Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
The preliminary amendment filed 07/10/2024 is entered. Claims 1-19 are pending and under consideration in this action.
Priority
The instant claims are entitled to an effective filing date of 06/02/2021.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
The list of references can be found in the specification spanning pages 22-24.
Drawings
The black and white drawings filed 11/30/2023 are objected to under 37 CFR 1.83(a) because they fail to show:
in figure 4(A) the OMT variant (blue) as described on p. 2 line 24;
in figure 4(B) variants shown in orange as describe on p. 2 line 29;
in figure 4(B) the substrate NOR and cofactor S-adenosyl-methionine in pink and green respectively, as described on p. 2 lines 28-29;
in figure 5(a-e) the variant genotype highlighted in green as described on p.3 line 5;
in figures 6A-B the substrate norlaudanosoline shown in pink as described in p. 3 line 11;
in figures 6A-B the co-factor S-adenosyl-methionine shown in green and mutations shown in orange as described on p. 3 lines 11-12; and
in figures 6A-B the monomer that is colored blue as described on p. 3 line 13 in the specification. Any structural detail that is essential for a proper understanding of the disclosed invention should be shown in the drawing. MPEP § 608.02(d). 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.
The drawings are objected to because the following drawings include unreadable text:
Figures 1A-F;
Figure 2A;
Figure 5A and 5C.
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.
Claim Objections
Claims 4, 12 and 17 are objected to because of the following informalities:
Claims 4 recites “represented a protein” and claim 17 recites “represented as a protein”, which are grammatically incorrect and convoluted recitations. Claims 4 and 17 may be amended to: wherein the methyltransferase has 90% or more identity to SEQ ID NO:6.
Claim 12 recites a., b., and c. which should be amended to recite a), b) and c) respectively or something to that effect because MPEP 608.01(m) states that periods may not be used elsewhere in the claims except for abbreviations.
Appropriate correction is required.
Applicant is advised that should claim 5 be found allowable, claim 18 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
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 1-19 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 claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor, at the time the application was filed, had possession of the claimed invention.
The claims are drawn to a genus of methyltransferases that can methylate more than two positions in a benzylisoquinoline alkaloid (BIA) molecule. Claims 1-19 do not substantially limit the structure of the methyltransferase. Claims 6 and 17 require the methyltransferase to be represented by a protein with 90% or more identity to SEQ ID NO: 6 (length: 353aa), which encompasses sequences with up 36 mutations compared to SEQ ID NO:6. Claims 5 and 18 require the methyltransferase to have 95% or more identity to SEQ ID NO: 6, which encompasses sequences with up 18 mutations compared to SEQ ID NO:6. The specification does not disclose a representative number of species of the claimed genus by reduction to practice, and does not provide adequate guidance with regard to the structural features of the methyltransferase that is required to provide the required methylation at more than two positions in a BIA molecule. Therefore, one of skill cannot immediately envision which methyltransferases will have the required functional characteristics, and one could not conclude that Applicant was in possession of the claimed genus at the time the filing, as discussed more fully below.
For claims drawn to a genus, MPEP § 2163(3)(a)(ii) indicates 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. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406. 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 instant specification reduces to practice three examples of methyltransferases that can methylate four positions in one benzylisoquinoline alkaloid molecule, norlaudanosoline, in order to produce tetrahydropapaverine (THP). THP is produced by cells co-transformed with pThpR and grown in the presence of norlaudanosoline. See p. 2 lines 18-19. The benzylisoquinoline alkaloid molecule, norlaudanosoline (NOR), is methylated at four positions in order to form THP. See figure 4A. In example 1, the specification teaches evolving methyltransferase from Glaucium flavum, referred to as GfOMT1. See p. 17 line 26. The specification indicates that GfOMT1 (SEQ ID NO: 1) is a naturally occurring O-methyltransferase. See p. 13 line 16-17. The GfOMT1 produces the highest fluorescent signal in E. coli bearing pThpR when cultured with norlaudanosoline. Therefore, it is used as the starting point for evolution. See p. 17 lines 19-20. After five rounds of evolution, a GfOMT1 variant with seven substitutions, GEN 5, produces a 6- and 47-fold increase in fluorescent signal using pThpR compared to wild-type GfOMT1 when cultured with norlaudanosoline. See p. 17 lines 26-28. As shown in figure 4b, the evolved variants include: GEN1 with T178A and K146R mutations; GEN2 with a W22L mutation; GEN3 with a 1258V mutation; GEN4 with L177M and A291V mutations; and GEN5. See figure 4b. Figures 4d and 7 indicate that GEN3, GEN4 and GEN5 produce the THP benzylisoquinoline alkaloid molecule. Whereas the wild-type methyltransferase, and the evolved methyltransferases GEN1 and GEN2 do not produce THP. SEQ ID NO: 6 is disclosed as GfOMT_MUT5 (see p. 21 line 14), which suggests that SEQ ID NO: 6 is GEN5. Compared to SEQ ID NO: 1 (i.e. wild-type), SEQ ID NO: 6 includes the following seven mutations: T178A, K146R, W22L, I258V, L177M, A291V and M121V mutations. See p. 13 lines 27-29. SEQ ID NO: 1 (wild-type) is 98.3% identical to SEQ ID NO: 6. However, the specification does not teach or suggest that SEQ ID NO: 1 is capable of methylating a benzylisoquinoline alkaloid molecule at more than two positions. Thus, the specification reduces to practice three methyltransferases that can methylate four positions in one benzylisoquinoline alkaloid molecule, norlaudanosoline.
