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 .
DETAILED ACTION
Claims 40 and 62-80 are pending. Claims 1-39 and 41-61 are cancelled.
Election/Restrictions
Applicant’s election without traverse of Group II, claims 40 and 62-80, and the species of M. formatexigens thioesterase and CO2 in the reply filed on 10/31/2025 is acknowledged.
Claim 75 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 10/31/2025.
Claims 40, 62-74, and 76-80 are examined herein.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 66, 72, 74, and 79 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 66 recites that the fatty acid is esterified with acyl carrier protein (ACP) or with acetyl-CoA. Claim 66 depends from claim 62, which recites that the at least one functional acyltransferase gene encodes for an acyltransferase enzyme that catalyzes transesterification of the sn3 OH group, the sn2 OH group, or the sn1 OH group of a triacylglyceride precursor with a fatty acid. It is unclear whether claim 66 is further limiting the transesterification step recited in claim 62 or whether claim 66 is limiting the method by requiring an additional method step. It is further unclear whether this limitation refers to the structure of a precursor of the fatty acid in a previous step in the process. For example, the enzyme tes hydrolyzes acyl-ACP to a fatty acid. Acyl-ACP is a fatty acid precursor esterified with acyl carrier protein.
Claim 72 recites one or more of i) deletion of the entire coding sequence, ii) deletion of the promoter of the gene, iii) a frameshift mutation, iv) a nonsense mutation, v) a point mutation, vi) a deletion, vii) or an insertion. Claim 72 is indefinite because the claim does not present a closed group of alternatives (e.g. “one of more of i), ii), iii), iv), v), and vi)”). Furthermore, the placement of “or” after vii) and the absence of a conjunction before vii) further renders the claim indefinite.
Claim 74 recites wherein said engineered bacteria uses CO2 as its sole carbon source and/or said engineered bacteria uses H2 as its sole energy source. It is unclear whether this limitation is further limiting step a) of the method of claim 40 by requiring that the culture medium comprises CO2 and H2, by requiring that the culture medium consists of CO2 or H2, or is simply reciting a characteristic of the engineered bacterium.
Claim 79 is indefinite because the claim does not end in a period, thus the metes and bounds of the claim are undefined.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 73-74 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 73 does not further limit the subject matter of the claim upon which it depends because claim 40 already recites that the engineered bacterium is Cupriavidus necator, so the bacteria is already necessarily a chemoautotroph.
Claim 74 recites wherein said engineered bacteria uses CO2 as its sole carbon source and/or said engineered bacteria uses H2 as its sole energy source. When the claim is interpreted as only reciting a characteristic of the engineered bacteria, then the claim does not further limit the engineered bacteria because claim 40 already recites that the bacteria is an engineered Cupriavidus necator bacterium, which necessarily has these characteristics.
Applicant may cancel the claims, amend the claims to place the claim in proper dependent form, rewrite the claims in independent form, or present a sufficient showing that the dependent claims complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 40, 62-64, 66-74, 76, and 78 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502).
Chen teaches an engineered Ralstonia eutropha H16 (synonym for Cupriavidus necator) comprising a heterologous medium-chain-length-specific acyl-ACP thioesterase that produces fatty acids (Abstract). Chen cultures the engineered bacteria in rich broth (RB) to produce fatty acids (Figure 2A and 3A captions). The rich broth contains nutrient broth (bottom paragraph on page 3), which is a source of fatty acids as evidenced by Hardy Diagnostics (Summary paragraph 2). Chen also cultures the engineered Ralstonia eutropha H16 in culture medium comprising fructose; see Figure 4B. Chen also teaches knocking out PHB synthesis in the engineered Ralstonia eutropha (Abstract). Chen teaches that Ralstonia eutropha H16 encodes a fadD homologue (Figure 1 and caption).
Chen does not teach that the engineered Ralstonia eutropha H16 comprises an exogenous copy of an acyl transferase, specifically, a wax ester synthase/acyl-coenzyme A:diacylglycerolacyltransferase (WS/DGAT).
Chen does not teach that the engineered Ralstonia eutropha H16 comprises an exogenous phosphatidic acid phosphatase (PAP).
Chen does not teach culturing the engineered Ralstonia eutropha H16 to produce triacylglycerides or isolating the triacylglycerides.
