DETAILED ACTION
Applicant’s amendment submitted 2/3/2026 is acknowledged. Claims 1-283 and 293-303 are canceled. Claims 284-292 are pending in the instant application.
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 of Group I, claims 284-291 in the reply filed on 2/3/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). Claim 292 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 2/3/2026.
Priority
The instant application is a U.S. National Phase of PCT/US2022/028782, filed 5/11/2021, which claims Domestic Benefit to U.S. Provisional Application No. 63/186,949, filed 5/11/2021.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 11/10/2023 and 1/26/2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Drawings
The drawings are objected to because the chemical compound synthesized by deprotonation at C5 of intermediate 5 (i.e., Int5) is not labeled/identified in Figure 1A consistent with all other chemical species. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Page 11, line 17, of the instant specification comprises browser-executable code (i.e. http://) that should be removed.
Claim Objections
Claims 284, 287, and 289 are objected to because of the following informalities:
In claim 284, the recitation of “synthase enzyme” in the first and last lines is redundant. The superfluous recitation of “enzyme” should be deleted.
Claim 287 recites “substituted with” in line 2 whereas “substituted for” is the correct sentence structure that should be used.
Claim 289 recites “helices” whereas the singular “helix” should be recited instead because Table 13 identifies only a single helix for each species.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a)
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 284-291 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for making an α-Guaiene Synthase, comprising: providing an α-Guaiene Synthase encoded by SEQ ID NO: 1 that is capable of catalyzing cyclization of a prenyl diphosphate to produce α-Guaiene as a target sesquiterpene and one or more non-target sesquiterpene selected from the group consisting of: α-Bulnesene, β-Guajene, Guaia-9,11-diene, γ-Gurjunene, and Guai-4,11-diene through a series of cyclic carbocation intermediates by making one or more amino acid modifications within 10 Angstroms of the binding pocket of α-Guaiene Synthase encoded by SEQ ID NO: 1, a G2 helix, a D helix, a J helix, and/or a C helix, wherein the one or more amino acid modifications consist of substituting one or more aromatic amino acid or one or more aliphatic amino acid for one or more non-aromatic amino acid to stabilize a carbocation intermediate that deprotonates to α-Guaiene Synthase and/or substituting one or more non-aromatic amino acid for one or more aromatic amino acid or one or more aliphatic amino acid to destabilize a carbocation intermediate that deprotonates to at least one or more of the non-target sesquiterpene, does not reasonably provide enablement for making any other terpene synthase that produces any other target terpene and one or more non-target terpene by adding, positioning, removing, or shifting an aromatic side chain outside of the binding pocket, G2 helix, the D helix, the J helix, and the C helix. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
In making a determination that a disclosure does not satisfy the enablement requirement, the factors that may be considered include: (A) the breadth of the claims, (B) the nature of the invention, (C) the state of the prior art, (D) the level of one of ordinary skill, (E) the level of predictability in the art, (F) the amount of direction provided by the inventor, (G) the existence of working examples, and (H) the quantity of experimentation needed to make or use the invention based on the content of the disclosure. While it is not essential that every factor be examined in detail, those factors deemed most relevant should be considered.
Nature of the invention. The claimed invention is drawn to recombinantly producing a terpene synthase capable of catalyzing cyclization of a prenyl diphosphate to produce a target cyclic terpenoid and one or more non-target cyclic terpenoids through a series of cyclic carbocation intermediates. The one or more amino acid of the terpene synthase amino acid sequence is modified to add or position an aromatic side chain to stabilize a carbocation intermediate that deprotonates to the target cyclic terpenoid; and/or to remove or shift one or more aromatic side chains to destabilize a carbocation intermediate that deprotonates to at least one non-target cyclic terpenoid.
