DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 12/18/2025 has been entered.
Withdrawal of Rejections
The response and amendments filed on 11/13/2025 are acknowledged. Any previously applied minor objections and/or minor rejections (i.e., formal matters), not explicitly restated here for brevity, have been withdrawn necessitated by Applicant’s formality correction and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, and/or maintaining, if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner’s Response to Arguments section.
Briefly, the previous claim rejections under 35 U.S.C. 112(b) for indefiniteness have been withdrawn necessitated by Applicant’s amendments; however, new grounds of rejection are set forth below.
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
Claim Objections
Claim 23 is objected to because of the following informality: the gene names for ilvA and ilvE should be italicized. This is an objection, not a rejection, because this appears to be a typographical error. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b), Indefiniteness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 27, 31, 35-38, and 42-43 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 (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 27 recites b8) and b9); however, it is unclear after these limitations if the limitations pertaining to the genes encoding the bkdA, bkdB, and bkdC proteins are required because there is no and/or linking phrase between b9) and the recitation of “the gene encoding the bkdA protein is b11)…”. For the purposes of applying prior art, the Examiner has interpreted the limitations pertaining to the genes encoding the bkdA, bkdB, and bkdC proteins to be required. Furthermore, since the Examiner has interpreted these limitations as being required, it is further unclear if the limitations reciting “the gene encoding the bkdA protein is b11)”… and “the gene encoding the bkdB protein is…” to both be required because there is no and/or linking phrase. The Examiner has interpreted both of these limitations to be required because Applicant has recited “and” between the bkdB and bkdC sequence limitations.
Claim 31 depends on cancelled claim 30. For the purposes of applying prior art, the Examiner has made claim 31 dependent on independent claim 23.
Claim 31 recites amino acid and nucleotide sequences for ilvA and ilvE; however, it is unclear if all of these SEQ ID’s are required, or if just the sequences for ilvA (i.e., SEQ ID NOs: 14 and 15), and just the sequences for ilvE (i.e., SEQ ID NOs: 16 and 17) are required. This is unclear because there is no and/or linking phrase between these sequences. For the purposes of applying prior art, the Examiner has interpreted all the SEQ ID’s to be required because the ilvA and/or ilvE genes can be knocked out in the biological cell strain.
Claim 35 recites a “use” claim in step A1, which attempts to claim a process without setting forth any active steps involved in the process, which renders the claim indefinite (see, e.g., MPEP 2173.05(q)).
Claim 35 recites method A2 and A3; however, it is unclear if methods A2 and A3 are required, or if only one is required (i.e., method A2 or method A3). This is unclear because there is not and/or linking phrase between methods A2 and A3. For the purposes of applying prior art, the Examiner has interpreted only one (i.e., method A2 or method A3) to be required because Applicant has used the “or” linking phrase between method A1 and A2.
Claim 36 recites “(pH=7.0)” in parenthesis; however, it is unclear if what is recited in parenthesis is part of the claimed invention, or a preferred embodiment. For the purposes of applying prior art, the Examiner has interpreted the pH recited in parenthesis to be a preferred embodiment.
Claim 43 recites “the gene encoding the lpdA1 protein is the following b8) or b9)”; but then goes on to recite b8 and b9; therefore, it is unclear if either b8 or b9 is required, or if b8 and b9 are both required. For the purposes of applying prior art, the Examiner has interpreted b8 and b9 to be required.
Claims 37-38 and 42 are included in this rejection for depending on either rejected claim 35 or rejected claim 36, and these claims fail to rectify the noted deficiencies.
Examiner’s Response to Arguments
Regarding Applicant’s arguments pertaining to the 35 U.S.C. 112(b) rejections for indefiniteness (remarks, page 13), as stated above, all previous 112(b) rejection have been withdrawn necessitated by Applicant’s amendments; however, new grounds of rejection are set forth above.
Claim Rejections - 35 USC § 112(a), New Matter
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 23, 26-27, 31-38, and 42-43 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 23 has been amended to recite “wherein the recombinant cell strain remains viable in the absence of exogenously added branched-chain amino acids”; however, there is no support pertaining to the viability of the recombinant cell strain in the absence of exogenously added branched-chain amino acids. Moreover, the absence of exogenously added branched-chain amino acids must be positively recited in the originally-filed disclosure in order to be supported (see, e.g., MPEP 2173.05(i)).
Claims 26-27, 31-38, and 42-43 are included in this rejection for depending on independent claim 23 and failing to rectify the noted deficiency.
Claim Rejections - 35 USC § 101, Non-Statutory Subject Matter
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 35 is rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because “’Use’ claims that do not purport to claim a process, machine, manufacture, or composition of matter fail to comply with 35 U.S.C.101” (see, e.g., MPEP 2173.05(q)).
Broadest reasonable interpretation (BRI) of claim 35, step A1: the broadest scope of claim 35, step A1 is a branched-chain α-keto acid dehydrogenase complex that produces malonyl-CoA from oxaloacetate.
STEP 1: Is the claim directed to a process, machine, manufacture, or composition of matter?
NO, claim 35, step A1 is a “use” claim that does not set forth any active steps; therefore, it is unclear if this claim is a process (i.e., method) or composition claim. Therefore, this claim does not purport to claim a process, machine, manufacture, or composition of matter and fail to comply with 35 U.S.C. 101.
Claim Rejections - 35 USC § 103, Obviousness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 23, 26, and 34-36 are rejected under 35 U.S.C. 103 as being unpatentable over Cui (Avermectin biosynthesis: stable functional expression of branched chain α-keto acid dehydrogenase complex from Streptomyces avermitilis in Escherichia coli by selectively regulating individual subunit gene expression; 2017 – cited in the IDS filed on 02/14/2022 – previously cited) in view of Hu (US 2011/0244532; Date of Publication: October 6, 2011 – previously cited), Falb (WO 2016/201380; Date of Publication: December 15, 2016 – previously cited), and Franco (Bacterial Branched-Chain Amino Acid Biosynthesis: Structures, Mechanisms, and Drugability; 2018 – newly cited).
Cui’s general disclosure relates to improving “the production of short branched-chain acyl-coAs for avermectin biosynthesis” by selectively regulating expression of individual branched chain α-keto acid dehydrogenase complex subunit components in Escherichia coli (see, e.g., Cui, abstract).
Regarding claim 23 pertaining to introducing the branched chain α-keto acid dehydrogenase complex and culturing the microbial cell strain, Cui teaches the production of a recombinant E. coli cell that is capable of expressing the gene encoding the branched chain α-keto acid dehydrogenase complex (see, e.g., Cui, Methods, “Cultivation of recombinant E. coli strains and expression analysis, pg. 1568 & Supplemental Table 1). Additionally, Cui teaches the expression of lpdA1 from Streptomyces avermitilis in E. coli cells (see, e.g., Cui, abstract & Results and Discussion, pg. 1569). Moreover, Cui teaches that the recombinant E. coli cell strain expressing the branched chain α-keto acid dehydrogenase complex from S. avermitilis is viable in the absence of exogenously added branched-chain amino acids because Cui does not teach the addition of branched chain amino acids for this recombinant cell strain in order to maintain viability. Furthermore, Cui teaches that the recombinant E. coli cell strain is viable without the addition of exogenous branched-chain amino acids because Cui teaches culturing and expression of the E. coli recombinant cell (see, e.g., Cui, Methods – “Cultivation of recombinant E. coli strains and expression analysis & “Extraction of intracellular CoA-esters and PIVP”, pgs. 2-3) and these steps do not involve the addition of exogenous branched chain amino acids.
Regarding claim 34 pertaining to the biological cell strain, Cui teaches that “E. coli BW25113 was used as the parental strain for branched chain α-keto acid dehydrogenase (BKDH) complex gene expression” (see, e.g., Cui, “Bacterial strains and plasmids”, pg. 1568).
Regarding claim 35 pertaining to method A2, Cui teaches the synthesis of phlorisovalerophenone (PIVP) as the target product from generation of short branched-chain acyl-CoAs, such as malonyl-CoA, as an intermediate product (see, e.g., Cui, Introduction, 1568).
