DETAILED CORRESPONDENCE
Status of the Application
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 March 10, 2026 has been entered.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-10, 13-38, and 41-62 are pending in the application.
Applicant’s amendment to the claims, filed March 10, 2026, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Applicant’s remarks filed March 10, 2026 in response to the final rejection filed January 26, 2026 have been fully considered.
Claims 63 and 64 have been canceled by the amendment filed March 10, 2026 and rejections previously applied to these claims are withdrawn.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Restriction/Election
In response to requirements for restriction/election filed June 7, 2023, April 3, 2024, and May 21, 2025, applicant elected without traverse the following invention and species in responses filed August 3, 2023, September 3, 2024, and June 23, 2025:
Group I, pending claims 1-10, 13-14, 29-38, 41, 42, and 57-64,
Species A1) the carbon source is glycerol or a biomass comprising glycerol in claims 3, 4, 31, and 32,
Species B1) the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors in claims 5 and 33,
Species C3) the genetic modification is one or more genetic modification is expressing one or more non-native genes in the engineered host cells in claims 7 and 35,
Species D1) the engineered host cell comprises a nucleic acid sequence encoding tyrosine ammonia lyase activity in claims 9 and 37,
Species E1) the engineered host cell comprises a chalcone isomerase in claims 10 and 38,
Species F1) the engineered host cell comprises an exogenous nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate in claims 13 and 41,
Species G1), the malonyl-CoA synthetase comprises amino acid sequence at least 95% identical to SEQ ID NO: 17 in claims 57 and 59, and
Species H1), mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase.
Claims 15-28 and 43-56 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Claims 6, 34, 58, and 60 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected species, there being no allowable generic or linking claim.
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 57, 59, 61, and 62 are being examined on the merits with claims 3, 4, 7, 9, 10, 13, 31, 32, 35, 37, 38, and 41 being examined only to the extent the claims read on the elected subject matter.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on March 19, 2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDS has been considered by the examiner.
Claim Rejections - 35 USC § 112(b)
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 57, 59, 61, and 62 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claims 1 (claims 2-5, 7-10, 13, 14, 57, and 61 dependent therefrom) and 29 (claims 30-33, 35-38, 41, 42, 59, and 62 dependent therefrom) are confusing in the recitation of “wherein the one or more genetic modifications to decrease fatty acid biosynthesis comprise mutation of a gene encoding malonyl-CoA-ACP transacylase; and an exogenous nucleic acid sequence encoding a malonate transporter” because it is unclear as to whether the phrase “and an exogenous…malonate transporter” is intended to be interpreted as meaning a mutation in an exogenous nucleic acid sequence encoding a malonate transporter, or the phrase “and an exogenous…malonate transporter” is intended to be interpreted as meaning the engineered host cell comprises an exogenous nucleic acid sequence encoding a malonate transporter. It is suggested that applicant clarify the meaning of the noted phrase.
Claim Rejections - 35 USC § 103
The rejection of claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 under 35 U.S.C. 103 as being unpatentable over Stephanopoulos et al. (US 2012/0034661 A1; cited on the IDS filed October 21, 2022; hereafter “Stephanopoulos”) in view of Yang et al. (Metabolic Engineering 29:217-226, 2015; cited on Form PTO-892 filed July 8, 2025) and Rengby et al. (Appl. Environ. Microbiol. 7:432-441, 2007; cited on Form PTO-892 filed January 26, 2026; hereafter “Rengby”),
the rejection of claims 14 and 42 under 35 U.S.C. 103 as being unpatentable over Stephanopoulos in view of Yang and Rengby as applied to claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 above, and further in view of Naesby et al. (WO 2017/050853 A1; cited on Form PTO-892 filed September 21, 2023; hereafter “Naesby”), and
the rejection of claims 57 and 59 under 35 U.S.C. 103 as being unpatentable over Stephanopoulos in view of Yang and Rengby as applied to claims 1-5, 7-10, 13, 29-33, 35-38, 41, and 61-64 above, and further in view of Hughes et al. (Chemistry and Biology 18:165-176, 2011; cited on Form PTO-892 filed September 11, 2024; hereafter “Hughes”) and as evidenced by UniProt Database Accession Number Q9L0A2 (February 2020, 2 pages; cited on Form PTO-892 filed September 11, 2024; hereafter “UniProt”)
are withdrawn in view of applicant’s amendment to claim 1 to recite “an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.” The combination of cited prior art does not explicitly teach the sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Claims 1-5, 7-10, 13, 29-33, 35-38, 41, and 61-64 are rejected under 35 U.S.C. 103 as being unpatentable over Stephanopoulos in view of Yang and Rengby and as evidenced by GenBank Database Accession AF022387 (December 1998, 3 pages; cited on the attached Form PTO-892; hereafter “GenBank”).
As amended, claims 1-5, 7-10, 13, 61, and 63 are drawn to an engineered host cell that comprises genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell, wherein the engineered host cell is E. coli, and wherein the genetic modifications comprise expression of heterologous malonyl-CoA synthetase and one or more genetic modifications to decrease fatty acid biosynthesis as compared to a corresponding host cell without the one or more genetic modifications, wherein the one or more genetic modifications to decrease fatty acid biosynthesis comprise mutation of a gene encoding malonyl-CoA-ACP transacylase; and an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
As amended, claims 29-33, 35-38, 41, 62, and 64 are drawn to a plurality of engineered host cells, wherein each of the plurality of the engineered host cells comprises genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell, wherein at least one of the plurality of the engineered host cells is E. coli, and wherein the genetic modifications comprise expression of heterologous malonyl-CoA synthetase and one or more genetic modifications to decrease fatty acid biosynthesis as compared to a corresponding host cell without the one or more genetic modifications, wherein the one or more genetic modifications to decrease fatty acid biosynthesis comprise mutation of a gene encoding malonyl-CoA-ACP transacylase; and an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Regarding claims 1, 9, 10, 13, 29, 37, 38, and 41, Stephanopoulos teaches the production of flavonoids in a cell through recombinant expression of genes encoding the enzymes tyrosine ammonia lyase (TAL), 4-coumarate:CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) (Abstract; Figure 1; paragraph [0039]). TAL converts L-tyrosine to p-coumaric acid (i.e., 4-coumaric acid), 4CL converts p-coumaric acid to coumaroyl-CoA, CHS converts coumaroyl-CoA to naringenin chalcone, and CHI converts naringenin chalcone to naringenin (Figure 1).
Stephanopoulos teaches the cell is an E. coli cell (paragraph [0009]).
