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
Applicant’s election without traverse of Group I, claims 2-3, 6-7, 9, 11-14, 18, 22, 27, 54, 64, and 67, as well as the species option (i) in claim 6, and an exogenous polynucleotide encoding an acyl-CoA synthase in claim 22, in the reply filed on 03/16/2026 is acknowledged.
Priority
The instant application filed on 12/08/2023 is a 371 of PCT/AU2022/050580 filed on 06/10/2022 and claims priority to AU2021901766 filed on 06/11/2021 and AU2021903160 filed on 10/01/2021. AU2021901766 finds support for the instantly claimed invention; therefore, the effective filing date of the instant application is 06/11/2021.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 08/16/2024 and 03/16/2026 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objections
Claim 9 is objected to because of the following informalities: “is least about” should read “at least about”. Appropriate correction is required. This is an objection, not a rejection, because this appears to be a typographical error. See 112(b) rejection below regarding this.
Claim Rejections - 35 USC § 112(b), Indefiniteness
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.
Claims 2-3, 6-7, 9, 11-14, 18, 22, 27, 54, 64, and 67 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 2 recites “at least some”; however, “some” is not defined in the instant specification and one of ordinary skill in the art cannot readily ascertain what “some” or “at least some” would be without a definition of what amount of fatty acids is covered by the term “some” (see, e.g., MPEP 2173.05(b)).
Claim 3 recites “…or LA is essentially absent from the TFA of the lipid and/or of the TAG content” and “…or PUFA are essentially absent from the TFA content of the lipid and/or the TAG content…”; however, “essentially absent” is not defined in the instant specification and one of ordinary skill in the art cannot readily understand what “essentially absent” would be without a definition because it is unclear if “essentially absent” means that LA and PUFA is completely absent or if this means that there are trace amounts of LA and PUFA. One of ordinary skill in the art would not understand the metes and bounds of the claimed invention and not know if they are infringing on the claimed invention since “essentially absent” is not defined.
Claims 6, 9, and 11 recite “at least about”; however, this is indefinite because it is unclear what the lowest values for the TFA or TAG content is, and it is unclear what the lowest C20:0 fatty acid content is. The instant specification defines “about” as “+/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value” (see, e.g., instant specification, pg. 44, lines 17-18). Therefore, for claims 6 and 9, if the TFA or TAG content is at least about 1.5, it is unclear if the TFA or TAG is at least 1.35 or 1.65 for +/- 10% of 1.5. Based on this, it is unclear if the TFA or TAG content is at least 1.35 or at least 1.65; therefore, it is unclear what the lowest value is. For claim 11, it is unclear if the C20:0 content is at least 0.9% or 1.1% for +/-10% of 1%. Based on this, it is unclear if the C20:0 content is at least 0.9% of at least 1.1% by weight of the TFA or TAG; therefore, it is unclear what the lowest percentage is. One of ordinary skill in the art would not be able to determine what the lowest values are for these contents and therefore would not be able to determine the metes and bounds of the claimed invention for infringement.
Claims 6 and 18 recites the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. Therefore, it is unclear if oleic acid is part of the claimed invention. See MPEP § 2173.05(d).
Claim 18 recites “less than about”; however, this is unclear because the instant specification defines “about” as “+/- 10%, more preferably +/- 5%, more preferably +/- 1%, of the designated value” (see, e.g., instant specification, pg. 44, lines 17-18). Therefore, if the extracted lipid is less than about 0.50, it is unclear if this value would be less than 0.45, or less than 0.55, or between 0.45 and 0.55.
Claim 22 recites the limitation "the microbe". There is insufficient antecedent basis for this limitation in the claim. No microbe was previously recited.
Claims 3, 6-7, 12-14, 27, 54, 64, and 67 are included in this rejection for depending on independent claim 2 and failing to rectify the noted deficiencies.