Regarding claim 36 pertaining to the catalytic reaction, Cui teaches that the bacteria expressing the bkdF, bkdG, bkdH, and lpdA1 proteins are cultured at 37oC (see, e.g., Cui, “Cultivation of recombinant E. coli strains and expression analysis”, pg. 1568). Therefore, since the bacteria are cultured at this temperature with expression of proteins necessary for the production of malonyl-CoA (i.e., bkdD, bkdG, bkdH, and lpdA1) (see, e.g., Cui, Introduction, pg. 1568), one of ordinary skill in the art would understand that the reaction to produce malonyl-CoA occurs at 37oC.
However, Cui does not teach: the bkdA, bkdB, and bkdC genes and proteins from Streptomyces avermitilis (claims 23); or inhibiting the synthesis of branched chain α-keto acids by knocking out an ilvA gene or/and an ilvE gene in the biological cell strain, or reducing the content or the activity of the proteins encoded by the ilvA gene or/and the ilvE gene in the biological cell strain (claim 23); or wherein the lpdA1 protein is a protein having the amino acid sequence shown in SE ID NO: 13; the bkdA protein is a protein having the amino acid sequence show in SEQ ID NO: 7; the bkdB protein is a protein have the amino acid sequence shown in SEQ ID NO: 8; and the bkdC protein is a protein having the amino acid sequence shown in SEQ ID NO: 9 (claim 26).
Hu’s general disclosure relates to “novel recombinant microbial cells that produce branched chain fatty acid derivatives” through the fatty acid biosynthetic pathway (see, e.g., Hu, [0011]).
Regarding claim 23 pertaining to the bkdA, bkdB, and bkdC genes and proteins, Hu teaches bkdA, bkdB, and bkdC genes, which encode teaches bkdA, bkdB, and bkdC proteins, (see, e.g., Hu, [0025]-[0026]). Additionally, the teaches bkdA, bkdB, and bkdC genes are derived from S. avermitilis (see, e.g., Hu, Table 4).
Regarding claim 26 pertaining to the lpdA1 protein, bkdA protein, bkdB protein, and bkdC protein, Hu teaches SEQ ID NO: 65, which has 100% sequence identity to instant SEQ ID NO: 13 and encodes for the lpdA1 protein (see, e.g., Hu, [0027] & Office Action Appendix). Hu teaches SEQ ID NO: 3, which has 100% sequence identity to instant SEQ ID NO: 7 and encodes for the bkdA protein (see, e.g., Hu, [0025] & Office Action Appendix). Hu teaches SEQ ID NO: 24, which has 100% sequence identity instant SEQ ID NO: 8 and encodes for the bkdB protein (see, e.g., Hu, [0025] & Office Action Appendix). Hu teaches SEQ ID NO: 45, which has 100% sequence identity to instant SEQ ID NO: 9 and encodes for the bkdC protein (see, e.g., Hu, [0026] & Office Action Appendix).
Falb’s general disclosure relates to “recombinant bacterial cells that have been engineered with genetic circuitry which allow the recombinant bacterial cells to sense a patient's internal environment and respond by turning an engineered metabolic pathway on or off” (see, e.g., Falb, abstract). Furthermore, Falb disclosures “compositions and therapeutic methods for reducing one or more excess branched chain amino acids, and/or an accumulated metabolite(s) thereof, for example, by converting the one or more excess branched chain amino acid(s) or accumulated metabolite(s) into alternate by product(s)” (see, e.g., Falb, “Summary”, pg. 2).
Regarding claim 23 pertaining to knocking out the ilvA genes, Falb teaches that “the bacterial cell comprises a genetic mutation in one or more endogenous gene(s) encoding a branched chain amino acid biosynthesis gene, wherein the genetic mutation reduces biosynthesis of one or more branched chain amino acids in the bacterial cell” (see, e.g., Falb, [086]). Furthermore, Falb teaches that genes required for cell survival and/or growth may be targeted, such as ilvA (see, e.g., Falb, [087]). Additionally, Falb teaches that the ilvA gene may be disrupted or deleted to product an auxotrophic strain (see, e.g., Falb, [088], & Table 9, pg. 200).
Franco’s general disclosure relates to the eight enzymes responsible for the biosynthesis of the three branched-chain amino acids (L-isoleucine, L-leucine, and L-valine) (see, e.g., Franco, abstract). Moreover, Franco discloses “Four of the eight enzymes are shared for the synthesis of all three BCAAs (IlvB/N; IlvC, IlvD, and IlvE); three are responsible for only the synthesis of L-leucine (LeuA, LeuC/D, and LeuB), and one is specifically involved in L-isoleucine biosynthesis (IlvA)” (see, e.g., Franco, Introduction, pg. 2).
Regarding claim 23 pertaining to knocking out the ilvA gene, Franco teaches “The first committed step in the biosynthesis of L-isoleucine is catalyzed by the ilvA-encoded threonine dehydratase/deaminase (EC 4.3.1.19, TD). This enzyme plays a very important role in the biosynthetic pathway of BCAAs in microorganisms and plants” (see, e.g., Franco, “IlvA, Threonine Dehydratase/Deaminase”, pg. 2). Furthermore, Franco teaches that ilvA is responsible for the conversion of threonine to 2-ketobutyrate (see, e.g., Franco, Graphical Abstract).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Cui’s recombinant E. coli strain expressing lpdA1 from S. avermitilis, wherein the recombinant E. coli strain also expresses bkdA, bkdB, and bkdC, as taught by Hu. One would have been motivated to do so because Hu teaches that bkdA, bkdB, and bkdC are proteins that comprise the branched-chain α-ketoacid dehydrogenase complex for the biosynthesis of branched-chain fatty acids (see, e.g., Hu, [0025]-[0026], table 4). Furthermore, Cui teaches heterogeneously expressing a branched-chain α-ketoacid dehydrogenase complex for production of short branched-chain acyl-CoAs (see, e.g., Cui, Introduction, pg. 1568). Therefore, based on the teachings of Cui and Hu, it would have been obvious to express a branched-chain α-ketoacid dehydrogenase complex comprising bkdA, bkdB, and bkdC in order to produce branched-chain fatty acids. One would have expected success because Cui and Hu both teach heterogeneously expressing a branched-chain α-ketoacid dehydrogenase complex for production of branched-chain fatty acids.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Cui’s recombinant E. coli strain expressing lpdA1 from S. avermitilis, wherein the E. coli strain has the ilvA gene knocked out, as taught by Falb. One would have been motivated to do so because Falb teaches that ilvA is required for cell growth and/or survival and deletion of the ilvA gene results in an auxotrophic strain (see, e.g., Falb, [087]-[088], & Table 9, pg. 200). Furthermore, Franco teaches that ilvA is responsible for the conversion of threonine to 2-ketobutyrate (see, e.g., Franco, Graphical Abstract), which is a branched-chain keto acid; therefore, one of ordinary skill in the art would understand that a knockout of ilvA would inhibit the production of this branched-chain keto acid. Moreover, Cui teaches that the branched-chain α-ketoacid dehydrogenase complex produced short branched-chain acyl-CoAs by catalyzing the decarboxylation of branched-chain α-keto acids (see, e.g., Cui, Introduction, pg. 1568). Additionally, Cui teaches that L-isoleucine can be used to produce malonyl-CoA (see, e.g., Cui, Figure 1). Therefore, based on the teachings of Cui, Falb, and Franco, it would have been obvious to knockout the ilvA gene within the recombinant E. coli strain in order eliminate production of intermediates that do not directly produce malonyl-CoA. One would have expected success because Cui, Falb, and Franco all teach the branched-chain α-keto dehydrogenase complex.
