DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
After further consideration, the restriction requirement as set forth in the Office action mailed on 3/5/2026 is hereby withdrawn.
Claims 1-9 are pending and have been considered on the merits herein.
Claim Objections
Claims 6 and its dependent claims 7-9 are objected to because of the following informalities: claim 6 depends from withdrawn claim 3. Appropriate correction is required.
Claim Rejections - 35 USC § 102
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 (i.e., changing from AIA to pre-AIA ) 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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1, 2 is/are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Miyagawa et al. (US20150211033, IDS) supported by https://pubchem.ncbi.nlm.nih.gov/compound/Linoleic-acid#section=Crystal-Structures.
Miyagawa et al. (US20150211033) teaches a method for culturing a microorganism comprising culturing the microorganism in the presence of a carbon source, wherein the microorganism is a transformed microorganism comprising an introduced gene encoding a protein comprising the amino acid sequence of SEQ ID NO:1.
A sequence search conducted by the Office finds that SEQ ID NO:4 of Miyagawa has 100% sequence identity to SEQ ID NO:1.
Query Match 100.0%; Score 3468; Length 672;
Best Local Similarity 100.0%;
Matches 672; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSYPSLFAPLDLGFTTLKNRVLMGSMHTGLEEYPDGAERLAAFYAERARHGVALIVSGGI 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSYPSLFAPLDLGFTTLKNRVLMGSMHTGLEEYPDGAERLAAFYAERARHGVALIVSGGI 60
Qy 61 APDLTGVGMEGGAMLNDASQIPHHRTITEAVHQEGGKIALQILHTGRYSYQPHLVAPSAL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 APDLTGVGMEGGAMLNDASQIPHHRTITEAVHQEGGKIALQILHTGRYSYQPHLVAPSAL 120
Qy 121 QAPINRFVPHELSHEEILQLIDNFARCAQLAREAGYDGVEVMGSEGYLINEFLTLRTNQR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 QAPINRFVPHELSHEEILQLIDNFARCAQLAREAGYDGVEVMGSEGYLINEFLTLRTNQR 180
Qy 181 SDQWGGDYRNRMRFAVEVVRAVRERVGNDFIIIYRLSMLDLVEDGGTFAETVELAQAIEA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SDQWGGDYRNRMRFAVEVVRAVRERVGNDFIIIYRLSMLDLVEDGGTFAETVELAQAIEA 240
Qy 241 AGATIINTGIGWHEARIPTIATPVPRGAFSWVTRKLKGHVSLPLVTTNRINDPQVADDIL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AGATIINTGIGWHEARIPTIATPVPRGAFSWVTRKLKGHVSLPLVTTNRINDPQVADDIL 300
Qy 301 SRGDADMVSMARPFLADAELLSKAQSGRADEINTCIGCNQACLDQIFVGKVTSCLVNPRA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 SRGDADMVSMARPFLADAELLSKAQSGRADEINTCIGCNQACLDQIFVGKVTSCLVNPRA 360
Qy 361 CHETKMPILPAVQKKNLAVVGAGPAGLAFAINAAARGHQVTLFDAHSEIGGQFNIAKQIP 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CHETKMPILPAVQKKNLAVVGAGPAGLAFAINAAARGHQVTLFDAHSEIGGQFNIAKQIP 420
Qy 421 GKEEFYETLRYYRRMIEVTGVTLKLNHTVTADQLQAFDETILASGIVPRTPPIDGIDHPK 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GKEEFYETLRYYRRMIEVTGVTLKLNHTVTADQLQAFDETILASGIVPRTPPIDGIDHPK 480
Qy 481 VLSYLDVLRDKAPVGNKVAIIGCGGIGFDTAMYLSQPGESTSQNIAGFCNEWGIDSSLQQ 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 VLSYLDVLRDKAPVGNKVAIIGCGGIGFDTAMYLSQPGESTSQNIAGFCNEWGIDSSLQQ 540
Qy 541 AGGLSPQGMQIPRSPRQIVMLQRKASKPGQGLGKTTGWIHRTTLLSRGVKMIPGVSYQKI 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AGGLSPQGMQIPRSPRQIVMLQRKASKPGQGLGKTTGWIHRTTLLSRGVKMIPGVSYQKI 600
Qy 601 DDDGLHVVINGETQVLAVDNVVICAGQEPNRALAQPLIDSGKTVHLIGGCDVAMELDARR 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 DDDGLHVVINGETQVLAVDNVVICAGQEPNRALAQPLIDSGKTVHLIGGCDVAMELDARR 660
Qy 661 AIA QGTRLALEI 672
||||||||||||
Db 661 AIA QGTRLALEI 672
Regarding claim 1, the microorganism is E. coli which has been transformed to include the fadH gene encoding 2,4-dienoyl-CoA reductase (of SEQ ID NO:1), wherein the E. coli has increased 2,4-dienoyl-CoA reductase activity (0009, 0012-0021, 0142-0151, Ex. 1 and 2, 0266-0284).