Xu teaches a heterologous pathway for the biosynthesis of triacylglycerides in E. coli comprising the genes PAP, WS/DGAT (wax synthase/diglycerideacyltransferase hybrid), ACS, and MCFA specific TE (Fig. 1). Xu teaches that triacylglycerides rich in MCFA are of particular interest due to their lower freezing point and higher carbon conversion yield. (Intro Background, paragraph spanning left and right column on page 1).
Xu teaches bacterial WS/DGAT enzymes, including AtfA from Acinetobacter baylyi (page 2, left column, paragraph 2) as well as bacterial PAPs, including R. opacus (page 2, left column, paragraph 3).
Xu teaches that only a few bacterial PAPs have been characterized for TAG synthesis, which include PAP from R. jostii and PAP from R. opacus (page 2, left column, bottom paragraph). The heterologous expression in E. coli of WS/DGAT from Acinetobacter baylyi and PAP from R. opacus increases TAG yield in E. coli (page 3, left column, bottom paragraph).
Xu teaches isolating lipids (includes triacylglycerides) from the cell cultures (Lipid analysis page 10, right column, bottom paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify Chen’s engineered Ralstonia eutropha H16 by introducing AtfA from Acinetobacter baylyi and R. opacus PAP in order to convert the free fatty acids into triacylglycerides. The person of ordinary skill in the art would have been motivated by the teaching of Xu, which suggests that TAGs rich in MCFA are valuable. The person of ordinary skill in the art would have had a reasonable expectation of success in introducing AtfA from Acinetobacter baylyi encoding WS/DGAT and R. opacus PAP into Ralstonia eutropha H16 given that Ralstonia eutropha H16 is a bacteria, like Acinetobacter baylyi and R. opacus, and Ralstonia eutropha H16 is capable of expressing heterologous genes, as demonstrated by Chen. Furthermore, the person of ordinary skill in the art would have had a reasonable expectation of success in the production of triacylglycerides given that Chen already demonstrates that Ralstonia eutropha H16 is capable of producing fatty acids and Xu teaches that from fatty acids, only the enzymes ACS (FadD), WS/DGAT, and PAP are necessary to convert fatty acids to triacylglycerides (see Xu Fig. 1). Furthermore, Chen teaches that Ralstonia eutropha H16 encodes a FadD homologue and phosphatidic acid (substrate for PAP) is necessarily present in the bacteria R. eutropha because it is a key intermediate in bacterial membrane phospholipid synthesis as evidenced by Yao (see Abstract and Highlights bullet point 1).
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Chen Figure 1.
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Xu et al. Figure 1.
It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to collect the triacylglycerides from the culture medium per the teaching of Xu. The person of ordinary skill in the art would have had a reasonable expectation of success in collecting the TAGs given that Xu’s method is appropriate for collecting TAGs from a bacterial culture medium.
Regarding claim 62-63, Xu’s WS/DGAT catalyzes transesterification of the sn3 OH group of diacylglycerol with a fatty acid. See Xu Figure 1.
Regarding claim 64 and pertaining to embodiment a), Xu teaches bacterial WS/DGAT enzymes, including AtfA from Acinetobacter baylyi (page 2, left column, paragraph 2).
Regarding claim 66, the claim is interpreted as a structural limitation of a fatty acid precursor in the method of producing triacylglycerides. Chen teaches that the fatty acid precursor is esterified with ACP prior to the reaction catalyzed by the enzyme tes (see Chen Figure 1 and caption: “acyl-ACP thioesterase (test) enables the hydrolysis of acyl-ACPs and release of fatty acids,” so “acyl-ACP” necessarily contains a thioester bond between the acyl group and ACP).
Regarding claims 67-69, Xu teaches R. opacus PAP and R. jostii PAP (page 3, left column, bottom paragraph), which are both enzymes that catalyze dephosphorylation at the sn3 position of phosphatidic acid (see Figure 1 of Xu).
Chen does not teach that the engineered Ralstonia eutropha further comprises R. jostii PAP.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method of Chen and Xu by heterologously expressing R. jostii PAP in the engineered Ralstonia eutropha. The person of ordinary skill in the art would have been motivated to further increase the supply of diacylglycerol available for conversion into TAGs by expressing another bacterial PAP in the engineered bacterium. The person of ordinary skill in the art would have had a reasonable expectation of success given that R. jostii is a bacterial enzyme and R. eutropha is capable of expressing heterologous bacterial enzymes, as demonstrated by Chen.