Regarding claim 36’s reaction temperature being between 30-37oC, and more specifically 30oC, MPEP 2144.05 states that “Generally, differences in concentrations or temperatures will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. Where the general conditions of a claims are disclosure in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation”. Those working in the biological and/or pharmaceutical arts would understand that the adjustments of particular conventional working conditions (e.g., concentration or amount of a compound) is deemed a matter of judicious selection and routine optimization, which is within the purview of the skilled artisan. For example, the disclosure of Cui teaches that the bacteria are grown at 37oC in order to express branched-chain α-ketoacid dehydrogenase proteins within an E. coli host cell (see, e.g., Cui, “Cultivation of recombinant E. coli strains and expression analysis”, pg. 1568), wherein these proteins are used in a catalytic reaction to produce malonyl-CoA within the same host cell. Additionally, Hu teaches “Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein” (see, e.g., Hu, [0285]). Therefore, one of ordinary skill in the art would reasonably understand that the temperature by which the catalytic reaction occurs to produce malonyl-CoA using the branched-chain α-ketoacid dehydrogenase complex will affect the amount and time by which it takes to produce the malonyl-CoA. This is motivation for someone of ordinary skill in the art to practice or test the parameter widely to find those that are functional or optimal which then would be inclusive or cover the steps as instantly claimed. Absent any teaching of criticality by the Applicant concerning concentration, it would be prima facie obvious that one of ordinary skill in the art would recognize these limitations are result effective variables which can be met as a matter of routine optimization.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb and Franco as applied to claims 23, 26, and 34-36 above, and further in view of Rude (WO 2011/100667; Date of Publication: August 18, 2011 – previously cited), Gupta (Project Report Codon Optimization; 2003 – previously cited), Gustafsson (Codon bias and heterologous protein expression; 2004 – previously cited), and Denoya (WO 1995/004150; Date of Publication: February 9, 1995 – previously cited).
The teachings of Cui, Hu, Falb, and Franco, herein referred to as modified-Cui-Hu-Falb-Franco, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
Regarding claim 27 pertaining to the gene encoding the gene encoding the lpdA1 protein, modified-Cui-Hu-Falb-Franco teaches SEQ ID NO: 66, which has 99.9% sequence identity to instant SEQ ID NO: 4 (see, e.g., Hu, sequence listing & Office Action Appendix). Gupta teaches basic rationales for optimizing codons for specific living organisms (see, e.g., Gupta at §§ 2.1, 4.1, 4.2, 4.3) and identifies that circa 2003 web-based software was available for converting yeast and human codons (id. at §5). Gustafsson identifies that a “common strategy" to improve expression of heterologous proteins in a host is to alter rare codons in a gene to reflect codon usage of a host without modifying the amino acid sequence of the encoded protein (see, e.g., Gustafsson et al. at 348 col II §”Results from Codon Optimization”, Table 1 on 349). Therefore, based on the teachings of Gupta and Gustafsson, it is routine to one of ordinary skill in the art to use different nucleic acid sequences to express a protein in a different organism (i.e., codon optimization). Additionally, the nucleic acid encoding the protein would be optimized for expression in a particular organism. Furthermore, the transitional phrase “having” is synonymous to “comprising”, which is “inclusive or open-ended and does not exclude additional, unrecited elements or method steps” (see, e.g., MPEP 2111.03(I)); therefore, SEQ ID NO: 4 can have additional, unrecited elements. Additionally, 99.9% sequence similarity taught by modified-Cui-Hu-Falb-Franco is close to 100% sequence similarity, and a prima facie case of obviousness exists when “the claimed ranges or amounts do not overlap with the prior art but are merely close” (see, e.g., MPEP 2144.05(I)).
However, modified-Cui-Hu-Falb-Franco does not teach: the gene encoding the bkdA protein corresponding to nucleotide sequence positions 1-1146 of instant SEQ ID NO: 1 (claim 27); or the gene encoding the bkdB protein corresponding to nucleotide positions 1220-2224 of instant SEQ ID NO: 1 (claim 27); or the gene encoding the bkdC protein corresponding to nucleotide positions 2224-3591 of instant SEQ ID NO: 1 (claim 27).
Rude’s general disclosure relates to “a surfactant and/or a cleaning composition comprising a microbially produced branched fatty alcohol or a derivative thereof” (see, e.g., Rude, abstract).
Regarding claim 27 pertaining to the gene encoding the bkdB protein corresponding to nucleotide positions 1220-2224 of SEQ ID NO: 1, Rude teaches SEQ ID NO: 33, which has 100% sequence identity to nucleotide positions 1220-2224 of instant SEQ ID NO: 1 (see, e.g., Rude, [0012] & Office Action Appendix).
Regarding claim 27 pertaining to the gene encoding the bkdC protein corresponding to nucleotide positions 2224-3591 of SEQ ID NO: 1, Rude teaches SEQ ID NO: 56, which has 100% sequence identity to nucleotide positions 2224-3591 of instant SEQ ID NO: 1 (see, e.g., Rude, [0012] & Office Action Appendix).
Denoya’s general disclosure relates to “DNA sequence that encode for the branched-chain alpha-ketoacid dehydrogenase complex of an organism belonging to the genus Streptomyces and to polypeptides produced by the expression of such sequences” (see, e.g., Denoya, abstract).
Regarding claim 27 pertaining to the gene encoding the bkdA protein corresponding to nucleotide positions 1-1146 of SEQ ID NO: 1, Denoya teaches SEQ ID NO: 1, which has 100% identity to nucleotide positions 1-1146 of instant SEQ ID NO: 1 (see, e.g., Denoya, Description of Sequences, pgs. 14 and 16).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis in E. coli, wherein the genes sequences expressing bkdB and bkdC are taught by Rude, and the gene sequence expressing bkdA is taught by Denoya. One would have been motivated to do so because Rude teaches that the branched-chain alpha-keto dehydrogenase complex can be used to produce branched fatty alcohols from alpha keto acids, and that the “oxidative decarboxylation step, which converts the alpha keto acids to the corresponding branched-chain acyl-CoA involves a branched-chain α-keto acid dehydrogenase complex” (see, e.g., Rude, [00120]). Additionally, Rude teaches that for SEQ ID NO: 33, which has 100% sequence identity to nucleotide positions 1220-2224 of SEQ ID NO: 1, and for SEQ ID NO: 56, which has 100% sequence identity to nucleotide positions 2224-3591 of instant SEQ ID NO: 1, that these fragments produce a branched fatty alcohol (see, e.g., Rude, [0012]). Moreover, Denoya teaches that SEQ ID NO: 1, which has 100% identity to nucleotide positions 1-1146 of instant SEQ ID NO: 1 encodes for the “E1-alpha subunit of S. avermitilis BCKDH” (see, e.g., Denoya, Description of Sequences, pgs. 14 and 16). Furthermore, modified-Cui-Hu-Falb-Franco teaches expression of bkdA, bkdB, bkdC, and lpdA1 from S. avermitilis (see, e.g., Hu, [0025]-[0026] & Table 4) (see, e.g., Cui, abstract & Results and Discussion, pg. 1569). Therefore, based on the teachings of modified-Cui-Hu-Falb-Franco, Rude, and Denoya, it would have been obvious to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis in E. coli, wherein the genes sequences expressing bkdB and bkdC are taught by Rude, and the gene sequence expressing bkdA is taught by Denoya because Rude and Denoya both teach S. avermitilis gene sequences for bkdA, bkdB, and bkdC. One would have expected success because modified-Cui-Hu-Falb-Franco, Rude, an Denoya all teach components of the branched-chain alpha-keto dehydrogenase complex.
Claim 31 are rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb and Franco, as applied to claims 23, 29-30, and 34-36 above, and further in view of Katz (US 2012/0219998; Date of Publication: August 30, 2012 – previously cited) and Kodera (WO 2006/093322; Date of Publication: September 8, 2006 – previously cited).
The teachings of Cui, Hu, Falb, and Franco, herein referred to as modified-Cui-Hu-Falb-Franco, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
Regarding claim 31 pertaining to the ilvA protein and gene, modified-Cui-Hu-Falb-Franco teaches SEQ ID NO: 148, which has 100% sequence identity to instant SEQ ID NO: 15 and encodes the ilvA protein (see, e.g., Hu, [0036] & Office Action Appendix). Additionally, modified-Cui-Hu-Falb-Franco teaches SEQ ID NO: 149, which has 100% sequence identity to instant SEQ ID NO: 14 and encodes the ilvA gene (see, e.g., Hu, Table 1 & Office Action Appendix).
