DETAILED CORRESPONDENCE
Status of the Application
The present application is being examined under the pre-AIA first to invent provisions.
Applicant’s preliminary amendment to the claims, filed October 30, 2023, is acknowledged. This listing of the claims replaces all prior versions and listings of the claims.
Claims 1 and 39-54 are pending in the application and are being examined on the merits.
Priority
This application is a continuation application of U.S. non-provisional application number 17/348,363, filed June 15, 2021, now U.S. Patent No. 11,821,021, which is a continuation application of U.S. non-provisional application number 15/816,779, filed November 17, 2017, now U.S. Patent No. 11,041,176, which is a divisional application of U.S. non-provisional application number 14/416,633, filed January 22, 2015, now U.S. Patent No. 9,850,507, which is filed under 35 U.S.C. 371 as a national stage filing of international application number PCT/US2013/052069, filed July 25, 2013, which claims domestic priority under 35 U.S.C. 119(e) to U.S. provisional application number 61/675,788, filed on July 25, 2012.
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosure of the prior-filed application, Application No. 61/675,788, fails to provide adequate support or enablement in the manner provided by pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application.
The provisional application 61/675,788 fails to provide descriptive support for the limitations of claims 42-53.
The effective filing date of claims 1, 39-41, and 54 is July 25, 2012 and the effective filing date for claims 42-53 is July 25, 2013.
Information Disclosure Statement
The two information disclosure statements (IDSs) submitted on October 30, 2023 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the IDSs have been considered by the examiner.
Specification/Informalities
Applicant should correct the specification’s continuing data beginning line 5 of p. 1. As noted above, this application is a continuation application of U.S. non-provisional application number 17/348,363, filed June 15, 2021, now U.S. Patent No. 11,821,021, which is a continuation application of U.S. non-provisional application number 15/816,779, filed November 17, 2017, now U.S. Patent No. 11,041,176, which is a divisional application of U.S. non-provisional application number 14/416,633, filed January 22, 2015, now U.S. Patent No. 9,850,507.
Claim Objections
Claims 1, 40, and 42 are objected to because of the following informalities:
Claim 1 is objected to because of the recitation of the abbreviations “TCA” and “NAD(P)+” and in the interest of improving claim form, it is suggested that the entire phrase for which each of the abbreviations is used be recited at least once in the claims, e.g., tricarboxylic acid for TCA and nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide diphosphate for NAD(P)+.
Claim 40 is objected to in the recitation of “cells” in line 2 and in the interest of improving grammar, should be replaced with “cell.”
Claim 42 is objected to in the recitation of “Pichia sp. YB-4149 (NRRL designation)” and in the interest of improving claim form, it is suggested the noted phrase be amended to recite “Pichia sp. with NRRL designation YB-4149.”
Claim Rejections - 35 USC § 112, Second Paragraph
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1 and 39-54 are rejected under 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
Claim 1 (claims 39-54 dependent therefrom) is indefinite in the recitation of “one or more of” in line 3 because it is unclear as to intended interpretation of the noted phrase. In this case, the “one or more of” can be interpreted as meaning the claim requires one or more copies of all of the genes recited as (i), (ii), (iii), and (iv) or, alternatively, the noted phrase can be interpreted as meaning the claim requires any one or a combination of (i), (ii), (iii), and (iv). If the applicant intends for the phrase “one or more of” to be interpreted as meaning the claim requires any one or a combination of (i), (ii), (iii), and (iv), it is suggested that the phrase "one or more of" be amended to recite "an exogenous gene selected from the group consisting of…and combinations thereof”.
Claims 40-50 and 54 recite the limitation "the host cell.” There is insufficient antecedent basis for this limitation in the claim. If applicant intends for “the host cell” to refer to the “recombinant yeast cell,” applicant may consider replacing “host” with “recombinant.”
Claims 43-50 are confusing in the recitation of “The recombinant cell…as a wild-type strain” because it is unclear as to how the recombinant yeast cell of claims 43-50, which is genetically modified as recited in claim 1, can simultaneously also be a wild-type strain. It is suggested that applicant clarify the meaning of the claims.
