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 claims 1-3 and 6-14 in the reply filed on 11/23/2025 is acknowledged.
Claims 4-5 and 15 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 11/23/2025.
Priority
This application is a 371 of PCT/EP2022/053036 (2/8/2022) which claims priority to EP21155780.6 (2/8/2021); PT117340 K (7/13/2021); and EP21196276.6 (9/13/2021) as reflected in the filing receipt issued on 1/12/2024.
Information Disclosure Statement
The information disclosure statement (IDS) filed on 8/2/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claim 14 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, 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.
Regarding written description, 35 U.S.C. 112(a) and the first paragraph of pre-AlA 35 U.S.C. 112 require that the "specification shall contain a written description of the invention ...." This requirement is separate and distinct from the enablement requirement. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010). To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention (MPEP § 2163(I)).
MPEP 2163(II)(A)(3)(a)(i and ii) 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. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., .759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014). Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.
In the instant case, claim 14 recites the phenylpyruvate decarboxylase, phospho-2-dehydro-3-deoxyheptonate aldolase, prephenate dehydrogenase, and uridine diphosphate dependent glycosyltransferase having at least 60%, 65%, 70%, 75%, 80%, particularly 85%, more particularly 90%, even more particularly 95% or yet even more particularly >95% identity to SEQ ID NOs: 1-4 respectively, and having a catalytic activity of at least 75% of the activity of SEQ ID NOs: 1-4. There is not sufficient written description support in the disclosure for sequences having at least 60% identity to SEQ ID NOs: 1-4 and having the claimed catalytic activity. There is no disclosed structure-function relationship to indicate which residues or regions of SEQ ID NOs: 1-4 can be modified or differ from the wild type sequences and still retain the catalytic function, beyond the specific point mutations set forth in the specification in Tables 7 and 8. As claimed, up to 40% of any of the amino acid residues may differ between SEQ ID NOs: 1-4, encompassing a large number of potential sequences.
It is known in the art that sequence identity is not necessarily a reliable indicator of protein function (see Devos et al., p. 98 “Abstract”; “Sequence Similarity and the Prediction of Protein Structure and Function”). Thus, it is considered that modifications to an amino acid sequence are unpredictable, and a skilled artisan would not be aware of which of the many possible amino acid sequences would maintain the claimed catalytic function. Claim 14 encompasses a large genus of potential amino acid sequences, and the specification lacks sufficient variety of representative species to reflect the variation in this genus.
For these reasons, claim 14 fails to comply with the written description requirement, as the disclosure does not reasonably convey to a person having ordinary skill in the art that the inventors had possession of the entire scope of the claimed invention at the time of filing.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-3 and 6-14 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c).
In the present instance, claim 1 recites the broad recitation “a metabolic precursor”, and the claim also recites “particularly wherein the metabolic precursor is glucose” which is the narrower statement of the range/limitation.
Claim 2 recites the broad recitation “of the genus Escherichia”, and the claim also recites “particularly wherein the transgenic bacterial cell is of the species E. coli, more particularly wherein the transgenic bacterial cell is of the strain E. coli BL21” which is the narrower statement of the range/limitation.
Claim 3 recites the broad recitation “from yeast”, and the claim also recites “particularly from S. cerevisiae” which is the narrower statement of the range/limitation.
Claim 8 recites the broad recitation “one or several plasmid vector(s)”, and the claim also recites “particularly wherein phenylpyruvate decarboxylase, phospho-2-dehydro-3-deoxyheptonate aldolase and prephenate dehydrogenase are encoded by a medium-copy plasmid vector, and/or uridine diphosphate dependent glycosyltransferase is encoded by a low-copy plasmid vector” which is the narrower statement of the range/limitation.