However, modified-Cui-Hu-Falb-Franco does not teach: the ilvE gene encoding a protein having the amino acid sequence shown in instant SEQ ID NO: 17 (claim 31); or the ilvE gene having the nucleotide sequence shown in instant SEQ ID NO: 16 (claim 31).
Katz’s general disclosure relates to “iso-octane production using polyketide synthases” (see, e.g., Katz, [0003]). Furthermore, Katz discloses “a polyketide synthase (PKS), capable of synthesizing a trimethylpentanoic acid” within a host cell (see, e.g., Katz, [0005]). Moreover, Katz teaches overproduction of isobutyryl-CoA by overexpressing the E. coli gene ilvE, which encodes the enzyme branched-chain amino acid transaminase, and the genes pdhD, bfmBB, bfmAA, and bfmBB for the enzyme complex of branched-chain α-keto acid hydrogenase from Bacillus subtilis (see, e.g., Katz, [0077]).
Regarding claims 31 pertaining to the ilvE protein, Katz teaches an ilvE amino acid sequence from E. coli, which has 100% sequence identity to instant SEQ ID NO: 17 (see, e.g., Katz, [0055] & Office Action Appendix).
Kodera’s general disclosure relates to “A method for manufacturing 4-hydroxy-L-isoleucine or a salt thereof, characterized by comprising the steps of contacting the biocatalyst having activity in catalyzing a reaction producing 4-hydroxy-L-isoleucine from acetaldehyde and a-ketobutyric acid or a salt thereof in the presence of an amino group donor with acetaldehyde and a-ketobutyric acid or a salt thereof in the presence of an amino group donor, and producing 4-hydroxy-L-isoleucine” (see, e.g., Kodera, abstract).
Regarding claim 31 pertaining to the ilvE gene, Kodera teaches SEQ ID NO: 1, which has 100% sequence identity to instant SEQ ID NO: 16 (see, e.g., Kodera, pg. 13, lines 16-21 & Office Action Appendix).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis, wherein the E. coli strain encodes ilvE, as taught by Katz and Kodera. One would have been motivated to do so because Katz teaches that ilvE can be used to synthesize isobutyryl-CoA from valine (see, e.g., Katz, [0053]). Additionally, Kodera teaches that ilvE is a branched chain amino acid aminotransferase that can be used to produce 4-hydroxy-L-leucine from 4-hydroxy-3-methyl-3-keto-pentanoic acid (see, e.g., Kodera, “Description of Invention”, pg. 2). Moreover, modified-Cui-Hu-Falb-Franco teaches that ilvE is a branched chain amino acid aminotransferase (see, e.g., Falb, [04]) and is responsible for converting branched chain amino acids into their corresponding α-ketoacid, such as conversion of leucine to 2-oxo-4-methylpentanoate (see, e.g., Falb, [07] & Figure 7). Therefore, based on the teachings of modified-Cui-Hu-Falb-Franco, Katz, and Kodera, it would be obvious to knock out the ilvE gene within the recombinant E. coli cell in order to eliminate breakdown of specific branched chain alpha keto acids so that the end product is manipulated. One would have expected success because modified-Cui-Hu-Falb-Franco, Katz, and Kodera all teach production of α-ketoacids from branched chain amino acids.
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb and Franco as applied to claims 23, 26, and 34-36 above, and further in view of Chinen (US 2010/0099152; Date of Publication: April 22, 2010 – previously cited) and Sauer (The PEP–pyruvate–oxaloacetate node as the switch point for carbon flux distribution in bacteria; 2005 – newly cited).
The teachings of Cui, Hu, Falb, and Franco, herein referred to as modified-Cui-Hu-Falb-Franco, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
However, modified-Cui-Hu-Falb-Franco does not teach: wherein step 1) further comprises the step of introducing a gene encoding a ppc protein into the biological cell strain and expressing the encoding gene, or increasing the content of the ppc protein or enhancing the activity of the ppc protein in the biological cell strain; further, the ppc protein and its encoding gene are derived from Corynebacterium glutamicum (claim 32); or wherein the ppc protein has an amino acid sequence shown in SEQ ID NO: 19 (claim 32); or wherein the ppc gene is a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 18 (claim 32).
Chinen’s general disclosure relates to “a method for efficiently producing L-amino acids such as L-glutamic acid, L-glutamine, L-proline, L-arginine, L-leucine, and L-cysteine by fermentation” (see, e.g., Chinen, [0003]).
Regarding claim 32 pertaining to introducing the ppc gene into a biological cell strain, Chinen teaches “the ppc gene from C. glutamicum ATCC13032 strain” (see, e.g., Chinen, [0218]). Furthermore, Chinen teaches construction of a plasmid expressing the ppc gene, and wherein these plasmids can be transformed into cells (see, e.g., Chinen, [0281]-[0284] & Example 6). Moreover, Chinen teaches SEQ ID NO: 62, which has 100% sequence identity to instant SEQ ID NO: 18 and encodes for the ppc gene (see, e.g., Chinen, [0218] & Office Action Appendix). Additionally, Chinen taches SEQ ID NO: 63, which has 100% sequence identity to instant SEQ ID NO: 19 and encodes for the ppc protein (see, e.g., Chinen, [0218] & Office Action Appendix).
Sauer’s general disclosure relates to “what is known about the enzymes and the metabolic fluxes at the PEP–pyruvate–oxaloacetate node in bacteria” (see, e.g., Sauer, abstract). Moreover, Sauer discloses that “In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)–pyruvate–oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell” (see, e.g., Sauer, abstract).
Regarding claim 32 pertaining to ppc, Sauer teaches that phosphoenolpyruvate carboxylase is responsible for regeneration of oxaloacetate (see, e.g., Sauer, Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis, wherein the E. coli strain also expresses the ppc protein, as taught by Chinen. One would have been motivated to do so because Chinen teaches that the ppc protein encodes a phosphoenolpyruvate carboxylase (see, e.g., Chinen, [0135]) and Sauer teaches that phosphoenolpyruvate carboxylase is responsible for regeneration of oxaloacetate (see, e.g., Sauer, Figure 1). Moreover, modified-Cui-Hu-Falb-Franco teaches that oxaloacetate can be used to produce L-isoleucine, which is a precursor to malonyl-CoA (see, e.g., Cui, Figure 1). Therefore, based on the teachings of modified-Cui-Hu-Falb-Franco, Chinen, and Sauer, it would have been obvious to express the pcc gene within the recombinant E. coli strain in order to regenerate oxaloacetate within the TCA cycle in order to L-isoleucine to be produced from oxaloacetate. One would have expected success because modified-Cui-Hu-Falb-Franco, Chinen, and Sauer all teach metabolism pathways, as well as precursors and intermediates for malonyl-CoA production.
Claim 33 is rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb, and Franco as applied to claims 23, 26, and 34-36 above, and further in view of Falb (US 2017/0232043; Date of Publication: August 17, 2017 – newly cited – herein referred to as “Falb2017”) and Schwaneberg (WO 2009/013115; Date of Publication: January 29, 2009 – previously cited).
The teachings of Cui, Hu, Falb, and Franco, herein referred to as modified-Cui-Hu-Falb-Franco, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
However, modified-Cui-Hu-Falb-Franco does not teach: wherein step 1 further comprises the step of knocking out the gene encoding the outer membrane protease VII in the biological cell strain or reducing the content or activity of the outer membrane protease VII in the biological cell strain (claim 33); or wherein the outer membrane protease VII is an ompT protein (claim 33); or wherein the ompT protein has the amino acid sequence shown in SEQ ID NO: 28 (claim 33); or wherein the ompT gene has the nucleotide sequence shown in SEQ ID NO: 27 (claim 33).