The carbon source is linoleic acid, which is a carbon source comprising an oil containing an unsaturated fatty acid or comprises a free unsaturated fatty acid according to claim 1 (0011-0014).
Regarding claim 2, linoleic acid is known to have an iodine value of 181, see p. 39 at https://pubchem.ncbi.nlm.nih.gov/compound/Linoleic-acid#section=Crystal-Structures.
Thus, the reference anticipates the claimed subject matter.
Claim(s) 1, 2 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by He et al. (Eur. J. Biochem, 1997, IDS) supported by Sigma-Aldrich Ethyloleate (mixture of esters), stabilised, for synthesis 85049-36-1.
He teaches a method for culturing a microorganism comprising culturing the microorganism in the presence of a carbon source, wherein the microorganism is a transformed microorganism comprising an introduced gene encoding a protein comprising the amino acid sequence of SEQ ID NO:1.
The sequence of He (Fig.1) has 100% sequence identity to SEQ ID NO:1, as the sequence is that of the fadH gene.
Regarding claim 1, the microorganism is E. coli which has been transformed to include the fadH gene encoding 2,4-dienoyl-CoA reductase (p. 517, Overexpression of the cloned fadH gene section).
He teaches culturing the transformed E. coli in the presence of oleate as a carbon source (p. 518, Overproduction and purific. Section-p. 519 ).
Regarding claim 2, oleate is known to have an iodine value between 75-85, see Ethyloleate (mixture of esters), stabilised, for synthesis 85049-36-1.
Thus, the reference anticipates the claimed subject matter.
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 (i.e., changing from AIA to pre-AIA ) 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.
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.
Claim(s) 1-9 is/are rejected under 35 U.S.C. 103 as being unpatentable over the combination of Zhila et al. (Proc. Biochem, 2015, IDS), Riedel et al (Appl. Microb., Biotech, vol. 98, p.1469-1483, 2014), Kutralam-Muniasamy et al. (W. J. microb., and biotech., 2018, IDS) in view of Miyagawa et al. (US20150211033) and He et al. (Eur. J. Biochem, 1997, IDS) and Feng et al (J. Bacter., p. 4289-4299, 2010).
Regarding claims 6-9, Zhila teaches a Cupriavidus (formerly Ralstonia eutropha) strain comprising a gene encoding PHA synthase, which when cultured in the presence of oleic acid or sunflower seed oil, i.e. a carbon source comprising an oil containing unsaturated fatty acid or comprises a free unsaturated fatty acid, produces fatty acids and a poly(3-hydrooxyalkanoate), specifically 3-hydroxybutyric acid, 3-hydroxyvalerate and 3-hydroxyhexanoate according to claims 7-9 (p. 75, 2nd col., 1st full parag., Table 5). PHA’s are produced via β-oxidation of fatty acids and Cupriavidus are natural producers of PHA’s as they all genes necessary for fatty acid synthesis via the β-oxidation pathway (intro.). However, Zhila teaches that sunflower oil comprises 54% linoleic acid, which may inhibit PHA synthesis due to the inhibiting effect of unsaturated fatty acids (which becomes stronger as the double bonds of the molecule increases).