Stephanopoulos teaches the cell comprises a recombinantly-expressed malonate assimilation pathway, the assimilation pathway comprising genes encoding Rhizobium trifolii MatB and MatC (paragraph [00013]) for transport of malonate into the cell and subsequent conversion to malonyl-CoA (paragraph [0118]). Stephanopoulos does not teach malonyl-CoA synthetase, however, according to the instant specification, malonyl-CoA synthetase generates malonyl-CoA from malonate (p. 5, lines 19-20) and malonyl-CoA synthetase is abbreviated as matB (p. 118). Given that Stephanopoulos teaches recombinantly-expressed MatB for conversion of malonate to malonyl-CoA, MatB of Stephanopoulos is considered to be a malonyl-CoA synthetase. Stephanopoulos does not teach Rhizobium trifolii MatC comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. However, evidentiary reference GenBank, which discloses the amino acid sequence of Rhizobium trifolii MatC (p. 2, top), is cited to show that Rhizobium trifolii MatC comprises the amino acid sequence of instant SEQ ID NO: 81 (see Appendix for sequence alignment).
Stephanopoulos teaches inhibiting fatty acid biosynthesis by culturing cells in the presence of the fatty acid pathway inhibitor cerulenin, which limits the amount of malonyl-CoA lost to the synthesis of fatty acids (paragraph [0058]). However, Stephanopoulos does not teach decreasing fatty acid biosynthesis by genetic modification to mutate a gene encoding malonyl-CoA-ACP transacylase.
Yang teaches using antibiotics such as cerulenin to inhibit fatty acid biosynthesis is usually very costly and is infeasible for application in large-scale production (p. 218, column 1, middle). Instead of antibiotics to inhibit fatty acid biosynthesis, Yang teaches genetically modifying E. coli to knockdown (or downregulate) expression of the essential gene fabD encoding malonyl-CoA-ACP transacylase (p. 218, Figure 1 caption and column 2, bottom; p. 224, column 2), which resulted in a 4.5-fold increase in intracellular malonyl-CoA (p. 218, column 2). Yang teaches combining downregulation of fabD expression with a biosynthetic pathway for naringenin, which led to significant improvement in naringenin production (p. 218, column 2; paragraph bridging pp. 222-223).
While Yang teaches antisense RNA to knockdown (or downregulate) expression of the essential gene fabD, Rengby teaches methods to knockdown (or downregulate) expression of an essential gene using a PBAD promoter (p. 432, Abstract and column 1) by plasmid-driven complementation of a chromosomal gene deletion (p. 432, column 1) or replacing an endogenous promoter with a PBAD promoter (p. 432, Abstract).
In view of Stephanopoulos, Yang, and Rengby, it would have been obvious to one of ordinary skill in the art before the effective filing date to genetically modify the E. coli of Stephanopoulos to knockdown fabD expression with a PBAD promoter by plasmid-driven complementation or by replacing the endogenous promoter with a PBAD promoter as taught by Rengby. Given a broadest reasonable interpretation, knockdown of fabD expression with a PBAD promoter by plasmid-driven complementation or by replacing the endogenous promoter with a PBAD promoter each comprises mutation of a gene encoding malonyl-CoA-ACP transacylase and is considered to be encompassed by the recitation of “one or more genetic modifications to decrease fatty acid biosynthesis [that] comprise mutation of a gene encoding malonyl-CoA-ACP transacylase” in claims 1 and 29. One would have been motivated to do this because while Stephanopoulos taught inhibiting fatty acid biosynthesis with cerulenin, Yang taught cerulenin inhibition of fatty acid biosynthesis is usually very costly and is infeasible for application in large-scale production and taught down-regulating expression of the essential gene fabD to inhibit fatty acid biosynthesis, which led to significant improvement in intracellular malonyl-CoA and naringenin production, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter to control expression of the essential gene. One would have expected success because Yang taught downregulating expression of fabD with a biosynthetic pathway for naringenin led to significant improvement in naringenin production, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter.
Regarding claims 2-4 and 30-32, Stephanopoulos teaches the carbon source for producing the flavonoids is glucose (paragraph [0016]), which is a sugar and Yang teaches growing E. coli cells on a medium comprising glycerol (p. 219, column 1). According to the instant specification, glucose and glycerol are examples of carbon sources that are also energy sources (p. 48, lines 8-9).
Regarding claims 5 and 33, Stephanopoulos teaches L-tyrosine serves as the main precursor for the flavonoid naringenin, strains exhibiting an enhanced capacity for its synthesis provide a natural platform for exploring the potential of microbial flavonoid production, noting that the strains have a high flux through the aromatic amino acid pathway already in place and the next logical step is a functional pathway consisting of TAL, 4CL, CHS, and CHI in order to mediate conversion of L-tyrosine to naringenin (paragraph [0095]).
Regarding claims 7 and 35, Stephanopoulos teaches the genes encoding TAL, 4CL, CHS, and CHI for expression in E. coli are from organisms other than E. coli (paragraph [0096]) and thus are considered to be “non-native genes.”
Regarding claims 8 and 36, Stephanopoulos teaches the embodiment of culturing the cells in a medium supplemented with L-tyrosine (paragraph [0097]).
Regarding claims 61 and 62, Yang teaches the fabB gene encoding β-ketoacyl-ACP synthase I (p. 218, Figure 1 caption) is involved in malonyl-CoA consumption (p. 221, column 2, bottom) and teaches downregulating fabB (p. 217, Abstract).
Therefore, the invention of claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 14 and 42 are rejected under 35 U.S.C. 103 as being unpatentable over Stephanopoulos in view of Yang and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 above, and further in view of Naesby.
The relevant teachings of Stephanopoulos, Yang, and Rengby and evidentiary reference GenBank as applied to claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 are set forth above.
The combination of Stephanopoulos, Yang, and Rengby does not teach production of catechin.
Naesby teaches producing a catechin-producing strain by transforming a naringenin-producing strain with a vector comprising genes encoding CPR, a F3’H, F3H-1, a DFR, and a LAR (Example 1 and paragraphs [00149] and [00150] of Example 4).
In view of Naesby, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the naringenin-producing E. coli of Stephanopoulos with a vector comprising genes encoding CPR, F3’H, F3H-1, DFR, and LAR. One would have been motivated and would have expected success to do this because Stephanopoulos taught a naringenin-producing E. coli and Naesby taught including genes encoding CPR, F3’H, F3H-1, DFR, and LAR to a naringenin-producing strain for the production of catechin.
Therefore, the invention of claims 14 and 42 would have been obvious to one of ordinary skill in the art before the effective filing date.
Claims 57 and 59 are rejected under 35 U.S.C. 103 as being unpatentable over Stephanopoulos in view of Yang and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt.