Falb2017’s general disclosure relates to “recombinant bacterial cells that have been engineered with genetic circuitry which allow the recombinant bacterial cells to sense a patient's internal environment and respond by turning an engineered metabolic pathway on or off. When turned on, the recombinant bacterial cells complete all of the steps in a metabolic pathway to achieve a therapeutic effect in a host subject. These recombinant bacterial cells are designed to drive therapeutic effects throughout the body of a host from a point of origin of the microbiome. Specifically, the present disclosure provides recombinant bacterial cells comprising a heterologous gene encoding a branched chain amino acid catabolism enzyme. The disclosure further provides pharmaceutical compositions comprising the recombinant bacteria, and methods for treating disorders involving the catabolism of branched chain amino acids” (see, e.g., Falb2017, abstract).
Regarding claim 33 pertaining to knocking out ompT, Falb2017 teaches that the genetically engineered bacteria that are expressing a therapeutic polypeptide of interest, such as a branched chain amino acid catabolism enzyme, has periplasmic proteases deleted, wherein the periplasmic protease can be ompT (see, e.g., Falb2017, [0104]). Furthermore, Falb2017 teaches that deletion of ompT results in increased stability of the polypeptide in the periplasm (see, e.g., Falb2017, [0104]).
Schwaneberg’s general disclosure relates to “vesicles comprising at least one transmembrane triggered transport system for controlled transmembrane efflux and/or influx” (see, e.g., Schwaneberg, abstract). Additionally, Schwaneberg discloses the use of transmembrane protein proteins “as gates for selective compound passage” (see, e.g., Schwaneberg, [0005.1]).
Regarding claim 33 pertaining to amino acid and nucleic acid sequences encoding the ompT protein, Schwaneberg teaches SEQ ID NO: 48 (see, e.g., Schwaneberg, [0025.1], [0042.1]), which has 100% sequence identity to instant SEQ ID NO: 28 (see, e.g., Office Action Appendix). Furthermore, Schwaneberg teaches SEQ ID NO: 14 (see, e.g., Schwaneberg, [0025.1], [0026.1]), which has 100% sequence identity to instant SEQ ID NO: 27 (see, e.g., Office Action Appendix).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis, wherein the E. coli strain has ompT knocked out, as taught by Falb2017. One would have been motivated to do so because Falb2017 teaches that deletion of ompT results in increased stability of the polypeptide in the periplasm (see, e.g., Falb2017, [0104]). Furthermore, Schwaneberg teaches the gene and amino acid sequences for ompT with 100% sequence identity to the instantly claimed ompT sequences (see, e.g., Schwaneberg, [0025.1], [0026.1], and [0042.1]). Moreover, modified-Cui-Hu-Falb-Franco teaches that bkdA, bkdB, and bkdC are proteins that comprise the branched-chain α-ketoacid dehydrogenase complex for the biosynthesis of branched-chain fatty acids (see, e.g., Hu, [0025]-[0026], table 4). Furthermore, modified-Cui-Hu-Falb-Franco teaches heterogeneously expressing a branched-chain α-ketoacid dehydrogenase complex for production of short branched-chain acyl-CoAs (see, e.g., Cui, Introduction, pg. 1568). Therefore, based on the teachings of modified-Cui-Hu-Falb-Franco, Falb2017, and Schwaneberg, it would have been obvious to produce a recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis, wherein the E. coli strain has ompT knocked out because this would allow for the expression of lpdA1, bkdA, bkdB, and bkdC proteins, as well as the production of branched-chain fatty acids, without out proteolytic activity by ompT, thereby increasing stability of the polypeptides in the periplasm. One would have expected success because modified-Cui-Hu-Falb-Franco, Falb2017, and Schwaneberg all teach components related to branched chain amino acid catabolism and/or protein synthesis.
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb and Franco as applied to claims 23, 26, and 34-36 above, and further in view of Walther (US 2015/0159182; Date of Publication: June 11, 2015 – previously cited), Gupta (previously cited), Gustafsson (previously cited), Liu (WO 2018/188617; Date of Publication: October 18, 2018 – previously cited), and Liu (Dissection of Malonyl-Coenzyme A Reductase of Chloroflexus aurantiacus Results in Enzyme Activity Improvement; 2013 – newly cited – herein referred to as Liu2013).
The teachings of Cui, Hu, Falb, and Franco, herein referred to as modified-Cui-Hu-Falb-Franco, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
Regarding claim 37 pertaining to recombinant cell strain A, modified-Cui-Hu-Falb teaches the production of a recombinant E. coli cell that is capable of expressing the gene encoding the branched chain α-keto acid dehydrogenase complex (see, e.g., Cui, Methods, “Cultivation of recombinant E. coli strains and expression analysis, pg. 1568 & Supplemental Table 1).
However, modified-Cui-Hu-Falb-Franco does not teach: wherein the target product is 3- hydroxypropionic acid, and the method comprises the steps of: introducing into the recombinant cell strain A a gene encoding a mcr protein and expressing the encoding gene or increasing the content of the mcr protein or enhancing the activity of the mcr protein in the recombinant cell strain A, to obtain a recombinant cell strain, which is recorded as recombinant cell strain-mcr; culturing the recombinant cell strain-mcr to prepare the target product (claim 37); or wherein the mcr protein and its encoding gene are derived from Chloroflexus aurantiacus (claim 37); or wherein the mcr N-terminal domain is a protein having the amino acid sequence shown in SEQ ID NO: 22 (claim 37); or wherein the mcr C-terminal domain is a protein having the amino acid sequence shown in SEQ ID NO: 23 (claim 37); or wherein the gene encoding the mcr protein is composed of the gene encoding the mcr N-terminal domain and the gene encoding the mcr C-terminal domain, and the gene encoding the mcr N-terminal domain is a DNA molecule having the nucleotide sequence shown in positions 1-1689 of SEQ ID NO: 21 (claim 37); or wherein the gene encoding the mcr C-terminal domain is a DNA molecule having the nucleotide sequence shown in positions 1704-3749 of SEQ ID NO: 21 (claim 37); or wherein the gene encoding the mcr protein is a DNA molecule having the nucleotide sequence shown in SEQ ID NO: 21 (claim 37).
Walther’s general disclosure relates to “a novel method of production of 2,4-dihydroxybutyric acid from malate and/or glyoxylate and/or succinyl-CoA by the implementation of a synthetic pathway that converts malate and/or glyoxylate and/or succinyl-CoA into malylCoA, malylCoA into malate-4-semialdehyde, and then converting said malate-4-semialdehyde into 2,4-dihydroxybutyric acid (2,4-DHB)” (see, e.g., Walther, [0001]).
Regarding claim 37 pertaining to introducing the mcr gene into the recombinant cell strain A, Walther teaches “Construction of plasmids containing wild-type genes coding for malonyl-CoA reductase and succinyl-CoA reductase: The DNA sequence of the mcr gene coding for malyl-CoA reductase in Sulfolobus tokodaii str 7 (Alber et al., 2006) was optimized for the expression in Escherichia coli using the GENEius software (Eurofins). The optimized mcr sequence, and the natural DNA sequence of the sucD gene coding for succinyl-CoA reductase in Porphyromonas gingivalis W83 were synthesized by Eurofins MWG OPERON.RTM. adding NheI and EcoRI restriction sites upstream of the start codon and downstream of the stop codon of mcr, respectively, which allowed direct cloning of the synthesized DNA fragments into the pET28a+ vector (Novagen) using T4 DNA ligase (Biolabs). Ligation products were transformed into E. coli DH5.alpha. cells, amplified, and the plasmids, pET28-St-mcr (expressing the malonyl-CoA reductase from S. tokodaii), and pET28-Pgi-sucD (expressing the succinyl-CoA reductase from P. gingivalis), were isolated using standard genetic protocols (Sambrook et al., 1989)” (see, e.g., Walther, [0111]). Therefore, Walther teaches the production of a recombinant cell expressing the mcr gene that encodes the mcr protein. Furthermore, Walther teaches that the mcr gene and protein are derived from Chloroflexus aurantiacus (see, e.g., Walther, [0030]). Walther teaches SEQ ID NO: 190 (see, e.g., Walther, [0030]), which has 97.4% sequence identity to nucleotide positions 1-1689 of instant SEQ ID NO 21 (see, e.g., Office Action Appendix). Walther teaches SEQ ID NO: 190 (see, e.g., Walther, [0030]), which has 98% sequence identity to nucleotide positions 1704-3749 of instant SEQ ID NO 21 (see, e.g., Office Action Appendix). Walther teaches SEQ ID NO: 190 (see, e.g., Walther, [0030]), which has 95.7% sequence identity to instant SEQ ID NO 21 (see, e.g., Office Action Appendix).
