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, 62-72, 74-80 are pending. Claims 1-39 and 41-61 were cancelled previously. Claim 73 is newly cancelled. Claim 75 is withdrawn. Claims 40, 62-72, 74, and 76-80 are under examination on their merits.
Response to Arguments
Applicant's arguments filed 6/5/2026 have been fully considered but they are not persuasive.
Applicant argues against the rejection of claims under 35 U.S.C. 103 on the grounds that Chen and Xu do not teach that the culture medium comprises CO2 as the sole carbon source (Arguments, paragraph 2 on page 9).
In response, this argument is unpersuasive because Li is relied on to teach the culture medium comprising CO2 as the sole carbon source.
Applicant also argues that Yao does not specifically discuss the presence of phosphatidic acid in Cupravidus necator (Ralstonia eutropha) (Arguments, paragraph 3 on page 11).
In response, this argument is unpersuasive because 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). Cupravidus necator as a Gram-negative bacteria necessarily has an inner phospholipid membrane bilayer and thus phosphatidic acid is necessarily present in Cupravidus necator as it is required for the synthesis of the membrane bilayer.
Applicant argues against the nonstatutory double patenting rejection of the instant claims over claim 59 of copending Application No. 18/294,871 on the grounds that the instant application has the earlier patent term filing date and thus the provisional nonstatutory double patenting rejection should be withdrawn (Arguments, paragraph 4 on page 15).
In response, it would be improper to withdraw the provisional nonstatutory double patenting rejection unless it is the only remaining rejection. See MPEP 804(I)(B)(1)(b). Here, the claims are also rejected under 35 U.S.C. 103, thus the nonstatutory double patenting rejections are not the only remaining rejections.
Claim Rejections - 35 USC § 112
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.
(New Rejection Necessitated by the Amendment) Claim 74 is 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 74 does not further limit the culture medium recited in claim 40, upon which claim 74 depends. Applicant may consider amending the claim to recite the culture medium further comprises H2 as the sole energy source.
Applicant may cancel the claim, amend the claim to place the claim in proper dependent form, rewrite the claim in independent form, or present a sufficient showing that the dependent claim 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.
The following rejections are necessitated by the amendment.
Claims 40, 62-64, 66-72, 73, 76, and 78 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790; cited in the Non-Final Action mailed on 12/18/2025) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177; cited in the Non-Final Action mailed on 12/18/2025) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502; cited in the Non-Final Action mailed on 12/18/2025).
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 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 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 an acyl transferase, specifically, 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).
Li 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 an engineered E. coli comprising the genes PAP, WS/DGAT (wax synthase/diglycerideacyltransferase hybrid), ACS (acyl-CoA synthetase), 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 Li’s engineered Ralstonia eutropha H16 by introducing AtfA from Acinetobacter baylyi (exogenous acyltransferase) and R. opacus PAP (exogenous phosphatidic acid phosphatase) 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 Li. 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, WS/DGAT, and PAP are necessary to convert fatty acids to triacylglycerides (see Xu Fig. 1).
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Xu et al. Figure 1.
Furthermore, 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 because Xu discloses collecting TAGs from an engineered bacterial culture medium.
Regarding claim 62-63, Xu’s WS/DGAT (exogenous actyltransferase) 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, 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).
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 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 Li.
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 74, Li teaches culturing R. eutropha with CO2 as the sole carbon source and H2 as the sole electron donor (Abstract). Although O2 is present in Li’s medium, oxygen is an electron acceptor, not an electron donor (energy source).
Regarding claims 76 and 78, Li teaches that the engineered Ralstonia eutropha H16 produces greater than 50% C16 and C18 fatty acids (Fig. 3 E). The selection for greater than 50% C14, C16, and C18 fatty acids would have necessarily resulted in total TAGs isolated comprising at least 50% TAGs comprising C16 and C18 R group fatty acids in the method of Li modified by Xu. C14, C16 and C18 are all 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 Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 74, 76, and 78 above, further in view of NCBI (2016, website).
See discussion of Li, and Xu above, which is incorporated into this rejection as well.
