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 106-109 are not in compliance with 37 CFR 1.121(c), which states: the status of every claim must be indicated after its claim number by using one of the following identifiers in a parenthetical expression: (Original), (Currently amended), (Canceled), (Withdrawn), (Previously presented), (New), and (Not entered). Claims 106-109 should be identified as (Withdrawn) for being directed towards non-elected inventions.
Claims 1-3, 7-9, 13-15, 19, 22-26 and 106-109 are pending.
Priority
This application, filed on 11/14/2023, is a 371 of PCT/US2022/030364 filed on 5/20/2022, which claims benefit of 63/190,954 filed on 5/20/2021. The effective filing date of the current application is May 20, 2022.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-3, 7-9, 13-15, 19 and 22-26, in the reply filed on April 17, 2026 is acknowledged.
Claims 106-109 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on April 17, 2026.
Claims 1-3, 7-9 13-15, 19 and 22-26 are under examination.
Claim Objections
Claims 1 and 8-9 are objected to because of the following informalities:
Claim 1 recites “the amino acid sequence forth in” in line 5, which is missing the word “set” and should be amended to recite “the amino acid sequence set forth in”.
Claim 8 recites “The modified cell of any one of claim 1” in line 1, which contains the extraneous phrase “any one of”, which should be removed.
Claim 9 recites “transcription factor: (a)is ADR1, PIP2” in line 2, which is missing a space between “(a)” and “is”, and should be amended to recite “transcription factor is: (a) ADR1, PIP2”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 1-3, 7-9, 13-15, 19, and 22-26 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 regards as the invention.
Claim 1 recites “A genetically modified yeast cell of the genus Saccharomyces (modified cell)” in lines 1-2. Claim 2 recites “A genetically modified yeast cell (modified cell)” in line 1. The same abbreviated term is used for two different claim limitations of different scope, which renders claims 1-2 and dependent claims 3, 7-9, 13-15, 19, and 22-26 indefinite.
Claim 9 recites “The modified cell of claim 8, wherein the transcription factor: (a) is ADR1, PIP2, OAF1, OAF3, or ADR comprising a substitution mutation of serine at position 230; (b) comprises: (i) an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; or (ii) the amino acid sequence set forth in SEQ ID NO:24”. It is unclear whether the transcription factor is required to meet both limitations (a) and (b). It is further unclear whether the phrase “comprising a substitution mutation of serine at position 230” is modifying only the ADR transcription factor, or if it applies to any of “ADR1, PIP2, OAF1, OAF3, or ADR”.
Claim 23 recites “wherein the gene encoding the enzyme having acyl-CoA desaturase 1 (OLE1) activity, and/or the gene encoding the enzyme having alcohol-O-acyltransferase (AAT) activity” in lines 2-4. Claim 23 depends from claim 8, which depends from claim 1. There is insufficient antecedent basis for the limitations “the enzyme having acyl-CoA desaturase 1 (OLE1) activity” and “the gene encoding the enzyme having alcohol-O-acyltransferase (AAT) activity”, because neither limitation is recited previously either in claim 8 or in claim 1. It is noted that claim 14 recites these limitations.
Claim 26 recites “wherein the yeast cell is S. cerevisiae California Ale Yeast strain WLP001, EC-1118, Elegance, Red Star Côte de Blancs, or Epernay II”. Claim 26 contains the trademark/trade names California Ale Yeast WLP001®; EC-1118®; and Red Star®. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b). See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe specific strains of Saccharomyces cerevisiae yeast and, accordingly, the identification/description is indefinite.
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.
Claim 8 is rejected under 35 U.S.C. 112(d) 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 8 recites “the modified cell of claim 1, comprising a gene encoding a transcription factor that increases peroxisomal size and/or number and/or increases fatty acid beta-oxidation as compared to a cell that does not express the transcription factor”. Claim 8 depends from claim 1, which recites “a genetically modified yeast cell of the genus Saccharomyces comprising a heterologous gene conceding an enzyme having oleate 12-hydroxylase activity, wherein the enzyme comprises a sequence having at least 90% sequence identity to the amino acid sequence [set] forth in either SEQ ID NO:6 or SEQ ID NO:23; wherein the modified cell produces a fermented product having an increased level of γ-decalactone in the absence of fatty acid supplementation as compared to a level of γ-decalactone produced by a counterpart cell that does not comprise the enzyme having oleate 12-hydroxylase activity”. The scope of claim 8 is broader than the scope of claim 1 and does not further narrow claim 1 from which it depends, and thus is an improper dependent claim. Amending the claim to recite the word “further” before “comprising” in line 1 would obviate this rejection.