Claim Rejections - 35 USC § 103
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
Claims 1 and 39-54 are rejected under 35 U.S.C. 103(a) as being unpatentable over Verwaal et al. (WO 2009/065778 A1; cited on the IDS filed October 30, 2023; hereafter “Verwaal”) in view of
Molina, A. M. (“Design and Implementation of Metabolic Networks for the Improvement of Product Yields in Cofactor-Limiting Systems in Escherichia coli”, Doctoral Dissertation, University of Texas, Houston, 2005; cited on the IDS filed October 30, 2023; hereafter “Molina”),
Burgaard et al. (US 2011/0207189 A1; cited on the IDS filed October 30, 2023; hereafter “Burgaard”), and
Buelter et al. (WO 2010/051527 A2; cited on the IDS filed October 30, 2023; hereafter “Buelter”).
The claims are drawn to a recombinant yeast cell which has an active reductive TCA pathway from pyruvate or phosphoenolpyruvate to succinate and overexpresses a NAD(P)+ transhydrogenase enzyme and which further has integrated into its genome one or more of (i) an exogenous pyruvate carboxylase gene that encodes for an enzyme which catalyzes the conversion of pyruvate to oxaloacetate, (ii) an exogenous malate dehydrogenase gene which encodes for an enzyme that catalyzes the conversion of oxaloacetate to malate (iii) an exogenous fumarase gene that encodes for an enzyme which catalyzes the conversion of malate to fumarate and (iv) an exogenous fumarate reductase gene which encodes for an enzyme which catalyzes the conversion of fumarate to succinate.
Regarding claim 1, the reference of Verwaal teaches a yeast cell for producing succinate (p. 1, lines 29-34), comprising a polynucleotide encoding an NAD(H)-dependent fumarate reductase, which catalyzes the conversion of fumaric acid to succinate (p. 1, lines 31-34), and further comprises a polynucleotide encoding pyruvate carboxylase or phosphoenolpyruvate reductase, which catalyzes the conversion of pyruvate or phosphoenolpyruvate to oxaloacetate (p. 8, lines 24-33; p. 9, lines 3-17), a polynucleotide encoding a malate dehydrogenase, which catalyzes the conversion of oxaloacetate to malate (paragraph bridging pp. 8-9; p. 10, lines 5-22), and a polynucleotide encoding a fumarase, which catalyzes the conversion of malate to fumarate (p. 9, lines 1-2; p. 10, lines 23-26). Verwaal discloses each nucleic acid construct may be integrated into the cell's genome (p. 7, lines 13-15).
The differences between Verwaal and claim 1 are:
1) Verwaal does not teach an active reductive TCA pathway from pyruvate or phosphoenolpyruvate to succinate; and
2) Verwaal does not teach overexpressing an NAD(P)+ transhydrogenase enzyme.
Regarding difference 1), Verwaal teaches fermentative production of succinate (p. 1, lines 13-28), and Molina teaches succinate formation through the reductive branch of the TCA cycle is also known as the fermentative pathway (p. 114, middle). As such, one would have recognized that Verwaal’s teaching of fermentative production of succinic acid from pyruvate or phosphoenolpyruvate to succinic acid is the same as an active reductive TCA pathway from pyruvate or phosphoenolpyruvate to succinate.
Regarding difference 2), at the time of the invention, it was well-known in the prior art that maximum product yield in an engineered metabolic pathway, including a reductive TCA pathway, can limited by insufficient reducing co-factor NADH. For example, Burgaard teaches that in numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient or loss of reducing equivalents (paragraph 38). With regard to reductive TCA cycle, Molina teaches that a major obstacle to high succinate yield through the reductive TCA cycle is due to NADH limitation (p. 114, bottom). According to Molina, the succinate yield can be improved by increasing NADH availability, which can be accomplished by supplying NADH by, e.g., NADH generating pathways (p. 115, middle). Buelter teaches yeasts do not have transhydrogenases and the heterologous expression of a bacterial transhydrogenase in yeast can be used to provide cofactor balance (paragraph 317) and teaches using a transhydrogenase that catalyzes the conversion of NADPH to NADH under conditions in which the reduced cofactor NADH is limiting (paragraph 282). Buelter teaches the E. coli sthA gene encodes a soluble transhydrogenase and has been shown to convert NADPH to NADH (paragraph 288).