Regarding claims 70-72, Chen teaches deactivating the PHB (polyhydroxybutyrate, which is a specific polyhydroxyalkanoate) synthesis pathway by knocking out (i.e. deletion of the entire coding sequence) the phaCAB gene (first full paragraph on page 10).
Regarding claim 73, Chen teaches that Ralstonia eutropha H16 is a chemolithoautotrophic bacteria (Introduction lines 1-2 on page 1), which is a specific type of chemoautotroph.
Regarding claim 74, the claim is interpreted as reciting a characteristic of the engineered bacterium. Chen teaches that Ralstonia eutropha H16 is capable of using H2 as its sole energy source and CO2 as its sole carbon source (Chen, Introduction, lines 1-2 on page 1).
Regarding claims 76 and 78, Chen teaches the heterologous expression of medium-chain-length-specific acyl-ACP thioesterase UcFatB2 in Ralstonia eutropha H16 results in production of laurate (C12 fatty acid) (Abstract). Chen also teaches that the introduction of medium-chain-length-specific acyl-ACP thioesterase UcFatB2 results in selective production of lauric acid, as laurate production increased by 15 mg/L while all other detected fatty acids increased by less than 1 mg/L (Fig. 2B; page 12, Discussion, paragraph 2). Chen’s modification results in greater than 50% lauric acid (C12). The selection for greater than 50% lauric acid (C12 fatty acid) would have necessarily resulted in total TAGs isolated comprising at least 50% TAGs comprising C12 R-group fatty acids, which are within the claimed range of C4-C18 R-group fatty acids.
Claim 70 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of NCBI (2016, website).
See discussion of Chen and Xu above, which is incorporated into this rejection as well.
Regarding embodiment b) of claim 70, Chen does not teach deactivating an endogenous dgkA. However, Xu teaches that dgkA catalyzes the reverse reaction of PAP (see Figure 1), thus decreasing the supply of diacylglycerols.
NCBI teaches the genes diacylglycerol kinase (dgkA) in Cupriavidus necator: see Name.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to inactivate dgkA in Chen’s engineered Ralstonia eutropha in order to increase the supply of diacylglycerols, thus increasing a precursor in the conversion of fatty acids to TAGs. The person of ordinary skill in the art would have had a reasonable expectation of success given that the sequence of dgkA was known in Ralstonia eutropha.
Claim 77 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further evidenced by Jadhav et al. (J Food Sci Technol. 2023 Aug;60(8):2143-2152).
See discussion of Chen and Xu above, which is incorporated into this rejection as well.
Regarding claim 77, animal triglycerides (animal fats) comprise lauric acid as a constituent fatty acid of the triglyceride as evidenced by Jadhav (page 2145, left column, Sources of medium chain triglycerides, paragraph 1, lines 1-4). Therefore, the triglycerides produced by the method of Chen modified by Xu, which comprise lauric acid as a constituent, are necessarily animal fats.
Claims 65 and 80 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of Lazaro et al. PLoS One 12.4 (2017): e0176520).
See discussion of Chen and Xu above, which is incorporated into this rejection as well.
Chen and Xu do not teach that the WS/DGAT is from Thermomonospora curvata DGAT.
Lazaro teaches heterologously expressing the WS/DGAT from Thermomonospora curvata triggers triglyceride accumulation in E. coli (Title and Abstract). Lazaro teaches that the properties of biofuels, such as fatty acid methyl esters (obtained by chemical transesterification of TAGs with methanol or ethanol), are determined by the length and saturation degree of the acyl chains forming these TAGS (introduction, paragraph 2 on page 2). Lazaro teaches further that the Thermomonospora curvata WS/DGAT shifts the fatty acid profile toward medium unsaturated fatty acids (second section title on page 8). Table 2 of Lazaro illustrates the fatty acid distribution in the TAG fraction, which is 50% C16.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the Acinetobacter baylyi WS/DGAT of Xu with Lazaro’s WS/DGAT in the method of Chen modified by Xu. The person of ordinary skill in the art would have been motivated to control the chain length of the TAGs for further application in the chemical/biofuel sector. The person of ordinary skill in the art would have had a reasonable expectation of success given that WS/DGAT from Thermomonospora curvata is a bacterial enzyme and Ralstonia eutropha H16 is capable of heterologous expression of bacterial enzymes, as Chen demonstrates with thioesterase.
Regarding claim 80, heterologous expression of WS/DGAT from Thermomonospora curvata in the engineered Ralstonia eutropha would have resulted in TAGs isolated comprising at least 50% TAGs comprising C16 R-group fatty acids since Table 2 of Lazaro illustrates that the Thermomonospora curvata WS/DGAT produces TAGs with at least 50% C16 fatty acids.