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.
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 1, 3, 7-9, 13-15, 19 and 22-26 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claims contain 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, at the time the application was filed, had possession of the claimed invention.
Applicant is referred to MPEP 2163(II)(A)(3)(a)(i and ii), which states that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the claimed genus. A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. The disclosure of only one species encompassed within a genus adequately describes a claim directed to that genus only if the disclosure indicates that the patentee has invented species sufficient to constitute the genus. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.
Claim 1 is drawn to a genetically modified yeast cell of the genus Saccharomyces comprising a heterologous gene encoding an enzyme having oleate 12-hydroxylase activity, wherein the enzyme comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in either SEQ ID NO:6 or SEQ ID NO:23.
Claim 3 is drawn to the modified cell of claim 1, wherein the enzyme having oleate 12-hydroxylase activity comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NO:6 or 20-23.
Claim 9 is drawn to the modified cell of claim 8, wherein the transcription factor comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24.
Claim 15 is drawn to the modified cell of claim 14, wherein the enzyme having OLE1 activity comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.
Claim 19 is drawn to the modified cell of claim 14, wherein the enzyme having AAT activity comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID Nos: 1-5 or 25.
The current specification identifies that SEQ ID NO:6 or 20-23 are amino acid sequences for an enzyme having oleate 12-hydroxylase activity (p.2, lines 23-25). The current specification identifies that SEQ ID NO:24 is an amino acid sequence for a deregulated transcription factor (p.3, lines 2-4). The current specification identifies that SEQ ID NO:7 is an amino acid sequence for an enzyme having OLE1 activity. The current specification identifies that SEQ ID NO:1-5 or 25 are amino acid sequences for an enzyme having AAT activity. The current specification states that identification of additional enzymes having acyl-CoA desaturase activity may be performed based on similarity or homology with one or more domains of an acyl-CoA desaturase 1, such as SEQ ID NO:7, and an enzyme may be identified based on similarity or homology with an active domain such as a catalytic domain, but have a relatively low level of sequence identity to the reference enzyme based on analysis of a larger portion of the enzyme or across the full length of the enzyme (p.23, lines 3-14). The specification discusses general methods of genetic engineering, and using software programs and algorithms for aligning amino acid sequences (p.30, lines 16-19). However, the specification does not identify regions for any sequence that are critical to preserving the desired functionality of the enzyme or transcription factor for any of the SEQ ID NO:’s.
The current specification describes engineering a S. cerevisiae that overexpressed acyl-CoA desaturase 1 (OLE1) under transcriptional control of the strong promoter pENO2 (p.66, lines 29-32); overexpressed the fatty acid hydroxylase (FAH) enzyme from Claviceps purpurea (p.67, lines 2-3); and overexpressed an alcohol-O-acyltransferase from a peach plant (Prunus persica, PpAAT1) (p.67, lines 5-7). The specification further describes generating a modified microbe by targeting PpAAT1 to the peroxisome organelle (p.67, lines 14-17). The current specification describes a strain of S. cerevisiae expressing a deregulated mutant of the positive transcriptional activator of glucose-regulated genes, ADR1, along with oleate 12-hydroxylase and OLE1 enzymes (p.69, lines 3-9). The current specification also describes a S. cerevisiae Elegance strain expressing CpFAH, OLE1, MpAAT (N385D V62A) (y1185) (p.69, lines 19-20).
The current specification does not provide any guidance on the structure-function relationship of SEQ ID NO:1 – SEQ ID NO:7 or SEQ ID NO:20 – SEQ ID NO:25, and does not provide any information on which amino acids can be varied or deleted while preserving the desired enzymatic function of the polypeptide and meet the requirement of an enzyme having oleate 12-hydroxylase activity for SEQ ID NO:6 or SEQ ID NO: 20-23; a transcription factor that increases peroxisomal size or number or increases fatty acid beta-oxidation for SEQ ID NO:24; an enzyme having acyl-CoA desaturase 1 (OLE1) activity for SEQ ID NO:7; or an enzyme having alcohol-O-acyltransferase (AAT) activity for SEQ ID NO:1-5 or SEQ ID NO:25. The specification does not provide any structural guidance on required features or parameters including: whether the sequences must retain any particular functional features; and whether any portions of the sequence must be modified or must be preserved. Based on the lack of art-recognized structure-function relationships of sequences that comprise at least 90% sequence identity to the claimed SEQ ID NOs to other functional variants, it is highly unpredictable whether 90% sequence identity to the claimed sequences would provide functional enzymes having the desired activity, or a functional transcription factor that provides the desired functions.