Regarding embodiment b) of claim 70, Li 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 Li’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 Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 74, 76, and 78 above, further evidenced by Jadhav et al. (J Food Sci Technol. 2023 Aug;60(8):2143-2152).
See discussion of Li 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 Li 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 Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 74, 76, and 78 above, further in view of Lazaro et al. PLoS One 12.4 (2017): e0176520).
See discussion of Li and Xu above, which is incorporated into this rejection as well.
Li, 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 Li 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 Li 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 and 81 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 4, 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 Li 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.
Li 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 Li 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 Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 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 Li 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.
Li 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 Li 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 70-71 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 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 claim 70 in which the engineered Cupriavidus necator bacterium comprises a functional heterologous thioesterase.
This rejection applies to the embodiment (a) in claim 71 in which the engineered Cupriavidus necator bacterium further comprises a Marvinbryantia formatexigens TE gene.
See discussion of Li and Xu above, which is incorporated into this rejection as well.
Li 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 express Jawed’s TesBF (Marvinbryantia formatexigens thioesterase) in Li’s 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 lengths of 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. 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.
Claims 70-72 and 79 are rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790), Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177), and Jawed et al. (PloS one 11.7 (2016): e0160035) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 70-71 above, further in view of Chen et al. (PeerJ 3 (2015): e1468).
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 (embodiment (a)) and the gene FadE is deleted (embodiment (d)).
See discussion of Li, Xu, and Jawed above, which is incorporated into this rejection as well.
Li does not teach knocking out the gene FadE in the engineered Ralstonia eutropha.
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.
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).
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 Li 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).
Regarding 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).
Claim 82 is rejected under 35 U.S.C. 103 as being unpatentable over Li et al. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790; cited in the Non-Final Action mailed on 12/18/2025) in view of Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177; cited in the Non-Final Action mailed on 12/18/2025) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502; cited in the Non-Final Action mailed on 12/18/2025), as applied to claims 40, 62-64, 66-72, 73, 76, and 78 above, further in view of Riedel et al. (Applied microbiology and biotechnology 98.4 (2014): 1469-1483).
See discussion of Li and Xu above, which is incorporated into this rejection as well.
Li does not teach that the engineered Ralstonia eutropha comprises endogenous fadB with an engineered inactivating modification.
Xu teaches that FadB is part of the beta-oxidation cycle that takes away acyl-CoA from the production of triacylglycerol (Fig. 1).
Riedel teaches deleting fadB’ in R. eutropha (page 1470, right column, paragraph 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to delete the fadB’ in the engineered R. eutropha of Li modified by Xu in order to minimize the flux of acyl-CoA entering the beta-oxidation cycle. The person of ordinary skill in the art would have been motivated to increase the flux of acyl-CoA to fatty acids. The person of ordinary skill in the art would have had a reasonable expectation of success in deleting fadB’.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
The following rejections are necessitated by the amendment.
Claims 40, 62-72, 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and 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 at least 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).
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. 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, WS/DGAT, and PAP are necessary to convert fatty acids to triacylglycerides (see Xu Fig. 1). Furthermore, 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, and Xu 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 .
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, 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 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 Li 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 74, Li cultures Ralstonia eutropha H16 in minimal medium with a H2:CO2:O2 gas mixture (page 786, left column, top paragraph).
Regarding claims 76 and 78, Li teaches that the engineered Ralstonia eutropha H16 produces greater than 50% C16 and C18 fatty acids (Fig. 3 E). The selection for greater than 50% C14, C16, and C18 fatty acids would have necessarily resulted in total TAGs isolated comprising at least 50% TAGs comprising C16 and C18 R group fatty acids in the method of Li modified by Xu. C14, C16 and C18 are all 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 74, 76, and 78 above, further in view of NCBI (2016, website).
See discussion of claim 59 of ‘871, Li, and Xu above, which is incorporated into this rejection as well.
Regarding embodiment b) of claim 70, Li 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 and Xu 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 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 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 Li modified by Xu 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 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 and Xu above, which is incorporated into this rejection as well.
