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 .
Claim Objections
Claim 26 and 33-35 objected to because of the following informalities:
In claim 26, a period can only appear at the end of the claim such that “a.” should be “a)” or similar notation; same for “b.” and “c.”.
In claims 33-35, “rate of conversion” implies a temporal element since a “rate” is change over time. However, a “rate” cannot be expressed as a percentage such that claims 33-35 can only be understood as a final molar conversion or yield that is not a rate. Since claims 33-35 are not subject to multiple scope interpretations, they are not rejected under 35 U.S.C. 112(b) but should be corrected to reflect a conversion yield rather than a “rate.”
Appropriate correction is required.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 46, 49 and 50 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yamada et al. (Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida, Biotechnol. Lett. 30, 2008, 665-70) (see IDS) further in view of Uniprot, Accession No. A0A081CI87, 2020, www.uniprot.org.
Yamada, abstract, teaches:
“The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20°C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.”
“The transformant, E. coli/pT21IDE, was pre-cultured on 200 ml of LB medium containing 50 μg ampicillin/ml at 37°C for 8 h with reciprocal shaking at 90 rpm in a 500-ml conical flask. For the expression of isoeugenol monooxygenase, isopropyl thio-β-D-galactopyranoside [IPTG] was added to give 1 mM, and the cultivation was continued at 25°C for a further 16 h.”
“The E. coli/pT21IED cells, harvested by centrifugation at 9,000g for 20 min, were washed twice with 50 mM potassium phosphate buffer (pH 7.0). The washed cells were suspended in the same buffer and used for vanillin production.”
“The reaction mixture contained 300 mM isoeugenol, 100 mM glycine/NaOH buffer (pH 10.5), 10% (v/v) DMSO and cells of P. putida IE27 or E. coli/pT21IDE in a total volume of 10 ml. The reaction was aerobically agitated using a magnetic stirrer at 20°C in a 50-ml conical flask, started by the addition of isoeugenol, and stopped by adding 10 ml ethanol. After centrifugation at 13,000g, the supernatant was analyzed by HPLC for isoeugenol and vanillin. Vanillin-producing activity of the cells was defined as the amount of vanillin formed per min by 1 mg dry cell wt. Acetaldehyde was determined as acetaldehyde 2,4-dinitrophenylhydrazone as described previously.”
A recombinant E. coli cell in a culture as described above is understood as being “isolated.”
However, Yamada et al. does not teach E. coli transformed to a gene/polynucleotide gene encoding a monooxygenase gene having at least 70% identity to recited SEQ ID NO: 2.
“With respect to the microbial degradation of isoeugenol, we recently revealed for the first time that isoeugenol monooxygenase catalyzes the double-bond cleavage to form vanillin. The biotransformation of isoeugenol has been reported using several isoeugenol-degrading bacteria.” Yamada et al. page 665, right col.
Substitution of known elements is obvious upon a finding of:
(1) a finding that the prior art contained a device (method, product, etc.) which differed from the claimed device by the substitution of some components (step, element, etc.) with other components;
(2) a finding that the substituted components and their functions were known in the art;
(3) a finding that one of ordinary skill in the art could have substituted one known element for another, and the results of the substitution would have been predictable; and
(4) whatever additional findings based on the Graham factual inquiries may be necessary, in view of the facts of the case under consideration, to explain a conclusion of obviousness. (MPEP 2143(I)(B)).