Claim Rejections - 35 USC § 112(a), Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 74-75 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 74 recites “…a nucleotide sequence which is at least 70% identical to any one or more of SEQ ID NOs: 88 or 89”. Claim 75 recites “…a nucleotide sequence which is at least 70% identical to the sequence set forth in SEQ ID NO: 91”. The instant specification defines SEQ ID NO: 88 as a “Nucleotide sequence of the protein coding region cloned into pAT136 (PcACS-X1) from P. chlororaphis, flanked by BsaI sites. The start codon ATG is at nucleotides 12-14 and the stop codon at nucleotides 1647-1649. 1660nt” (see, e.g., instant specification, pg. 36, lines 11-13). The instant specification defines SEQ ID NO: 89 as a “Nucleotide sequence of the protein coding region cloned into pAT138 (PcACS-X2) from P. chlororaphis, flanked by BsaI sites; 1660nt (see, e.g., instant specification, pg. 36, lines 14-15). The instant specification defines SEQ ID NO: 91 as “Nucleotide sequence of the protein coding region of MtLPAAT from M. alpina; 945nt (see, e.g., instant specification, pg. 36, lines 18-19). Furthermore, the instant specification recites “In another embodiment, at least one DNA construct, or all DNA constructs, in the cell encoding an acyl-CoA synthetase (ACS) comprises nucleotides having a sequence as set forth in any one of SEQ ID NOs: 88 to 89, or a nucleotide sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95% or at least 97% identical, to any one or more of SEQ ID NOs: 88 to 89 (see, e.g., instant specification, pg. 28, lines 17-21). Moreover, the instant specification recites “In another embodiment, at least one DNA construct, or all DNA constructs, in the cell encoding a lysophosphatidic acid acyltransferase (LPAAT) comprises nucleotides having a sequence as set forth in SEQ ID NO: 91 or a nucleotide sequence which is at least 70% identical, preferably at least 80% identical, more preferably at least 90%, at least 95% or at least 97% identical, to SEQ ID NO: 91 (see, e.g., instant specification, pg. 28, lines 22-26). The instant specification does not teach nucleotide variations within SEQ ID NOs: 88, 89, and 91, especially when there is up to 30% variation in the sequences, nor does the instant specification teach what variations can occur within the nucleotide sequence to maintain expression of a ACS or LPAAT with activity once the polynucleotides are translated. As such, the scope of the claimed nucleotides encompasses a large array of nucleotides without any necessary “core” sequence that would be needed in order for the nucleotide to still encode a functioning ACS and/or LPAAT protein upon translation.
The written description may be met by providing a representative number of species for the genus. In the instant case, claim 74 states that the ACS nucleotide can have at least 70% sequence identity to SEQ ID NOs: 88 or 89, which accounts for up to 30% nucleotide variation (up to 498 nucleotides), which, in turn, would result in variation for up to 166 amino acids in the encoded ACS protein. Furthermore, in the instant case, claim 75 states that the LPAAT nucleotide can have at least 70% sequence identity to SEQ ID NO: 91, which accounts for up to 30% nucleotide variation (up to 283 nucleotides), which, in turn, would result in variation for up to 94 amino acids in the encoded LPAAT protein. Therefore, the specification fails to demonstrate a representative number of nucleotide species so that one of ordinary skill in the art can extrapolate to the claimed genus. Additionally, the specification does not set forth “core” nucleotides that are required within the nucleotide sequence so that the encoded protein maintains ACS and/or LPAAT activity. Therefore, the instant specification does not demonstrate which nucleotides are necessary or required in SEQ ID NOs: 88, 89, and 91 when there is 30% sequence variation.
Furthermore, as discussed above, variation can exist in up to 498 nucleotides for SEQ ID NOs: 88 or 89, and up to 283 nucleotides for SEQ ID NO: 91. This results in a large number of combinations of different nucleotide variants or fragments that can be produced due to this variation; however the instant specification does not teach or suggest any nucleotide variants or fragments that can be produced in SEQ ID NOs: 88, 89, and 91. Therefore, the instant specification lacks written description for which nucleotides can be changed, which would result in production of a large number of combination of LPAAT and ACS nucleotide variants or fragments. The instant specification only teaches the full length sequences for SEQ ID NOs: 88, 89, and 91, and does not provide written description or guidance for up 30% sequence variation in SEQ ID NOs: 88, 89, and 91.
Claim Rejections - 35 USC § 102, Anticipation
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.