Regarding claim 37’s sequence identities for instant SEQ ID NO: 21, Gupta teaches basic rationales for optimizing codons for specific living organisms (see, e.g., Gupta at §§ 2.1, 4.1, 4.2, 4.3) and identifies that circa 2003 web-based software was available for converting yeast and human codons (id. at §5). Gustafsson identifies that a “common strategy" to improve expression of heterologous proteins in a host is to alter rare codons in a gene to reflect codon usage of a host without modifying the amino acid sequence of the encoded protein (see, e.g., Gustafsson et al. at 348 col II §”Results from Codon Optimization”, Table 1 on 349). Therefore, based on the teachings of Gupta and Gustafsson, it is routine to one of ordinary skill in the art to use different nucleic acid sequences to express a protein in a different organism (i.e., codon optimization). Additionally, the nucleic acid encoding the protein would be optimized for expression in a particular organism
Liu’s general disclosure pertains to “a method for construction of recombinant bacteria for producing 3-hydroxy propionic acid”, wherein the method comprises “knocking out fadR, fabF and fabH genes of receptor bacteria, introducing acc genes or gene clusters, alKL and Mcr genes, and enhancing the expression of fadL, fadD, sthA genes and atoSC gene clusters in the receptor bacteria” (see, e.g., Liu, abstract).
Regarding claim 37 pertaining to the mcr N-terminal domain protein sequence, Liu teaches SEQ ID NO: 37, which as 100% sequence identity to instant SEQ ID NO: 22 (see, e.g., Office Action Appendix).
Regarding claim 37 pertaining to the mcr C-terminal domain protein sequence, Liu teaches SEQ ID NO: 23, which has 100% sequence identity to instant SEQ ID NO: 23 (see, e.g., Office Action Appendix).
Liu2013’s general disclosure relates to “The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain” (see, e.g., Liu2013, abstract).
Regarding claim 37 pertaining to the target product and the introduction of the mcr protein, Liu2013 teaches that “The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical” (see, e.g., Liu2013, abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis, wherein the E. coli strain also expresses the mcr protein, as taught by Walther and Liu. One would have been motivated to do so because Walther teaches that the mcr protein expresses a malonyl-CoA reductase (see, e.g., Walther, [0029]) and Liu2013 teaches that “The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical” (see, e.g., Liu2013, abstract). Moreover, Liu2013 teaches that “the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher K cat/K m value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain” (see, e.g., Liu2013, abstract). Moreover, modified-Cui-Hu-Falb-Franco teaches the production of malonyl-CoA through the branched chain α-keto acid dehydrogenase complex (see, e.g., Cui, Figure 1). Therefore, based on the teachings of modified-Cui-Hu-Falb-Franco, Walther, Liu, and Liu2013, it would have been obvious to introduce the mcr protein into the recombinant E. coli cell in order to produce 3-hydroxypropionate from malonyl-CoA, wherein the malonyl-CoA is produced via through the branched chain α-keto acid dehydrogenase complex. One would have expected success because modified-Cui-Hu-Falb-Franco, Walther, Liu, and Liu2013 all teach precursors, intermediates, and/or final products from the branched chain α-keto acid dehydrogenase complex, and how the branched chain α-keto acid dehydrogenase complex can be used to produce malonyl-CoA, which can be used to produce other downstream products.
Claim 38 is rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb, Walther, Gupta, Gustafsson, Liu, and Liu2013 as applied to claims 23, 29-30, and 34-37 above, and further in view of Wang (CN 104404058; Date of Publication: March 11, 2015 – previously cited – herein referred to as “Wang2015”) and Wang (CN 102618509; Date of Publication: August 1, 2012 – previously cited – herein referred to as “Wang2012”).
The teachings of Cui, Hu, Falb, Walther, Gupta, Gustafsson, Liu, and Liu2013 herein referred to as modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu2013, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
Regarding claim 38 pertaining to recombinant cell strain A, modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu2013 teaches the production of a recombinant E. coli cell that is capable of expressing the gene encoding the branched chain α-keto acid dehydrogenase complex (see, e.g., Cui, Methods, “Cultivation of recombinant E. coli strains and expression analysis, pg. 1568 & Supplemental Table 1).
However, modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu2013 does not teach: wherein the target product picric acid or an intermediate product from malonyl-CoA to picric acid in the picric acid synthesis pathway, and the method comprises the steps of: introducing into the recombinant cell strain A a gene encoding a vps protein and expressing the encoding gene, or increasing the content of the vps protein or enhancing the activity of the vps protein in the recombinant cell strain A, to obtain a recombinant cell strain, which is recorded as recombinant cell strain-vps; culturing the recombinant cell strain-vps to prepare the target product (claim 38); or wherein the vps protein and its encoding gene are derived from Humulus lupulus (claim 38); or wherein the vps protein has the amino acid sequence shown in SEQ ID NO: 26 (claim 38); or wherein the gene encoding the vps protein has the nucleotide sequence shown in SEQ ID NO: 25 (claim 38).
Wang2015’s general disclosure relates to a protein for preparing beta-picric acid (see, e.g., Wang, abstract).
Regarding claim 38 pertaining to encoding the vps protein in a recombinant cell strain, Wang2015 teaches transforming vectors “pESC-LEU/CCL2/CCL4. pESC-HIS/VPS and PESC-URA/PT1/PT2 into the yeast cell DD104, to obtain CCL2, CCL4, VPS, PTl and PT2 nucleotide sequence of recombinant yeast cells, the recombinant yeast cell is named as DD104-H1. the pESC-LEU/CCL2/CCL4, DD104, to obtain the CCL2, pESC-URA/PTl-At/PT2-At pESC-HIS/VPS and CCL4 into the yeast cell, VPS PT1-At and PT2-At nucleotide sequence of recombinant yeast cells, the recombinant yeast cells named DD104 -At. the pESC-LEU/CCL2/CCL4, pESC-HIS/VPS and PESC-URA/PT1-S/PT2-S into the yeast cell DD104, to obtain CCL2, CCL4, VPS, PTl-S and PT2-S nucleotide sequence of recombinant yeast cells, the recombinant yeast cell is named as DD104-S” (see, e.g., Wang2015, [0110]). Furthermore, Wang2015 teaches “the pESC-URA carrier by BamH I and Sal I recognition sequence sites is SEQ ID No. DNA molecules (picric acid synthesis-related gene) nucleotide at position 121-1245 1, keeping the other sequence change to obtain recombinant vector pESC-URA/PTl lambda 40-At, lambda pESC-URA/PTl embodiment 40-At, in the step PESC-LEU/CCL2/CCL4 and pESC-HIS/VPS into the yeast cell DD104, to obtain a CCL2, CCL4, VPS and PTl nucleotide sequences of 40 -At of recombinant yeast cells, the recombinant yeast cell is named as a DDKM-At Lambda 40” (see, e.g., Wang, [0131]). Therefore, Wang2015 teaches the production of a recombinant cell strain expressing vps. Furthermore, Wang2015 teaches that the vps protein and its encoded gene are derived from Humulus lupulus (see, e.g., Wang2015, [0002]). Additionally, Wang2015 teaches SEQ ID NO: 6 (see, e.g., Wang2015, [0102]), which has 100% sequence identity to instant SEQ ID NO: 25 (see, e.g., Office Action Appendix). Moreover, Wang2015 teaches SEQ ID NO: 4 (see, e.g., Wang2015, [0020]), which has 100% sequence identity to instant SEQ ID NO: 26 (see, e.g., Office Action Appendix).