The relevant teachings of Stephanopoulos, Yang, and Rengby and evidentiary reference GenBank as applied to claims 1-5, 7-10, 13, 29-33, 35-38, 41, 61, and 62 are set forth above.
The combination of Stephanopoulos, Yang, and Rengby does not teach a malonyl-CoA synthetase comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 17.
Hughes teaches Streptomyces coelicolor MatB shares characteristics with R. trifoli MatB (p. 170, column 2), teaches S. coelicolor MatB produces malonyl-CoA (p. 173, column 2, bottom), and teaches expression of the S. coelicolor MatB in an E. coli host cell (p. 174, column 1, bottom). Hughes does not teach the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application, however, evidentiary reference UniProt is cited to show that the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application (see Appendix of the Office action filed September 11, 2024).
In view of Hughes, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the R. trifolii MatB of Stephanopoulos with the S. coelicolor MatB of Hughes. One of ordinary skill would have expected success and could have substituted the R. trifolii MatB of Stephanopoulos with the S. coelicolor MatB of Hughes because Stephanopoulos taught expression of R. trifolii MatB in E. coli for production of malonyl-CoA, Hughes taught S. coelicolor MatB shares characteristics with R. trifoli MatB, Hughes taught S. coelicolor MatB produces malonyl-CoA, and Hughes taught expression of the S. coelicolor MatB in an E. coli host cell. One of ordinary skill in the art would have found it obvious to make the substitution because, based on the relevant teachings of Stephanopoulos and Hughes, an ordinarily skilled artisan would have predicted that the R. trifolii MatB of Stephanopoulos can be substituted with the S. coelicolor MatB of Hughes.
Therefore, the invention of claims 57 and 59 would have been obvious to one of ordinary skill in the art before the effective filing date.
RESPONSE TO REMARKS: In summary, applicant argues that replacing the endogenous promoter of fabD with a PBAD promoter is not a “mutation of a gene encoding malonyl-CoA-ACP transacylase” as recited in the claims and the claims have been amended to recite “an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83,” which is neither taught nor suggested by the cited prior art.
Applicant’s argument is not found persuasive. Contrary to applicant’s position, given that the endogenous fabD promoter is part of the fabD gene, which encodes a malonyl-CoA-ACP transacylase, replacing the endogenous fabD promoter with a PBAD promoter is encompassed by a “mutation of a gene encoding malonyl-CoA-ACP transacylase.” The newly added limitation “an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83” is addressed above.
For these reasons, it is the examiner’s position that the claimed invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim Rejections - 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.
The provisional rejection of claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 4, 6-10, 13, and 27 of co-pending application 17/720,020 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby,
the provisional rejection of claim 57 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 4, 6-10, 12, 13, and 27 of co-pending application 17/720,020 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt,
the provisional rejection of claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, and 5 of co-pending application 17/720,036 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby,
the provisional rejection of claim 57 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, and 5 of co-pending application 17/720,036 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt,
the provisional rejection of claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of co-pending application 17/720,031 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby,
the provisional rejection of claim 57 on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of co-pending application 17/720,031 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt,
the rejection of claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. 12,203,104 (cited on Form PTO-892 filed July 8, 2025) in view of Naesby, Stephanopoulos, and Yang, and
the rejection of claim 57 on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. 12,203,104 (cited on Form PTO-892 filed July 8, 2025) in view of Naesby, Stephanopoulos, Yang, and Rengby as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt
are withdrawn in view of applicant’s amendment to claim 1 to recite “an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.” The claims of the reference application or patent do not recite and the combination of cited prior art does not explicitly teach the sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 6, 8-10, and 27 of co-pending application 17/720,020 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank.
The claims of the reference application are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictoyl (EDL), and/or pentahydroxyflavaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR), cytochrome b5, and flavanone-3'-hydroxylase (F3'H), wherein the engineered host cell is E. coli.
The claims of the reference application do not recite one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, wherein the one or more genetic modifications comprise expression of heterologous malonyl-CoA synthetase, mutation of a gene encoding malonyl-CoA-ACP transacylase, and an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Regarding instant claims 1-4, 7, 9, 10, 29-32, 35, 37, and 38, Naesby teaches a microorganism, comprising an operative metabolic pathway capable of producing an anthocyanin from a simple sugar, the operative metabolic pathway comprising:
a 4-coumaric acid-CoA ligase (4CL);
a chalcone synthase (CHS);
a flavanone 3-hydroxylase (F3H);
a dihydroflavonol-4-reductase (DFR);
an anthocyanidin synthase (ANS);
an anthocyanidin 3-O-glycosyltransferase (A3GT);
a chalcone isomerase (CHI); and
at least one of
a) a tyrosine ammonia lyase (TAL); or
b) a phenylalanine ammonia lyase (PAL) and a trans-cinnamate 4- monooxygenase (C4H),
wherein at least one enzyme of the operative metabolic pathway is encoded by a gene heterologous to the microorganism (claim 1 of Naesby).
The anthocyanin biosynthetic pathway is shown in FIG. 1. of Naesby (paragraph [0003]), which proceeds through multiple chemical intermediates.
According to the specification of this application, glucose and glycerol are examples of a carbon sources that is also an energy source (p. 48, lines 8-9). Naesby teaches the simple sugar is glucose or other simple carbon sources such as glycerol (paragraph [0061] and claim 29 of Naesby).
Naesby teaches the microorganism is Escherichia coli (claim 9 of Naesby).
Naesby teaches that previous demonstration of anthocyanin production from sugar in E. coli could have been due to a lack of the precursor malonyl-CoA (paragraph [0057]) and Naesby teaches the recombinant host cell is capable of producing malonyl-CoA (paragraph [0072]).
Similar to Naesby, Stephanopoulos teaches production of flavonoids by a genetically modified E. coli (Abstract; Figure 1; paragraph [0039]) through enzymatic conversion of multiple chemical intermediates (Figure 1; paragraph [0039]). Stephanopoulos teaches the cell comprises a recombinantly-expressed malonate assimilation pathway, the assimilation pathway comprising genes encoding Rhizobium trifolii MatB and MatC (paragraph [00013]) for transport of malonate into the cell and subsequence conversion to malonyl-CoA (paragraph [0118]). According to the instant specification, malonyl-CoA synthetase generates malonyl-CoA from malonate (p. 5, lines 19-20) and malonyl-CoA synthetase is abbreviated as matB (p. 118). Given that Stephanopoulos teaches recombinantly-expressed MatB for conversion of malonate to malonyl-CoA, MatB of Stephanopoulos is considered to be a malonyl-CoA synthetase. Stephanopoulos does not teach Rhizobium trifolii MatC comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. However, evidentiary reference GenBank, which discloses the amino acid sequence of Rhizobium trifolii MatC (p. 2, top), is cited to show that Rhizobium trifolii MatC comprises the amino acid sequence of instant SEQ ID NO: 81 (see Appendix for sequence alignment).