Claims 2-3, 6-7, 9, 11-12, 14, 54, 64, and 73 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Moseley (US 2020/0392470; Date of Publication: December 17, 2020 – cited in the IDS filed on 08/16/2024).
Moseley’s general disclosure relates to “microalgal cells having an ablated or downregulated fatty acyl-ACP thioesterase (FATA) gene, wherein the cell is modified to express a heterologous lysophosphatidic acid acyltransferase (LPAAT)” and “wherein the modified microalgal cell produces an oil with an elevated ratio of saturated-unsaturated-saturated triglycerides over trisaturated triglycerides as compared to a corresponding unmodified cell” (see, e.g., Moseley, abstract).
Regarding claim 2 pertaining to the extracted microbial lipid, Moseley teaches the production of various genetically modified microbial strains that produce lipids in varying amounts (see, e.g., Moseley, Table 23). Moreover, Moseley teaches that the lipids extracted from these genetically modified microbes contain saturated fatty acids (SFA) such as stearic acid, palmitic acid, myristic acid, arachidic acid, behenic acid, and lignoceric acid (see, e.g., Moseley, Table 23), and that the SFAs are over 50% by weight of the total fatty acid content for all genetically modified microbial strains (see, e.g., Moseley, Table 23). Moseley teaches that for the lipids derived from the microbial strains have 45.8-56.5% stearic acid by weight of the total fatty acid content (see, e.g., Moseley, Table 23). Moseley teaches that the monounsaturated fatty acid (MUFA) content of the extracted microbial lipid comprises oleic acid and palmitoleic acid (see, e.g., Moseley, Table 23). Moseley teaches that the polyunsaturated fatty acid (PUFA) content, which comprises linoleic acid, is 0.8-1.4% by weight of the total fatty acid content (see, e.g., Moseley, Table 23). Moseley teaches that phospholipids may be removed (see, e.g., Moseley, [0089]), therefore, the extracted microbial lipid lacks polar lipid. Moseley teaches “In this example we describe genetically engineered Prototheca moriformis strains in which we have modified fatty acid and triacylglycerol biosynthesis to maximize the accumulation of Stearoyl-Oleoyl-Stearoyl (SOS) TAGs, and minimize the production of trisaturated TAGs. Tailored oils from these strains resemble plant seed oils known as “structuring fats”, which have high proportions of Saturated-Oleate-Saturated TAGs and low levels of trisaturates. These structuring fats (often called “butters”) are generally solid at room temperature but melt sharply between 35-40° C” (see, e.g., Moseley, Example 7, [0161]). Furthermore, Moseley teaches genetically engineering Prototheca moriformis strains that have “modified fatty acid and triacylglycerol (TAG) biosynthesis in order to maximize accumulation of Stearoyl-Oleoyl-Stearoyl (SOS) TAGs, and minimize the production of trisaturated TAGs” (see, e.g., Moseley, [0161], Example 7). Therefore, there are TAGs produced from MUFAs by the modified microbial strains (see, e.g., Moseley, Table 24).
Regarding claim 3 pertaining to content of linoleic acid and oleic acid, Moseley teaches that the content of linoleic acid is 0.8-1.4% by weight and the content of oleic acid is 35.4-45.9% by weight (see, e.g., Moseley, Table 23).
Regarding claim 6 pertaining to the method by which the microbial lipid was extracted, this is considered a product-by-process limitation (see, e.g., MPEP 2113); therefore, patentability is based on the product itself (i.e., the extracted microbial lipid). Additionally, the product-by-process limitation recited in claim 6 does not impart structural limitation(s) on the product (i.e., the extracted microbial lipid). Therefore, since patentability is based on the product, claim 6 is anticipated by Moseley, as discussed in claim 2 above.
Regarding claim 7 pertaining to the TFA content, Moseley teaches that the TAG profile of the extracted microalgal oils comprise 82.0-83.9% of TAG molecules which comprise oleic acid esterified at their sn-2 position (see, e.g., Moseley, Table 24, ‘Sat-O-Sat’).
Regarding claim 9 pertaining to the ratio of total saturated fatty acids comprising 18 carbons or more to total saturated fatty acids comprising 16 carbons or less of the TFA content, Moseley teaches that the ratio is calculated to be 12.3-13.9 (see, e.g., Moseley, Table 23).