Here, (1) Yamada teaches an isolated recombinant having all of the features of claim 46 except for expression a polynucleotide encoding a different isoeugenol monooxygenase (i.e. not having at least 70% identity to SEQ ID NO: 2.). (2) However, an isoeugenol monooxygenase having at least 70% identity to SEQ ID NO: 2 is known in the prior art as taught by GenBank A0A081CI87. That is, an isoeugenol monooxygenase by definition utilizes isoeugenol as a substate wherein Yamada teaches that the product of monooxygenation is vanillin. (3) An ordinarily skilled artisan at the time of filing could have replaced the specific polynucleotide encoding isoeugenol monooxygenase taught by Yamada with another known isoeugenol monooxygenase with an expectation of maintaining some ability to convert isoeugenol to vanillin, wherein GenBank A0A081CI87 teaches such an isoeugenol monooxygenase that can be substituted into embodiments of Yamada. (4) No other additional findings are deemed to be necessary. As such, in view of MPEP 2143(I)(B), an ordinarily skilled artisan at time of filing would have motivated to substitute a gene encoding the isoeugenol monooxygenase taught by GenBank A0A081CI87 into embodiment E. coli of Yamada in view of the guidance of MPEP 2143(I)(B), which reaches the features of claims 46, 49 and 50.
An alignment between recited SEQ ID NO: 2 and GenBank A0A081CI87 is as follows showing greater than 80% identity to SEQ ID NO: 2:
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Claim(s) 26, 27, 30-37, 40-44, 46, 49 and 50 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yamada et al. (Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida, Biotechnol. Lett. 30, 2008, 665-70) and Uniprot, Accession No. A0A081CI87, 2020, www.uniprot.org as applied to claims 46, 49 and 50 above, and further in view of Goldsmith et al. (U.S. 2017/0172184 A1).
Regarding claims 26 and 36, as set forth above, as discussed above, an ordinarily skilled artisan at the time of filing would have been motivated to practice embodiments of Yamada with other known isoeugenol monooxidases included as taught by GenBank A0A081CI87. Upon making such a combination, practice of the methods of Yamada, results in the following method:
A bioconversion method of producing vanillin, comprising: a. expressing a VpIEM gene in E. coli cells in a mixture, wherein the expressed protein of the VpIEM gene has an amino acid sequence with at least 70% identity to SEQ ID NO: 2 in the mixture; b. placing the E. coli cells in a second mixture; and c. feeding isoeugenol to the second mixture; and d. converting isoeugenol to vanillin.
That is, Yamada teaches expressing a IEM gene in a cell culture, collecting the cells and placing the cells into a reaction mixture for converting isoeugenol to vanillin.
Regarding the requirement of claim 26 that expressing a VpIEM gene and feeding isoeugenol is done with the same “the mixture,” Goldsmith, abstract, also relates to “Methods for recombinant production of vanillin and compositions containing vanillin are provided by this invention.” “Recombinant hosts described herein can be used in methods to produce vanillin. For example, if the recombinant host is a microorganism, the method can include growing the recombinant microorganism in a culture medium under conditions in which vanillin biosynthesis genes are expressed. The recombinant microorganism can be grown in a fed batch or continuous process. Typically, the recombinant microorganism is grown in a fermentor at a defined temperature(s) for a desired period of time. In certain embodiments, microorganisms include, but are not limited to S. cerevisiae, A. niger, A. oryzae, E. coli, L. lactis and B. subtilis. The constructed and genetically engineered microorganisms provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, continuous perfusion fermentation, and continuous perfusion cell culture.” Goldsmith, para. [0216]. Goldsmith, para. [0219], further provides:
“In some embodiments, vanillin can be produced using whole cells that are fed raw materials that contain precursor molecules. The raw materials may be fed during cell growth or after cell growth. The whole cells may be in suspension or immobilized. The whole cells may be in fermentation broth or in a reaction buffer. In some embodiments a permeabilizing agent may be required for efficient transfer of substrate into the cells.”