In view of Verwaal, Burgaard, Molina, and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention to modify the recombinant yeast cell of Verwaal to overexpress a NAD(P)+ transhydrogenase enzyme. One would have been motivated to modify the recombinant yeast cell of Verwaal to overexpress a NADP+ transhydrogenase enzyme because Molina teaches succinate production by reductive TCA cycle can be increased by an NADH generating pathway and Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production. One would have had a reasonable expectation of success to modify the recombinant yeast cell of Verwaal to overexpress a NADP+ transhydrogenase enzyme because Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production and Verwaal, Buelter, and Molina each teaches recombinant methods for overexpressing enzymes in a microorganism.
Regarding claim 39, Verwaal discloses a pyruvate decarboxylase negative yeast for production of succinic acid (p. 1, lines 22-25).
Regarding claims 40 and 41, Verwaal discloses the yeast is Saccharomyces cerevisiae (p. 8, lines 14-19).
Regarding claims 42 and 54, at the time of the invention, it was known in the prior art that Issatchenkia orientalis is alternatively referred to as Candida krusei. Verwaal discloses the yeast belongs to the genus Candida (p. 8, line 14-16) and Buelter acknowledges that Issatchenkia orientalis is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (Abstract; paragraph [0047]).
In view of Verwaal and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention for the recombinant yeast cell of Verwaal to be Issatchenkia orientalis. One would have been motivated and would have expected success because Verwaal taught the yeast belongs to the genus Candida, Issatchenkia orientalis is alternatively referred to as Candida krusei, and Buelter acknowledges that Issatchenkia orientalis is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product.
Regarding claim 43, Buelter acknowledges that a crabtree-negative yeast is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (paragraph [0047]).
Regarding claims 44-50, the combination of Verwaal, Burgaard, Molina, and Buelter does not teach the properties recited in claims 44-50. However, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 44-50 have been included in the instant rejection. Also, given that the structure of the recombinant yeast cell of Verwaal modified to overexpress a NADP+ transhydrogenase enzyme is encompassed by claims 44-50, it is presumed that the recombinant yeast cell of claim 1 of the patent exhibits the recited properties of instant claims 44-50 (see MPEP 2112.01.I).
Regarding claims 51-53, the combination of Verwaal, Burgaard, Molina, and Buelter does not teach the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of Verwaal modified to overexpress a NADP+ transhydrogenase enzyme is encompassed by claim 1, it is presumed that the recombinant yeast cell of Verwaal modified to overexpress a NADP+ transhydrogenase enzyme exhibits the recited properties of claims 51-53 (see MPEP 2112.01.I).
Therefore, the recombinant yeast cell of claims 1 and 39-54 would have been obvious to one of ordinary skill in the art at the time of the invention.
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.
Claims 1 and 39-54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 8, 9, and 12 of U.S. Patent No. 9,850,507 B2 (cited on the IDS filed on October 20, 2023). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claim 1, claim 1 of the patent recites a recombinant yeast cell engineered to produce succinate through an active reductive tricarboxylic acid (TCA) pathway from pyruvate or phosphoenolpyruvate to succinate, wherein the recombinant yeast cell is modified from a parent yeast cell by having integrated into its genome an exogenous gene encoding a soluble nicotinamide adenine dinucleotide phosphate (NAD(P)+) transhydrogenase enzyme, wherein the soluble NAD(P)+transhydrogenase enzyme is expressed in the cytosol of the recombinant yeast cell, wherein the recombinant yeast cell is further modified from the parent yeast cell by having integrated into its genome at least one of:
(i) an exogenous pyruvate carboxylase gene that encodes an enzyme which catalyzes the conversion of pyruvate to oxaloacetate;
(ii) an exogenous malate dehydrogenase gene which encodes an enzyme that catalyzes the conversion of oxaloacetate to malate;
(iii) an exogenous fumarase gene that encodes an enzyme which catalyzes the conversion of malate to fumarate; and
(iv) an exogenous fumarate reductase gene which encodes an enzyme which catalyzes the conversion of fumarate to succinate, and wherein the recombinant yeast cell produces more succinate through the active reductive TCA pathway as compared to the parent cell.
Regarding instant claim 39, claim 8 of the patent recites the recombinant yeast cell of claim 1, wherein the recombinant yeast cell is further modified from the parent yeast cell by having a deletion or disruption of a native pyruvate decarboxylase gene.
Regarding instant claims 40-42 and 54, claim 9 of the patent recites the recombinant yeast cell of claim 1, wherein the recombinant yeast cell is a yeast of the species Issatchenkia orientalis.