Claims 64-65 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of Wei et al. (Microbial cell factories 17.1 (2018): 11) and Payá-Milans et al. (Phytochemistry 111 (2015): 27-36).
See discussion of Chen and Xu above, which is incorporated into this rejection as well.
This rejection applies to the embodiment in which the acyltransferase is a functional heterologous Theobroma cacao GPAT gene.
Chen does not teach that the engineered Ralstonia eutropha bacterium further comprises a functional heterologous Theobroma cacao GPAT gene.
Xu teaches that glycerol-3-phosphate acyl transferase (GPAT) converts glycerol-3-phosphate to lysophosphatidic acid, which is a precursor to phosphatidic acid (Figure 1).
Wei teaches the heterologous expression of GPAT and LPAT genes from cocoa in S. cerevisiae increases total fatty acid TAG production and increases cocoa butter lipids (Abstract Results).
Unlike Ralstonia eutropha, which is a prokaryote, S. cerevisiae is a eukaryote.
However, Payá-Milans teaches the heterologous production of a GPAT from sunflower in E. coli (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Chen and Xu by expressing Theobroma cacao (cocoa) GPAT in the engineered Ralstonia eutropha. The person of ordinary skill in the art would have been motivated to further increase the production of TAG, specifically high-value TAGs similar to cocoa butter. The person of ordinary skill in the art would have had a reasonable expectation of success in expressing Theobroma cacao GPAT in the engineered Ralstonia eutropha because Payá-Milans already demonstrated that GPATs from plants can be successfully produced in bacteria.
Claims 64-65 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of Wei et al. (Microbial cell factories 17.1 (2018): 11) and Arroyo-Caro et al. (Plant Science 199 (2013): 29-40).
See discussion of Chen and Xu above, which is incorporated into this rejection as well.
This rejection applies to the embodiment in which the acyltransferase is a functional heterologous Theobroma cacao LPAT gene.
Chen does not teach that the engineered Ralstonia eutropha bacterium further comprises a functional heterologous Theobroma cacao LPAT gene.
Xu teaches that the expression of plant LPAT enzymes increases MCFA content in plants (page 1, right column, bottom paragraph).
Wei teaches the heterologous expression of GPAT and LPAT genes from cocoa in S. cerevisiae increases total fatty acid TAG production and increases cocoa butter lipids (Abstract Results).
Unlike Ralstonia eutropha, which is a prokaryote, S. cerevisiae is a eukaryote.
However, Arroyo-Caro teaches the heterologous production of LPAT from castor seed in E. coli (Abstract, Title, and page 31, right column, bottom paragraph, 2.8. Determination of LPAT enzymatic activity).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Chen and Xu by expressing Theobroma cacao (cocoa) LPAT in the engineered Ralstonia eutropha. The person of ordinary skill in the art would have been motivated to further increase the production of TAGs, specifically high-value TAGs similar to cocoa butter. The person of ordinary skill in the art would have had a reasonable expectation of success in expressing Theobroma cacao LPAT in the engineered Ralstonia eutropha because Arroyo-Caro already demonstrated that LPAT from plants can be successfully produced in bacteria.
Claims 70-72 and 79 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (PeerJ 3 (2015): e1468) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Hardy Diagnostics (2020, website) and by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of Jawed et al. (PloS one 11.7 (2016): e0160035).
This rejection applies to the embodiment of claims 70 and 72 in which the engineered Cupriavidus necator bacterium comprises a functional heterologous thioesterase and an endogenous beta-oxidation gene comprising at least one engineered inactivating modification, which is deletion of the entire coding sequence.
This rejection applies to the embodiment in claim 71 in which the engineered Cupriavidus necator bacterium further comprises a Marvinbryantia formatexigens TE gene.
See discussion of Chen and Xu above, which is incorporated into this rejection as well. Chen does not teach that the engineered Ralstonia eutropha H16 (Cupriavidus necator) further comprises a Marvinbryantia formatexigens TE gene.
