferase comprises an amino acid sequence which is at least 80% identity to the amino acid sequence according to SEQ ID NO: 11.
The features of the claims are anticipated by patented claims except for recitation of an NADH-dependent amino acid dehydrogenase having recited SEQ ID NO: 6 as follows: claims 1, 10 and 13 are met by patented claims 1 and 15 (SEQ ID NO: 2 is identical to SEQ ID NO: 11 of the patented claims); claims 4-9 met by patented claims 6-14; claim 11 is met by patented claim 13; claim 12 is met by patented claim 14; claim 15 is met by patented claim 17; claims 16-17 and 20-21 are met by patented claims 23-26; and claims 18-19 are met by patented claim 21-22.
Regarding the recited requirement for a NADH-dependent amino acid dehydrogenase having recited SEQ ID NO: 6, patented claim 4 recites “comprising at least one gene encoding a protein having an enzymic activity of a glyoxylate aminotransferase is overexpressed.”
As discussed above, Schneider expressly teaches that a recombinant microorganism engineered to express L-arginine:glycine amidinotransferase for the purpose of production of GAA should desirably express a glyoxylate aminotransferase enzyme for the purpose of converting glyoxylate to glycine, which is a substrate of L-arginine:glycine amidinotransferase.
Koch, abstract, teaches: The present invention provides biochemical pathways, glyoxylate producing recombinant microorganisms, and methods for the production and yield improvement of glycolic acid and/or glycine via a reverse glyoxylate shunt.
“To increase the production of glycine, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more genes encoding enzymes that catalyze the glyoxylate to glycine conversion. For example, the recombinant microorganism of any one of the embodiments disclosed herein may comprise a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine transaminase, a gene encoding serine-glyoxylate transaminase, and/or a gene encoding glycine oxidase. In one embodiment, the recombinant microorganism of any one of the embodiments disclosed herein may over-express one or more of these genes to increase the yield of glycine.” Koch, para. [0113].
“The overall stoichiometry is glyoxylate+NH3+1 NAD(P)H->glycine. Other enzymes that can facilitate the conversion of glyoxylate to glycine include glycine dehydrogenase (E.C. 1.4.1.10), glycine transaminase (E.C. 2.6.1.4), serine-glyoxylate transaminase (E.C. 2.6.1.45) and glycine oxidase (E.C. 1.4.3.19). Accordingly, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more gene selected from the group consisting of: a gene encoding alanine-glyoxylate aminotransferase (EC 2.6.1.44), a gene encoding glycine dehydrogenase (E.C. 1.4.1.10), a gene encoding glycine transaminase (E.C. 2.6.1.4), a gene encoding serine-glyoxylate transaminase (E.C. 2.6.1.45) and/or a gene encoding glycine oxidase (E.C. 1.4.3.19).” Koch, para. [0156].
All of an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19), are all enzymes that catalyze the conversion of glyoxylate to glycine but differ in the source of an amino group (NH4+ for glycine dehydrogenase and glycine oxidase; another amino acid for transaminases) and/or a source of reducing equivalents (NAD(P)H for dehydrogenases, H2O2 for glycine oxidase, for transaminases the amino acid reactant is the source of both an amino group and reducing equivalents). Regardless, Koch teaches that the enzymes an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19) can interchangeably function to catalyze the desired conversion of glyoxylate to glycine, wherein Koch, Fig. 1, show he operation of a glycine dehydrogenase employing NAD(P)H and NH4+:
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CN’560 as discussed above further teaches by means of working example that expression of a NADH-dependent glycine dehydrogenase having recited SEQ ID NO: 6 (SEQ ID NO: 7 of CN’560) supports increased production of glycine in E. coli.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the patented claims to express an enzyme appropriate for the conversion of glyoxylate to glycine as expressly taught by Schneider, Schneider teaching a glyoxylate aminotransferase as such an enzyme. Further, Koch and CN’560 directly teaches that a glycine dehydrogenase is substitutable for a glyoxylate aminotransferase for the purpose of converting glyoxylate to glycine such that at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the patented claims express a glycine dehydrogenase for the purpose of achieving the desirable benefit of conversion of glyoxylate to glycine as otherwise taught by Schneider.