In view of Naesby and Stephanopoulos, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the engineered host cell of the claims of the reference application according to Naesby and Stephanopoulos. One would have been motivated to and would have had a reasonable expectation of success to do so because the claims of the reference application are drawn to an engineered host cell with genetic modifications to produce DHQ, DHM, and/or or PHF, Naesby teaches genetic modifications for production of DHQ, DHM, or PHF from a simple sugar, yet acknowledges that E. coli may lack malonyl-CoA for anthocyanin production, while Stephanopoulos teaches genetic modification to express heterologous MatB and MatC genes for the synthesis of malonyl-CoA.
Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway (paragraph [0060]).
Yang teaches using antibiotics such as cerulenin to inhibit fatty acid biosynthesis is usually very costly and is infeasible for application in large-scale production (p. 218, column 1, middle). Instead of antibiotics to inhibit fatty acid biosynthesis, Yang teaches genetically modifying E. coli to knockdown (or downregulate) expression of the essential gene fabD encoding malonyl-CoA-ACP transacylase (p. 218, Figure 1 caption and column 2, bottom; p. 224, column 2), which resulted in a 4.5 fold increase in intracellular malonyl-CoA (p. 218, column 2). Yang teaches combining downregulation of fabD expression with a biosynthetic pathway for naringenin, which led to significant improvement in naringenin production (p. 218, column 2; paragraph bridging pp. 222-223).
While Yang teaches antisense RNA to knockdown (or downregulate) expression of the essential gene fabD, Rengby teaches methods to knockdown (or downregulate) expression of an essential gene using a PBAD promoter (p. 432, Abstract and column 1) by plasmid-driven complementation of a chromosomal gene deletion (p. 432, column 1) or replacing an endogenous promoter with a PBAD promoter (p. 432, Abstract).
In view of Naesby, Yang, and Rengby, it would have been obvious to one of ordinary skill in the art before the effective filing date to further genetically modify the engineered host cell of the claims of the reference application to knockdown fabD expression with a PBAD promoter by plasmid-driven complementation or by replacing the endogenous promoter with a PBAD promoter as taught by Rengby. One would have been motivated and would have expected success because Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway, Yang teaches down-regulating expression of the essential gene fabD to inhibit fatty acid biosynthesis and increase intracellular malonyl-CoA, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter. One would have expected success because Yang taught downregulating expression of fabD with a biosynthetic pathway for naringenin led to significant improvement in naringenin production, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter.
Regarding claims 8 and 36, Stephanopoulos teaches the embodiment of culturing the cells in a medium supplemented with L-tyrosine (paragraph [0097]).
Regarding claims 13 and 41, Naesby teaches p-coumaric acid (i.e., 4-coumaric acid) is formed directly from tyrosine by the action of tyrosine ammonia lyase (TAL) (paragraph [0003]; Figure 1).
Regarding claims 14 and 42, Naesby teaches a (+)-catechin-producing engineered host cell comprising a PAL, a C4H, a 4CL, a CHS, a CHI, a CPR, a F3’H, F3H-1, a DFR, and a LAR (Example 1 and paragraphs [00149] and [00150] of Example 4).
Regarding claims 29-33, 35-38, 41, and 42, Naesby teaches culturing the microorganism (claim 18 of Naesby), which results in growth of the microorganism (paragraph [0121]). One would have recognized that Naesby’s culture of the microorganism necessarily comprises a plurality (i.e., more than one) of the microorganisms.
Regarding claims 61 and 62, Yang teaches the fabB gene encoding β-ketoacyl-ACP synthase I (p. 218, Figure 1 caption) is involved in malonyl-CoA consumption (p. 221, column 2, bottom) and teaches downregulating fabB (p. 217, Abstract).
Therefore, claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 of this application are unpatentable over the claim(s) of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 57 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, 6, 8-10, and 27 of co-pending application 17/720,020 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt.
The claims of the reference application and the relevant teachings of Naesby, Stephanopoulos, Yang, and Rengby and evidentiary reference GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are set forth above.
The claims of the reference application do not recite and Naesby, Stephanopoulos, Yang, and Rengby do not teach a malonyl-CoA synthetase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17.
Hughes teaches Streptomyces coelicolor MatB shares characteristics with R. trifoli MatB (p. 170, column 2), teaches S. coelicolor MatB produces malonyl-CoA (p. 173, column 2, bottom), and teaches expression of the S. coelicolor MatB in an E. coli host cell (p. 174, column 1, bottom). Hughes does not teach the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application, however, evidentiary reference UniProt is cited to show that the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application (see Appendix of the Office action mailed on September 11, 2024).
In view of Hughes, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the R. trifolii MatB of Stephanopoulos for with the S. coelicolor MatB of Hughes. One of ordinary skill would have expected success and could have substituted the R. trifolii MatB of Stephanopoulos with the S. coelicolor MatB of Hughes because Stephanopoulos taught expression of R. trifolii MatB in E. coli for production of malonyl-CoA, Hughes taught S. coelicolor MatB shares characteristics with R. trifoli MatB, Hughes taught S. coelicolor MatB produces malonyl-CoA, and Hughes taught expression of the S. coelicolor MatB in an E. coli host cell. One of ordinary skill in the art would have found it obvious to make the substitution because, based on the relevant teachings of Stephanopoulos and Hughes, an ordinarily skilled artisan would have predicted that the R. trifolii MatB of Stephanopoulos can be substituted with the S. coelicolor MatB of Hughes.
Therefore, claim 57 of this application is unpatentable over the claim(s) of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, and 5 of co-pending application 17/720,036 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank.
The claims of the reference application are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3- glucoside (Cy3G).
The claims of the reference application do not recite one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, wherein the one or more genetic modifications comprise expression of heterologous malonyl-CoA synthetase, mutation of a gene encoding malonyl-CoA-ACP transacylase, and an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Regarding claims 1-4, 7, 9, 10, 29-32, 35, 37, and 38, Naesby teaches a microorganism, comprising an operative metabolic pathway capable of producing an anthocyanin from a simple sugar, the operative metabolic pathway comprising:
a 4-coumaric acid-CoA ligase (4CL);
a chalcone synthase (CHS);
a flavanone 3-hydroxylase (F3H);
a dihydroflavonol-4-reductase (DFR);
an anthocyanidin synthase (ANS);
an anthocyanidin 3-O-glycosyltransferase (A3GT);
a chalcone isomerase (CHI); and
at least one of
a) a tyrosine ammonia lyase (TAL); or
b) a phenylalanine ammonia lyase (PAL) and a trans-cinnamate 4- monooxygenase (C4H),
wherein at least one enzyme of the operative metabolic pathway is encoded by a gene heterologous to the microorganism (claim 1 of Naesby).