Breadth of the claims. A terpene synthase, target cyclic terpenoid, and non-target cyclic terpenoid are recited very broadly. The specification broadly defines a target cyclic terpenoid as a desired product of the terpene synthase reaction and defines a non-target cyclic terpenoid as side products of the same reaction (see Instant Specification – paragraph bridging p.31-32). The one or more amino acid modifications to add or position an aromatic side chain and/or to remove or shift one or more aromatic side chains are recited at a high level of generality. Claim 285 limits the target cyclic terpenoid to a sesquiterpenoid, triterpenoid, monoterpenoid, or a diterpenoid. Claim 286 recites that an aromatic side chain is added to stabilize a cation-π-interaction; and an aromatic side chain is removed to destabilize a cation-π-interaction. Claim 287 recites an aromatic side chain in the terpene synthase is substituted with a non-aromatic side chain, wherein the aromatic chain forms a cation-π-interaction with the carbocation that deprotonates to a non-target cyclic terpenoid and wherein the aromatic side chain that is added to stabilize a cation-π-interaction is phenylalanine. Claim 288 recites the amino acid modifications results in removal of all aromatic or aliphatic residues to a distance that is at least about 6 Angstroms from the carbocation that deprotonates to a non-target terpenoid. Claim 289 broadly recites one or more amino acid modifications are made to secondary structure elements selected from the G2 helices, the D helices, the J helices, and the C helices. Claim 290 broadly recites the amino acid modifications are guided by a structural model of the terpene synthase. Claim 291 broadly recites the terpene synthase is expressed in a host cell that produces the prenyl diphosphate.
State of the prior art and predictability of the art. The state of the art at the time of the invention reveals a general understanding of terpene synthase carbocations and the interaction of terpene synthase active site aromatic side chains of Phe, Tyr, and Trp to stabilize transient cations through cation-π-interaction. Furthermore, several novel and unconventional terpene synthases that perform terpene synthase-like reactions but do not resemble canonical terpene synthases in sequence or structure have been discovered.
Rudolf et al. (Nat. Prod. Rep., 2020, Vol. 37, pp.425-463; of record) teaches that all terpenoids are constructed from the same two C5 activated isoprene units: the allylic dimethylallyl diphosphate (DMAPP) and the homoallylic isopentenyl diphosphate (IPP) (see p.426, left column, 1st passage, and Fig. 1). These building blocks are combined in successive reactions to generate linear chains such as farnesyl diphosphate (FPP) that are catalyzed into various terpenoids classified based on carbon numbers (see p.426, left column, 1st passage, and Fig. 1). Figure 1 demonstrates that sesquiterpenoids are synthesized from a FPP precursor although other linear allylic diphosphates are required to generate other terpenoids. Terpene synthases mediate these reactions through a combination of substrate preference and folding, transient carbocations stabilization, and controlled carbocation quenching (see p.426, right column, 1st passage). Two canonical classes of terpene synthases have been recognized for decades based on their sequences, structures, functions, and associated mechanisms. More recently, non-canonical terpene synthases have been discovered, some of which generate carbocations while other forego carbocation chemistry (see p.427, left column, 1st paragraph). The two canonical classes of terpene synthases are divided by how the initial carbocation is generated (see p.427, left column, 1st passage). In both classes of canonical terpene synthases, carbocation intermediates must be protected from solvent due to their intrinsic reactivity (see p.427, passage bridging left and right columns). Most of the active site cavity is required to be lined with side chains of hydrophobic amino acids because of the nature of the hydrophobic terpenoid substrates, which also assists in the prevention of bulk solvent from reaching the carbocation. In many terpene synthase active sites, the aromatic side chains of Phe, Tyr, and Trp use cation-π-interaction to stabilize transient cations (see p.427, right column, 1st paragraph). While this is known, it is still unclear how terpene synthases precisely control these complex cyclization reactions, which appear to be influenced by factors such as inherent reactivity of the terpenoid substrate, substrate folding, and enzyme induced fits. Canonical terpene synthases are generally typified by the presence of highly conserved Asp-rich motifs, which is used to identify and confirm the functions of putative terpene synthases from genomes, gene clusters, and sequence databases (see p.427, right column, 2nd passage). These Asp-rich motifs play completely different roles in class I and class II terpene synthases (see p.427, right column, 2nd paragraph,-p.429, left column, 1st passage). Class I and Class II terpene synthases comprises a variety of structural domains including α, β, and γ domains, some terpene synthases being bifunctional and some adopting various combinations of these domains (see p.430, left column, 1st paragraph, and Figs. 2C-E). Rudolf et al. describe 12 additional types of terpene synthases that are non-canonical, extremely diverse, and largely uncharacterized (see p.456, right column, 2nd paragraph). Some of the 12 enzyme types mimic class 1 terpene synthases, some mimic class II terpene synthases, while others have completely distinct mechanisms. Nearly half of the enzymes are disguised as completely different enzymes since their primary sequences suggest they are not terpene synthases but their primary role is to perform terpene synthase cyclizations or as a secondary function. These non-canonical terpene synthases have a vast range of substrates and structural domains, some of which resemble canonical domains or are radically different. Lastly, few groups of the non-canonical terpene synthases are considered to be prototypes of completely novel terpene synthase families (see paragraph bridging pp.456-457).