Jawed teaches that the thioesterase TesBF from Bryantella formatexigens (synonym for Marvinbryantia formatexigens) is specific mainly towards C6-ACP substrates (Table 2, TesBF column). Jawed heterologously expresses TesBF in E. coli (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace Chen’s UcFatB2 thioesterase with Jawed’s TesBF (Marvinbryantia formatexigens thioesterase) in the engineered Ralstonia eutropha H16. The person of ordinary skill in the art would have been motivated by the teachings of Xu, who suggests that TAGs rich in MCFA (e.g. C6) are valuable, and further, that heterologously expressing thioesterases with specificity toward medium chain length fatty acid precursors results in greater yields of TAGs rich in MCFA. The person of ordinary skill in the art would have been motivated to express Jawed’s TesBF in Ralstonia eutropha because of its specificity towards C6-ACP, which is a medium chain fatty acid precursor. The person of ordinary skill in the art would have had a reasonable expectation of success given that Jawed demonstrates expressing TesBF in E. coli, which is another bacteria.
Regarding embodiment d) of claim 71 and claim 79, Jawed teaches knocking out (i.e. deleting) FadE in order to eliminate beta-oxidation of fatty acids (Figure 4A, ΔFadE, page 11, Role of host fatty acid synthesis and degradation pathway on production of butyric acid, paragraph 2). Jawed teaches that the yield of free fatty acids from C4 to C6 is approximately 50% of the total free fatty acid in the ΔFadE mutant (Figure 4(A).
Chen teaches that Ralstonia eutropha has a gene performing the same function as FadE. See Chen Figure 1 and Xu Figure 1, which illustrates that FadE catalyzes the first enzymatic reaction in the beta-oxidation cycle, which is the dehydrogenation of acyl-CoA.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to knock out the FadE homolog of Ralstonia eutropha in order to eliminate the beta-oxidation cycle, thus increasing the amount of medium chain length fatty acids available for conversion to TAGs in the method of Chen modified by Xu and Jawed. The person of ordinary skill in the art would have had a reasonable expectation of success given that Ralstonia eutropha is genetically tractable and the FadE homolog in Ralstonia eutropha was previously identified (see Figure 1 of Chen).
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 40, 62-74, 76, and 78 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 59 of copending Application No. 18/294,871 (‘871) in view of Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790), Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and Chen et al. (PeerJ 3 (2015): e1468) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502).
Claim 1 of ‘871 is drawn to a system for producing a bioproduct comprising at least one reactor chamber containing at least one solution selected from carbon dioxide, hydrogen, and oxygen, at least one production solution comprising carbon dioxide, hydrogen, and oxygen, wherein the last one reactor chamber contains at least one bacterium that produces a bioporudct.
Claim 59 of ‘871 is drawn to the system of claim 1, wherein the bioproduct is triglyceride (b).
Claim 59 of ‘871 does not recite culturing an engineered Cupriavidus necator bacterium in a culture medium comprising CO2 and H2, wherein the engineered Cupriavidus necator bacterium comprises at least one exogenous copy of at least one functional acyltransferase gene and/or at least one exogenous copy of at least one functional phosphatidic acid phosphatase gene, and isolating, collecting, or concentrating TAGs from the engineered Cupriavidus necator bacterium or the culture medium.
Li teaches an engineered Ralstonia eutropha H16 (synonym for Cupriavidus necator) that produces fatty acids from H2, CO2, and O2 (page 784, right column, paragraph 1). Li engineers the Ralstonia eutropha bacterium as follows: Li overexpresses endogenous acetyl-CoA carboxylase complex and cytoplasmic thioesterase in order to increase the precursor supply of malonyl-CoA and catalyze the hydrolysis of acyl-ACP to release free fatty acids, respectively (page 786, right column, paragraph 2). Li also heterologously expresses the type-I polyketide synthase gene fas and holo-ACP synthase gene acpS from Corynebacterium glutamicum (page 786, right column, paragraph 2). Li also deactivates the PHA synthesis pathway by knocking out the phaC1 gene (page 786, right column, paragraph 1).
Li cultures Ralstonia eutropha H16 in minimal medium with a H2:CO2:O2 gas mixture (page 786, left column, top paragraph).
Li does not teach that the engineered Ralstonia eutropha H16 comprises an exogenous copy of a wax ester synthase/acyl-coenzyme A:diacylglycerolacyltransferase (WS/DGAT).
Li does not teach that the engineered Ralstonia eutropha H16 comprises an exogenous phosphatidic acid phosphatase (PAP).