Regarding claim 11 pertaining to the content of C20:0, C22:0, and C24:0 fatty acids, Moseley teaches that the content of C20:0 fatty acid is 1.5-2% by weight of the TFA content (see, e.g., Moseley, Table 23).
Regarding claim 12 pertaining to the C20:0, C22:0, and C24:0 fatty acids, Moseley teaches that the sum of the contents of the C20:0, C22:0, and C24:0 fatty acids is calculated to be 1.9-2.3% (see, e.g., Moseley, Table 23). Moseley does not teach fatty acids longer than C24:0; therefore, C20:0, C22:0, and C24:0 fatty acids comprise 100% of the fatty acids of the TFA content of the extracted lipid which are at least 20 carbons or longer (see, e.g., Moseley, Table 23).
Regarding claim 14 pertaining to the TAG content, Moseley teaches the TAG profile of the extracted microalgal oils comprise 82.0-83.9% of TAG molecules which comprise oleic acid esterified at their sn-2 position (see, e.g., Moseley, Table 24, ‘Sat-O-Sat’).
Regarding claims 54, 64, and 67 pertaining to compositions comprising the extracted lipid, Moseley teaches “there is a process for producing an oil, triglyceride, fatty acid, or derivative of any of these, comprising transforming a cell with any of the nucleic acids discussed herein. In another embodiment, the transformed cell is cultivated to produce an oil and, optionally, the oil is extracted. Oil extracted in this way can be used to produce food, oleochemicals or other products” (see, e.g., Moseley, [0107]). Moreover, Moseley teaches “The oils discussed above alone or in combination are useful in the production of foods, fuels and chemicals (including plastics, foams, films, etc)” (see, e.g., Moseley, [0108]).
Regarding claim 73 pertaining to the microbial cell, Moseley teaches “the cells used are optionally cells having a type II fatty acid biosynthetic pathway such as plant cells, yeast cells, microalgal cells including heterotrophic or obligate heterotrophic microalgal cells” (see, e.g., Moseley, [0091]).
Claim Rejections - 35 USC § 103, Obviousness
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.
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.
Claims 13, 18, and 72 are rejected under 35 U.S.C. 103 as being unpatentable over Moseley (previously cited) as applied to claims 2-3, 6-7, 9, 11-12, 14, 54, 64, and 73 above, and further in view of Ngantung (US 2016/0194584; Date of Publication: July 7, 2016 – cited in the IDS filed on 08/16/2024).
The teachings of Moseley as it pertains to an extracted microbial lipid are discussed above.
Regarding claim 72 pertaining to the MUFA content of the extracted microbial lipid, Moseley teaches that the MUFA content of the extracted microbial lipid comprises oleic acid and palmitoleic acid (see, e.g., Moseley, Table 23).
However, Moseley does not teach: wherein the extracted microbial lipid comprises a polar lipid, wherein the TFA content of the polar lipid has one or more features as defined in claims 2 to 11 for the TFA content of the extracted lipid or the TAG content (claim 13); or wherein the lipid comprises TAG molecules which comprise a MUFA, preferably oleic acid, esterified at their sn-2 position, wherein the ratio of the number of TAG molecules which comprise a MUFA esterified at the sn-2 position to the number of TAG molecules which comprise a fatty acid other than a MUFA esterified at their sn-2 position (MUFA:other FA ratio at sn-2) in the extracted lipid is less than about 0.50, less than about 0.30, less than about 0.20, less than about 0.10, less than about 0.05, less than about 0.04, less than about 0.03 or less than about 0.02 (claim 18); or wherein the MUFA content comprises C16:1
∆
7 and/or C17:1 (claim 72).
Ngantung’s general disclosure relates to “microalgal oils having a low polyunsaturated fatty acid profile and derivatives of the oils, including acids, esters, epoxides, hydroxylated acids and esters, urethanes, amides, and polymers thereof” (see, e.g., Ngantung, abstract). Moreover, Ngantung discloses “Embodiments of the present invention relate to oils/fats, fuels, foods, and oleochemicals and their production from cultures of genetically engineered cells. Specific embodiments relate to oils with a high content of triglycerides bearing fatty acyl groups upon the glycerol backbone in particular regiospecific patterns, highly stable oils, oils with high levels of oleic or mid-chain fatty acids, and products produced from such oils” (see, e.g., Ngantung, [0003]).