Claim 9 recites the broad recitation “a promoter sequence”, and the claim also recites “particularly a T7 promoter (SEQ ID NO. 31), a lac promoter (SEQ ID NO. 32), a tac promoter
(SEQ ID NO. 33) or a trc promoter (SEQ ID NO. 34), more particularly wherein the gene encoding uridine diphosphate dependent glycosyltransferase is under control of a trc promoter, and/or the gene encoding phenylpyruvate decarboxylase is under control of a T7 promoter, and/or the gene encoding phospho-2-dehydro-3-deoxyheptonate aldolase is under control of a T7 promoter, and/or the gene encoding prephenate dehydrogenase is under control of a T7 promoter” which is the narrower statement of the range/limitation.
Claim 10 recites the broad recitation “adding isopropyl-β-d-thiogalactopyranoside (IPTG)”, and the claim also recites “particularly at a concentration of ~0.1 mM IPTG” which is the narrower statement of the range/limitation.
Claim 11 recites the broad recitation “10 to 50 g/L of glucose”, and the claim also recites “particularly 15 to 30 g/L of glucose” which is the narrower statement of the range/limitation.
Claim 13 recites the broad recitation “antibiotics”, and the claim also recites “particularly wherein the antibiotics are 50-200 μg/mL ampicillin, 10-50 μg/mL kanamycin and 25-45 μg/mL chloramphenicol” which is the narrower statement of the range/limitation.
Claim 14 recites the broad recitation “at least 60%, 65%, 70%, 75%, 80%”, and the claim also recites “particularly 85%, more particularly 90%, even more particularly 95% or yet even more particularly >95% sequence identity” which is the narrower statement of the range/limitation.
The claims are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims.
For the purposes of examination, only the limitations of the broad recitations are deemed to be required.
Claim 7 recites the limitation "wherein the method is directed at the production of salidroside". There is insufficient antecedent basis for this limitation in the claim. Claim 7 depends on claim 1. Claim 1 is directed to a method for production of tyrosol. There is no recitation in claim 1 of production of salidroside. Thus, it is unclear what this limitation in claim 7 is referring to.
Similarly, claims 8, 9, and 14 recite “uridine diphosphate dependent glycosyltransferase is encoded” and “the gene encoding uridine diphosphate dependent glycosyltransferase”. Claims 8, 9, and 14 depend on claim 1, which does not recite a uridine diphosphate dependent glycosyltransferase. Therefore, it is unclear what these limitations in claims 8, 9, and 14 refer to.
Claim 12 recites “the transgenes”. There is insufficient antecedent basis for this limitation in the claim. Claim 12 depends on claim 1. Claim 1 does not recite a “transgene”. From the specification, it appears that transgene refers to a gene derived from a different organism than the host cell (see specification p. 1). However, as this term is not used in claim 1, and claim 1 recites multiple different genes that are expressed in the bacterial cell, it is not clear what this limitation in claim 12 refers to. It is suggested that the claims are amended to utilize consistent terminology for clarity.
Claim 1 recites: “(ARO10)”, “(aroF)”, “(tyrA)”, “(bifunctional chorismate mutase/prephenate dehydratase)”, and “(phenylacetaldehyde dehydrogenase)”. The parentheses in these instances make it unclear if these limitations are required or optional. For example, it is not clear if claim 1 requires the expression of any phenylpyruvate decarboxylase, or if it requires specifically ARO10.
Claim 6 recites “(tyrR)”. As stated above, the parentheses make it unclear if this limitation is required or optional, i.e. any DNA-binding transcriptional regulatory protein or specifically tyrR.
Claim 9 recites “(SEQ ID NO: 31)”, “(SEQ ID NO: 32)”, “(SEQ ID NO: 33)”, and “(SEQ ID NO: 34)”. As stated above, the parentheses make it unclear if these limitations are required or optional, i.e. any T7/lac/tac/trc promoter, or specifically those having the recited sequences.
Claim 13 recites “1-3 % (w/v) glucose” and “0.01-0.05% (w/v) yeast extract”. The parentheses around w/v make it unclear if this is a required or optional claim limitation.