The anthocyanin biosynthetic pathway is shown in FIG. 1. of Naesby (paragraph [0003]), which proceeds through multiple chemical intermediates.
According to the specification of this application, glucose and glycerol are examples of a carbon sources that is also an energy source (p. 48, lines 8-9). Naesby teaches the simple sugar is glucose or other simple carbon sources such as glycerol (paragraph [0061] and claim 29 of Naesby).
Naesby teaches the microorganism is Escherichia coli (claim 9 of Naesby).
Naesby teaches that previous demonstration of anthocyanin production from sugar in E. coli could have been due to a lack of the precursor malonyl-CoA (paragraph [0057]) and Naesby teaches the recombinant host cell is capable of producing malonyl-CoA (paragraph [0072]).
Similar to Naesby, Stephanopoulos teaches production of flavonoids by a genetically modified E. coli (Abstract; Figure 1; paragraph [0039]) through enzymatic conversion of multiple chemical intermediates (Figure 1; paragraph [0039]). Stephanopoulos teaches the cell comprises a recombinantly-expressed malonate assimilation pathway, the assimilation pathway comprising genes encoding Rhizobium trifolii MatB and MatC (paragraph [00013]) for transport of malonate into the cell and subsequence conversion to malonyl-CoA (paragraph [0118]). According to the instant specification, malonyl-CoA synthetase generates malonyl-CoA from malonate (p. 5, lines 19-20) and malonyl-CoA synthetase is abbreviated as matB (p. 118). Given that Stephanopoulos teaches recombinantly-expressed MatB for conversion of malonate to malonyl-CoA, MatB of Stephanopoulos is considered to be a malonyl-CoA synthetase. Stephanopoulos does not teach Rhizobium trifolii MatC comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. However, evidentiary reference GenBank, which discloses the amino acid sequence of Rhizobium trifolii MatC (p. 2, top), is cited to show that Rhizobium trifolii MatC comprises the amino acid sequence of instant SEQ ID NO: 81 (see Appendix for sequence alignment).
In view of Naesby and Stephanopoulos, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the engineered host cell of the claims of the reference application according to Naesby and Stephanopoulos. One would have been motivated to and would have had a reasonable expectation of success to do so because the claims of the reference application are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of catechin to Cy3G, Naesby teaches genetic modifications for production of catechin from a simple sugar, yet acknowledges that E. coli may lack malonyl-CoA for anthocyanin production, while Stephanopoulos teaches genetic modification to express heterologous MatB and MatC genes for the synthesis of malonyl-CoA.
Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway (paragraph [0060]).
Yang teaches using antibiotics such as cerulenin to inhibit fatty acid biosynthesis is usually very costly and is infeasible for application in large-scale production (p. 218, column 1, middle). Instead of antibiotics to inhibit fatty acid biosynthesis, Yang teaches genetically modifying E. coli to knockdown (or downregulate) expression of the essential gene fabD encoding malonyl-CoA-ACP transacylase (p. 218, Figure 1 caption and column 2, bottom; p. 224, column 2), which resulted in a 4.5 fold increase in intracellular malonyl-CoA (p. 218, column 2). Yang teaches combining downregulation of fabD expression with a biosynthetic pathway for naringenin, which led to significant improvement in naringenin production (p. 218, column 2; paragraph bridging pp. 222-223).
While Yang teaches antisense RNA to knockdown (or downregulate) expression of the essential gene fabD, Rengby teaches methods to knockdown (or downregulate) expression of an essential gene using a PBAD promoter (p. 432, Abstract and column 1) by plasmid-driven complementation of a chromosomal gene deletion (p. 432, column 1) or replacing an endogenous promoter with a PBAD promoter (p. 432, Abstract).
In view of Naesby, Yang, and Rengby, it would have been obvious to one of ordinary skill in the art before the effective filing date to further genetically modify the engineered host cell of the claims of the reference application to knockdown fabD expression with a PBAD promoter by plasmid-driven complementation or by replacing the endogenous promoter with a PBAD promoter as taught by Rengby. One would have been motivated and would have expected success because Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway, Yang teaches down-regulating expression of the essential gene fabD to inhibit fatty acid biosynthesis and increase intracellular malonyl-CoA, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter. One would have expected success because Yang taught downregulating expression of fabD with a biosynthetic pathway for naringenin led to significant improvement in naringenin production, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter.
Regarding claims 8 and 36, Stephanopoulos teaches the embodiment of culturing the cells in a medium supplemented with L-tyrosine (paragraph [0097]).
Regarding claims 13 and 41, Naesby teaches p-coumaric acid (i.e., 4-coumaric acid) is formed directly from tyrosine by the action of tyrosine ammonia lyase (TAL) (paragraph [0003]; Figure 1).
Regarding claims 14 and 42, Naesby teaches a (+)-catechin-producing engineered host cell comprising a PAL, a C4H, a 4CL, a CHS, a CHI, a CPR, a F3’H, F3H-1, a DFR, and a LAR (Example 1 and paragraphs [00149] and [00150] of Example 4).
Regarding claims 29-33, 35-38, 41, and 42, Naesby teaches culturing the microorganism (claim 18 of Naesby), which results in growth of the microorganism (paragraph [0121]). One would have recognized that Naesby’s culture of the microorganism necessarily comprises a plurality (i.e., more than one) of the microorganisms.
Regarding claims 61 and 62, Yang teaches the fabB gene encoding β-ketoacyl-ACP synthase I (p. 218, Figure 1 caption) is involved in malonyl-CoA consumption (p. 221, column 2, bottom) and teaches downregulating fabB (p. 217, Abstract).
Therefore, claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 of this application are unpatentable over the claim(s) of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 57 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 4, and 5 of co-pending application 17/720,036 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt.
The claims of the reference application and the relevant teachings of Naesby, Stephanopoulos, Yang, and Rengby and evidentiary reference GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are set forth above.
The claims of the reference application do not recite and Naesby, Stephanopoulos, Yang, and Rengby do not teach a malonyl-CoA synthetase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17.
Hughes teaches Streptomyces coelicolor MatB shares characteristics with R. trifoli MatB (p. 170, column 2), teaches S. coelicolor MatB produces malonyl-CoA (p. 173, column 2, bottom), and teaches expression of the S. coelicolor MatB in an E. coli host cell (p. 174, column 1, bottom). Hughes does not teach the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application, however, evidentiary reference UniProt is cited to show that the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application (see Appendix of the Office action mailed on September 11, 2024).