Riedel teach that the major precursor of fatty acid biosynthesis is acetyl-CoA, which is also the major product of β-oxidation, i.e. fatty acid degradation. Ralstonia eutropha (renamed Cupriavidus) is a native producer of PHA’s including PHB’s, and it utilizes acetyl-CoA from fatty acid metabolism to produce PHA’s. The microorganism can be engineered to produce a variety of PHA’s (abstract, Intro. p. 1469-1470, whole page). Riedel teaches the β-oxidation cycle which reduces fatty acyl chains in a cycle by removing a two-carbon acetyl-CoA group at every turn of the cycle, finally producing acetyl-CoA, which is the precursor for PHA and PHB synthesis in R. eutropha (p. 1470, Lipid and fatty acid met. Section, Fig. 1). Riedel teaches that recombinant strains of R. eutropha can produce PHA precursors including (R)HA-CoA from β-oxidation and fatty acid synthesis using the phaJ gene encoding a hydratase catalyzing the conversion of CoA’s to (R)3-HA-CoA.
Regarding claim 5, Kutralam-Muniasamy (K-M) teach the Cupriavidus necator H16 strain to be one which comprises a gene encoding a poly(3-hydroxyalkanoate) synthase gene (p. 2-3, Cupriavidus necator section-p. 79). K-M teaches that C. necator also contains operons encoding genes for fatty acid oxidation to produce PHA’s and PHB’s and genes encoding hydratases for the conversion of (S)HA-CoA to (R )-3HA-CoA, which they believe could participate in the unsaturated fatty acid oxidation pathway; however, the phaJ genes are identified to have an affinity for medium chain length fatty acids and have the ability to generate (R )-3HA-CoA, which can be used in engineering strategies. K-M suggests that 2,4-dienoyl-coA reductase fadH is required to convert (S)-3-HA CoA to R-3-HA CoA (unsaturated fatty acid oxidation enzyme), which increases PHA and PHB synthesis (P. 6, whole second col. ) yet not identified in C. necator.
The above references are relied upon for teach microorganisms having a gene encoding a PHA synthase and when cultured in the presence of a carbon source comprising an oil containing unsaturated fatty acid or comprises a free unsaturated fatty acid, produces fatty acids and a poly(3-hydrooxyalkanoate) via β-oxidation.
Regarding claims 3-9, the references do not teach a transformed microorganism comprising both a PHA synthase gene and an introduced foreign gene encoding the protein of SEQ ID NO:1.
Miyagawa teaches a transformed microorganism, i.e. E. coli, which has been transformed to include the fadH gene encoding 2,4-dienoyl-CoA reductase (of SEQ ID NO:1), wherein the E. coli has increased 2,4-dienoyl-CoA reductase activity (0009, 0012-0021, 0142-0151, Ex. 1 and 2, 0266-0284). Miyagawa teaches a method for culturing a microorganism comprising culturing the microorganism in the presence of a carbon source, i.e., linoleic acid, which is a carbon source comprising an oil containing an unsaturated fatty acid or comprises a free unsaturated fatty acid according to claim 1 (0011-0014).
The microorganism is a transformed microorganism comprising an introduced gene encoding a protein comprising the amino acid sequence of SEQ ID NO:1.
A sequence search conducted by the Office finds that SEQ ID NO:4 of Miyagawa has 100% sequence identity to SEQ ID NO:1.