The disclosure of single examples of modified yeast cells comprising genes encoding enzymes that have 100% sequence identity to the amino acid sequences identified as SEQ ID NO:1-6 and SEQ ID NO:20-25 is not considered to constitute a representative number of species to sufficiently describe the genus of enzymes having oleate 12-hydroxylase activity and at least 90% sequence homology to SEQ ID NO:6 and SEQ ID NO:20-23; the genus of transcription factors that increase peroxisomal size or number or increases fatty acid beta-oxidation and having at least 90% sequence identity to SEQ ID NO:24; the genus of enzymes having OLE1 activity and at least 90% sequence identity to SEQ ID NO:7; or the genus of enzymes having AAT activity and at least 90% sequence identity to SEQ ID NO:1-5 or SEQ ID NO:25. This disclosure does not describe a representative number of species of the specified genera in view of the potential breadth and variability of the genera, and therefore fails to satisfy the written description requirement for the genera.
Claim Rejections - 35 USC § 102
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 1, 7 and 25 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262), as evidenced by the GenBank accession number CDG23429, deposited on February 27, 2014 and referred to as GenBank 01.
Regarding claim 1, Beopoulos teaches heterologous expression of the castor bean hydroxylase, encoded by the Δ12 (oleate) hydroxylase gene FAH12 (p.252, 1st column – 3rd paragraph). Beopoulos teaches S. cerevisiae does not contain genes coding for Δ12 desaturases, and heterologous expression of the C. purpurea FAH12 hydroxylase resulted in ricinoleic acid (RA) accumulation to 8% of total fatty acid (p.252, 1st column last sentence to 2nd column top sentence).
Beopoulos discloses the sequence of the FAH12 gene as a nucleotide sequence of codon-optimized heterologous hydroxylase gene that was submitted to the European Nucleotide Archive (EMBL-EBI) under the accession no. HG326650 for CpFAH12 (p. 255, left column, first paragraph under Table 1). As evidenced by GenBank 01, the sequence of the fatty acid hydroxylase from Beopoulos with the EMBL accession no. HG326650 is the same as the fatty acid hydroxylase sequence under the GenBank accession no. CDG23429, which has 100% identity to instant SEQ ID NO:6. Therefore, Beopoulos teaches an “enzyme comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:6”. The alignment of instant SEQ ID NO:6 (Query) and GenBank 01 (Subject) is shown below.
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Beopoulos does not disclose whether the modified cell produces a fermented product having an increased level of γ-decalactone in the absence of fatty acid supplementation as compared to a level of γ-decalactone produced by a counterpart cell that does not comprise the enzyme having oleate 12-hydroxylase activity. However, this limitation is a desired property that necessarily results from the structure of the genetically modified Saccharomyces cell comprising the heterologous oleate 12-hydroxylase. Thus, the Saccharomyces cerevisiae comprising the C. purpurea FAH12 gene taught by Beopoulos would necessarily produce a fermented product having an increased level of γ-decalactone.
Regarding claim 7, Beopoulos teaches the enzyme having oleate 12-hydroxylase activity comprises the amino acid sequence set forth in SEQ ID NO:6, as evidenced by GenBank 01, and discussed in the rejection of claim 1 above.
Regarding claim 25, Beopoulos teaches Saccharomyces cerevisiae (p.252, 1st column last sentence to 2nd column top sentence).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 2-3, 8-9 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262) as evidenced by the GenBank accession number CDG23429 (GenBank 01), as applied to claim 1 above, and further in view of Ratushny et al. (“Control of Transcriptional Variability by Overlapping Feed-Forward Regulatory Motifs”, Biophysical Journal, 2008, Vol. 95, pp.3715-3723).