Regarding claim 13 pertaining to the polar lipid, Ngantung teaches that the polar lipid, which are free fatty acids, comprise oleic acid at 0.02% in an oil sample named “RBD469”, which was obtained from strain D (see, e.g., Ngantung, Example 4, [0375], Table 9).
Regarding claim 18 pertaining to the MUFA:other FA ratio at sn-2, Ngantung teaches Strain C, which has a MUFA:other FA ratio at sn-2 of 0.21 (see, e.g., Ngantung, Example 3, Table 7).
Regarding claim 72 pertaining to the MUFA content of the extracted microbial lipid, Ngantung teaches that the fatty acid profile of oil derived from a microbial strain comprised C17:1 and C16:1
∆
7 (see, e.g., Ngantung, Example 58 & Table 63). Additionally, Ngantung teaches that the fatty acid profile of oil derived from a microbial strain also comprised oleic acid and palmitoleic acid (see, e.g., Ngantung, Example 58 & Table 63).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Moseley’s microbial lipid, wherein the lipid comprises a polar lipid, such as a free fatty acid that is oleic acid, as taught by Ngantung. One would have been motivated to do so because Ngantung teaches that free fatty acids, which are polar lipids, as defined in the instant specification (see, e.g., instant specification, pg. 44, lines 29-30), can be derivatized to produce other useful chemicals or products, such as highly sulfated fatty acids, cleansing compositions, fabric softener compositions, emulsions for treating skin, water repellant compositions, paint additives, lipid-enriched ruminant feedstock, and surfactants for detergents and cleaners (see, e.g., Ngantung, [0286]). Moreover, Moseley teaches “The pool of acyl-CoAs in the ER can be utilized for the synthesis of TAGs as well as phospholipids and long chain fatty acids. The enzymes involved in the synthesis of TAGS and phospholids actively compete against each other for the same substrates. Acyl-CoAs can associate with lysophosphatidate to form phosphatidate which is converted to phosphatidylcholine (PC) and other phospholipid species” (see, e.g., Moseley, [0151]). Therefore, based on the teachings of Moseley and Ngantung, it would be obvious to produce polar lipids because they can be formulated into useful chemicals and products, and because the polar lipids can be formulated using acyl-CoAs.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention or produce Moseley’s microbial lipid, wherein the lipid comprises TAG molecules that comprise MUFAs esterified as the sn-2 position, as taught by Ngantung. One would have been motivated to do so because Ngantung teaches that the fatty acid profile of triglycerides is altered by altering the sn-2 profile (see, e.g., Ngantung, [0136]). Additionally, Ngantung teaches that “For example, by virtue of expressing an exogenous active LPAAT in an oleaginous cell, the percent of unsaturated fatty acid at the sn-2 position is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90% or more. For example, a cell may produce triglycerides with 30% unsaturates (which may be primarily 18:1 and 18:2 and 18:3 fatty acids) at the sn-2 position. In this example, introduction of the LPAAT activity increases the unsaturates at the sn-2 position by 20% so that 36% of the triglycerides comprise unsaturates at the sn-2 position” (see, e.g., Ngantung, [0136]). Moreover, Moseley teaches altering the sn-2 profile of triglycerides in host cells (see, e.g., Moseley, [0014]), and also teaches driving the transfer of unsaturated fatty acids towards the sn-2 positions through expression of LPAT2 (see, e.g., Moseley, [0097]). Therefore, based on the teachings of Moseley and Ngantung, it would have been obvious to produce TAG molecules with MUFAs at the sn-2 position in order to alter the sn-2 profile of the TAG.