Regarding instant claims 43-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 43-50 have been included in the instant rejection. Also, the claims of the patent do not recite the properties recited in claims 43-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent is encompassed by claims 43-50, it is presumed that the recombinant yeast cell of claim 1 of the patent exhibits the recited properties of instant claims 43-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, claim 12 of the patent recites (in relevant part) the recombinant yeast cell of claim 1, wherein the recombinant yeast cell exhibits a volumetric glucose consumption rate of at least 0.9 gram of glucose per liter of broth per hour…
Therefore, claims 1 and 39-54 of this application are unpatentable over claims 1, 8, 9, and 12 of the patent.
Claims 1 and 43-53 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,041,176 B2 (cited on Form PTO-892). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claim 1, claim 1 of the patent recites a fermentation broth comprising at least one carbon source and recombinant yeast cells engineered to produce succinate through an active reductive tricarboxylic acid (TCA) pathway from pyruvate or phosphoenolpyruvate, wherein the recombinant yeast cells are genetically modified to express a soluble nicotinamide adenine dinucleotide phosphate (NAD(P)+) transhydrogenase enzyme in the cytosol of the yeast cells by having integrated into their genomes an exogenous gene encoding the soluble NAD(P)+ transhydrogenase enzyme, wherein the recombinant yeast cells are further modified by having integrated into their genomes at least one of:
(i) an exogenous pyruvate carboxylase gene that encodes an enzyme which catalyzes the conversion of pyruvate to oxaloacetate;
(ii) an exogenous malate dehydrogenase gene which encodes an enzyme that catalyzes the conversion of oxaloacetate to malate;
(iii) an exogenous fumarase gene that encodes an enzyme which catalyzes the conversion of malate to fumarate; and
(iv) an exogenous fumarate reductase gene which encodes an enzyme which catalyzes the conversion of fumarate to succinate, and
wherein the recombinant yeast cells produce more succinate through the active reductive TCA pathway as compared to a corresponding parent yeast cell lacking the soluble NAD(P)+ transhydrogenase enzyme.
Regarding instant claims 43-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 43-50 have been included in the instant rejection. Also, the claim of the patent does not recite the properties recited in claims 43-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent is encompassed by claims 43-50, it is presumed that the recombinant yeast cell of claim 1 of the patent exhibits the recited properties of instant claims 43-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, the claim of the patent does not recite the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of claim 1 of the patent is encompassed by claims 51-53, it is presumed that the recombinant yeast cell of claim 1 of the patent exhibits the recited properties of instant claims 51-53 (see MPEP 2112.01.I).
Therefore, claims 1 and 43-53 of this application are unpatentable over claim 1 of the patent.
Claims 39-42 and 54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 11,041,176 B2 (cited on Form PTO-892) in view of Verwaal and Buelter.
Claim 1 of the patent does not recite the limitations of instant claims 39-42 and 54.
Regarding instant claim 39, Verwaal discloses a pyruvate decarboxylase negative yeast for production of succinic acid (p. 1, lines 22-25).
Regarding instant claims 40 and 41, Verwaal discloses the yeast is Saccharomyces cerevisiae (p. 8, lines 14-19).
Regarding instant claims 42 and 54, at the time of the invention, it was known in the prior art that Issatchenkia orientalis is alternatively referred to as Candida krusei. Verwaal discloses the yeast belongs to the genus Candida (p. 8, line 14-16) and the reference of Buelter acknowledges that Issatchenkia orientalis is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (Abstract; paragraph [0047]).
In view of Verwaal and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention for the recombinant yeast cell of claim 1 of the patent to be a pyruvate decarboxylase negative yeast, Saccharomyces cerevisiae, or Issatchenkia orientalis. One would have been motivated and would have expected success for the recombinant yeast cell of claim 1 of the patent to be a pyruvate decarboxylase negative yeast, Saccharomyces cerevisiae, or Issatchenkia orientalis because Verwaal and Buelter acknowledge that a pyruvate decarboxylase negative yeast, Saccharomyces cerevisiae, or Issatchenkia orientalis is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product.
Therefore, claims 39-42 and 54 of this application are unpatentable over claim 1 of the patent in view of Verwaal and Buelter.