Regarding recitation of a specific NADH-dependent glycine dehydrogenase having SEQ ID NO: 6, “In an exemplary embodiment, a gene encoding glycine dehydrogenase (EC 1.4.1.10) can be from Mycobacterium sp. (e.g. Mycobacterium tuberculosis, Mycobacterium smegmatis), Pseudomonas sp., Xanthobacter sp., or Bacillus sp.” Koch, para. [0157]. The sequence of glycine dehydrogenase from M. tuberculosis is known in the prior art as taught by Uniprot A5U6D3. As such, since Koch expressly states that glycine dehydrogenase from M. tuberculosis is an appropriate embodiment of glycine dehydrogenase, an ordinarily skilled artisan at the time of filing would have been motivated to employ a known M. tuberculosis sequence as taught by Uniprot A5U6D3, which is a NADH-dependent glycine dehydrogenase identical to recited SEQ ID NO: 6. As discussed, CN’560 also teaches a NADH-dependent glycine dehydrogenase identical to recited SEQ ID NO: 6 as suitable for conversion of glyoxylate to glycine in E. coli by working example.
Regarding recitation of a specific NADH-dependent glycine or alanine dehydrogenase having SEQ ID NO: 18, the rejections above under 35 U.S.C. 103 relying upon Phogosee are incorporated herein by reference. As discussed above, just as an ordinarily skilled artisan would have substituted a dehydrogenase having SEQ ID NO: 6 with known ability to convert glyoxylate to glycine, and ordinarily skilled artisan at time of filing would have had an expectation of success in substituting a dehydrogenase having SEQ ID NO: 18 catalyzing the same reaction in combination with a L-arginine:glycine amidinotransferase as recited in the patented claims.
Claims 1, 2, 4, 11, 14, 15, 16-17 and 20-21 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-15 of U.S. Patent No. 12,312,622 B2 in view of Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22), Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org, Phogosee et al. (Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties, Archives Microbiol. 200, 2018, 719-27) and GenBank, Accession No. MG430510.1, 2018, www.ncib.nlm.nih.gov.
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference with respect to the teachings of the references stated above.
Patented claim 1 recites:
1. A microorganism, comprising:
at least one heterologous gene coding for a protein having a function of a L-arginine:glycine amidinotransferase belonging to E.C. 2.1.4.1;
at least one heterologous gene coding for a protein having a function of a carbamate kinase belonging to E.C. 2.7.2.2; and
further comprising at least one gene coding for a protein having a function of a NADH-dependent amino acid dehydrogenase belonging to E.C. 1.4.1.
10. A method for a fermentative production of guanidino acetic acid (GAA), the method comprising:
a) cultivating the microorganism as defined in claim 1 in a medium, and
b) accumulating GAA in the medium to form a GAA containing fermentation broth.
The patented claims explicitly require a combination of a L-arginine:glycine amidinotransferase belonging to E.C. 2.1.4.1 and a NADH-dependent amino acid dehydrogenase E.C. 1.4.1, but do not specify a specific glycine dehydrogenase or alanine dehydrogenase including recited SEQ ID NO: 6 or 18.
Regarding the recited requirement for a NADH-dependent amino acid dehydrogenase having recited SEQ ID NO: 6 or 18, some specific NADH-dependent amino acid dehydrogenase must be used in forming any embodiment of the patented claims. From the context of the claims, the recited NADH-dependent amino acid dehydrogenase is for promoting formation of substrates for L-arginine:glycine amidinotransferase being arginine and glycine
As discussed above, Koch, abstract, “To increase the production of glycine, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more genes encoding enzymes that catalyze the glyoxylate to glycine conversion. For example, the recombinant microorganism of any one of the embodiments disclosed herein may comprise a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine transaminase, a gene encoding serine-glyoxylate transaminase, and/or a gene encoding glycine oxidase. In one embodiment, the recombinant microorganism of any one of the embodiments disclosed herein may over-express one or more of these genes to increase the yield of glycine.” Koch, para. [0113].
“The overall stoichiometry is glyoxylate+NH3+1 NAD(P)H->glycine. Other enzymes that can facilitate the conversion of glyoxylate to glycine include glycine dehydrogenase (E.C. 1.4.1.10), glycine transaminase (E.C. 2.6.1.4), serine-glyoxylate transaminase (E.C. 2.6.1.45) and glycine oxidase (E.C. 1.4.3.19). Accordingly, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more gene selected from the group consisting of: a gene encoding alanine-glyoxylate aminotransferase (EC 2.6.1.44), a gene encoding glycine dehydrogenase (E.C. 1.4.1.10), a gene encoding glycine transaminase (E.C. 2.6.1.4), a gene encoding serine-glyoxylate transaminase (E.C. 2.6.1.45) and/or a gene encoding glycine oxidase (E.C. 1.4.3.19).” Koch, para. [0156].