In view of Hughes, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the R. trifolii MatB of Stephanopoulos for with the S. coelicolor MatB of Hughes. One of ordinary skill would have expected success and could have substituted the R. trifolii MatB of Stephanopoulos with the S. coelicolor MatB of Hughes because Stephanopoulos taught expression of R. trifolii MatB in E. coli for production of malonyl-CoA, Hughes taught S. coelicolor MatB shares characteristics with R. trifoli MatB, Hughes taught S. coelicolor MatB produces malonyl-CoA, and Hughes taught expression of the S. coelicolor MatB in an E. coli host cell. One of ordinary skill in the art would have found it obvious to make the substitution because, based on the relevant teachings of Stephanopoulos and Hughes, an ordinarily skilled artisan would have predicted that the R. trifolii MatB of Stephanopoulos can be substituted with the S. coelicolor MatB of Hughes.
Therefore, claim 57 of this application is unpatentable over the claim(s) of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, and 61-64 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of co-pending application 17/720,031 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank.
The claims of the reference application are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine, wherein the one or more genetic modification is downregulation or deletion of lactate dehydrogenase and/or pyruvate oxidase.
The claims of the reference application do not recite one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, wherein the one or more genetic modifications comprise expression of heterologous malonyl-CoA synthetase, mutation of a gene encoding malonyl-CoA-ACP transacylase, and an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Regarding claims 1-4, 7, 9, 10, 29-32, 35, 37, and 38, Naesby teaches a microorganism, comprising an operative metabolic pathway capable of producing an anthocyanin from a simple sugar, the operative metabolic pathway comprising:
a 4-coumaric acid-CoA ligase (4CL);
a chalcone synthase (CHS);
a flavanone 3-hydroxylase (F3H);
a dihydroflavonol-4-reductase (DFR);
an anthocyanidin synthase (ANS);
an anthocyanidin 3-O-glycosyltransferase (A3GT);
a chalcone isomerase (CHI); and
at least one of
a) a tyrosine ammonia lyase (TAL); or
b) a phenylalanine ammonia lyase (PAL) and a trans-cinnamate 4- monooxygenase (C4H),
wherein at least one enzyme of the operative metabolic pathway is encoded by a gene heterologous to the microorganism (claim 1 of Naesby).
The anthocyanin biosynthetic pathway is shown in FIG. 1. of Naesby (paragraph [0003]), which proceeds through multiple chemical intermediates.
According to the specification of this application, glucose and glycerol are examples of a carbon sources that is also an energy source (p. 48, lines 8-9). Naesby teaches the simple sugar is glucose or other simple carbon sources such as glycerol (paragraph [0061] and claim 29 of Naesby).
Naesby teaches the microorganism is Escherichia coli (claim 9 of Naesby).
Naesby teaches that previous demonstration of anthocyanin production from sugar in E. coli could have been due to a lack of the precursor malonyl-CoA (paragraph [0057]) and Naesby teaches the recombinant host cell is capable of producing malonyl-CoA (paragraph [0072]).
Similar to Naesby, Stephanopoulos teaches production of flavonoids by a genetically modified E. coli (Abstract; Figure 1; paragraph [0039]) through enzymatic conversion of multiple chemical intermediates (Figure 1; paragraph [0039]). Stephanopoulos teaches the cell comprises a recombinantly-expressed malonate assimilation pathway, the assimilation pathway comprising genes encoding Rhizobium trifolii MatB and MatC (paragraph [00013]) for transport of malonate into the cell and subsequence conversion to malonyl-CoA (paragraph [0118]). According to the instant specification, malonyl-CoA synthetase generates malonyl-CoA from malonate (p. 5, lines 19-20) and malonyl-CoA synthetase is abbreviated as matB (p. 118). Given that Stephanopoulos teaches recombinantly-expressed MatB for conversion of malonate to malonyl-CoA, MatB of Stephanopoulos is considered to be a malonyl-CoA synthetase. Stephanopoulos does not teach Rhizobium trifolii MatC comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. However, evidentiary reference GenBank, which discloses the amino acid sequence of Rhizobium trifolii MatC (p. 2, top), is cited to show that Rhizobium trifolii MatC comprises the amino acid sequence of instant SEQ ID NO: 81 (see Appendix for sequence alignment).
In view of Naesby and Stephanopoulos, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the engineered host cell of the claims of the reference application according to Naesby and Stephanopoulos. One would have been motivated to and would have had a reasonable expectation of success to do so because the claims of the reference application are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine, Naesby teaches genetic modifications for production of anthocyanin from tyrosine using a simple sugar, yet acknowledges that E. coli may lack malonyl-CoA for anthocyanin production, while Stephanopoulos teaches genetic modification to express heterologous MatB and MatC genes for the synthesis of malonyl-CoA.
Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway (paragraph [0060]).
Yang teaches using antibiotics such as cerulenin to inhibit fatty acid biosynthesis is usually very costly and is infeasible for application in large-scale production (p. 218, column 1, middle). Instead of antibiotics to inhibit fatty acid biosynthesis, Yang teaches genetically modifying E. coli to knockdown (or downregulate) expression of the essential gene fabD encoding malonyl-CoA-ACP transacylase (p. 218, Figure 1 caption and column 2, bottom; p. 224, column 2), which resulted in a 4.5 fold increase in intracellular malonyl-CoA (p. 218, column 2). Yang teaches combining downregulation of fabD expression with a biosynthetic pathway for naringenin, which led to significant improvement in naringenin production (p. 218, column 2; paragraph bridging pp. 222-223).
While Yang teaches antisense RNA to knockdown (or downregulate) expression of the essential gene fabD, Rengby teaches methods to knockdown (or downregulate) expression of an essential gene using a PBAD promoter (p. 432, Abstract and column 1) by plasmid-driven complementation of a chromosomal gene deletion (p. 432, column 1) or replacing an endogenous promoter with a PBAD promoter (p. 432, Abstract).
In view of Naesby, Yang, and Rengby, it would have been obvious to one of ordinary skill in the art before the effective filing date to further genetically modify the engineered host cell of the claims of the reference application to knockdown fabD expression with a PBAD promoter by plasmid-driven complementation or by replacing the endogenous promoter with a PBAD promoter as taught by Rengby. One would have been motivated and would have expected success because Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway, Yang teaches down-regulating expression of the essential gene fabD to inhibit fatty acid biosynthesis and increase intracellular malonyl-CoA, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter. One would have expected success because Yang taught downregulating expression of fabD with a biosynthetic pathway for naringenin led to significant improvement in naringenin production, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter.
Regarding claims 8 and 36, Stephanopoulos teaches the embodiment of culturing the cells in a medium supplemented with L-tyrosine (paragraph [0097]).