Query Match 100.0%; Score 3468; Length 672;
Best Local Similarity 100.0%;
Matches 672; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MSYPSLFAPLDLGFTTLKNRVLMGSMHTGLEEYPDGAERLAAFYAERARHGVALIVSGGI 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSYPSLFAPLDLGFTTLKNRVLMGSMHTGLEEYPDGAERLAAFYAERARHGVALIVSGGI 60
Qy 61 APDLTGVGMEGGAMLNDASQIPHHRTITEAVHQEGGKIALQILHTGRYSYQPHLVAPSAL 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 APDLTGVGMEGGAMLNDASQIPHHRTITEAVHQEGGKIALQILHTGRYSYQPHLVAPSAL 120
Qy 121 QAPINRFVPHELSHEEILQLIDNFARCAQLAREAGYDGVEVMGSEGYLINEFLTLRTNQR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 QAPINRFVPHELSHEEILQLIDNFARCAQLAREAGYDGVEVMGSEGYLINEFLTLRTNQR 180
Qy 181 SDQWGGDYRNRMRFAVEVVRAVRERVGNDFIIIYRLSMLDLVEDGGTFAETVELAQAIEA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 SDQWGGDYRNRMRFAVEVVRAVRERVGNDFIIIYRLSMLDLVEDGGTFAETVELAQAIEA 240
Qy 241 AGATIINTGIGWHEARIPTIATPVPRGAFSWVTRKLKGHVSLPLVTTNRINDPQVADDIL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 AGATIINTGIGWHEARIPTIATPVPRGAFSWVTRKLKGHVSLPLVTTNRINDPQVADDIL 300
Qy 301 SRGDADMVSMARPFLADAELLSKAQSGRADEINTCIGCNQACLDQIFVGKVTSCLVNPRA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 SRGDADMVSMARPFLADAELLSKAQSGRADEINTCIGCNQACLDQIFVGKVTSCLVNPRA 360
Qy 361 CHETKMPILPAVQKKNLAVVGAGPAGLAFAINAAARGHQVTLFDAHSEIGGQFNIAKQIP 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CHETKMPILPAVQKKNLAVVGAGPAGLAFAINAAARGHQVTLFDAHSEIGGQFNIAKQIP 420
Qy 421 GKEEFYETLRYYRRMIEVTGVTLKLNHTVTADQLQAFDETILASGIVPRTPPIDGIDHPK 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GKEEFYETLRYYRRMIEVTGVTLKLNHTVTADQLQAFDETILASGIVPRTPPIDGIDHPK 480
Qy 481 VLSYLDVLRDKAPVGNKVAIIGCGGIGFDTAMYLSQPGESTSQNIAGFCNEWGIDSSLQQ 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 VLSYLDVLRDKAPVGNKVAIIGCGGIGFDTAMYLSQPGESTSQNIAGFCNEWGIDSSLQQ 540
Qy 541 AGGLSPQGMQIPRSPRQIVMLQRKASKPGQGLGKTTGWIHRTTLLSRGVKMIPGVSYQKI 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 AGGLSPQGMQIPRSPRQIVMLQRKASKPGQGLGKTTGWIHRTTLLSRGVKMIPGVSYQKI 600
Qy 601 DDDGLHVVINGETQVLAVDNVVICAGQEPNRALAQPLIDSGKTVHLIGGCDVAMELDARR 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 DDDGLHVVINGETQVLAVDNVVICAGQEPNRALAQPLIDSGKTVHLIGGCDVAMELDARR 660
Qy 661 AIA QGTRLALEI 672
||||||||||||
Db 661 AIA QGTRLALEI 672
He teaches a transformed microorganism, i.e., E. coli, which has been transformed to include the fadH gene encoding 2,4-dienoyl-CoA reductase (p. 517, Overexpression of the cloned fadH gene section) which play an important role in the β-oxidation of unsaturated fatty acids by removing even-numbered double bonds (intro., 1st parag.) and a method for culturing the microorganism in the presence of a carbon source, i.e. oleate (p. 518, Overproduction and purific. Section-p. 519 ), wherein the microorganism is a transformed microorganism comprising an introduced gene encoding a protein comprising the amino acid sequence of SEQ ID NO:1.
The sequence of He (Fig.1) has 100% sequence identity to SEQ ID NO:1, as the sequence is that of the fadH gene.