All of an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19), are all enzymes that catalyze the conversion of glyoxylate to glycine but differ in the source of an amino group (NH4+ for glycine dehydrogenase and glycine oxidase; another amino acid for transaminases) and/or a source of reducing equivalents (NAD(P)H for dehydrogenases, H2O2 for glycine oxidase, for transaminases the amino acid reactant is the source of both an amino group and reducing equivalents). Regardless, Koch teaches that the enzymes an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19) can interchangeably function to catalyze the desired conversion of glyoxylate to glycine, wherein Koch, Fig. 1, show he operation of a glycine dehydrogenase employing NAD(P)H and NH4+:
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CN’560 as discussed above further teaches by means of working example that expression of a NADH-dependent glycine dehydrogenase having recited SEQ ID NO: 6 (SEQ ID NO: 7 of CN’560) supports increased production of glycine in E. coli.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the patented claims to express a NADH-dependent amino acid dehydrogenase belonging to E.C. 1.4.1 enzyme appropriate for the conversion of glyoxylate to glycine already shown and taught to be appropriate for formation of glycine and specifically being a NADH-dependent glycine dehydrogenase having recited SEQ ID NO: 6 (SEQ ID NO: 7 of CN’560) supports increased production of glycine in E. coli. That is, a NADH-dependent amino acid dehydrogenase belonging to E.C. 1.4.1 as recited in the patented claims must have some specific identity wherein CN’560 (and Koch) teach that a dehydrogenase with recited SEQ ID NO: 6 is a particularly appropriate dehydrogenase for expression.
Regarding recitation of a specific NADH-dependent glycine or alanine dehydrogenase having SEQ ID NO: 18, the rejections above under 35 U.S.C. 103 relying upon Phogosee are incorporated herein by reference. As discussed above, just as an ordinarily skilled artisan would have substituted a dehydrogenase having SEQ ID NO: 6 with known ability to convert glyoxylate to glycine, and ordinarily skilled artisan at time of filing would have had an expectation of success in substituting a dehydrogenase having SEQ ID NO: 18 catalyzing the same reaction in combination with a L-arginine:glycine amidinotransferase as recited in the patented claims.
Regarding claims 4 and 11, the same are met by patented claims 7 and 8. Claims 16 and 17 are met by the methods of patented claims 10 and 12. Claims 20 and 21 are met by patented claims 14 and 15.
Claims 1, 2 and 4-23 (all pending claims) are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-15 of U.S. Patent No. 12,312,622 B2 in view of Schneider et al. (WO 2022/008276 A1), Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22), Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org, Phogosee et al. (Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties, Archives Microbiol. 200, 2018, 719-27) and GenBank, Accession No. MG430510.1, 2018, www.ncib.nlm.nih.gov.
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference. The non-statutory double rejection of claims 1, 2, 4, 11, 14, 15, 16-17 and 20-21 over claims of U.S. Patent No. 12,312,622 B2 above are incorporated herein by reference.
Regarding remaining features of the claims not addressed above, Schneider, as discussed above, teach that such features as addressed above are advantageous for a microorganism modified for producing GAA and/or creatine such that an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the patented claims to have all of the features of the rejected claims as addressed above under the rejections under 35 U.S.C. 103.
Claims 1, 2 and 4-23 (all pending claims) are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-21 of copending Application No. 18/562,157 in view of Schneider et al. (WO 2022/008276 A1), Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22), Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org, Phogosee et al. (Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties, Archives Microbiol. 200, 2018, 719-27) and GenBank, Accession No. MG430510.1, 2018, www.ncib.nlm.nih.gov.
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference.
Copending claim 1 recites:
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Regarding the recited requirement for a NADH-dependent amino acid dehydrogenase having recited SEQ ID NO: 6, as discussed above, Schneider expressly teaches that a recombinant microorganism engineered to express L-arginine:glycine amidinotransferase for the purpose of production of GAA should desirably express a glyoxylate aminotransferase enzyme for the purpose of converting glyoxylate to glycine, which is a substrate of L-arginine:glycine amidinotransferase.