Xu teaches a heterologous pathway for the biosynthesis of triacylglycerides in E. coli comprising the genes PAP, WS/DGAT (wax synthase/diglycerideacyltransferase hybrid), ACS, and MCFA specific TE (Fig. 1). Xu teaches that triacylglycerides rich in MCFA are of particular interest due to their lower freezing point and higher carbon conversion yield (Intro Background, paragraph spanning left and right column on page 1).
Xu teaches bacterial WS/DGAT enzymes, including AtfA from Acinetobacter baylyi (page 2, left column, paragraph 2) as well as bacterial PAPs, including R. opacus (page 2, left column, paragraph 3).
Xu teaches that only a few bacterial PAPs have been characterized for TAG synthesis, which include PAP from R. jostii and PAP from R. opacus (page 2, left column, bottom paragraph). The heterologous expression in E. coli of WS/DGAT from Acinetobacter baylyi and PAP from R. opacus increases TAG yield in E. coli (page 3, left column, bottom paragraph).
Xu teaches isolating lipids (includes triacylglycerides) from the cell cultures (Lipid analysis (page 10, right column, bottom paragraph).
Li and Xu do not teach that Ralstonia eutropha H16 encodes a fadD homologue (ACS).
Chen teaches an engineered Ralstonia eutropha H16 comprising a heterologous medium-chain-length-specific acyl-ACP thioesterase that produces fatty acids (Abstract). Chen teaches knocking out PHB synthesis (Abstract). Chen teaches that Ralstonia eutropha H16 encodes a fadD homologue (Figure 1 and caption).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify Li’s engineered Ralstonia eutropha H16 by introducing AtfA from Acinetobacter baylyi and R. opacus PAP in order to convert the free fatty acids into triacylglycerides. The person of ordinary skill in the art would have been motivated by the teaching of Xu, who suggests that MCFA-rich TAGs are value-added products. The person of ordinary skill in the art would have had a reasonable expectation of success in introducing AtfA from Acinetobacter baylyi encoding WS/DGAT and R. opacus PAP into Ralstonia eutropha H16 given that Ralstonia eutropha H16 is a bacteria, like Acinetobacter baylyi and R. opacus, and Ralstonia eutropha H16 is capable of expressing heterologous genes, as demonstrated by both Li and Chen. Furthermore, the person of ordinary skill in the art would have had a reasonable expectation of success in the production of triacylglycerides given that Li already demonstrates that Ralstonia eutropha H16 is capable of producing fatty acids and Xu teaches that from fatty acids, only the enzymes ACS (FadD), WS/DGAT, and PAP are necessary to convert fatty acids to triacylglycerides (see Xu Fig. 1). Furthermore, Chen teaches that Ralstonia eutropha H16 encodes a FadD homologue and phosphatidic acid (substrate for PAP) is necessarily present in the bacteria R. eutropha because it is a key intermediate in bacterial membrane phospholipid synthesis as evidenced by Yao (see Abstract and Highlights bullet point 1).
It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to collect the triacylglycerides from the culture medium per the teaching of Xu. The person of ordinary skill in the art would have had a reasonable expectation of success in collecting the TAGs given that Xu’s method is appropriate for collecting TAGs from a bacterial culture medium.
It would have been further obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to culture the engineered Ralstonia eutropha bacterium of Li, Xu, and Chen in the system of claim 59 of ‘571 and the person of ordinary skill in the art would have had a reasonable expectation of success in doing so. The system of claim 59 of ‘571 is specifically designed for producing triglycerides from H2, CO2, and O2, which are the required substrates in the method of Li modified by Xu and Chen.
Regarding claim 62-63, Xu’s WS/DGAT catalyzes transesterification of the sn3 OH group of diacylglycerol with a fatty acid. See Xu Figure 1.
Regarding claim 64 and pertaining to embodiment a), Xu teaches bacterial WS/DGAT enzymes, including AtfA from Acinetobacter baylyi (page 2, left column, paragraph 2).
Regarding claim 66, the claim is interpreted as a structural limitation of a fatty acid precursor in the method of producing triacylglycerides. Li teaches that the fatty acid precursor is esterified with ACP prior to the reaction catalyzed by the enzyme tes (see Li Supplementary Figure 1, “acyl-ACP”).
Regarding claims 67-69, Xu teaches R. opacus PAP and R. jostii PAP (page 3, left column, bottom paragraph), which are both enzymes that catalyze dephosphorylation at the sn3 position of phosphatidic acid (see Figure 1 of Xu).