Claims 1 and 39-54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 12, and 15 of U.S. Patent No. 11,821,021 B2 (cited on the attached Form PTO-892). Although the claims at issue are not identical, they are not patentably distinct from each other.
Regarding instant claim 1, claim 1 of the patent recites a process for producing succinate, comprising culturing a recombinant yeast cell under fermentation conditions in a fermentation broth that includes a sugar that is fermentable by the cell, wherein the recombinant yeast cell has an active reductive tricarboxylic acid (TCA) pathway from pyruvate or phosphoenolpyruvate to succinate,
wherein the recombinant yeast cell is modified from a parent yeast cell by having integrated into its genome an exogenous gene encoding a soluble nicotinamide adenine dinucleotide phosphate (NAD(P)+) transhydrogenase enzyme, wherein the soluble NAD(P)+ transhydrogenase enzyme is expressed in the cytosol of the recombinant yeast cell,
wherein the recombinant yeast cell is further modified from the parent yeast cell by having integrated into its genome an exogenous gene selected from the group consisting of:
(i) an exogenous pyruvate carboxylase gene that encodes an enzyme which catalyzes the conversion of pyruvate to oxaloacetate,
(ii) an exogenous malate dehydrogenase gene which encodes an enzyme that catalyzes the conversion of oxaloacetate to malate,
(iii) an exogenous fumarase gene that encodes an enzyme which catalyzes the conversion of malate to fumarate,
(iv) an exogenous fumarate reductase gene which encodes an enzyme which catalyzes the conversion of fumarate to succinate, and
(v) combinations thereof,
wherein the recombinant yeast cell produces more succinate through the active reductive TCA pathway as compared to the parent cell.
Regarding instant claim 39, claim 12 of the patent recites wherein the recombinant yeast cell has a deletion or disruption of a native pyruvate decarboxylase gene.
Regarding instant claims 40-42 and 54, claim 15 of the patent recites wherein the recombinant yeast cell is Issatchenkia orientalis.
Regarding instant claims 43-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 43-50 have been included in the instant rejection. Also, the claim of the patent does not recite the properties recited in claims 43-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent is encompassed by claims 43-50, it is presumed that the recombinant yeast cell of claim 1 of the patent exhibits the recited properties of instant claims 43-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, the claim of the patent does not recite the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of claim 1 of the patent is encompassed by claims 51-53, it is presumed that the recombinant yeast cell of claim 1 of the patent exhibits the recited properties of instant claims 51-53 (see MPEP 2112.01.I).
Therefore, claims 1 and 39-54 of this application are unpatentable over claims 1, 12, and 15 of the patent.
Claims 1 and 39-54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 14, 17, and 18 of U.S. Patent No. 9,605,285 B2 (cited on the IDS filed October 30, 2023) in view of Molina, Burgaard, and Buelter.
Regarding instant claim 1, claim 1 of the patent recites a genetically modified yeast cell having an active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate, wherein the active succinate fermentation pathway comprises an exogenous malate dehydrogenase gene encoding a polypeptide comprising an amino acid sequence with at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:172.
Claim 1 of the patent does not recite overexpressing an NAD(P)+ transhydrogenase enzyme.
At the time of the invention, it was well-known in the prior art that maximum product yield in an engineered metabolic pathway, including a reductive TCA pathway, can limited by insufficient reducing co-factor NADH. For example, Burgaard teaches that in numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient or loss of reducing equivalents (paragraph 38). With regard to reductive TCA cycle, Molina teaches that a major obstacle to high succinate yield through the reductive TCA cycle is due to NADH limitation (p. 114, bottom). According to Molina, the succinate yield can be improved by increasing NADH availability, which can be accomplished by supplying NADH by, e.g., NADH generating pathways (p. 115, middle). Buelter teaches yeasts do not have transhydrogenases and the heterologous expression of a bacterial transhydrogenase in yeast can be used to provide cofactor balance (paragraph 317) and teaches using a transhydrogenase that catalyzes the conversion of NADPH to NADH under conditions in which the reduced cofactor NADH is limiting (paragraph 282). Buelter teaches the E. coli sthA gene encodes a soluble transhydrogenase and has been shown to convert NADPH to NADH (paragraph 288).