Koch, abstract, teaches: The present invention provides biochemical pathways, glyoxylate producing recombinant microorganisms, and methods for the production and yield improvement of glycolic acid and/or glycine via a reverse glyoxylate shunt.
“To increase the production of glycine, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more genes encoding enzymes that catalyze the glyoxylate to glycine conversion. For example, the recombinant microorganism of any one of the embodiments disclosed herein may comprise a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine transaminase, a gene encoding serine-glyoxylate transaminase, and/or a gene encoding glycine oxidase. In one embodiment, the recombinant microorganism of any one of the embodiments disclosed herein may over-express one or more of these genes to increase the yield of glycine.” Koch, para. [0113].
“The overall stoichiometry is glyoxylate+NH3+1 NAD(P)H->glycine. Other enzymes that can facilitate the conversion of glyoxylate to glycine include glycine dehydrogenase (E.C. 1.4.1.10), glycine transaminase (E.C. 2.6.1.4), serine-glyoxylate transaminase (E.C. 2.6.1.45) and glycine oxidase (E.C. 1.4.3.19). Accordingly, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more gene selected from the group consisting of: a gene encoding alanine-glyoxylate aminotransferase (EC 2.6.1.44), a gene encoding glycine dehydrogenase (E.C. 1.4.1.10), a gene encoding glycine transaminase (E.C. 2.6.1.4), a gene encoding serine-glyoxylate transaminase (E.C. 2.6.1.45) and/or a gene encoding glycine oxidase (E.C. 1.4.3.19).” Koch, para. [0156].
All of an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19), are all enzymes that catalyze the conversion of glyoxylate to glycine but differ in the source of an amino group (NH4+ for glycine dehydrogenase and glycine oxidase; another amino acid for transaminases) and/or a source of reducing equivalents (NAD(P)H for dehydrogenases, H2O2 for glycine oxidase, for transaminases the amino acid reactant is the source of both an amino group and reducing equivalents). Regardless, Koch teaches that the enzymes an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19) can interchangeably function to catalyze the desired conversion of glyoxylate to glycine, wherein Koch, Fig. 1, show he operation of a glycine dehydrogenase employing NAD(P)H and NH4+:
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CN’560 as discussed above further teaches by means of working example that expression of a NADH-dependent glycine dehydrogenase having recited SEQ ID NO: 6 (SEQ ID NO: 7 of CN’560) supports increased production of glycine in E. coli.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the copending claims to express an enzyme appropriate for the conversion of glyoxylate to glycine as expressly taught by Schneider, Schneider teaching a glyoxylate aminotransferase as such an enzyme. Further, Koch and CN’560 directly teaches that a glycine dehydrogenase is substitutable for a glyoxylate aminotransferase for the purpose of converting glyoxylate to glycine such that at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the copending claims express a glycine dehydrogenase for the purpose of achieving the desirable benefit of conversion of glyoxylate to glycine as otherwise taught by Schneider.
Regarding recitation of a specific NADH-dependent glycine dehydrogenase having SEQ ID NO: 6, “In an exemplary embodiment, a gene encoding glycine dehydrogenase (EC 1.4.1.10) can be from Mycobacterium sp. (e.g. Mycobacterium tuberculosis, Mycobacterium smegmatis), Pseudomonas sp., Xanthobacter sp., or Bacillus sp.” Koch, para. [0157]. The sequence of glycine dehydrogenase from M. tuberculosis is known in the prior art as taught by Uniprot A5U6D3. As such, since Koch expressly states that glycine dehydrogenase from M. tuberculosis is an appropriate embodiment of glycine dehydrogenase, an ordinarily skilled artisan at the time of filing would have been motivated to employ a known M. tuberculosis sequence as taught by Uniprot A5U6D3, which is a NADH-dependent glycine dehydrogenase identical to recited SEQ ID NO: 6. As discussed, CN’560 also teaches a NADH-dependent glycine dehydrogenase identical to recited SEQ ID NO: 6 as suitable for conversion of glyoxylate to glycine in E. coli by working example.
Regarding recitation of a specific NADH-dependent glycine or alanine dehydrogenase having SEQ ID NO: 18, the rejections above under 35 U.S.C. 103 relying upon Phogosee are incorporated herein by reference. As discussed above, just as an ordinarily skilled artisan would have substituted a dehydrogenase having SEQ ID NO: 6 with known ability to convert glyoxylate to glycine, and ordinarily skilled artisan at time of filing would have had an expectation of success in substituting a dehydrogenase having SEQ ID NO: 18 catalyzing the same reaction in combination with a L-arginine:glycine amidinotransferase as recited in the copending claims.