Claim 59 of ‘871 does not recite and Li does not teach that the engineered Ralstonia eutropha further comprises R. jostii PAP.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method of Li modified by Xu and Chen performed in the system of claim 59 of ‘871 by heterologously expressing R. jostii PAP in the engineered Ralstonia eutropha bacteria. The person of ordinary skill in the art would have been motivated to further increase the supply of diacylglycerol available for conversion into TAGs by expressing another bacterial PAP in the engineered R. eutropha bacterium. The person of ordinary skill in the art would have had a reasonable expectation of success given that R. jostii is a bacterial enzyme and Chen demonstrates heterologous expression of bacterial enzymes in R. eutropha.
Regarding claims 70-72, Li teaches deactivating the PHA synthesis pathway by knocking out the phaC1 gene (page 786, right column, paragraph 1). Li applies a homologous recombination technique in order to completely delete the phaC1 coding sequence (Li Supplementary Figure 2).
Regarding claim 73, Li teaches that Ralstonia eutropha H16 is a chemolithoautotrophic bacteria (page 784, left column, lines 1-2), which is a specific type of chemoautotroph.
Regarding claim 74, the claim is interpreted as reciting a characteristic of the engineered bacterium. Chen teaches that Ralstonia eutropha H16 is capable of using H2 as its sole energy source and CO2 as its sole carbon source (Chen, Introduction, lines 1-2 on page 1).
Regarding claims 76 and 78, claim 59 of ‘871 does not recite and Li does not teach producing animal triglycerides, wherein the total TAGs comprise at least 50% TAGs comprising C4-C18 R-group fatty acids.
Chen teaches the heterologous expression of medium-chain-length-specific acyl-ACP thioesterase UcFatB2 in Ralstonia eutropha H16 results in production of laurate (C12 fatty acid) (Abstract). Chen also teaches that the introduction of medium-chain-length-specific acyl-ACP thioesterase UcFatB2 results in selective production of lauric acid, as laurate production increased by 15 mg/L while all other detected fatty acids increased by less than 1 mg/L (Fig. 2B; page 12, Discussion, paragraph 2). Chen’s modification results in greater than 50% lauric acid (C12).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method of Li, Xu, and Chen performed in the system of claim 59 of ‘871 by adding a step of expressing the exogenous medium-chain-length-specific acyl-ACP thioesterase UcFatB2 in the engineered Ralstonia eutropha H16 in order to increase the precursor supply of medium-chain-length fatty acids and thus produce more medium-chain-length triacylglycerides. The person of ordinary skill in the art would have had a reasonable expectation of success given that Chen demonstrates that expressing medium-chain-length-specific acyl-ACP thioesterase UcFatB2 in Ralstonia eutropha H16 produces laurate, which is a C12 (medium chain length) fatty acid. Furthermore, the selection for greater than 50% lauric acid (C12 fatty acid) would have necessarily resulted in total TAGs isolated comprising at least 50% TAGs comprising C12 R group fatty acids, which are within the claimed range of C4-C18 R-group fatty acids.
This is a provisional nonstatutory double patenting rejection.
Claim 70 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 59 of copending Application No. 18/294,871 (‘871) in view of Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790), Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177), and Chen et al. (PeerJ 3 (2015): e1468) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of NCBI (2016, website).
See discussion of claim 59 of ‘871, Li, Xu, and Chen above, which is incorporated into this rejection as well.
Regarding embodiment b) of claim 70, Chen does not teach deactivating an endogenous dgkA. However, Xu teaches that dgkA catalyzes the reverse reaction of PAP (see Figure 1), thus decreasing the supply of diacylglycerols.
NCBI teaches the gene diacylglycerol kinase (dgkA) in Cupriavidus necator: see Name.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to inactivate dgkA in Li’s engineered Ralstonia eutropha in the method of Li, Xu, and Chen performed in the system of claim 59 of ‘871. The person of ordinary skill in the art would have been motivated to increase the supply of diacylglycerols, thus increasing a required precursor in the conversion of fatty acids to TAGs. The person of ordinary skill in the art would have had a reasonable expectation of success given that the sequence of dgkA was known in Ralstonia eutropha.
This is a provisional nonstatutory double patenting rejection.
Claim 77 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 59 of copending Application No. 18/294,871 (‘871) in view of Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790), Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and Chen et al. (PeerJ 3 (2015): e1468) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further evidenced by Jadhav et al. (J Food Sci Technol. 2023 Aug;60(8):2143-2152).
See discussion of claim 59 of ‘871, Li, Xu, and Chen above, which is incorporated into this rejection as well.