Regarding claims 13 and 41, Naesby teaches p-coumaric acid (i.e., 4-coumaric acid) is formed directly from tyrosine by the action of tyrosine ammonia lyase (TAL) (paragraph [0003]; Figure 1).
Regarding claims 14 and 42, Naesby teaches a (+)-catechin-producing engineered host cell comprising a PAL, a C4H, a 4CL, a CHS, a CHI, a CPR, a F3’H, F3H-1, a DFR, and a LAR (Example 1 and paragraphs [00149] and [00150] of Example 4).
Regarding claims 29-33, 35-38, 41, and 42, Naesby teaches culturing the microorganism (claim 18 of Naesby), which results in growth of the microorganism (paragraph [0121]). One would have recognized that Naesby’s culture of the microorganism necessarily comprises a plurality (i.e., more than one) of the microorganisms.
Regarding claims 61 and 62, Yang teaches the fabB gene encoding β-ketoacyl-ACP synthase I (p. 218, Figure 1 caption) is involved in malonyl-CoA consumption (p. 221, column 2, bottom) and teaches downregulating fabB (p. 217, Abstract).
Therefore, claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 of this application are unpatentable over the claim(s) of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 57 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of co-pending application 17/720,031 (reference application) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, and 61-64 above, and further in view of Hughes and as evidenced by UniProt.
The claims of the reference application and the relevant teachings of Naesby, Stephanopoulos, Yang, and Rengby and evidentiary reference GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are set forth above.
The claims of the reference application do not recite and Naesby, Stephanopoulos, Yang, and Rengby do not teach a malonyl-CoA synthetase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17.
Hughes teaches Streptomyces coelicolor MatB shares characteristics with R. trifoli MatB (p. 170, column 2), teaches S. coelicolor MatB produces malonyl-CoA (p. 173, column 2, bottom), and teaches expression of the S. coelicolor MatB in an E. coli host cell (p. 174, column 1, bottom). Hughes does not teach the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application, however, evidentiary reference UniProt is cited to show that the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application (see Appendix of the Office action mailed on September 11, 2024).
In view of Hughes, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the R. trifolii MatB of Stephanopoulos for with the S. coelicolor MatB of Hughes. One of ordinary skill would have expected success and could have substituted the R. trifolii MatB of Stephanopoulos with the S. coelicolor MatB of Hughes because Stephanopoulos taught expression of R. trifolii MatB in E. coli for production of malonyl-CoA, Hughes taught S. coelicolor MatB shares characteristics with R. trifoli MatB, Hughes taught S. coelicolor MatB produces malonyl-CoA, and Hughes taught expression of the S. coelicolor MatB in an E. coli host cell. One of ordinary skill in the art would have found it obvious to make the substitution because, based on the relevant teachings of Stephanopoulos and Hughes, an ordinarily skilled artisan would have predicted that the R. trifolii MatB of Stephanopoulos can be substituted with the S. coelicolor MatB of Hughes.
Therefore, claim 57 of this application is unpatentable over the claim(s) of the reference application. This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. 12,203,104 (cited on Form PTO-892 filed July 8, 2025) in view of Naesby, Stephanopoulos, and Yang and as evidenced by GenBank.
The claims of the patent are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to an endogenous gene to increase the production and/or availability of malonyl-CoA in comparison to a wild-type host cell, wherein the engineered host cell expresses acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 85% identical to the polypeptide set forth in SEQ ID NO: 15.
The claims of the reference application do not recite one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, wherein the one or more genetic modifications comprise expression of heterologous malonyl-CoA synthetase, mutation of a gene encoding malonyl-CoA-ACP transacylase, and an exogenous nucleic acid sequence encoding a malonate transporter, wherein the malonate transporter comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83.
Regarding claims 1-4, 7, 9, 10, 29-32, 35, 37, and 38, Naesby teaches a microorganism, comprising an operative metabolic pathway capable of producing an anthocyanin from a simple sugar, the operative metabolic pathway comprising:
a 4-coumaric acid-CoA ligase (4CL);
a chalcone synthase (CHS);
a flavanone 3-hydroxylase (F3H);
a dihydroflavonol-4-reductase (DFR);
an anthocyanidin synthase (ANS);
an anthocyanidin 3-O-glycosyltransferase (A3GT);
a chalcone isomerase (CHI); and
at least one of
a) a tyrosine ammonia lyase (TAL); or
b) a phenylalanine ammonia lyase (PAL) and a trans-cinnamate 4- monooxygenase (C4H),
wherein at least one enzyme of the operative metabolic pathway is encoded by a gene heterologous to the microorganism (claim 1 of Naesby).
The anthocyanin biosynthetic pathway is shown in FIG. 1. of Naesby (paragraph [0003]), which proceeds through multiple chemical intermediates.
According to the specification of this application, glucose and glycerol are examples of a carbon sources that is also an energy source (p. 48, lines 8-9). Naesby teaches the simple sugar is glucose or other simple carbon sources such as glycerol (paragraph [0061] and claim 29 of Naesby).
Naesby teaches the microorganism is Escherichia coli (claim 9 of Naesby).
Naesby teaches that previous demonstration of anthocyanin production from sugar in E. coli could have been due to a lack of the precursor malonyl-CoA (paragraph [0057]) and Naesby teaches the recombinant host cell is capable of producing malonyl-CoA (paragraph [0072]).
Similar to Naesby, Stephanopoulos teaches production of flavonoids by a genetically modified E. coli (Abstract; Figure 1; paragraph [0039]) through enzymatic conversion of multiple chemical intermediates (Figure 1; paragraph [0039]). Stephanopoulos teaches the cell comprises a recombinantly-expressed malonate assimilation pathway, the assimilation pathway comprising genes encoding Rhizobium trifolii MatB and MatC (paragraph [00013]) for transport of malonate into the cell and subsequence conversion to malonyl-CoA (paragraph [0118]). According to the instant specification, malonyl-CoA synthetase generates malonyl-CoA from malonate (p. 5, lines 19-20) and malonyl-CoA synthetase is abbreviated as matB (p. 118). Given that Stephanopoulos teaches recombinantly-expressed MatB for conversion of malonate to malonyl-CoA, MatB of Stephanopoulos is considered to be a malonyl-CoA synthetase. Stephanopoulos teaches recombinantly-expressed MatB for conversion of malonate to malonyl-CoA, MatB of Stephanopoulos is considered to be a malonyl-CoA synthetase. Stephanopoulos does not teach Rhizobium trifolii MatC comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:18, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83. However, evidentiary reference GenBank, which discloses the amino acid sequence of Rhizobium trifolii MatC (p. 2, top), is cited to show that Rhizobium trifolii MatC comprises the amino acid sequence of instant SEQ ID NO: 81 (see Appendix for sequence alignment).