As such, it is known in the prior art to provide a precursor material, which in embodiments of Yamada is isoeugenol, during cell growth; that is, in the culture/mixture in which vanillin biosynthetic genes are expressed. Yamada clearly indicates that E. coli cells can be combined in a mixture with isoeugenol to produce vanillin. In view of Goldsmith, at the time of filing it would have been obvious to modify the methods of Yamada such that isoeugenol is added/fed to a culture of E. coli expressing an IEM gene, i.e. to the following: “The transformant, E. coli/pT21IDE, was pre-cultured on 200 ml of LB medium containing 50 μg ampicillin/ml at 37°C for 8 h with reciprocal shaking at 90 rpm in a 500-ml conical flask. For the expression of isoeugenol monooxygenase, isopropyl thio-β-D-galactopyranoside [IPTG] was added to give 1 mM, and the cultivation was continued at 25°C for a further 16 h,” since Goldsmith teaches that it is a known approach to provide precursors to vanillin synthesis to a cell culture during cell growth without a need to first harvest the cells for placement in a separate reaction mixture. Since IEM is necessary for conversion of isoeugenol to vanillin, an ordinarily skilled artisan at the time of filing would have readily understood that addition (i.e. feeding) of isoeugenol should be done after at least some IEM is expressed after IPTG addition.
The above also meets the features of claims 32 and 36 except for extracting/collecting vanillin from the medium. However, an ordinarily skilled artisan at the time of filing would have readily understood that vanillin must be recovered after production in order to make use of the same. “After the recombinant microorganism has been grown in culture for the desired period of time, vanillin can then be recovered from the culture using various techniques known in the art. “ Goldsmith, para. [0218]. For this reason, an ordinarily skilled artisan would have been motivated to extract vanillin from any medium or composition in which vanillin is produced.
The features of claims 26, 27, 32, 36 and 37 are met for these reasons.
Regarding claim 30, Yamada teaches that any gene encoding IEM is expressed in E. coli bacterium.
Regarding claims 33-35, Yamada, abstract, states that “The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20[Symbol font/0xB0]C after 6 h.” Yamada teaches that amount of conversion of isoeugenol to vanillin is dependent upon specific reaction conditions. For example, Fig. 3 of Yamada shows the percent amount of conversion of isoeugenol to vanillin is dependent upon an initial concentration of isoeugenol. Fig. 4 shows a similar effect for concentration of cells expressing IEM. “[T]he vanillin producing reaction using E. coli/pT21IDE cells was optimized. The maximal vanillin-producing activity was obtained at 20[Symbol font/0xB0]C in 100 mM glycine/NaOH buffer (pH 10.5) containing 10% (v/v) DMSO, as well as in the case using P. putida IE27 cells. The addition of DMSO was effective in enhancing the solubility of isoeugenol in the reaction mixture.”
“"[T]he PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his [or her] claimed product. Whether the rejection is based on ‘inherency’ under 35 U.S.C. 102, on ‘prima facie obviousness’ under 35 U.S.C. 103, jointly or alternatively, the burden of proof is the same." MPEP 2112(V). Here, Yamada teaches an IEM that is capable of converting greater than 80% of isoeugenol to vanillin and that the conversion conditions can be optimized to increase the amount/rate of conversion. The specification evidences that an IEM identical to SEQ ID NO: 2 is capable of greater than 80%, 85% or 90% conversion of isoeugenol to vanillin as recited in claims 33-35. While the prior art does not directly indicate whether the IEM taught in Uniprot A0A081CI87 is capable of the same greater than 80%, 85% or 90% conversion of isoeugenol to vanillin, in view of MPEP 2112(V) the applicant has the burden of proving that the prior art products do not necessarily or inherently possess the characteristics of isoeugenol to vanillin conversion as recited in claims 33-35.
Regarding claim 31, as discussed, claim 26 from which claim 31 depends requires that that expression of the VpIEM gene be done in the same mixture as to which later “feeding isoeugenol to the mixture.” Regardless, Yamada, page 667, right col. and Fig. 2, teaches purification of IEM from E. coli “using simple purification procedures.” Since an IEM enzyme is clearly a valuable product, at the time of filing it would have been obvious to purify an IEM (including the IEM taught by Uniprot A0A081CI87) from any mixture or composition in which the same is found as taught by Yamada.