Claim 59 of ‘871 does not recite and Li 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 Li modified by Xu 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 Li 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 and 81 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 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 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.
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 L and Xu 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. (Journal of Industrial Microbiology & Biotechnology (2019) 46:783–790) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 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 claim 59 of ‘871, Li 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.
Claim 59 of ‘871 does not recite and Li and Xu do not teach that the engineered Ralstonia eutropha further comprises a functional heterologous Theobroma cacao LPAT 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, 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 Li and Xu performed in the system of claim 59 of ‘871 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.
This is a provisional nonstatutory double patenting rejection.
Claims 70-71 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502)., as applied to claims 40, 62-72, 74, 76, and 78 above, further in view of Jawed et al. (PloS one 11.7 (2016): e0160035).
See discussion of claim 59 of ‘871, Li and Xu above, which is incorporated into this rejection as well.
This rejection applies to the embodiment of claim 70 in which the engineered Cupriavidus necator bacterium comprises (a) a functional heterologous thioesterase.
This rejection applies to the embodiment in claim 71 in which the engineered Cupriavidus necator (synonym for Ralstonia eutropha) bacterium further comprises a Marvinbryantia formatexigens TE gene.
Claim 59 of ‘871 does not recite and Li and Xu do not teach that the engineered Ralstonia eutropha bacterium further comprises a Marvinbryantia formatexigens TE gene.
Xu teaches expressing castor thioesterase RcFatB in E. coli to tailor fatty acid chain length (Abstract Results). Xu teaches that RcFatB is a medium chain-specific TE gene (page 5, right column, Overexpression of the castor TE gene RcFatB-triggered MCFA flux into TAG backbones). 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).
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 modify the method of Li and Xu performed in the system of claim 59 of ‘871 by expressing Jawed’s TesBF (Marvinbryantia formatexigens thioesterase) in Li’s 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 lengths of 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. 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.
This is a provisional nonstatutory double patenting rejection.
Claims 70-72 and 79 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 Jawed et al. (PloS one 11.7 (2016): e0160035) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502)., as applied to claims 70-71 above, further in view of Chen et al. (PeerJ 3 (2015): e1468).
See discussion of claim 59 of ‘871, Li, and Xu above, which is incorporated into this rejection as well.
This rejection applies to the embodiment of claims 70 and 72 in which the engineered Cupriavidus necator bacterium comprises (a) a functional heterologous thioesterase and (d) 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 (embodiment (a)) and the gene FadE is deleted (embodiment (d)).
Regarding claim 79, claim 59 of ‘871 does not recite and Li and Xu do not teach an engineered Ralstonia eutropha in which the FadE gene has been deleted.
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.
Jawed teaches knocking out 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).
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 knock out the FadE homolog in 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 Li modified by Xu and Jawed performed in the system of claim 59 of ‘871. 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).
Regarding 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).
This is a provisional nonstatutory double patenting rejection.
Claim 82 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) and Xu et al. (Biotechnology for Biofuels 11.1 (2018): 177) and as evidenced by Yao et al. (Biochim Biophys Acta. 2013 Mar;1831(3):495-502), as applied to claims 40, 62-72, 74, 76, and 78 above, further in view of Riedel et al. (Applied microbiology and biotechnology 98.4 (2014): 1469-1483).
See discussion of claim 59 of ‘871, Li and Xu above, which is incorporated into this rejection as well.
Claim 59 of ‘871 does not recite and Li does not teach that the engineered Ralstonia eutropha comprises endogenous fadB with an engineered inactivating modification.
Xu teaches that FadB is part of the beta-oxidation cycle that takes away acyl-CoA from the production of triacylglycerol (Fig. 1).
Riedel teaches deleting fadB’ in R. eutropha (page 1470, right column, paragraph 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to delete the fadB’ in the engineered R. eutropha of claim 59 of ‘871 modified by Li and Xu in order to minimize the flux of acyl-CoA entering the beta-oxidation cycle. The person of ordinary skill in the art would have been motivated to increase the flux of acyl-CoA to fatty acids. The person of ordinary skill in the art would have had a reasonable expectation of success in deleting fadB’.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657
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