Wang2012’s general disclosure relates to the CCL2 protein derived from Humulus lupulus, wherein the CCL2 gene is co-expressed with the VPS gene in yeast cells to produce the picric acid precursor, PIVP (see, e.g., Wang2012, abstract).
Regarding claim 38 pertaining to synthesis of picric acid and expression of the vps protein, Wang2012 teaches “picric acid was formed by the reaction of one molecule of isovaleryl-CoA and three molecules of malonyl-CoA catalyzed by valerophenone synthase (vps)”; therefore, vps expression has good correlation to picric acid levels in different tissues (see, e.g., Wang2012, English translation, pg. 2). Furthermore, Wang2012 teaches “VPS may interact with other proteins in the metabolic pathway (possible candidate proteins include isopentenyl transferase, P450, etc.) to form metabolic channels (Metabolic channeling) to directly generate end products such as picric acid, predicted intermediate metabolites (such as PIVP), etc. Not released during metabolic reactions” (see, e.g., Wang2012, English translation, pg. 2).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu2013’s recombinant E. coli strain expressing lpdA1, bkdA, bkdB, and bkdC from S. avermitilis, wherein the E. coli strain also expresses mcr, and wherein the E. coli strain expresses vps, as taught by Wang2012 and Wang2015. One would have been motivated to do so because Wang2015 teaches that the vps protein is upstream of picric acid synthesis in yeast cells (see, e.g., Wang2015, English Translation, pg 5, “Description of drawings”). Furthermore, Wang2012 teaches “picric acid was formed by the reaction of one molecule of isovaleryl-CoA and three molecules of malonyl-CoA catalyzed by valerophenone synthase (vps)”; therefore, vps expression has good correlation to picric acid levels in different tissues (see, e.g., Wang2012, English translation, pg. 2). Moreover, modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu2013 teaches production of malonyl-CoA through expression of the branched-chain α-keto acid dehydrogenase complex (see, e.g., Cui, Introduction, 1568 & Figure 1). Therefore, based on the teachings of modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu201, Wang2012, and Wang2015, it would have been obvious to utilize the branched-chain α-keto acid dehydrogenase complex to produce malonyl-coA, which can be used to produce picric acid as an end product. One would have expected success because modified-Cui-Hu-Falb-Walther-Gupta-Gustafsson-Liu-Liu201, Wang2012, and Wang2015 all teach components, intermediates, and end-products related to the branched-chain α-keto acid dehydrogenase complex.
Claims 42-43 are rejected under 35 U.S.C. 103 as being unpatentable over Cui, Hu, Falb and Franco as applied to claims 23, 26, and 34-36, and further in view of Rude (previously cited), Gupta (previously cited), Gustafsson (previously cited).
The teachings of Cui, Hu, Falb, and Franco, herein referred to as modified-Cui-Hu-Falb-Franco, are discussed above as it pertains to a biological cell strain encoding the branched-chain α-ketoacid dehydrogenase complex.
Regarding claim 42 pertaining to the lpdA protein, modified-Cui-Hu-Falb-Franco teaches SEQ ID NO: 65, which has 100% sequence identity to instant SEQ ID NO: 13 (see, e.g., Hu, [0027] & Office Action Appendix).
Regarding claim 43 pertaining to the gene encoding the gene encoding the lpdA1 protein, modified-Cui-Hu-Falb-Franco teaches SEQ ID NO: 66, which has 99.9% sequence identity to instant SEQ ID NO: 4 (see, e.g., Hu, sequence listing & Office Action Appendix). Gupta teaches basic rationales for optimizing codons for specific living organisms (see, e.g., Gupta at §§ 2.1, 4.1, 4.2, 4.3) and identifies that circa 2003 web-based software was available for converting yeast and human codons (id. at §5). Gustafsson identifies that a “common strategy" to improve expression of heterologous proteins in a host is to alter rare codons in a gene to reflect codon usage of a host without modifying the amino acid sequence of the encoded protein (see, e.g., Gustafsson et al. at 348 col II §”Results from Codon Optimization”, Table 1 on 349). Therefore, based on the teachings of Gupta and Gustafsson, it is routine to one of ordinary skill in the art to use different nucleic acid sequences to express a protein in a different organism (i.e., codon optimization). Additionally, the nucleic acid encoding the protein would be optimized for expression in a particular organism. Furthermore, the transitional phrase “having” is synonymous to “comprising”, which is “inclusive or open-ended and does not exclude additional, unrecited elements or method steps” (see, e.g., MPEP 2111.03(I)); therefore, SEQ ID NO: 4 can have additional, unrecited elements. Additionally, 99.9% sequence similarity taught by modified-Cui-Hu-Falb is close to 100% sequence similarity, and a prima facie case of obviousness exists when “the claimed ranges or amounts do not overlap with the prior art but are merely close” (see, e.g., MPEP 2144.05(I)).
However, modified-Cui-Hu-Falb-Franco does not teach: the bkdF protein corresponding to SEQ ID NO: 10 (claim 42); or the bkdG protein corresponding to SEQ ID NO: 11 (claim 42); or the bkdH protein corresponding to SEQ ID NO: 12 (claim 42); or the gene encoding the bkdF protein corresponding to the nucleotide sequence shown in positions 1-1221 of SEQ ID NO: 2 (claim 43); or the gene encoding the bkdG protein corresponding to the nucleotide sequence shown in positions 1223-2200 of SEQ ID NO: 2 (claim 43); the gene encoding the bkdH protein corresponding to the nucleotide sequence shown in positions 2220-3608 of SEQ ID NO: 2 (claim 43).
Rude’s general disclosure is discussed above.
Regarding claim 42 pertaining to the bkdF, bkdG, and bkdH proteins, Rude teaches SEQ ID NO: 3 (see, e.g., Rude, [095]), which has 100% sequence identity to instant SEQ ID NO: 10 encoding the bkdF protein (see, e.g., Office Action Appendix). Rude teaches SEQ ID NO: 26 (see, e.g., Rude, [095]), which has 99.2% sequence identity to instant SEQ ID NO: 11 encoding the bkdG protein (see, e.g., Office Action Appendix). The transitional phrase “having” is synonymous to “comprising”, which is “inclusive or open-ended and does not exclude additional, unrecited elements or method steps” (see, e.g., MPEP 2111.03(I)); therefore, SEQ ID NO: 11 can have additional, unrecited elements. Additionally, 99.2% sequence similarity taught by Rude is close to 100% sequence similarity, and a prima facie case of obviousness exists when “the claimed ranges or amounts do not overlap with the prior art but are merely close” (see, e.g., MPEP 2144.05(I)). Additionally, Rude teaches SEQ ID NO: 49 (see, e.g., Rude, [095]), which has 100% sequence identity to instant SEQ ID NO: 12 (see, e.g., Office Action Appendix).