It would have been thirdly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Mosely’s microbial lipid, wherein the lipid contains C17:1 and/or C16:1
∆
7 as part of the MUFA content, as taught by Ngantung. One would have been motivated to do so because Ngantung teaches increasing mid-chain fatty acids, such as C16:0 moieties, alters fatty acids profiles (see, e.g., Ngantung, [0136]). Additionally, Ngantung teaches an embodiment with a saturated fatty acids at the sn-2 position of a TAG (see, e.g., Ngantung, [0144]), which would be C16:1
∆
7. Moreover, Moseley teaches producing an oil with an elevated ratio of unsaturated fatty acids at the sn-2 position (see, e.g., Moseley, abstract), which could alter the sn-2 profile of triglycerides in host cells (see, e.g., Moseley, [0014]). Therefore, based on the teachings of Moseley and Ngantung, it would be obvious to produce a microbial lipid that contains C17:1 and/or C16:1
∆
7 as part of the MUFA content because C17:1 and/or C16:1
∆
7 can be used to alter the sn-2 profile. One would have expected success because Moseley and Ngantung both teach production of oils by modified microbial cells, wherein the oils have TAGs with altered sn-2 profiles.
Claims 22 and 74 are rejected under 35 U.S.C. 103 as being unpatentable over Moseley (previously cited) as applied to claims 2-3, 6-7, 9, 11-12, 14, 54, 64, and 73 above, and further in view of Eastham (U.S. Patent No. 10,704,063; Date of Publication: July 7, 2020), Gupta (Project Report: Codon Optimization; 2003), Gustafsson (Codon bias and heterologous protein expression; 2004), and Watkins (Evidence for 26 distinct acyl-coenzyme A synthetase genes in the human genome; 2007).
The teachings of Moseley as it pertains to an extracted microbial lipid are discussed above.
However, Moseley does not teach: wherein the microbe further comprises an exogenous polynucleotide encoding an acyl-CoA synthetase (ACS) (claim 22); or wherein the ACS comprises nucleotides having a sequence as set forth as any one of SEQ ID NOs: 88 to 89, or a nucleotide sequence which is at least 70% identical to any one or more of SEQ ID NOs: 88 to 89 (claim 74).
Eastham’s general disclosure relates to “producing methacrylic acid and/or derivatives thereof including the following steps: (a) biologically converting isobutyryl-CoA into methacrylyl-CoA by the action of an oxidase; and (b) converting methacrylyl-CoA into methacrylic acid and/or derivatives thereof. The invention also extends to microorganisms adapted to conduct the steps of the process” (see, e.g., Eastham, abstract).
Regarding claims 22 and 74 pertaining to an exogenous polynucleotide encoding ACS, Eastham teaches SEQ ID NO: 12, which is an acyl-CoA synthetase from Pseudomonas chloroaphis, and which has 62.3% sequence identity to instant SEQ ID NO: 88 (see, e.g., Eastham, col 29, lines 57-59 & Office Action Appendix). Gupta teaches basic rationales for optimizing codons for specific living organisms (see, e.g., Gupta at §§§ 2.1, 4.1, 4.2, 4.3) and identifies that circa 2003 web-based software was available for converting yeast and human codons (id. at §5). Gustafsson identifies that a "common strategy" to improve expression of heterologous proteins in a host is to alter rare codons in a gene to reflect codon usage of a host without modifying the amino acid sequence of the encoded protein (see, e.g., Gustafsson et al. at 348 col II §"Results from Codon Optimization", Table 1 on 349). Therefore, based on the teachings of Gupta and Gustafsson, it is routine to one of ordinary skill in the art to use different nucleic acid sequences to express a protein in a different organism (i.e., codon optimization). Additionally, the nucleic acid encoding the protein would be optimized for expression in a particular organism. Moreover, optimization of codons in the polynucleotide sequence of acyl-CoA synthetase would result in a different polynucleotide sequence than what is in the art, but may lead to the same amino acid sequence encoding the acyl-CoA synthetase. Therefore, one of ordinary skill in the art would be motivated to optimize the codons for the polynucleotide sequence encoding acyl-CoA synthetase in order to be able to express the polynucleotide sequence in a different organism without modifying the amino acid sequence. Furthermore, the transitional phrase “having” is synonymous to “comprising”, which is “is inclusive or open-ended and does not exclude additional, unrecited elements or method steps” (see, e.g., MPEP 2111.03(I)); therefore, instant SEQ ID NO: 88 can have additional, unrecited elements and still read on the instantly claimed invention.