In view of Naesby and Stephanopoulos, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the engineered host cell of the claims of the patent according to Naesby and Stephanopoulos. One would have been motivated to and would have had a reasonable expectation of success to do so because the claims of the patent are drawn to an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA, Naesby teaches genetic modifications for production of anthocyanin from tyrosine using a simple sugar, yet acknowledges that E. coli may lack malonyl-CoA for anthocyanin production, while Stephanopoulos teaches genetic modification to express heterologous MatB and MatC genes for the synthesis of malonyl-CoA.
Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway (paragraph [0060]).
Yang teaches using antibiotics such as cerulenin to inhibit fatty acid biosynthesis is usually very costly and is infeasible for application in large-scale production (p. 218, column 1, middle). Instead of antibiotics to inhibit fatty acid biosynthesis, Yang teaches genetically modifying E. coli to knockdown (or downregulate) expression of the essential gene fabD encoding malonyl-CoA-ACP transacylase (p. 218, Figure 1 caption and column 2, bottom; p. 224, column 2), which resulted in a 4.5 fold increase in intracellular malonyl-CoA (p. 218, column 2). Yang teaches combining downregulation of fabD expression with a biosynthetic pathway for naringenin, which led to significant improvement in naringenin production (p. 218, column 2; paragraph bridging pp. 222-223).
While Yang teaches antisense RNA to knockdown (or downregulate) expression of the essential gene fabD, Rengby teaches methods to knockdown (or downregulate) expression of an essential gene using a PBAD promoter (p. 432, Abstract and column 1) by plasmid-driven complementation of a chromosomal gene deletion (p. 432, column 1) or replacing an endogenous promoter with a PBAD promoter (p. 432, Abstract).
In view of Naesby, Yang, and Rengby, it would have been obvious to one of ordinary skill in the art before the effective filing date to further genetically modify the engineered host cell of the claims of the patent to knockdown fabD expression with a PBAD promoter by plasmid-driven complementation or by replacing the endogenous promoter with a PBAD promoter as taught by Rengby. One would have been motivated and would have expected success because Naesby teaches that heterologous compound production via heterologous biosynthetic pathways often faces competition from host enzymes capable of degrading or modifying intermediates, or otherwise shunting them away from the main pathway, Yang teaches down-regulating expression of the essential gene fabD to inhibit fatty acid biosynthesis and increase intracellular malonyl-CoA, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter. One would have expected success because Yang taught downregulating expression of fabD with a biosynthetic pathway for naringenin led to significant improvement in naringenin production, and Rengby taught methods for downregulating expression of an essential gene using a PBAD promoter.
Regarding claims 8 and 36, Stephanopoulos teaches the embodiment of culturing the cells in a medium supplemented with L-tyrosine (paragraph [0097]).
Regarding claims 13 and 41, Naesby teaches p-coumaric acid (i.e., 4-coumaric acid) is formed directly from tyrosine by the action of tyrosine ammonia lyase (TAL) (paragraph [0003]; Figure 1).
Regarding claims 14 and 42, Naesby teaches a (+)-catechin-producing engineered host cell comprising a PAL, a C4H, a 4CL, a CHS, a CHI, a CPR, a F3’H, F3H-1, a DFR, and a LAR (Example 1 and paragraphs [00149] and [00150] of Example 4).
Regarding claims 29-33, 35-38, 41, and 42, Naesby teaches culturing the microorganism (claim 18 of Naesby), which results in growth of the microorganism (paragraph [0121]). One would have recognized that Naesby’s culture of the microorganism necessarily comprises a plurality (i.e., more than one) of the microorganisms.
Regarding claims 61 and 62, Yang teaches the fabB gene encoding β-ketoacyl-ACP synthase I (p. 218, Figure 1 caption) is involved in malonyl-CoA consumption (p. 221, column 2, bottom) and teaches downregulating fabB (p. 217, Abstract).
Therefore, claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 of this application are unpatentable over the claim(s) of the patent.
Claim 57 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of U.S. Patent No. 12,203,104 (cited on Form PTO-892 filed July 8, 2025) in view of Naesby, Stephanopoulos, Yang, and Rengby and as evidenced by GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 above, and further in view of Hughes and as evidenced by UniProt.
The claims of the patent and the relevant teachings of Naesby, Stephanopoulos, and Yang and evidentiary reference GenBank as applied to claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 61, and 62 are set forth above.
The claims of the patent do not recite and Naesby, Stephanopoulos, Yang, and Rengby do not teach a malonyl-CoA synthetase comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 17.
Hughes teaches Streptomyces coelicolor MatB shares characteristics with R. trifoli MatB (p. 170, column 2), teaches S. coelicolor MatB produces malonyl-CoA (p. 173, column 2, bottom), and teaches expression of the S. coelicolor MatB in an E. coli host cell (p. 174, column 1, bottom). Hughes does not teach the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application, however, evidentiary reference UniProt is cited to show that the amino acid sequence of S. coelicolor MatB has at least 95% sequence identity to SEQ ID NO: 17 of this application (see Appendix of the Office action mailed on September 11, 2024).
In view of Hughes, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the R. trifolii MatB of Stephanopoulos for with the S. coelicolor MatB of Hughes. One of ordinary skill would have expected success and could have substituted the R. trifolii MatB of Stephanopoulos with the S. coelicolor MatB of Hughes because Stephanopoulos taught expression of R. trifolii MatB in E. coli for production of malonyl-CoA, Hughes taught S. coelicolor MatB shares characteristics with R. trifoli MatB, Hughes taught S. coelicolor MatB produces malonyl-CoA, and Hughes taught expression of the S. coelicolor MatB in an E. coli host cell. One of ordinary skill in the art would have found it obvious to make the substitution because, based on the relevant teachings of Stephanopoulos and Hughes, an ordinarily skilled artisan would have predicted that the R. trifolii MatB of Stephanopoulos can be substituted with the S. coelicolor MatB of Hughes.
Therefore, claim 57 of this application is unpatentable over the claim(s) of the patent.
RESPONSE TO REMARKS: Applicant requests the provisional rejections be held in abeyance until allowable subject matter is identified. Applicant’s request is acknowledged.
Conclusion
Status of the claims:
Claims 1-10, 13-38, and 41-62 are pending.
Claims 6, 15-28, 34, 43-56, 58, and 60 are withdrawn from consideration.
Claims 1-5, 7-10, 13, 14, 29-33, 35-38, 41, 42, 57, 59, 61, and 62 are rejected.
No claim is in condition for allowance.
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/David Steadman/Primary Examiner, Art Unit 1656
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