MPEP 2163(3)(a)(ii) states that “the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are "representative of the full variety or scope of the genus," or by the establishment of "a reasonable structure-function correlation. Such correlations may be established "by the inventor as described in the specification," or they may be "known in the art at the time of the filing date”. Considering the lack of guidance provided in the specification, one would appraise support from the state of the art to extrapolate the correlation between the methyltransferase structure of the required benzylisoquinoline alkaloid (BIA) methylation function.
With respect to the state of the art on methyltransferases, Lashley (Molecules. 2022 Dec 21;28(1):43), a past filing date reference, teaches that methyltransferases are categorized based on the substrate atoms O-, N-, C-, S-, and Se- that accept the methyl group. Among all plant natural product methyltransferases, O-directed methyltransferases (OMTs) are the most abundant to date. See Lashley p. 2 section 2 first paragraph. The abundance of OMTs is also seen through the incredible diversity of their methoxylated products. Notably, out of all known alkaloid OMTs to date, the majority of them are involved in benzylisoquinoline alkaloid (BIA) biosynthesis. See Lashley p. 2 section 2 last passage. Morris (Front Plant Sci. 2019 Aug 30;10:1058) adds that a tremendous number of BIA methyltransferases (MTs) have been characterized in plant extracts. See p. 4 left column last passage. Morris suggests that despite the ever-growing list of characterized and cloned methyltransferases, there remain many biosynthetic pathways to explore. See p. 2 right column last full paragraph.
With respect to the state of the art on the methylation of BIAs by methyltransferases, Morris suggests that there is a lack of specificity reported for BIA MTs occurring on two levels: substrate promiscuity, wherein the enzyme can methylate a number of different molecules, and product promiscuity, wherein a single substrate is methylated one or more times at various positions to yield different products. See Morris p. 6 right column first full paragraph. Jamil (Proc. Natl. Acad. Sci. 2022, U.S.A. 119 (33)), a past filing date reference, cites a study in which an engineered variant of an OMT from Glaucium flavum exhibits 3’OMT activity on a BIA scaffold. Jamil discloses that the authors of the study report that the variant of GfOMT1 is able to methylate four different positions on the BIA scaffold including the 3’position to produce THP from fed norlaudanosaline in E. coli. Jamil teaches examining the 3’OMT activity of the enzyme [from the cited study]. However, Jamil suggests that the THP product of 3’-O-methylation is not detectable in the media at levels above background. See p. 5 right column. The study cited by Jamil is d’Oelsnitz (bioRxiv [preprint] 2021). D’Oelsnitz teaches identifying and broadening the substrate specificity of GfOMT1 to streamline a more complex set of methylations for the production of THP to a single enzyme. See p. 12 last passage. D’Oelsnitz reasons that it might be possible to evolve a methyltransferase that can uniquely methylate all four positions, thereby reducing a four-enzyme pathway into a single multifunctional enzyme. See p. 10 first full paragraph.