Watkin’s general disclosure relates to identifying the number of ACS genes present in the human genome (see, e.g., Watkins, abstract). Moreover, Watkins discloses that “The existence of many ACSs suggests that each plays a unique role, directing the acyl-CoA product to a specific metabolic fate” (see, e.g., Watkins, abstract). Furthermore, Watkins discloses that fatty acids have many essential functions in living organisms, such as being the building blocks of lipids, storage molecules, structural lipids, and signaling molecules, as well as being degraded for energy production; however, all of the metabolic processes involving fatty acids have a common initial step, which is “activation” of the fatty acid by forming a thioester with CoA, which is catalyzed by ACS (see, e.g., Watkins, Introduction, pg. 2736). Moreover, Watkins discloses that “Using highly conserved amino acid sequence motifs, 26 proven or likely human ACS genes were detected” in their studies (see, e.g., Watkins, Introduction, pg. 2737).
Regarding claims 22 and 74 pertaining to the ACS, Watkins teaches “Acyl-coenzyme A synthetases (ACSs) catalyze the fundamental, initial reaction in fatty acid metabolism. “Activation” of fatty acids by thioesterification to CoA allows their participation in both anabolic and catabolic pathways” (see, e.g., Watkins, abstract). Furthermore, Watkins teaches all metabolic processes for catabolism and anabolism of fatty acids have a common initial step, which is the “activation” of the fatty acid by forming a thioester with CoA, which is catalyzed by ACS (see, e.g., Watkins, Introduction, pg. 2736).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a microbial cell that expresses lipids, as taught by Moseley, wherein the cell expresses ACS, as taught by Eastham and Watkins. One would have been motivated to do so because Watkins teaches that fatty acids have many essential functions in living organisms, such as being the building blocks of lipids, storage molecules, structural lipids, and signaling molecules, as well as being degraded for energy production; however, all of the metabolic processes involving fatty acids have a common initial step, which is “activation” of the fatty acid by forming a thioester with CoA, which is catalyzed by ACS (see, e.g., Watkins, Introduction, pg. 2736). Furthermore, Eastham teaches microorganisms expressing acyl-CoA synthetase (see, e.g., Eastham, col 12, line 29). Moreover, Moseley teaches the production of oil by microalgal cells (see, e.g., Moseley, abstract), wherein the oil comprises fatty acids with altered profiles (see, e.g., Moseley, abstract & [0003], [0041], [0042]). Therefore, based on the teachings of Moseley, Eastham, and Watkins, one would have been motivated to express ACS in a microbial cell that is producing fatty acids in order to activate the fatty acids for downstream metabolic processes. One would have expected success because Mosely, Eastham, and Watkins all teach the function and expression of enzymes involved in metabolic processes.
Claims 75-76 are rejected under 35 U.S.C. 103 as being unpatentable over Moseley, Eastham, Gupta, Gustafsson, and Watkins as applied to claims 2-3, 6-7, 9, 11-12, 14, 22, 54, 64, and 73-74 above, and further in view of Damude (U.S. Patent No. 7,879,591; Date of Publication: February 1, 2011).
The teachings of Mosely, Eastham, Gupta, Gustafsson, and Watkins, herein referred to as modified-Moseley-Eastham-Gupta-Gustafsson-Watkins, are discussed above is it pertains to an extracted microbial lipid.
However, modified-Moseley-Eastham-Gupta-Gustafsson-Watkins does not teach: the exogenous polynucleotide encoding a lysophosphatidic acid acyltransferase (LPAAT), wherein the LPAAT comprises nucleotides having the sequence as set forth in SEQ ID NO: 91, or a nucleotide sequence which is at least 70% identical to the sequence as set forth in SEQ ID NO: 91 (claim 75); or wherein the yeast cells are Yarrowia lipolytica (claim 76).