Chappell et al. (US2003/0087406) describes novel synthases that generate terpenoids that were designed based on the 3-dimensional coordinates of tobacco 5-epi-aristolochene synthase (TEAS), with or without a substrate bound in the active site (see Abstract, paragraphs [0006], [0010], [0146], and [0155]-[0158], Fig. 1, and Tables 10-11). Chappell et al. specifically generated mutants of the TEAS enzyme having SEQ ID NO: 2 by targeted substitutions to active site α-carbons mapped in Tables 1-6 (see paragraphs [0149]-[0157]). Chappell et al. generated various synthases based on TEAS SEQ ID NO: 2 that can produce monoterpenes, diterpenes, and sesquiterpenes (see paragraph [0159] and Examples 4-14).
Therefore, the state of the art demonstrates that terpene synthases are not “one-type-fits-all” and require characterization and crystallization to elucidate active sites and enzyme domains, since sequences and structures can be extremely varied across terpene synthases.
Guidance in the specification. The examples in the specification demonstrate the construction of seven α-Guaiene Synthases from the Aquilaria crassna α-Guaiene Synthase (AcaGS) encoded by SEQ ID NO: 1. The seven α-Guaiene Synthases are encoded by SEQ ID NOs: 2-5, 28, and 31-32 and were generated by targeted point mutations (see p.73, 1st paragraph). Point mutation sites were determined by selecting residues that were within 10 Angstroms of a binding pocket in a homology model of AcaGS with intermediate molecule 5 docked in the active site (see p.73, 1st paragraph and Figs. 1A and 19C). Mutations were also targeted to the hydrophobic bottom of the substrate binding pocket formed by helices C, D, G2, and J, since these domains play roles in stabilizing and destabilizing specific intermediates that lead to altered product profiles (see p.73, 2nd paragraph, Table 13, and Fig. 15). With reference to SEQ ID NO: 1, Helix C spans A267 to A276, Helix D spans S283 to V305, Helix G2 spans Y404 to M411, and Helix J spans V507 to D522. SEQ ID NO: 2 has 99.4% sequence identity to SEQ ID NO:1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: S375A, F407L, and Y443L. SEQ ID NO: 3 has 99.2% sequence identity to SEQ ID NO: 1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: N290T, S375A, F407L, and Y443L. SEQ ID NO: 4 has 99.0% sequence identity to SEQ ID NO: 1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: N290A, I293F, S375A, F407L, and Y443L. SEQ ID NO: 5 has 98.7% sequence identity to SEQ ID NO: 1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: M273I, N290A, I293F, S375A, I400V, F407L, Y443L, and L447V. SEQ ID NO: 28 has 98.3% sequence identity to SEQ ID NO: 1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: M273I, N290A, I293F T296V, E325T, S375A, I400V, F407L, Y443L, and L447V. SEQ ID NO: 31 has 97.5% sequence identity to SEQ ID NO: 1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: Y21F, G269S, M273I, N290A, I293F T296V, E325T, S375A, I400V, F407L, Y443L, L447V, Q448V, and A545P. SEQ ID NO: 32 has 97.5% sequence identity to SEQ ID NO: 1 and comprises the following amino acid substitutions with reference to SEQ ID NO: 1: Y21F, G269S, M273I, N290A, I293F T296V, E325T, S375A, I400V, F407L, Y443L, L447V, I487D, and A545P. In all seven mutant α-Guaiene Synthases constructed, the synthesis of the target sesquiterpenoid α-Guaiene was measured compared to the non-target sesquiterpenoid α-Bulnesene (see paragraph bridging pp.12-13, Examples 1 and 3, Tables 1-5, and Figs. 4, 6, 8, 10-12, 14, and 25-26). Figure 1A demonstrates the pathway for synthesis of α-Guaiene from FPP precursor. Various intermediate molecules are depicted with specific deprotonations driving the production of a variety of sesquiterpenes. Since α-Guaiene was the target sesquiterpenoid in the examples, intermediate 5 was utilized for binding in the active site in the homology model of AcaGS to discover key residues to mutate. Many mutations were screened for improved α-Guaiene synthesis over 6 rounds of mutations (see p.37, 1st paragraph,-p.48, 2nd paragraph). The selected mutations (i.e., those in SEQ ID NOs: 2-5, 28, and 31-32) largely substituted aliphatic amino acids for non-aliphatic amino acids in the identified sites of importance, few substituted aliphatic amino acids for aromatic amino acids, and some substituted aromatic amino acids for aliphatic amino acids. These mutations are considered by Applicant to stabilize intermediate molecule 5 to selectively deprotonate toward α-Guaiene synthesis and to destabilize intermediate molecule 5 to prevent deprotonation events that drive synthesis away from α-Guaiene (see paragraph bridging pp.74-75 and p.75, 1st paragraph, and Figs. 1 Therefore, the specification only provides guidance for the specific modification of AcaGS encoded by SEQ ID NO: 1 by targeted mutations to substitute aliphatic amino acids and aromatic amino acids for residues that are within 10 Angstroms of the active site when bound to intermediate 5 or to the hydrophobic bottom of the substrate binding pocket formed by helices C, D, G2, and J discovered from multiple screening rounds.