Regarding remaining features of the claims, Schneider, as discussed above, teach that such features as addressed above are advantageous for a microorganism modified for producing GAA such that an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the copending claims to have all of the features of the rejected claims as addressed above under the rejections under 35 U.S.C. 103.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 2 and 4-23 (all pending claims) are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-4, 10-21of copending Application No. 17/757,441 in view of Schneider et al. (WO 2022/008276 A1), Koch et al. (U.S. 2020/00263210 A1), CN 114058560 A (CN’560, published 02/18/22), Uniprot, Accession No. A5U6D3, 2018, www.uniprot.org, Phogosee et al. (Bifunctional alanine dehydrogenase from the halotolerant cyanobacterium Aphanothece halophytica: characterization and molecular properties, Archives Microbiol. 200, 2018, 719-27) and GenBank, Accession No. MG430510.1, 2018, www.ncib.nlm.nih.gov.
The rejections under 35 U.S.C. 103 stated above are incorporated herein by reference.
Copending claim 1 recites:
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Regarding the recited requirement for a NADH-dependent amino acid dehydrogenase having recited SEQ ID NO: 6, as discussed above, Schneider expressly teaches that a recombinant microorganism engineered to express L-arginine:glycine amidinotransferase for the purpose of production of GAA should desirably express a glyoxylate aminotransferase enzyme for the purpose of converting glyoxylate to glycine, which is a substrate of L-arginine:glycine amidinotransferase.
Koch, abstract, teaches: The present invention provides biochemical pathways, glyoxylate producing recombinant microorganisms, and methods for the production and yield improvement of glycolic acid and/or glycine via a reverse glyoxylate shunt.
“To increase the production of glycine, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more genes encoding enzymes that catalyze the glyoxylate to glycine conversion. For example, the recombinant microorganism of any one of the embodiments disclosed herein may comprise a gene encoding alanine-glyoxylate aminotransferase, a gene encoding glycine dehydrogenase, a gene encoding glycine transaminase, a gene encoding serine-glyoxylate transaminase, and/or a gene encoding glycine oxidase. In one embodiment, the recombinant microorganism of any one of the embodiments disclosed herein may over-express one or more of these genes to increase the yield of glycine.” Koch, para. [0113].
“The overall stoichiometry is glyoxylate+NH3+1 NAD(P)H->glycine. Other enzymes that can facilitate the conversion of glyoxylate to glycine include glycine dehydrogenase (E.C. 1.4.1.10), glycine transaminase (E.C. 2.6.1.4), serine-glyoxylate transaminase (E.C. 2.6.1.45) and glycine oxidase (E.C. 1.4.3.19). Accordingly, the recombinant microorganism of any one of the embodiments disclosed herein may comprise one or more gene selected from the group consisting of: a gene encoding alanine-glyoxylate aminotransferase (EC 2.6.1.44), a gene encoding glycine dehydrogenase (E.C. 1.4.1.10), a gene encoding glycine transaminase (E.C. 2.6.1.4), a gene encoding serine-glyoxylate transaminase (E.C. 2.6.1.45) and/or a gene encoding glycine oxidase (E.C. 1.4.3.19).” Koch, para. [0156].
All of an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19), are all enzymes that catalyze the conversion of glyoxylate to glycine but differ in the source of an amino group (NH4+ for glycine dehydrogenase and glycine oxidase; another amino acid for transaminases) and/or a source of reducing equivalents (NAD(P)H for dehydrogenases, H2O2 for glycine oxidase, for transaminases the amino acid reactant is the source of both an amino group and reducing equivalents). Regardless, Koch teaches that the enzymes an alanine-glyoxylate aminotransferase (EC 2.6.1.44), a glycine dehydrogenase (E.C. 1.4.1.10), a glycine transaminase (E.C. 2.6.1.4), a serine-glyoxylate transaminase (E.C. 2.6.1.45) and a gene encoding glycine oxidase (E.C. 1.4.3.19) can interchangeably function to catalyze the desired conversion of glyoxylate to glycine, wherein Koch, Fig. 1, show he operation of a glycine dehydrogenase employing NAD(P)H and NH4+:
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CN’560 as discussed above further teaches by means of working example that expression of a NADH-dependent glycine dehydrogenase having recited SEQ ID NO: 6 (SEQ ID NO: 7 of CN’560) supports increased production of glycine in E. coli.