Damude’s general disclosure relates to “Lysophosphatidic acid acyltransferase ["LPAAT"] participates in the second step of oil biosynthesis and is expected to play a key role in altering the quantity of long-chain polyunsaturated fatty acids ["LC-PUFAs"] produced in oils of oleaginous organisms. An LPAAT isolated from Mortierella alpina ["MaLPAAT1"] that is suitable for use in the manufacture of oils enriched in LC-PUFAs in oleaginous organisms is disclosed. Most desirably, the substrate specificity of the instant MaLPAAT1 will be particularly useful to enable increased C18 to C20 elongation conversion efficiency and increased
∆
4 desaturation conversion efficiency in recombinant host cells producing LC-PUFAs” (see, e.g., Damude, abstract).
Regarding claim 75 pertaining to the LPAAT having the sequence set forth in SEQ ID NO: 91, Damude teaches SEQ ID NO: 3, which encodes a nucleotide sequence for LPAAT, and which has 74.7% sequence identity to instant SEQ ID NO: 91 (see, e.g., Damude, col 20, lines 60-61 & Office Action Appendix).
Regarding claim 76 pertaining to the yeast cell, Damude teaches that the host cell is Yarrowia lipolytica (see, e.g., Damude, col 4, lines 25-26).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Moseley-Eastham-Gupta-Gustafsson-Watkins’ microbial cell producing lipids, wherein the microbial cell expresses LPAAT, as taught by Damude. One would have been motivated to do so because Damude teaches “Lysophosphatidic acid acyltransferase ["LPAAT"] participates in the second step of oil biosynthesis and is expected to play a key role in altering the quantity of long-chain polyunsaturated fatty acids ["LC-PUFAs"] produced in oils of oleaginous organisms” (see, e.g., Damude, abstract). Moreover, modified-Moseley-Eastham-Gupta-Gustafsson-Watkins teaches that LPAAT “enzymes are responsible for the transfer of acyl groups to the sn-2 position on the glycerol backbone. We disclose here that we can reduce the accumulation of excessive amounts of trisaturates in our high SOS strains by expressing heterologous LPAAT genes which were better than the endogenous acyltransferases at discriminating against saturated fatty acids” (see, e.g., Moseley, [0140]). Therefore, based on the teachings of modified-Moseley-Eastham-Gupta-Gustafsson-Watkins and Damude, it would be obvious to express LPAAT within the microbial cell expressing lipids because LPAAT is responsible for transferring acyl groups to the sn-2 position of the glycerol backbone, which allows for production of phosphatidic acid, and also allows for a reduction in the accumulation of excess trisaturates.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Moseley-Eastham-Gupta-Gustafsson-Watkins’ microbial cell producing lipids, wherein the microbial cell is Yarrowia lipolytica, as taught by Damude. One would have been motivated to do so because Damude teaches Y. lipolytica can be transformed to express LPAAT from Mortierella alpina (see, e.g., Damude, col 10, lines 1-6), and Damude teaches that the codon usage profile for Y. lipolytica has previously been established; therefore, allowing for identification of preferred codons based on a survey of genes derived from the host cell (see, e.g., Damude, col 16, lines 62-67 & col. 17, lines 1-3). Furthermore, Damude teaches that LPAAT from Mortierella alpina was codon-optimized for expression in Y. lipolytica “based on the previous determination of the Y. lipolytica codon usage profile, identification of those codons that were preferred, and determination of the consensus sequence around the `ATG` initiation codon” (see, e.g., Damude, col 20, lines 43-49). Moreover, modified-Moseley-Eastham-Gupta-Gustafsson-Watkins teaches that the cells utilized for production of microbial lipids “are optionally cells having a type II fatty acid biosynthetic pathway such as plant cells, yeast cells, microalgal cells including heterotrophic or obligate heterotrophic microalgal cells” (see, e.g., Moseley, [0091]). Therefore, based on the teachings of modified-Moseley-Eastham-Gupta-Gustafsson-Watkins and Damude, it would have been obvious to produce microbial lipids using Y. lipolytica as the host cell because the codon usage profile of this microorganism has already been established, and Y. lipolytica has already been shown to be capable of producing lipids. One would have expected success because modified-Moseley-Eastham-Gupta-Gustafsson-Watkins and Damude both teach production of lipids via expression by microbial host cells.
Conclusion
Claims 2-3, 6-7, 9, 11-14, 18, 22, 27, 54, 64, 67, and 72-76 are rejected.
No claims are allowed.
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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