The parenthetical limitations listed above render the scope of these claims unclear. It is suggested that the claims be amended to remove these instances of parentheses or restated to clarify what is a required element.
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.
(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.
Claims 1-3, 6-11, and 13-14 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Yang et al., Journal of Agricultural and Food Chemistry; 67(14):3900-8, as evidenced by Sigma-Aldrich M9 Minimal Salts Medium and NCBI phenylpyruvate decarboxylase aro10 gene, NP_010668.3.
Regarding claim 1, Yang teaches a method of producing tyrosol using transgenic bacteria (Yang “Abstract”). Yang teaches modified strain E. coli BFPG2, which produces tyrosol (p. 3905 Fig. 4).
Yang teaches that E. coli BFPG2 heterologously expresses a phenylpyruvate decarboxylase, ARO10 (p. 3900 para. 4; p. 3902 Table 3).
Yang teaches that E. coli BFPG2 additionally overexpresses a phospho-2-dehydro-3-deoxyheptonate aldolase gene, aroG*; and a prephenate dehydratase gene, tyrA* (p. 3901 first partial para.; p. 3902 Table 3 E. coli BFPG2). Yang teaches that the phospho-2-dehydro-3-deoxyheptonate aldolase AroG is the isoenzyme (i.e., catalyzes same reaction) of AroF (p. 3904 “Initial Pathway Construction”). Yang teaches that the prephenate dehydrogenase activity of TyrA can be inhibited by aromatic amino acids, but mutations of Met53Ile and Ala354Val of TyrA eliminate the inhibitory effect (p. 3906 “Discussion” para. 1). The gene tyrA* encodes the enzyme having these mutations, and tyrA* is overexpressed in E. coli BFPG2.
Yang teaches that E. coli BFPG2 has deletions of, or does not express, pheA (Tables 1 and 3, GenBank ID 947081; chorismate mutase/prephenate dehydratase) and feaB (Tables 1 and 3, GenBank ID 945933; phenylacetaldehyde dehydrogenase).
Yang teaches that this strain is grown in a medium comprising glucose, which is a metabolic precursor of PEP and E4P (p. 3901 “Media and Culture Conditions”). Yang teaches that tyrosol is extracted from the fermentation broth or culture medium (p. 3901-3902 “Media and Culture Conditions”; “HPLC Analysis”).
Regarding claim 2, Yang teaches that the transgenic bacterial cell is E. coli BL21 (p. 3901 “Strains and Plasmids”).
Regarding claim 3, Yang teaches that the ARO10 gene encoding the phenylpyruvate decarboxylase originates from the yeast S. cerevisiae (p. 3901 Table 1).
Regarding claim 6, the strain E. coli BFPG2 of Yang does not overexpress genes encoding alcohol dehydrogenase, DNA-binding transcriptional regulatory protein (tyrR), and tyrosine aminotransferase (p. 3902 Table 3, showing all genes which are overexpressed in BFPG2).
Regarding claim 7, “heterologously expressed” as defined on p. 1 of the instant specification “means that the gene is derived from a source other than the host species in which it is said to be heterologously expressed”. E. coli BFPG2 as taught by Yang expresses aro10 from S. cerevisiae, and aroA, tyrA*, aroE, and aroG derived from E. coli (Yang p. 3901 Table 1; p. 3902 Table 3). Thus, the only heterologously expressed gene in the cell is phenylpyruvate decarboxylase, aro10.
Regarding claim 8, Yang teaches that the overexpressed genes are introduced into the transgenic cell via plasmid vectors (p. 3901 “Strains and Plasmids”)
Regarding claim 9, Yang teaches that the genes encoding phenylpyruvate decarboxylase, phospho-2-dehydro-3-deoxyheptonate aldolase, and prephenate dehydrogenase are under the control of a T7 promoter (p. 3901 “Strains and Plasmids”).
Regarding claim 10, Yang teaches that the expression of heterologous and/or overexpressed genes is induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) (p. 3902 “Media and Culture Conditions”).