In view of Burgaard, Molina, and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention to modify the recombinant yeast cell of claim 1 of the patent to overexpress a NAD(P)+ transhydrogenase enzyme. One would have been motivated to modify the recombinant yeast cell of claim 1 of the patent to overexpress a NADP+ transhydrogenase enzyme because Molina teaches succinate production by reductive TCA cycle can be increased by an NADH generating pathway and Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production. One would have had a reasonable expectation of success to modify the recombinant yeast cell of claim 1 of the patent to overexpress a NADP+ transhydrogenase enzyme because Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production and Buelter and Molina each teaches recombinant methods for overexpressing enzymes in a microorganism.
Regarding instant claim 39, claim 14 of the patent recites wherein the cell comprises a deletion or disruption of a native pyruvate decarboxylase gene.
Regarding instant claims 40-42 and 54, claim 17 of the patent recites wherein the yeast cell comprises the species Issatchenkia orientalis.
Regarding instant claim 43, Buelter acknowledges that a crabtree-negative yeast is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (paragraph [0047]).
Regarding instant claim 44, claim 18 of the patent recites wherein the yeast cell is succinate resistant.
Regarding instant claims 45-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 45-50 have been included in the instant rejection. Also, the claim of the patent does not recite the properties recited in claims 45-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 45-50, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 45-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, the claim of the patent does not recite the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 51-53, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 51-53 (see MPEP 2112.01.I).
Therefore, claims 1 and 39-54 of this application are unpatentable over claims 1, 14, 17, and 18 of the patent in view of Burgaard, Molina, and Buelter.
Claims 1 and 39-54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 2, and 18 of U.S. Patent No. 9,885,065 B2 (cited on the IDS filed October 30, 2023) in view of Molina, Burgaard, and Buelter.
Regarding instant claim 1, claim 1 of the patent recites a method of producing a succinate, succinic acid, or succinate/succinic acid-containing fermentation broth, the method comprising:
(i) culturing in a fermenter a recombinant yeast in the presence of at least one carbon source to produce succinate, wherein the recombinant yeast is genetically engineered to produce succinate through a reductive tricarboxylic acid (TCA) active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate, wherein the active succinate fermentation pathway comprises the reactions:
(a) pyruvate to oxaloacetate;
(b) oxaloacetate to malate;
(c) malate to fumarate;
(d) fumarate to succinate; and
(e) export of succinate from inside the cell to the extracellular environment, and
(ii) introducing into the fermenter sufficient oxygen to provide an oxygen uptake rate (OUR) of greater than 8 mmol/L/h,
wherein the recombinant yeast produces more succinate when cultured under an OUR of greater than 8 mmol/L/h as compared to when the recombinant yeast is cultured at an OUR of 5 mmol/L/h, and
wherein a final concentration of succinate, succinic acid, or succinate/succinic acid in the fermentation broth is greater than 20 g/L.
Claim 1 of the patent does not recite overexpressing an NAD(P)+ transhydrogenase enzyme.
At the time of the invention, it was well-known in the prior art that maximum product yield in an engineered metabolic pathway, including a reductive TCA pathway, can limited by insufficient reducing co-factor NADH. For example, Burgaard teaches that in numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient or loss of reducing equivalents (paragraph 38). With regard to reductive TCA cycle, Molina teaches that a major obstacle to high succinate yield through the reductive TCA cycle is due to NADH limitation (p. 114, bottom). According to Molina, the succinate yield can be improved by increasing NADH availability, which can be accomplished by supplying NADH by, e.g., NADH generating pathways (p. 115, middle). Buelter teaches yeasts do not have transhydrogenases and the heterologous expression of a bacterial transhydrogenase in yeast can be used to provide cofactor balance (paragraph 317) and teaches using a transhydrogenase that catalyzes the conversion of NADPH to NADH under conditions in which the reduced cofactor NADH is limiting (paragraph 282). Buelter teaches the E. coli sthA gene encodes a soluble transhydrogenase and has been shown to convert NADPH to NADH (paragraph 288).
In view of Burgaard, Molina, and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention to modify the recombinant yeast cell of claim 1 of the patent to overexpress a NAD(P)+ transhydrogenase enzyme. One would have been motivated to modify the recombinant yeast cell of the claims of the patent to overexpress a NADP+ transhydrogenase enzyme because Molina teaches succinate production by reductive TCA cycle can be increased by an NADH generating pathway and Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production. One would have had a reasonable expectation of success to modify the recombinant yeast cell of the claims of the patent to overexpress a NADP+ transhydrogenase enzyme because Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production and Buelter and Molina each teaches recombinant methods for overexpressing enzymes in a microorganism.