Feng teaches that fadH encodes 2, 4-dieoyl CoA reductase which is required to reduce cis-2-alkenoyyl-CoA formed by β-oxidation of unsaturated fatty acids (p. 4293, fadH promoter section) and therefore plays an auxiliary role in β-oxidation of certain unsaturated fatty acids (abstract). The genes encoding enzymes of β-oxidation (fad regulon) are responsible for transport, activation and β-oxidation of medium and long-chain fatty acids (intro., 1st parag.). FadH is responsible for reducing CoA’s to intermediates of β-oxidation (intro. 2nd col. Last parag.). Feng teaches transformed microorganisms expressing the fadH gene (SEQ ID NO:1) (p. 4290, plasmids and genetic manip. Section, p. 4291, Table 1 (strains FYJ55, 62,64,68,71-75, 79-81, for example). Feng teaches that fadH activities in cells grown on linoleic acid were higher than those grown on oleate because complete degradation of linoleic acid would require fadH, while oleate can be degraded without fadH (p. 4296, whole 1st col., Fig. 5).
Before the effective filing date of the claimed invention, the fadH gene encoding the protein 2,4-dienoyl-CoA reductase was known and recognized for its important role in the β-oxidation of unsaturated fatty acids and for catalyzing the reduction of CoA, a major precursor of fatty acid biosynthesis. The art teaches transformed microorganisms encoding this gene have an increased reductase activity when grown in the presence of a carbon source, particularly unsaturated fatty acids including oleate and linoleic acid.
It is also disclosed in the prior art references of record that the precursor for the production of PHA’s and PHB’s is acetyl-CoA, which is also the major product of β-oxidation, i.e. fatty acid degradation. The art teaches that microorganisms, particularly Cupriavidus (formerly Ralstonia eutropha), comprise a gene encoding PHA synthase, which when cultured in the presence of oleic acid or sunflower seed oil, i.e. a carbon source comprising an oil containing unsaturated fatty acid or comprises a free unsaturated fatty acid, produces fatty acids and a poly(3-hydrooxyalkanoate), specifically 3-hydroxybutyric acid, 3-hydroxyvalerate and 3-hydroxyhexanoate, thus producing PHA’s and PHB’s.
The presence of the fadH gene is critical in the β-oxidation of unsaturated fatty acids and for catalyzing the reduction of CoA, a major precursor of fatty acid biosynthesis, used by PHA synthase containing microorganisms to produce PHA’s and PHB’s. Additionally important for the conversion of CoA’s to PHA’s and PHB’s are enzymes for the conversion of (S)HA-CoA to (R )-3HA-CoA, believed to participate in the unsaturated fatty acid oxidation pathway; and the art suggests that 2,4-dienoyl-coA reductase fadH is required to convert (S)-3-HA CoA to R-3-HA CoA (unsaturated fatty acid oxidation enzyme), which increases PHA and PHB synthesis yet not identified in C. necator.
Therefore, before the effective filing date of the claimed invention, it would have been obvious to a posita to make a transformed microorganism comprising both a PHA synthase gene and a fadH encoding gene and using said transformed microorganism in a method of making PHA’s and PHB’s according the claims because the presence of both genes allow for increased precursor conversion which thereby increases PHA and PHB synthesis, and when culturing said transformed microorganisms in the presence of a carbon source comprising an oil containing unsaturated fatty acid or comprises a free unsaturated fatty acid, particularly oils comprising linoleic acid, which may inhibit PHA synthesis due to the inhibiting effect of unsaturated fatty acids, fadH activity is significantly increased and thus, allowing one to utilize unsaturated fatty acids as a carbon source for PHA synthesis, free of inhibitions.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIFFANY MAUREEN GOUGH whose telephone number is (571)272-0697. The examiner can normally be reached M-Thu 8-5.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Melenie Gordon can be reached at 571-272-8037. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/TIFFANY M GOUGH/ Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651
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