As such, at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the copending claims to express an enzyme appropriate for the conversion of glyoxylate to glycine as expressly taught by Schneider, Schneider teaching a glyoxylate aminotransferase as such an enzyme. Further, Koch and CN’560 directly teaches that a glycine dehydrogenase is substitutable for a glyoxylate aminotransferase for the purpose of converting glyoxylate to glycine such that at the time of filing an ordinarily skilled artisan would have been motivated to modify embodiments of the copending claims express a glycine dehydrogenase for the purpose of achieving the desirable benefit of conversion of glyoxylate to glycine as otherwise taught by Schneider.
Regarding recitation of a specific NADH-dependent glycine dehydrogenase having SEQ ID NO: 6, “In an exemplary embodiment, a gene encoding glycine dehydrogenase (EC 1.4.1.10) can be from Mycobacterium sp. (e.g. Mycobacterium tuberculosis, Mycobacterium smegmatis), Pseudomonas sp., Xanthobacter sp., or Bacillus sp.” Koch, para. [0157]. The sequence of glycine dehydrogenase from M. tuberculosis is known in the prior art as taught by Uniprot A5U6D3. As such, since Koch expressly states that glycine dehydrogenase from M. tuberculosis is an appropriate embodiment of glycine dehydrogenase, an ordinarily skilled artisan at the time of filing would have been motivated to employ a known M. tuberculosis sequence as taught by Uniprot A5U6D3, which is a NADH-dependent glycine dehydrogenase identical to recited SEQ ID NO: 6. As discussed, CN’560 also teaches a NADH-dependent glycine dehydrogenase identical to recited SEQ ID NO: 6 as suitable for conversion of glyoxylate to glycine in E. coli by working example.
Regarding recitation of a specific NADH-dependent glycine or alanine dehydrogenase having SEQ ID NO: 18, the rejections above under 35 U.S.C. 103 relying upon Phogosee are incorporated herein by reference. As discussed above, just as an ordinarily skilled artisan would have substituted a dehydrogenase having SEQ ID NO: 6 with known ability to convert glyoxylate to glycine, and ordinarily skilled artisan at time of filing would have had an expectation of success in substituting a dehydrogenase having SEQ ID NO: 18 catalyzing the same reaction in combination with a L-arginine:glycine amidinotransferase as recited in the copending claims.
Regarding remaining features of the claims, Schneider, as discussed above, teach that such features as addressed above are advantageous for a microorganism modified for producing GAA such that an ordinarily skilled artisan at time of filing would have been motivated to modify embodiments of the copending claims to have all of the features of the rejected claims as addressed above under the rejections under 35 U.S.C. 103.
This is a provisional nonstatutory double patenting rejection.
Response to arguments
The last submitted remarks are dated 09/05/2025 and were addressed in the advisory action dated 09/17/2025, which are summarized below.
Schneider teaches a cells with a glyoxylate shunt that provides glyoxylate for the production of glycine. “The so called glyoxylate shunt pathway, naturally occurring in microorganisms, such as E. coli or C. glutamicum, is a side reaction of the tri-carbonic acid (TCA) cycle (Krebs cycle) and includes the formation of glyoxylate and succinate from isocitrate by isocitrate lyase and the formation of malate from glyoxylate and acetyl-CoA by malate synthase.” Schneider, page 2, lines 30-34. While Koch teaches that a reverse glyoxylate shunt has certain advantages, the obviousness rejections of record do NOT propose modification of embodiments of Schneider to have a reverse glyoxylate shunt. The motivation for modification is solely the teachings of Koch, para. [0113], that a glycine dehydrogenase can perform an equivalent function of a transaminase for conversion of glyoxylate to glycine. The same is a teaching of Koch, para. [0113], are contained in a single paragraph of the prior art and are not hindsight bias. “Any judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper.” MPEP 2145(X)(A).
There is no technical reason why an ordinarily skilled artisan at the time of filing would believe that the provision of glyoxylate via a glyoxylate shunt or a reverse glyoxylate shunt would be of any consequence to basic operability of producing glycine provided that glyoxylate is available in the cell, which is further evidenced by CN’560.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652