Regarding instant claim 39, claim 18 of the patent recites wherein the cell comprises a deletion or disruption of a native pyruvate decarboxylase gene.
Regarding instant claims 40-42 and 54, claim 2 of the patent recites wherein the yeast cell of the species Issatchenkia orientalis.
Regarding instant claim 43, Buelter acknowledges that a crabtree-negative yeast is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (paragraph [0047]).
Regarding instant claims 44-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 44-50 have been included in the instant rejection. Also, the claims of the patent do not recite the properties recited in claims 44-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 44-50, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 44-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, the claims of the patent do not recite the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 51-53, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 51-53 (see MPEP 2112.01.I).
Therefore, claims 1 and 39-54 of this application are unpatentable over claims 1, 2, and 18 of the patent in view of Burgaard, Molina, and Buelter.
Claims 1 and 39-54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10,066,246 B2 (cited on the IDS filed on October 30, 2023) in view of Verwaal, Molina, Burgaard, and Buelter.
Regarding instant claims 1, 40-42, and 54, claim 1 of the patent recites a recombinant I. orientalis cell having an active reductive TCA pathway from pyruvate to succinate and which further metabolizes succinate to one or more succinate metabolization products, which reductive TCA pathway includes a reaction that oxidizes NADPH to NADP+ comprising a conversion of fumarate to succinate catalyzed by an NADPH-dependent fumarate reductase enzyme and the recombinant cell has integrated into its genome an heterologous fumarate reductase gene that encodes for the NADPH-dependent fumarate reductase enzyme.
Claim 1 of the patent does not recite overexpressing an NAD(P)+ transhydrogenase enzyme.
At the time of the invention, it was well-known in the prior art that maximum product yield in an engineered metabolic pathway, including a reductive TCA pathway, can limited by insufficient reducing co-factor NADH. For example, Burgaard teaches that in numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient or loss of reducing equivalents (paragraph 38). With regard to reductive TCA cycle, Molina teaches that a major obstacle to high succinate yield through the reductive TCA cycle is due to NADH limitation (p. 114, bottom). According to Molina, the succinate yield can be improved by increasing NADH availability, which can be accomplished by supplying NADH by, e.g., NADH generating pathways (p. 115, middle). Buelter teaches yeasts do not have transhydrogenases and the heterologous expression of a bacterial transhydrogenase in yeast can be used to provide cofactor balance (paragraph 317) and teaches using a transhydrogenase that catalyzes the conversion of NADPH to NADH under conditions in which the reduced cofactor NADH is limiting (paragraph 282). Buelter teaches the E. coli sthA gene encodes a soluble transhydrogenase and has been shown to convert NADPH to NADH (paragraph 288).
In view of Burgaard, Molina, and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention to modify the recombinant cell of claim 1 of the patent to overexpress a NAD(P)+ transhydrogenase enzyme. One would have been motivated to modify the recombinant cell of the patent to overexpress a NADP+ transhydrogenase enzyme because Molina teaches succinate production by reductive TCA cycle can be increased by an NADH generating pathway and Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production. One would have had a reasonable expectation of success to modify the recombinant cell of the patent to overexpress a NADP+ transhydrogenase enzyme because Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production and Buelter and Molina each teaches recombinant methods for overexpressing enzymes in a microorganism.
Regarding instant claim 39, Verwaal discloses a pyruvate decarboxylase negative yeast for production of succinic acid (p. 1, lines 22-25).
Regarding instant claim 43, Buelter acknowledges that a crabtree-negative yeast is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (paragraph [0047]).
Regarding instant claims 44-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 44-50 have been included in the instant rejection. Also, the claims of the patent do not recite the properties recited in claims 44-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 44-50, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 44-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, the claims of the patent do not recite the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 51-53, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 51-53 (see MPEP 2112.01.I).
Therefore, claims 1 and 39-54 of this application are unpatentable over claim 1 of the patent in view of Verwaal, Burgaard, Molina, and Buelter.
Claims 1 and 39-54 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 and 15 of U.S. Patent No. 11,390,873 B2 (cited on the IDS filed October 30, 2023) in view of Verwaal, Molina, Burgaard, and Buelter.