Amount of experimentation necessary. In view of the breadth of the claims, a person of skill in the art would need to perform extensive research to develop models for all possible terpene synthases. Given the fact that many non-canonical terpene synthases exist, the disclosed homology model of AcaGS can only reasonable be used to generate mutants of α-Guaiene Synthase encoded by SEQ ID NO: 1. One of skill in the art would need to characterize the terpene synthase, determine key sites and domains of importance in the terpene synthase, know the synthesis pathway to the desired and undesired terpenes, and assess the various intermediate molecules within the determined sites of importance in the model of the characterized terpene synthase to practice the full scope of the claimed invention.
Taking these factors into account, undue experimentation would be required by one of ordinary skill in the art to practice the full scope of the claimed invention. Thus, the claims are not fully enabled by the disclosure.
Claim Rejections - 35 USC § 112(b)
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 287-288 and 290 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.
Claim 287 recites “wherein an aromatic side chain in the terpene synthase is substituted with a non-aromatic side chain, wherein the aromatic side chain forms a cation-π interaction with the carbocation that deprotonates to a non-target cyclic terpenoid and wherein the aromatic side chain that is added to stabilize a cation-π interaction is phenylalanine.” The recitation “an aromatic side chain the terpene synthase” seems to suggest that the terpene synthase already comprises an aromatic side chain, however, the recitation “the aromatic side chain that is added to stabilize a cation-π interaction is phenylalanine” suggests that a phenylalanine is added to the terpene synthase. Since the claim recites the aromatic side chain is substituted for a non-aromatic side chain and that phenylalanine is added, it is not clear if the aromatic side chain is in the terpene synthase already as recited. One of ordinary skill in the art would not be able to determine the metes and bounds of the claim.
Claim 288 recites “the amino acid modifications result in removal of all aromatic or aliphatic residues to a distance that is at least about 6 Angstroms from the carbocation that deprotonates to a non-target terpenoid.” It is not clear if “at least about 6 Angstroms from the carbocation that deprotonates to a non-target terpenoid” is intended to refer to a distance of at least about 6 Angstroms from an intermediate carbocation molecule when bound/docked within a terpene synthase. The instant specification appears to indicate that the distance is to be measured when the carbocation intermediate is bound in the active site of Aquilaria crassna α-Guaiene Synthase, but this is not recited in the claim (see Instant Specification – p.12, 2nd paragraph, p.13, 1st paragraph, p.33, 1st paragraph, and Fig. 19C). Therefore, the metes and bounds of the claim limitation cannot be determined by one of ordinary skill in the art.
Claim 290 recites “wherein amino acid modifications are guided by a structural model of the terpene synthase.” It is not clear what active method step is required by the claim limitation “guided” and the specification does not provide a clear definition for performing this claimed step. Consequently, one of ordinary skill in the art would not be able to determine the metes and bounds of the claim limitation.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(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 284-287 and 289-291 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by WO2020/051488 to Philippe et al.; of record in the IDS filed 11/10/2023.