Regarding claim 77, animal triglycerides (animal fats) comprise lauric acid as a constituent fatty acid of the triglyceride as evidenced by Jadhav (page 2145, left column, Sources of medium chain triglycerides, paragraph 1, lines 1-4). Therefore, the triglycerides produced by the method of Li modified by Xu and Chen performed in the system of claim 59 of ‘871, which comprise lauric acid as a constituent, are necessarily animal fats.
This is a provisional nonstatutory double patenting rejection.
Claims 65 and 80 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 59 of copending Application No. 18/294,871 (‘871) in view of Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790), Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and Chen et al. (PeerJ 3 (2015): e1468) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of Lazaro et al. PLoS One 12.4 (2017): e0176520).
See discussion of claim 59 of ‘871, Li, Xu, and Chen above, which is incorporated into this rejection as well.
Claim 59 of ‘871 does not recite and Li, Xu, and Chen do not teach that the WS/DGAT is from Thermomonospora curvata DGAT.
Lazaro teaches heterologously expressing the WS/DGAT from Thermomonospora curvata triggers triglyceride accumulation in E. coli (Title and Abstract). Lazaro teaches that the properties of biofuels, such as fatty acid methyl esters (obtained by chemical transesterification of TAGs with methanol or ethanol), are determined by the length and saturation degree of the acyl chains forming these TAGS (introduction, paragraph 2 on page 2). Lazaro teaches further that the Thermomonospora curvata WS/DGAT shifts the fatty acid profile toward medium unsaturated fatty acids (second section title on page 8). Table 2 of Lazaro illustrates the fatty acid distribution in the TAG fraction, which is 50% C16.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the Acinetobacter baylyi WS/DGAT of Xu with Lazaro’s WS/DGAT in the method of Li modified by Xu and Chen performed in the system of claim 59 of ‘871. The person of ordinary skill in the art would have been motivated to control the chain length of the TAGs for further application in the chemical/biofuel sector. The person of ordinary skill in the art would have had a reasonable expectation of success given that WS/DGAT from Thermomonospora curvata is a bacterial enzyme and Ralstonia eutropha H16 is capable of heterologous expression of bacterial enzymes, as Chen demonstrates with thioesterase.
Regarding claim 80, heterologous expression of WS/DGAT from Thermomonospora curvata in the engineered Ralstonia eutropha would have resulted in TAGs isolated comprising at least 50% TAGs comprising C16 R-group fatty acids since Table 2 of Lazaro illustrates that the Thermomonospora curvata WS/DGAT produces TAGs with at least 50% C16 fatty acids.
This is a provisional nonstatutory double patenting rejection.
Claims 64-65 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 59 of copending Application No. 18/294,871 (‘871) in view of Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790), Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and Chen et al. (PeerJ 3 (2015): e1468) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-74, 76, and 78 above, further in view of Wei et al. (Microbial cell factories 17.1 (2018): 11) and Payá-Milans et al. (Phytochemistry 111 (2015): 27-36).
See discussion of claim 59 of ‘871, Li, Xu, and Chen above, which is incorporated into this rejection as well.
This rejection applies to the embodiment in which the acyltransferase is a functional heterologous Theobroma cacao GPAT gene.
Claim 59 of ‘871 does not recite and Li does not teach that the engineered Ralstonia eutropha further comprises a functional heterologous Theobroma cacao GPAT gene.
Wei teaches the heterologous expression of GPAT and LPAT genes from cocoa in S. cerevisiae increases total fatty acid TAG production and increases cocoa butter lipids (Abstract Results).
Unlike Ralstonia eutropha, which is a prokaryote, S. cerevisiae is a eukaryote.
However, Payá-Milans teaches the heterologous production of a GPAT from sunflower in E. coli (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of Li, Xu, and Chen performed in the system of claim 59 of ‘871 by expressing Theobroma cacao (cocoa) GPAT in the engineered Ralstonia eutropha. The person of ordinary skill in the art would have been motivated to further increase the production of TAGs, specifically high-value TAGs similar to cocoa butter. The person of ordinary skill in the art would have had a reasonable expectation of success in expressing Theobroma cacao GPAT in the engineered Ralstonia eutropha because Payá-Milans already demonstrated that GPATs from plants can be successfully produced in bacteria.
This is a provisional nonstatutory double patenting rejection.
Claims 64-65 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 59 of copending Application No. 18/294,871 (‘871) in view of Li et al. (J