DETAILED ACTION
All objections and rejections stated in prior Office Actions are withdrawn unless restated below.
Claims 12-15 remain withdrawn.
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 08/20/2025 has been entered.
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.
Claims 1-6, 8-11, 20-21, 23, 25 and 27 (all non-withdrawn claims) are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement.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.
The purpose of the written description requirement is to ensure that the inventor had possession, at the time the invention was made, of the specific subject matter claimed. For a broad generic claim, the specification must provide adequate written description to identify the genus of the claim.
“A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) (“In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus.”). Regents of the University of California v. Eli Lilly & Co., 119, F.3d 1559, 1568, 43 USPQ2d 1398, 1405 (Fed. Cir. 1997).
MPEP § 2163 further states that if a biomolecule is described only by a functional characteristic, without any disclosed correlation between function and structure of the biomolecule, it is "not sufficient characteristic for written description purposes, even when accompanied by a method of obtaining the claimed biomolecule.”
“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.” MPEP 2163.
Furthermore, 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 gen[us].’ See Enzo Biochem, 323 F.3d at 966, 63 USPQ2d at 1615; Noelle v. Lederman, 355 F.3d 1343, 1350, 69 USPQ2d 1508, 1514 (Fed. Cir. 2004) (Fed. Cir. 2004) (‘[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated.’). ‘A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when … the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed.’ In re Curtis, 354 F.3d 1347, 1358, 69 USPQ2d 1274, 1282 (Fed. Cir. 2004).” MPEP 2163.
Scope of claims
The rejected claims recite a genetically modified host cell capable of production a hydroxylated methyl ketone (HMK). Independent claim 1 recites the presence in the modified host cell of specific nucleic acid constructs and gene deletions consistent with the pathway shown in Fig. 1 of the specification for production of methyl ketone labeled as “Prior Art.”
An HMK is understood to be produced by the conversion of a substrate by activity of a cytochrome P450 (CYP) that is recited to be CYP170A1. Applicant has elected to act as their own lexicographer as set forth in MPEP 2111.01(IV): The only exceptions to giving the words in a claim their ordinary and customary meaning in the art are (1) when the applicant acts as their own lexicographer; and (2) when the applicant disavows or disclaims the full scope of a claim term in the specification. To act as their own lexicographer, the applicant must clearly set forth a special definition of a claim term in the specification that differs from the plain and ordinary meaning it would otherwise possess.
Applicant clearly sets forth the following in para. [0010] of the specification: “In some embodiments, the CYP is an epi-isozizaene 5-monooxygenase, such as Streptomyces coelicolor epi-isozizaene 5-monooxygenase (CYP170A1).”
As such, the specification clearly sets forth a special definition of the claim term CYP170A1 as being Streptomyces coelicolor epi-isozizaene 5-monooxygenase, which the specification indicates has the amino acid sequence of SEQ ID NO: 3.
Claim 1 states that the substrate is a fatty acid. The disclosure of the specification appears to describe conversion of methyl ketones (lacking a carboxylic acid group) to HMKs by CYP as shown, or conversion of fatty acid to hydroxy fatty acid (HFA) by CYP and then to HMK as shown in Figs. 2 and 3 as excerpted below. For this reason, fatty acid as recited is considered to include methyl ketones produced from fatty acids (with applicant acting as their own lexicographer). Regardless, claim 1 explicitly requires CYP170A1 to have activity to convert a fatty acid to HFA.
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As such, claim 1 recites a genus of substrates of CYP170A1 being at least fatty acids convertible to HFA.
Analysis
As discussed, claim 1 recites a pathway in which a methyl ketone/fatty acid is converted to HMK by activity of CP170A1 defined by the specification as Streptomyces coelicolor epi-isozizaene 5-monooxygenase having SEQ ID NO: 3. Fasan et al. (U.S. 2014/0038850 A1), para. [0007], describe that “Heme-dependent monooxygenases, also referred to as cytochrome P450 monooxygenases or CYPs are a large class of enzymes found in both eukaryotic and prokaryotic organisms, including bacteria, fungi, plants, insects, and mammals.” “While many different monooxygenases can be isolated from natural sources or produced by protein engineering, a bottleneck remains, namely the time and screening effort required to identify the variant(s) with the suitable level of catalytic activity (i.e., turnover number and turnover rate) and selectivity (i.e., chemo-, regio- and stereoselectivity) for the intended synthetic application. Typically, this requires the screening of large libraries of natural or engineered monooxygenases by GC- or HPLC-based methods, which are inherently low throughput and involve extensive sample manipulation.” Fasan et al., para. [0010]. As such, Fasan et al. teach that the reactivity (i.e. substrate specificity) of CYP enzymes are not readily predictable. Fasan et al. proposes methods for better predicting the substrate specificity of CYPs particularly to terpenes (see Fasan et al., claim 6); however, Fasan et al. provides no guidance regarding which CYPs may be effective for conversion of methyl ketones/fatty acids to HMKs.