Regarding claim 11, Yang teaches that the medium comprises 1% w/v of glucose, which is equal to 1g/100mL, or 10 g/L (p. 3901 “Media and Culture Conditions”).
Regarding claim 13, Yang teaches M9Y minimal medium containing 1x M9 minimal salts, 5 mM MgSO4, 0.1 mM CaCl2, 1% w/v glucose, and 0.025% w/v yeast extract, with kanamycin (antibiotic) final concentration of 50 μg/mL (p. 3901 “Media and Culture Conditions”).
0.1 mM CaCl2, which has a molar mass of 110.98 g/mol, is equivalent to 0.011 g/L CaCl2.
5x M9 minimal salts from Sigma-Aldrich (see Sigma-Aldrich reference, p. 3) contains:
33.9 g/L Na2HPO4-
15 g/L KH2PO4
2.5 g/L NaCl
5 g/L NH4Cl
Thus, a 1x solution of M9 minimal salts, as used by Yang, comprises:
6.78 g/L Na2HPO4-
3 g/L KH2PO4
0.5 g/L NaCl
1 g/L NH4Cl
All of these values are within the claimed concentration ranges. As such, the medium
taught by Yang anticipates the claimed medium.
Regarding claim 14, Yang teaches that the phenylpyruvate decarboxylase aro10 gene has GenBank ID 851987 (Yang Table 1). The associated protein sequence is NCBI Reference Sequence: NP_010668.3 (see attached NCBI reference). This sequence is 100% identical to instant SEQ ID NO: 1 and is thus considered to have the same catalytic activity (see Sequence Alignment in OA appendix).
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.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Yang as applied to claims 1-3, 6-11, and 13-14 above, in view of Menzella, Microbial cell factories; 10(1):15.
Yang teaches the method according to claim 1, as set forth above. Yang does not teach that the transgenes are codon-optimized for expression in the transgenic bacterial cell as set forth in claim 12.
A “transgene” as set forth in the instant specification refers to a gene derived from a different organism than the host cell (see specification p. 1), i.e. aro10 from S. cerevisiae.
Regarding claim 12, Menzella teaches that for expressing enzymes in host cells, such as the preferred host cell, E. coli, species-specific variations in codon usage must be considered (Menzella “Background”). Menzella teaches that variations in codon usage between species are one of the major causes affecting recombinant protein expression levels, with a significant impact on the economy of industrial enzyme production processes (Menzella “Abstract”). Menzella teaches common strategies for codon optimization to allow for optimal expression in E. coli (Menzella “Abstract”).
It would have been obvious to a skilled artisan, before the effective filing date, to modify the method of Yang and codon-optimize the transgene, i.e. the aro10 gene from S. cerevisiae, for expression in E. coli. This is well-established technique, as taught by Menzella, and a skilled artisan would be aware of the differences in codon usage between species and the need for codon optimization of DNA sequences for efficient protein expression. It would have been obvious for an ordinary artisan to use a well-known codon-optimization technique for expression of a gene in the transgenic cell of Yang.
A person of ordinary skill in the art would have been motivated to modify the teachings of Yang and codon-optimize the aro10 gene from S. cerevisiae because, as taught by Menzella, differences in codon usage between species are a major issue in recombinant protein expression that can lead to poor expression of protein/enzymes in industrial settings. Further, it is desirable to express proteins in E. coli as a host (Menzella “Background”). As Yang teaches expression of S. cerevisiae aro10 in an E. coli host, an ordinary artisan would have been motivated to improve the expression of this gene derived from a different species using a common technique of codon optimization.
A skilled artisan would have a reasonable expectation of success in codon-optimizing the transgene of Yang. As codon optimization is a well-established and known technique for adapting DNA sequences to be expressed in various host species, particularly in E. coli, as taught by Menzella, a skilled artisan could expect to successfully codon optimize the transgene sequence of Yang for expression in E. coli.
Conclusion
Claims 1-3 and 6-14 are rejected. No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653