Regarding claim 40, Yamada, page 665, left col., states “Eugenol and isoeugenol are great potential phenylpropanoides as starting materials for the synthesis of aromatic flavorings and aromas” indicating that vanillin is a flavor and aroma compound. Further, Goldsmith, claims 1, 18 and 17, teach that vanillin, including vanillin produced by a recombinant microorganism, should be present or placed in a food product (i.e. consumable product). As such, at the time of filing, an ordinarily skilled artisan at the time of filing would have been motivated to incorporate any vanillin produced by a microorganism as discussed above into a consumable produce (i.e. food) since at least Goldsmith teaches that the same is the primary application for such vanillin.
Further regarding claims 40-44, “For example, a vanillin composition produced by a recombinant organism can be incorporated into a cold confectionary (e.g., ice cream), hard candy, or chocolate such that the food product has a maximum of about 95 mg/kg, 200 mg/kg, or 970 mg vanillin/kg food on a dry weight basis, respectively. A vanillin composition produced by a recombinant microorganism can be incorporated into a baked good (e.g., a biscuit) such that the food product has a maximum of about 200 mg vanillin/kg food on a dry weight basis. A vanillin composition produced by a recombinant microorganism can be incorporated into a beverage (e.g., a carbonated beverage) such that the beverage has a maximum of about 100 mg vanillin/kg. Vanillin sugar sold in supermarkets contains about 12500 mg vanillin/kg” Goldsmith, para. [0229].
An ordinarily skilled artisan at the time of filing would understand that these various food products including vanillin sugar are produced by mixing (admixing) the vanillin into the food product, such as sugar, as recited in claim 41. That is, Goldsmith does not teach that a producing microorganism is placed in the food product that then makes vanillin in situ. Rather, “Vanillin obtained by the methods disclosed herein” (i.e. vanillin collected from the microorganism or a composition of the microorganism) is employed to produce food products that necessarily requires admixing vanillin with some component of a consumable/food product. Regarding claims 42 and 44, “Vanilla is recognized as one of the most popular flavors and aromas around the world.” Goldsmith, para. [0004]. “Vanillin (CAS#121-33-5) is most responsible for the flavor and fragrance profiles of vanilla.” Goldsmith, para. [0005]. As such, the addition of vanillin to any consumable product is in an amount sufficient to impart a flavor and/or fragrance (aroma) note since the purpose of addition of vanillin is to impart flavor and/or fragrance, which cannot be achieved unless in a sufficient amount including the amounts stated in Goldsmith, para. [0229].
Claim(s) 26, 27, 30-37, 40-46, 49 and 50 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yamada et al. (Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida, Biotechnol. Lett. 30, 2008, 665-70), Uniprot, Accession No. A0A081CI87, 2020, www.uniprot.org, and Goldsmith et al. (U.S. 2017/0172184 A1) as applied to claims 26, 27, 30-37, 40-44, 46, 49 and 50 above, and further in view of Hiramoto et al. (U.S. 2014/0271522 A1).
Regarding claim 45, as discussed, vanillin is described as an aroma compound. Just as Goldsmith teaches that vanillin is used in food products in a similar manner in food products as vanillin derived from vanilla (natural plant source), an ordinarily skilled artisan at the time of filing would have been motivated to apply vanillin produced as discussed above, since vanillin has the same structure regardless of how it is produced. Hiramoto discussed deodorant compositions that can include vanillin. Hiramoto, abstract and para. [0023]. As such, an ordinarily skilled artisan at the time of filing would have been motivated to include vanillin produced as discussed above in a deodorant (fragrant product) as taught by Hiramoto.
Allowable Subject Matter
Claims 28-29, 38-39 and 47-48 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter:
The closest identified prior art is discussed above. No reasonable teaching regarding an isoeugenol monooxygenase having at least 90% identity to SEQ ID NO: 2 has been identified. As indicated in the Written Opinion of the ISA, SEQ ID NO: 2 is present in EMBL/GenBank database but is not characterized as an isoeugenol monooxygenase.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652