Peters et al. (Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3, J. Am. Chem. Soc. 125 (2003): 13442-49) further evidences the unpredictability of regioselectivity of CYP enzymes. Fig. 1 of Peters et al. evidences that a wild-type Bacillus megaterium (BM-3) CYP is unable to hydroxylate a fatty acid at the omega position. However, point mutation mutants of the BM-3 CYP is able to achieve limited omega hydroxylation as shown in Tables 1 and 2 of Peters et al.
Uniprot, Accession No. Q9K498, 2019, www.uniprot.org, and Zhao et al. (Crystal structure of albaflavenone monooxygenase containing a moonlighting terpene synthase active site, J. Biol. Chem. (2009): 36711-19) describe a cytochrome P450 enzyme identical to recited SEQ ID NO: 3 as an epi-isozizaene 5-monooxygenase from Streptomyces coelicolor, wherein epi-isozizaene is a terpene.
“More than 300,000 distinct CYP proteins are known.” Specification, para. [0009]. “Different CYP enzymes can install hydroxyl groups at the omega position, or at the omega-1 position with either R- or S- stereochemistry into saturated or unsaturated MKs, yielding the library of HMK compounds 1-6 (Figure 3).” Specification, para. [0027].
The specification, para. [0010], describes “In some embodiments, the CYP is an epi-isozizaene 5-monooxygenase, such as Streptomyces coelicolor epi-isozizaene 5-monooxygenase (CYP170A1).” “HMKs 1-6 are also produced through a second process, which operates independently or simultaneously in the recombinant cells comprising the invention. In Process 2 (Figure 3), saturated or unsaturated fatty acids are produced endogenously from glucose as described in Process 1 (Figure 2). In Process 2, these fatty acids are converted to hydroxy fatty acids (HFAs) by overexpression of CYP.” Specification, para. [0085].
It is unclear if the specification describes any working embodiments. An “Example 1” is provided in the specification. However, no details regarding specific host cells (e.g. E. coli, S. cerevisiae) are described, no specific recombinant vectors encoding specific enzymes are described, no methods for culturing a specific genetically modified microorganism are described (e.g. media, carbon source, temperature, time), and the production of any specific HMK is not described. As such, the specification does not describe a working example that can be reproduced by an ordinarily skilled artisan at the time of the invention.
The specification, in paras. [0009] and [0027], admits that at least 300,000 CYP enzymes are known in the art and that such CYP enzymes vary in at least regiospecificity and stereospecificity.
McLean et al. (Microbial cytochromes P450, Chapter 6 in Cytochrome P450: Structure, Mechanism, and Biochemistry, 4th Ed., 2015), page 265, right col., teach “The profound influence of mutations on P450 properties is evident from the fact that even a single amino acid change can alter substrate specificity or have other dramatic effects on catalysis.” “Microbial P450s have diverse catalytic functions (with numerous isoforms remaining uncharacterized) and divergent sequences, but adopt the same general structural fold as their eukaryotic counterparts.” McLean et al., page 265, left col.
McLean et al., Table 6.2, reviews the function of various microbial CYP450 family proteins. The specification, para. [0010], describes the CYP enzyme of SEQ ID NO: 3 as being of family CYP170A1. Table 6.2 of McLean et al. describes CYP170A1 from S. coelicolor A3(2) as having a function of “2-step allylic oxidations of epi-isozizaene to albaflavonols(s) in albaflavenone biosynthesis,” which is consistent with the function described by Zhao et al. Other classes of CYP450, such as CYP152A1 described in Table 6.2, is described as being a fatty acid hydroxylase.
Van Bogaert et al. (The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism, FEBS J. 278, 2011, 206-21) teach: “More than 3800 microbial P450s are known to date and we estimate that ~ 10–17% of them display activity towards fatty acids or related structures.” Van Bogaert, page 207, left col. As such, Van Bogaert evidences that the large majority of cytochrome P450 enzymes do not have activity to utilize fatty acids (and presumably methyl ketones as well), and an even smaller number of cytochrome P450s enzymes will have the specific activity recited in claim 2 of hydroxylation at the omega or omega-1 position wherein Peters evidence that among P450 enzymes capable of hydroxylation of fatty acids that not all are capable of performing omega hydroxylation.