Regarding claim 2, Beopoulos teaches heterologous expression of the castor bean hydroxylase, encoded by the Δ12 (oleate) hydroxylase gene FAH12 (p.252, 1st column – 3rd paragraph). Beopoulos teaches S. cerevisiae does not contain genes coding for Δ12 desaturases, and heterologous expression of the C. purpurea FAH12 hydroxylase resulted in ricinoleic acid (RA) accumulation to 8% of total fatty acid (p.252, 1st column last sentence to 2nd column top sentence).
Beopoulos does not teach a genetic modification that increases peroxisome size and/or number, or wherein the modified cell produces a fermented product having a level of γ-decalactone greater than 35 µg/L in the absence of fatty acid supplementation.
However, Ratushny teaches in yeast, β-oxidation of fatty acids takes place in the peroxisome (abstract). Ratushny teaches the expression of genes required for peroxisome assembly and function is controlled by a transcriptional regulatory network that is induced by fatty acids such as oleate (abstract). Ratushny teaches that in the budding yeast Saccharomyces cerevisiae, peroxisomes are induced in response to oleic acid, and the transcription of many peroxisomal proteins and proteins required for assembly and growth of the organelle is controlled by oleate response elements recognized by the fatty-acid bound heterodimer Oaf1p-Pip2p (p.3715, 1st column, 2nd paragraph). Ratushny further teaches four core transcription factor genes (ADR1, OAF1, PIP2 and OAF3) that are indicative of peroxisome induction (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of Beopoulos to further include a gene encoding a transcription factor such as ADR1, OAF1, PIP2 and OAF3 that increases peroxisomal size and fatty acid beta-oxidation, because Ratushny teaches that these four core transcription factors regulate oleate-responsive genes, and are responsible for inducing expression of genes required for peroxisome assembly and function. One of ordinary skill in the art would have found it beneficial to modify the yeast to include genes encoding transcription factors known to increase peroxisome number to improve beta-oxidation of fatty acids, which takes place in the peroxisome.
The limitation “wherein the modified cell produces a fermented product having a level of γ-decalactone greater than 35 µg/L in the absence of fatty acid supplementation” is a desired property that results from the structure of a genetically modified yeast cell comprising a heterologous gene encoding an enzyme having oleate 12-hydroxylase activity and a genetic modification that increases peroxisome size and/or number. One of ordinary skill in the art would reasonably expect that the engineered Saccharomyces cerevisiae yeast taught by Beopoulos and Ratushny would predictably result in a genetically modified yeast that produces a fermented product having a level of γ-decalactone greater than 35 µg/L in the absence of fatty acid supplementation.
Regarding claim 3, Beopoulos teaches Claviceps purpurea hydroxylase CpFAH12 (abstract), which is the enzyme having oleate 12-hydroxylase activity is from Claviceps purpurea. Beopoulos discloses the sequence of the FAH12 gene as a nucleotide sequence of codon-optimized heterologous hydroxylase gene that was submitted to the European Nucleotide Archive (EMBL-EBI) under the accession no. HG326650 for CpFAH12 (p. 255, left column, first paragraph under Table 1). As evidenced by GenBank 01, the sequence of the fatty acid hydroxylase from Beopoulos with the EMBL accession no. HG326650 is the same as the fatty acid hydroxylase sequence under the GenBank accession no. CDG23429, which has 100% identity to instant SEQ ID NO:6. Therefore, Beopoulos teaches an enzyme comprising a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. The alignment of instant SEQ ID NO:6 (Query) and GenBank 01 (Subject) is shown below.
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Claim 9 is being interpreted as comprising three alternatives, with only one alternative required for the transcription factor; i.e. the transcription factor is required to be one of (a) ADR1, PIP2, OAF1, OAF3, or ADR comprising a substitution mutation of serine at position 230; or (b)(i) comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:24; or (b)(ii) the amino acid sequence set forth in SEQ ID NO:24.
Regarding claims 8 and 9, Beopoulos does not teach a gene encoding a transcription factor that increases fatty acid beta-oxidation as compared to a cell that does not express the transcription factor (claim 8) or wherein the transcription factor is ADR1, PIP2, OAF1 or OAF3 (claim 9).