Regarding claim 284, Philippe teaches α-Guaiene Synthases comprising an amino acid sequence having one or more of the amino acid substitutions selected from T72I, M273L, R290K, F368M, I371L, S374A, R3775V, Y381W, F382L, I399V, F406L, L419T, V433I, Y442L, I443M, E454K, F512L, and K522D relative to SEQ ID NO: 8, an Aquilaria crassna Guaiene Synthase (see p.6, line 10, p.7, lines 26-30, and Claim 7). According to the instant specification, SEQ ID NO: 1 of the instant invention corresponds to SEQ ID NO: 8 of Philippe and Philippe identifies the amino acids substitutions S375A, F407L, and Y443L, which are identified as S374A, F406L, and Y442L in Philippe (see Instant Specification – p.36, 2nd and last paragraphs). Philippe embraces an enzyme that comprises all of the aforementioned mutations with relative to SEQ ID NO: 8 that includes the amino acids substitutions S375A, F407L, and Y443L. Philippe indicates that the substitutions F407L, Y443L, and S375A result in a 1.71, 1.16, and 1.13 fold improvement in α-Guaiene production, respectively (see p.20, last paragraph and Fig. 4). The instant specification indicates that the substitution F407L likely destabilizes intermediate 4 to favor production of α-Guaiene, while the mutation S375A stabilizes intermediate 5 to disfavor deprotonation into α-Bulnesene and favor production of α-Guaiene (see Instant Specification – p.36, last paragraph). Figure 2 of Philippe demonstrates that the prenyl diphosphate farnesyl diphosphate (FPP) is catalyzed into α-Guaiene by α-Guaiene Synthase (see also p.20, last paragraph). Therefore, Phillipe teaches making an α-Guaiene Synthase with an amino acid sequence of SEQ ID NO: 8 comprising the amino acid substitutions T72I, M273L, R290K, F368M, I371L, S374A, R375V, Y381W, F382L, I399V, F406L, L419T, V433I, Y442L, I443M, E454K, F512L, and K522D, which is capable of catalyzing cyclization of a prenyl diphosphate to produce α-Guaiene, reading on a recombinantly produced terpene synthase. According to the instant specification, the substitution F407L likely destabilizes intermediate 4 to favor production of α-Guaiene, while the mutation S375A stabilizes intermediate 5 to disfavor deprotonation into α-Bulnesene and favor production of α-Guaiene, which reads on adding an aromatic side chain to stabilize a carbocation intermediate that deprotonates to the target cyclic terpenoid and removing an aromatic side chain to destabilize a carbocation intermediate that deprotonates to at least one non-target cyclic terpenoid. Thus, Phillipe reads on the method of claim 284 for making a terpene synthase.
Regarding claim 285, Phillipe teaches producing the sesquiterpene α-Guaiene, reading on the target cyclic terpenoid is a sesquiterpenoid (see p.19, 4th paragraph,-paragraph bridging pp.20-21).
Regarding claim 286, according to the instant specification, the substitution F407L likely destabilizes intermediate 4 to favor production of α-Guaiene, while the mutation S375A stabilizes intermediate 5 to disfavor deprotonation into α-Bulnesene and favor production of α-Guaiene (see Instant Specification – p.36, last paragraph). Furthermore, the instant specification indicates F407L removes the cation-π-interaction, reading on removing an aromatic side chain, i.e., F, phenylalanine, to destabilize a cation-π-interaction (see Instant Specification – p.75, 1st passage). Philippe teaches the amino acid substitution Y381W which reads on adding an aromatic side chain, which is expected to stabilize a cation-π-interaction, since the substitution results in a 1.05 fold improvement in α-Guaiene production, absent evidence to the contrary (see Fig. 4).
In light of claim 287 being indefinite, the claim is being interpreted as substituting a phenylalanine for a non-aromatic amino acid. Regarding claim 287, Philippe teaches the substitution A451F results in a 0.91 fold improvement in α-Guaiene production (see Fig. 4). This is interpreted to read on substituting a phenylalanine aromatic side chain for a non-aromatic side chain.
Regarding claim 289, the instant specification indicates that F407L is within the G2 helix (see Table 13 and paragraph bridging pp.74-75). Therefore, Philippe teaches the amino acid modification is made to a G2 helix.
Regarding claim 290, Philippe teaches modifying terpene synthase enzymes from information obtained by a construction of a homology model (see p.6, last paragraph).
Regarding claim 291, Philippe teaches expressing the terpene synthase in an E. coli host organism that produces isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) that are converted to farnesyl diphosphate by a recombinant farnesyl diphosphate synthase (see Example 1).
Therefore, Philippe anticipates claims 284-287 and 289-291 of the instant invention.
Conclusion
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/J.P.S./Examiner, Art Unit 1651
/MELENIE L GORDON/Supervisory Patent Examiner, Art Unit 1651