Regarding claim 43 pertaining to nucleotide positions 1-1221 of SEQ ID NO: 2, Rude teaches SEQ ID NO: 4 (see, e.g., Rude, pg. 31, [012]), which has 99.9% sequence identity to nucleotide positions 1-1221 of instant SEQ ID NO: 2 (see, e.g., Office Action Appendix). Moreover, regarding nucleotide positions 1223-2200 of SEQ ID NO: 2, Rude teaches SEQ ID NO: 4 (see, e.g., Rude, pg. 31, [012]), which has 99.7% sequence identity to nucleotide positions 1223-2200 of instant SEQ ID NO: 2 (see, e.g., Office Action Appendix). Regarding nucleotide positions 2220-3608 of SEQ ID NO: 2, Rude teaches SEQ ID NO: 4 (see, e.g., Rude, pg. 31, [012]), which has 100% sequence identity to nucleotide positions 2220-3608 of instant SEQ ID NO: 2 (see, e.g., Office Action Appendix). In regards to nucleotide positions 1-1221 and 1223-2200 of SEQ ID NO: 2, the teachings of Gupta and Gustafsson are discussed above, and based on the teachings of Gupta and Gustafsson, it is routine to one of ordinary skill in the art to use different nucleic acid sequences to express a protein in a different organism (i.e., codon optimization). Additionally, the nucleic acid encoding the protein would be optimized for expression in a particular organism. Furthermore, transitional phrase “having” is synonymous to “comprising”, which is “inclusive or open-ended and does not exclude additional, unrecited elements or method steps” (see, e.g., MPEP 2111.03(I)); therefore, nucleotide positions 1-1221 of instant SEQ ID NO: 2 can have additional, unrecited elements. Additionally, 99.2% and 99.7% sequence similarity taught by Rude is close to 100% sequence similarity, and a prima facie case of obviousness exists when “the claimed ranges or amounts do not overlap with the prior art but are merely close” (see, e.g., MPEP 2144.05(I)).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the instant invention to produce modified-Cui-Hu-Falb-Franco’s recombinant E. coli strain expressing lpdA1, bkdF, bkdG, and bkdH, wherein the protein and gene sequences for lpdA1, bkdF, bkdG, and bkdH are taught by Rude. One would have been motivated to do so because Rude teaches that the branched-chain alpha-keto dehydrogenase complex can be used to produce branched fatty alcohols from alpha keto acids, and that the “oxidative decarboxylation step, which converts the alpha keto acids to the corresponding branched-chain acyl-CoA involves a branched-chain α-keto acid dehydrogenase complex” (see, e.g., Rude, [00120]). Additionally, Rude teaches SEQ ID NOs: 3 and 26 (see, e.g., Rude, [0095]), which have 100% and 99.2% sequence identity to instant SEQ ID NOs: 10 and 11, respectively, and which both together have alpha-keto acid decarboxylase activity (see, e.g., Rude, [0096]). Furthermore, Rude teaches SEQ ID NO: 49, which has 100% sequence identity to instant SEQ ID NO: 12, and which encodes a polypeptide with lipoamide acyltransferase activity (see, e.g., Rude, [0096]). Moreover, modified-Cui-Hu-Falb-Franco teaches that the branched chain α-keto acid dehydrogenase complex can comprise BkdH, BkdF, BkdG and LpdA1 from S. avermitilis and be expressed in E. coli (see, e.g., Cui, abstract). Furthermore, modified-Cui-Hu-Falb-Franco teaches that BkdH, BkdF, BkdG and LpdA1 from S. avermitilis can be expressed in E. coli in order to produce short branched-chain acyl-CoA (see, e.g., Cui, Introduction, pgs. 1567-1568). Therefore, based on the teachings of modified-Cui-Hu-Falb-Franco and Rude, it would have been obvious to express that BkdH, BkdF, BkdG and LpdA1 from S. avermitilis can be expressed in E. coli in order to produce short branched-chain acyl-CoA. One would have expected success because modified-Cui-Hu-Falb-Franco and Rude both teach the branched chain α-keto acid dehydrogenase complex.
Examiner’s Response to Arguments
Applicant's arguments filed 11/13/2025 have been fully considered but they are not persuasive.
Applicant has traversed the previous 35 U.S.C 103 rejections in light of the amendment of claim 23 which introduces new limitations (remarks, pages 13-15). As discussed above, all rejections have been withdrawn and new rejections are presented due to Applicant’s amendment on 11/13/2025. Therefore, Applicant’s arguments pertaining to the newly inserted limitation in independent claim 23 is moot.
Regarding Applicant’s argument pertaining to Cui and Hu not addressing a more efficient synthesis pathway for malonyl-CoA (remarks, page 15), this argument is not persuasive because the instantly claimed invention does not claim a more efficient synthesis pathway for malonyl-CoA. Moreover, the instantly claimed invention merely claims a method for preparing malonyl-CoA, which Cui and Hu teach. Cui teaches the production of a recombinant E. coli cell that is capable of expressing the gene encoding the branched chain α-keto acid dehydrogenase complex (see, e.g., Cui, Methods, “Cultivation of recombinant E. coli strains and expression analysis, pg. 1568 & Supplemental Table 1). Additionally, Cui teaches the expression of lpdA1 from Streptomyces avermitilis in E. coli cells (see, e.g., Cui, abstract & Results and Discussion, pg. 1569). Hu teaches bkdA, bkdB, and bkdC genes, which encode teaches bkdA, bkdB, and bkdC proteins, (see, e.g., Hu, [0025]-[0026]). Additionally, the teaches bkdA, bkdB, and bkdC genes are derived from S. avermitilis (see, e.g., Hu, Table 4). Therefore, based on the teachings of Cui and Hu, it would have been obvious to one of ordinary skill in the art to produce Cui’s recombinant E. coli strain expressing lpdA1 from S. avermitilis, wherein the recombinant E. coli strain also expresses bkdA, bkdB, and bkdC, as taught by Hu, because Hu teaches that bkdA, bkdB, and bkdC are proteins that comprise the branched-chain α-ketoacid dehydrogenase complex for the biosynthesis of branched-chain fatty acids (see, e.g., Hu, [0025]-[0026], table 4). Furthermore, Cui teaches heterogeneously expressing a branched-chain α-ketoacid dehydrogenase complex for production of short branched-chain acyl-CoAs (see, e.g., Cui, Introduction, pg. 1568). Therefore, based on the teachings of Cui and Hu, it would have been obvious to express a branched-chain α-ketoacid dehydrogenase complex comprising bkdA, bkdB, and bkdC in order to produce branched-chain fatty acids.
Regarding Applicant’s argument pertaining to the teachings of Wang and Walther not being directly coupled to the function of the branched-chain α-ketoacid dehydrogenase complex (remarks, page 16), this argument is not persuasive because Walther teaches construction of plasmids containing wild-type genes coding for malonyl-CoA reductase (mcr) in E. coli (see, e.g., Walther, [0111]). Furthermore, Liu2013 was cited as motivation for encoding the mcr gene into E. coli and this is because “The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical” (see, e.g., Liu2013, abstract). Moreover, Wang2015 teaches encoding the vps protein in a recombinant cell strain (see, e.g., Wang2015, [0110]). Furthermore, Wang2012 was cited as motivation for encoding the vps gene in E. coli and this is because “picric acid was formed by the reaction of one molecule of isovaleryl-CoA and three molecules of malonyl-CoA catalyzed by valerophenone synthase (vps)”; therefore, vps expression has good correlation to picric acid levels in different tissues (see, e.g., Wang2012, English translation, pg. 2). Therefore, Walther, Liu2013, Wang2012, and Wang2015 teach end products that can be produced from malonyl-CoA. Furthermore, malonyl-CoA can be produced via the branched-chain α-ketoacid dehydrogenase complex (see, e.g., Cui, Introduction & Figure 1, Hu, [0025]-[0026], table 4). Therefore, the teachings of Walther, Liu2013, Wang2012, and Wang2015 read upon the instantly claimed invention.
Regarding Applicant’s argument that the data in Example 1 shows that the engineered strain can catalyze the conversion of OAA to malonyl-CoA without any branched-chain amino acids (remarks, page 16), this argument is not persuasive because, as discussed above, Cui does not teach the exogenous addition of branched-chain amino acids to the branched-chain α-ketoacid dehydrogenase complex for the formation of malonyl-CoA. Therefore, Cui reads upon the instantly claimed invention. Moreover, as discussed above, this newly inserted limitation is considered new matter because the instant specification does not provide support pertaining to the viability of the recombinant cell strain in the absence of exogenously added branched-chain amino acids. Moreover, the absence of exogenously added branched-chain amino acids must be positively recited in the originally-filed disclosure in order to be supported (see, e.g., MPEP 2173.05(i)).
Conclusion
Claims 23, 26-27, 31-38, and 42-43 are rejected.
No claims are allowed.
Correspondence Information
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