While the specification asserts that the CYP450 of SEQ ID NO: 3 being CYP170A (as defined by the specification as discussed above) has activity to hydroxylate methyl ketones and/or fatty acids, such an activity is not consistent with the description of Streptomyces coelicolor epi-isozizaene 5-monooxygenase (CYP170A1) in the prior art that is not associated with such activity wherein Van Bogaert evidences that the large majority of cytochrome P450 enzymes do not have activity to utilize fatty acids (and presumably methyl ketones as well). Out of the “More than 300,000 distinct CYP proteins” referenced by the specification and several families of CYP450 enzymes described in Table 6.2 of McLean et al., it is not clear why the specification proposes that Streptomyces coelicolor epi-isozizaene 5-monooxygenase (CYP170A1) having has activity to hydroxylate methyl ketones and/or fatty acids, and it is unpredictable that Streptomyces coelicolor epi-isozizaene 5-monooxygenase (CYP170A1) characterized as a epi-isozizaene 5-monooxygenase will also have activity to hydroxylate methyl ketones and/or fatty acids in the absence of any objective evidence of the same wherein “[t]he profound influence of mutations on P450 properties is evident from the fact that even a single amino acid change can alter substrate specificity or have other dramatic effects on catalysis.” Further, “More than 3800 microbial P450s are known to date and we estimate that ~ 10–17% of them display activity towards fatty acids or related structures.” Van Bogaert et al., page 207. That is, “A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when … the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed.”
That is, in review of above, MPEP 2163(II)(A)(3)(a) provides:
i) For Each Claim Drawn to a Single Embodiment or Species:
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(A) Determine whether the application describes an actual reduction to practice of the claimed invention.
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(B) If the application does not describe an actual reduction to practice, determine whether the invention is complete as evidenced by a reduction to drawings or structural chemical formulas that are sufficiently detailed to show that inventor was in possession of the claimed invention as a whole.
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(C) If the application does not describe an actual reduction to practice or reduction to drawings or structural chemical formula as discussed above, determine whether the invention has been set forth in terms of distinguishing identifying characteristics as evidenced by other descriptions of the invention that are sufficiently detailed to show that inventor was in possession of the claimed invention.
The specification as discussed above does not reduce to practice the ability of CYP170A1 to convert any species of fatty acid to any species of HFA. As set forth above, the ability of CYP170A1 to have any additional hydroxylation activity in addition to being a coelicolor epi-isozizaene 5-monooxygenase, and specifically to hydroxylate fatty acids or methyl ketones, is unpredictable in the art wherein the large majority of CYP enzymes do not have such activity. See Van Bogaert et al. As such, the unsupported statements of the specification that CYP170A1 has activity to hydroxylate fatty acids or methyl ketones does not show that inventor(s) were in possession of the claimed invention wherein such activity is required by the claims.
Here, in the absence of any evidence that Streptomyces coelicolor epi-isozizaene 5-monooxygenase (CYP170A1) characterized as a epi-isozizaene 5-monooxygenase will also have activity to hydroxylate methyl ketones and/or fatty acids, including any evidence of omega or omega-1 hydroxylase activity on methyl ketone of fatty acid substrates, an ordinarily skilled artisan cannot predict the operability of CYP170A1 in embodiments of claims 1 and 2 and claims depending therefrom.
It is emphasized that a genus of enzymes having monooxygenase activity with 100% identity or otherwise significant identity to SEQ ID NO: 3 is supported by the as-filed specification. What is not supported by the as-filed specification is a genus of substrates for the recited CYP170A1 being at least fatty acids convertible to HFA by CYP170A1.
Comment on prior art
Gonzalez et al. (U.S. 2018/0135059 A1) is the only significant identified prior art concerning a modified host cell for production of a hydroxylated methyl ketone. As discussed, such hydroxylated methyl ketone are formed from an initial omega-hydroxy functionalized CoA to that is extended by condensation with acetyl-CoA by activity of a thiolase. However, Gonzalez et al. supplies no particular motivation to pursue production of an unsaturated and/or branched HMK, which would require the use of a hydroxylated unsaturated or branched CoA primer. However, the prior art is not considered to be enabling for production of such a hydroxylated unsaturated or branched CoA primer. Gonzalez et al. (U.S. 9,994,881 B2), in Figure 2 and related text, describes the production of isobutyryl-CoA and 3-metyl-butyryl-CoA that would form a branched product if applied to the methods of U.S. 2018/0135059 A1. However, the same is not considered to be enable or suggestive of, for example, a hydroxylated isobutyryl-CoA.
Response to arguments
Applicant argues:
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Applicant’s amendment is acknowledged. While applicant states a conclusion of “Claim 1 complies with the written description requirement,” applicant’s remarks do not address the grounds of rejection, which is that applicant has not shown possession of a genus of substrates for an enzyme having SEQ ID NO: 3 that are fatty acids.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652