However, Ratushny teaches in yeast, β-oxidation of fatty acids takes place in the peroxisome (abstract). Ratushny teaches the expression of genes required for peroxisome assembly and function is controlled by a transcriptional regulatory network that is induced by fatty acids such as oleate (abstract). Ratushny teaches that in the budding yeast Saccharomyces cerevisiae, peroxisomes are induced in response to oleic acid, and the transcription of many peroxisomal proteins and proteins required for assembly and growth of the organelle is controlled by oleate response elements recognized by the fatty-acid bound heterodimer Oaf1p-Pip2p (p.3715, 1st column, 2nd paragraph). Ratushny further teaches four core transcription factor genes [ADR1, OAF1, PIP2 and OAF3] that are indicative of peroxisome induction (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the yeast of Beopoulos to further include a gene encoding a transcription factor, ADR1, OAF1, PIP2 or OAF3, that increases peroxisomal size and fatty acid beta-oxidation, because Ratushny teaches that these four core transcription factors regulate oleate-responsive genes, and are responsible for inducing expression of genes required for peroxisome assembly and function. One of ordinary skill in the art would have found it beneficial to modify the yeast to include genes encoding transcription factors known to increase peroxisome number to improve beta-oxidation of fatty acids, which takes place in the peroxisome.
Regarding claim 24, Beopoulos teaches Saccharomyces cerevisiae (p.252, 1st column last sentence to 2nd column top sentence).
Claim 13 and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262) as evidenced by the GenBank accession number CDG23429 (GenBank 01) and Ratushny et al. (“Control of Transcriptional Variability by Overlapping Feed-Forward Regulatory Motifs”, Biophysical Journal, 2008, Vol. 95, pp.3715-3723) as applied to claim 8 above, and further in view of Feng et al. (“Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era”, Biology, 2021, Vol. 10, No. 6, article 504, 19 pages).
The teachings of Beopoulos et al. and Ratushny et al. are discussed above.
Regarding claim 13, Beopoulos and Ratushny do not teach the gene encoding the transcription factor is operably linked to a promoter, pPGK1.
Feng teaches Saccharomyces cerevisiae promoter engineering (title). Feng teaches the construction of artificial promoters for the yeast S. cerevisiae (abstract). Feng teaches a biosensor can be adopted to regulate protein expression according to the availability of fatty acid intermediates inside yeast cells (p.10, last paragraph). Feng further teaches using the constitutive PGK1 yeast promoter, which was used to detect fatty acids in media containing glucose (p.11, top paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to link the PGK1 promoter taught by Feng to the transcription factor taught by Ratushny in the Saccharomyces cerevisiae yeast taught by Beopoulos, because Feng teaches the PGK1 promoter can be used to detect fatty acids in media. One of ordinary skill in the art would have been motivated to use a promoter that was tied to fatty-acid sensing for increasing fatty acid beta-oxidation, because increased fatty acid concentration would further increase the expression of the gene tied to the promoter, i.e. the gene encoding an enzyme having oleate 12-hydroxylase activity as in claim 1; or the gene encoding a transcription factor that increases peroxisomal size and fatty acid beta-oxidation as in claim 8.
Regarding claim 23, Beopoulos does not teach a gene encoding a transcription factor that increases fatty acid beta-oxidation as compared to a cell that does not express the transcription factor, or teach the gene encoding the transcription factor is operably linked to a promoter selected from pPGK1.
Ratushny teaches that, in yeast, β-oxidation of fatty acids takes place in the peroxisome (abstract). Ratushny teaches the expression of genes required for peroxisome assembly and function is controlled by a transcriptional regulatory network that is induced by fatty acids such as oleate (abstract). Ratushny teaches that in the budding yeast Saccharomyces cerevisiae, peroxisomes are induced in response to oleic acid, and the transcription of many peroxisomal proteins and proteins required for assembly and growth of the organelle is controlled by oleate response elements recognized by the fatty-acid bound heterodimer Oaf1p-Pip2p (p.3715, 1st column, 2nd paragraph). Ratushny further teaches four core transcription factor genes (ADR1, OAF1, PIP2 and OAF3) that are indicative of peroxisome induction (abstract).
Feng teaches Saccharomyces cerevisiae promoter engineering (title). Feng teaches the construction of artificial promoters for the yeast S. cerevisiae (abstract). Feng teaches a biosensor can be adopted to regulate protein expression according to the availability of fatty acid intermediates inside yeast cells (p.10, last paragraph). Feng further teaches using the constitutive PGK1 yeast promoter, which was used to detect fatty acids in media containing glucose (p.11, top paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to link the PGK1 promoter taught by Feng to the transcription factor taught by Ratushny in the Saccharomyces cerevisiae yeast taught by Beopoulos, because Feng teaches the PGK1 promoter can be used to detect fatty acids in media. One of ordinary skill in the art would have been motivated to use a promoter that was tied to fatty-acid sensing for increasing fatty acid beta-oxidation, because increased fatty acid concentration would further increase the expression of the gene tied to the promoter, i.e. the gene encoding an enzyme having oleate 12-hydroxylase activity as in claim 1; or the gene encoding a transcription factor that increases peroxisomal size and fatty acid beta-oxidation as in claim 8.