Regarding instant claims 1, 40-42, and 54, claim 1 of the patent recites a method of producing succinate comprising culturing genetically modified yeast cells in the presence of at least one carbon source and isolating succinate from the culture, wherein the genetically modified yeast cells are from the Pichia fermentans/Issatchenkia orientalis clade and have an active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate, wherein the active succinate fermentation pathway comprises the reactions:
(a) pyruvate to oxaloacetate;
(b) oxaloacetate to malate;
(c) malate to fumarate; and
(d) fumarate to succinate,
wherein the cells comprise an exogenous succinate exporter gene that catalyzes export of succinate from inside the cell to the extracellular environment.
Claim 1 of the patent does not recite overexpressing an NAD(P)+ transhydrogenase enzyme.
At the time of the invention, it was well-known in the prior art that maximum product yield in an engineered metabolic pathway, including a reductive TCA pathway, can limited by insufficient reducing co-factor NADH. For example, Burgaard teaches that in numerous engineered pathways, realization of maximum product yields based on carbohydrate feedstock is hampered by insufficient or loss of reducing equivalents (paragraph 38). With regard to reductive TCA cycle, Molina teaches that a major obstacle to high succinate yield through the reductive TCA cycle is due to NADH limitation (p. 114, bottom). According to Molina, the succinate yield can be improved by increasing NADH availability, which can be accomplished by supplying NADH by, e.g., NADH generating pathways (p. 115, middle). Buelter teaches yeasts do not have transhydrogenases and the heterologous expression of a bacterial transhydrogenase in yeast can be used to provide cofactor balance (paragraph 317) and teaches using a transhydrogenase that catalyzes the conversion of NADPH to NADH under conditions in which the reduced cofactor NADH is limiting (paragraph 282). Buelter teaches the E. coli sthA gene encodes a soluble transhydrogenase and has been shown to convert NADPH to NADH (paragraph 288).
In view of Burgaard, Molina, and Buelter, it would have been obvious to one of ordinary skill in the art at the time of the invention to modify the recombinant cell of claim 1 of the patent to overexpress a NAD(P)+ transhydrogenase enzyme. One would have been motivated to modify the recombinant cell of the patent to overexpress a NADP+ transhydrogenase enzyme because Molina teaches succinate production by reductive TCA cycle can be increased by an NADH generating pathway and Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production. One would have had a reasonable expectation of success to modify the recombinant cell of the patent to overexpress a NADP+ transhydrogenase enzyme because Buelter teaches expressing a NAD(P)+ transhydrogenase in yeast as an NADH generating pathway to thereby increase NADH production and Buelter and Molina each teaches recombinant methods for overexpressing enzymes in a microorganism.
Regarding instant claim 39, claim 15 of the patent recites wherein the cell comprises a deletion or disruption of an endogenous pyruvate decarboxylase gene.
Regarding instant claim 43, Buelter acknowledges that a crabtree-negative yeast is a suitable yeast for genetically engineering a metabolic pathway to produce a desired product (paragraph [0047]).
Regarding instant claims 44-50, in view of the indefiniteness of the phrase “The recombinant cell…as a wild-type strain” and the lack of antecedent basis for “the host cell,” claims 44-50 have been included in the instant rejection. Also, the claims of the patent do not recite the properties recited in claims 44-50. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 44-50, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 44-50 (see MPEP 2112.01.I).
Regarding instant claims 51-53, the claims of the patent do not recite the properties recited in claims 51-53. However, given that the structure of the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme is encompassed by claims 51-53, it is presumed that the recombinant yeast cell of claim 1 of the patent modified to overexpress a NAD(P)+ transhydrogenase enzyme exhibits the recited properties of instant claims 51-53 (see MPEP 2112.01.I).
Therefore, claims 1 and 39-54 of this application are unpatentable over claims 1 and 15 of the patent in view of Verwaal, Burgaard, Molina, and Buelter.
Claims 1 and 39-54 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 21, 27, 38, and 39 of co-pending application 17/812,875 (reference application) in view of Verwaal, Molina, Burgaard, and Buelter.
Regarding instant claims 1 and 40, claim 21 of the reference application recites a genetically modified yeast cell of the genus Issatchenkia or Candida having an active succinate fermentation pathway from phosphoenolpy