Claim 22 is rejected under 35 U.S.C. 103 as being unpatentable over Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262) as evidenced by the GenBank accession number CDG23429 (GenBank 01) as applied to claim 1 above, and further in view of Feng et al. (“Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era”, Biology, 2021, Vol. 10, No. 6, article 504, 19 pages).
The teachings of Beopoulos et al. are discussed above.
Regarding claim 22, Beopoulos does not teach the gene encoding the enzyme having oleate 12-hydroxylase is operably linked to a promoter, pPGK1.
Feng teaches Saccharomyces cerevisiae promoter engineering (title). Feng teaches the construction of artificial promoters for the yeast S. cerevisiae (abstract). Feng teaches a biosensor can be adopted to regulate protein expression according to the availability of fatty acid intermediates inside yeast cells (p.10, last paragraph). Feng further teaches using the constitutive PGK1 yeast promoter, which was used to detect fatty acids in media containing glucose (p.11, top paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to link the PGK1 promoter taught by Feng to the enzyme having oleate 12-hydroxylase activity in the Saccharomyces cerevisiae yeast taught by Beopoulos, because Feng teaches the PGK1 promoter can be used to detect fatty acids in media. One of ordinary skill in the art would have been motivated to use a promoter that was tied to fatty-acid sensing for increasing fatty acid beta-oxidation, because increased fatty acid concentration would further increase the expression of the gene tied to the promoter, i.e. the gene encoding an enzyme having oleate 12-hydroxylase activity.
Claims 14-15 are rejected under 35 U.S.C. 103 as being unpatentable over Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262) as evidenced by the GenBank accession number CDG23429 (GenBank 01), as applied to claim 1 above, and further in view of Poutre et al. (EP 0 561 569 A2, published on September 22, 1993).
The teachings of Beopoulos et al. are discussed above.
Regarding claims 14 and 15, Beopoulos does not teach the modified cell further comprising a gene encoding an enzyme having acyl-CoA desaturase 1 (OLE1) activity (claim 14) or wherein the enzyme having OLE1 activity is derived from Saccharomyces cerevisiae and comprises the amino acid sequence set forth in SEQ ID NO:7 (claim 15).
However, Poutre teaches a yeast delta-9 desaturase gene (abstract). Poutre teaches the yeast delta-9 desaturase gene has been isolated from Saccharomyces cerevisiae and used to transform the same yeast strain under conditions in which it is apparently overexpressed, resulting in increased storage lipid accumulation in the transformed yeast cells (p.3, lines 20-23). Poutre teaches that typically the fatty acid composition of vegetable oils has been modified through traditional breeding techniques, for example to increase the amount of the monounsaturated fatty acid oleate in corn oil (p.2, lines 29-30 and 34-35). Poutre further teaches that saturated fatty acids may be desaturated, by an enzyme known as delta-9 saturase, and specifically stearate may be rapidly desaturated by a plastidial delta-9 desaturase enzyme to yield oleate (p.3, lines 3-5). Poutre teaches that in most oils, oleate is the major fatty acid synthesized, as the saturated fatty acids are present in much lower proportions (p.3, lines 8-10). Poutre teaches sequence listing 2, an amino acid sequence of 510 amino acids long (p.20-21, sequence for SEQ ID NO:2) that is 100% identical to instant SEQ ID NO:7, as shown in the alignment below, wherein “Query” is instant SEQ ID NO:7 and “Subject” is Poutre SEQ ID NO:2. Therefore, Poutre’s delta-9 desaturase is the same as the claimed OLE1.
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It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further include a gene for the yeast delta-9 desaturase taught by Poutre in the modified Saccharomyces yeast cell taught by Beopoulos, because Poutre teaches that Saccharomyces yeast transformed to include the gene overexpressed the gene with increased storage lipid accumulation. One of ordinary skill in the art would have been motivated to further include a gene for desaturase because Poutre teaches that in most oils, oleate is the major fatty acid synthesized. One of ordinary skill in the art would have found it beneficial to include an enzyme that rapidly desaturates stearate to yield oleate, as taught by Poutre.
Claims 14 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262) as evidenced by the GenBank accession number CDG23429 (GenBank 01), as applied to claim 1 above, and further in view of Aharoni et al. (WO 2000/032789 A1, published on June 8, 2000).
The teachings of Beopoulos et al. are discussed above.
Regarding claims 14 and 19, Beopoulos teaches S. cerevisiae do not contain genes coding for Δ12 desaturases, and heterologous expression of C. purpurea FAH12 hydroxylase resulted in ricinoleic acid (RA) accumulation to 8% of total fatty acid (p.252, 1st column last paragraph – 2nd column top paragraph). Beopoulos further teaches that when the ricinoleic-specific CpDGAT2 acyltransferase was co-expressed with the hydroxylase in S. cerevisiae, the RA content was slightly higher, at 10% of total fatty acids (p.252, 2nd column top paragraph).
Beopoulos does not teach the modified cell further comprising a gene encoding an enzyme having alcohol-O-acyltransferase (AAT) activity (claim 14) or wherein the enzyme having AAT activity comprises a sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:25 (claim 19).
However, Aharoni teaches DNA sequences encoding enzymes involved in the biosynthetic pathway for aliphatic and/or ester production in fruit (abstract). Aharoni teaches the enzymes have alcohol acyl transferase activities (abstract). Aharoni teaches the invention disclosed relates to strawberry (Fragaria ananassa) fruit derived genes and enzymes specifically involved in the formation of aliphatic and/or aromatic esters and other aroma and flavour compounds in fruits (p.1, lines 1-5). Aharoni teaches that the major components of strawberry flavour and aroma may be grouped into several chemical classes including lactones (p.1, lines 16-19). Aharoni teaches SEQ ID NO:18B, an amino acid sequence for apple alcohol acyl transferase of 454 amino acids long (p.111, sequence for SEQ ID NO:18B) that is 99% identical to instant SEQ ID NO:25, as shown in the alignment below, wherein “Query” is instant SEQ ID NO:25 and “Subject” is Aharoni SEQ ID NO:18B.
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It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the acyltransferase taught by Beopoulos with an alcohol-O-transferase having the sequence of SEQ ID NO:18B taught by Aharoni to be co-expressed with the hydroxylase in S. cerevisiae. Each of Beopoulos and Aharoni teach the use of acyltransferases in the production of fatty acids. One of ordinary skill would reasonably expect that replacing one known acyltransferase with another would predictably result in a genetically modified yeast cell comprising a gene encoding an enzyme having alcohol-O-acyltransferase activity, because it was known in the art at the time of invention that yeast engineered to express both oleate 12-hydroxylase enzymes and acyltransferase enzymes resulted in increased fatty acid production.
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over Beopoulos et al. (“Metabolic engineering for ricinoleic acid production in the oleaginous yeast Yarrowia lipolytica”, Applied Microbiology and Biotechnology, 2014, Vol. 98, pp.251-262) as evidenced by the GenBank accession number CDG23429 (GenBank 01), as applied to claim 1 above, and further in view of Berner et al. (“The impact of different ale brewer’s yeast strains on the proteome of immature beer”, BMC Microbiology, 2013, Vol. 13, article 215, 8 pages). The rejection of claim 26 is further evidenced by White Labs (WLP001 California Ale Yeast https://www.whitelabs.com/yeast-single?id=101&type=YEAST).
The teachings of Beopoulos are discussed above.
Regarding claim 26, Beopoulos does not teach wherein the yeast cell is S. cerevisiae California Ale Yeast WLP001.
However, Berner teaches two ale brewer’s yeast (Saccharomyces cerevisiae) strains (abstract). Berner teaches yeast strain WLP001 obtained from White Labs (p.2, 1st column – Yeast strains and media). As evidenced by White Labs, the WLP001 strain is known as WLP001 California Ale Yeast.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the S. cerevisiae strain taught by Beopoulos with the Saccharomyces cerevisiae strain WLP001 taught by Berner, because it would amount to a simple substitution of one known Saccharomyces cerevisiae strain for another, and the Saccharomyces cerevisiae WLP001 strain was known in the art at the time of invention.
Conclusion
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/DEEPA MISHRA/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
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