In view of the prior art, the instant disclosure does not satisfy the written description requirement because the species disclosed do not adequately represent the substantial variation within the claimed genus. As suggested by Lashley and Morris, the breadth of potential methyltransferase structures encompassed by the claims is substantial. As evidenced by Morris, the product promiscuity of methyltransferases is unpredictable due to a lack of reporting in the art. Furthermore, Jamil teaches examining the activity of a methyltransferase reported to methylate four different positions on a BIA; however Jamil suggests that the expected methylation product was undetectable. Considering the unpredictability in the art as illustrated by Lashley, Morris and Jamil, one of skill in the art at the time of filing could not have reasonably predicted which methyltransferases would have been capable of methylating more than two positions in any BIA molecule without undue experimentation. The instant specification reduces to practice three examples of methyltransferases capable of methylating norlaudanosoline (NOR), in order to produce tetrahydropapaverine (THP). This represents a very small fraction of the possible number of species within the breadth of the claims. Accordingly, one of skill could not conclude that Applicant was in possession of the claimed genus at the time the application was filed.
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 8-17 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 8 recites “the cell” in line 1 and “the host cell” in line 2, which renders the claim indefinite because “the cell” is a broad limitation recited in the same claim with “the host cell”, which is a narrower limitation. Claim 8 depends from claim 7, which requires a host cell. Therefore, it is unclear whether “the cell” recited in claim 8 refers to the host cell or a separate cell. To obviate this rejection, claim 8 can be amended to:
Claims 9-11 depend from claim 8 and are rejected for the reason set forth above.
Claim 12 recites “wherein the benzylisoquinoline alkaloid (BIA) composition requires methylation in its final form” in lines 2-3, which renders the claim indefinite because it is unclear which BIA final form requires methylation. Methylation is a process where a methyl group is added to a molecule. Claim 12 entails a method comprising “b. exposing the methyltransferase to the precursor of the BIA composition; and c. allowing the methyltransferase to methylate the precursor to the BIA composition, wherein the methyltransferase methylates more than two positions in the precursor of the BIA composition, thereby producing a methylated composition of interest”. As such, claim 12 indicates that the BIA precursor undergoes the methylation process, and that BIA in its final form (i.e. composition of interest) is methylated. Therefore, in one interpretation, the recitation “wherein the benzylisoquinoline alkaloid (BIA) composition requires methylation in its final form” is requiring the final form of the BIA composition to be methylated that is the result of performing step c; and under an alternative interpretation, the recitation requires step d, an additional methylation of a final form of a BIA after step c. To obviate this rejection, “wherein the benzylisoquinoline alkaloid (BIA) composition requires methylation in its final form” in lines 2-3 can be deleted. Furthermore, claim 12 recites the limitation “the precursor of the BIA composition” in line 6. There is insufficient antecedent basis for this limitation in the claim. Claim 12 is further indefinite because it is uncertain whether “a methylated composition of interest” (line 9) produced in step c is the same as “a benzylisoquinoline alkaloid (BIA) composition” (line 1) recited in the method preamble. The instant specification neither defines the term nor describes any methylated compositions of interest, and not all BIAs are methylated as evidenced by the structure of norlaudanosoline in figure 4A. Therefore, it is unclear whether claim 12 is drawn to a method of preparing any BIA, a method of preparing a methylated compound of interest, or a method of preparing a methylated BIA.
Claims 13-17 depend from claim 12 and are rejected for the reason set forth above.
Claim 15 recites “the BIA composition”, which renders the claim indefinite because it is unclear whether the BIA composition refers to the BIA composition before or after methylation. Claim 15 depends from claim 12, which requires preparing a BIA composition, wherein the BIA composition requires methylation in its final form. Therefore, in one interpretation the BIA composition of claim 15 refers to the prepared BIA composition, and under an alternative interpretation the BIA composition of claim 15 refers to the BIA composition that is methylated in its final form.
Claim 16 recites “the BIA is norlaudanosoline”, which renders the claim indefinite because it is unclear which BIA is required to be norlaudanosoline. Claim 16 depends from claim 12, which recites “a benzylisoquinoline alkaloid (BIA) composition” in line 1 and “the precursor of the BIA composition” in line 6. Norlaudanosoline is not methylated. Therefore, in one interpretation claim 16 is requiring the precursor of the BIA composition to be norlaudanosoline, such the methyltransferase methylates the norlaudanosoline precursor. However, claim 12 also indicates that the BIA composition requires methylation in its final form (see lines 2-3 of claim 12). Therefore, under an alternative interpretation the claim 16 is requiring the BIA composition in its final form to be norlaudanosoline. To obviate this rejection, “the BIA” can be amended to recite “the precursor of the BIA”.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1-7 and 18 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural product, which is a judicial exception, without significantly more. Each step described below is in reference to the subject matter eligibility test for products and processes (MPEP 2106).
Claim 1 recites a “methyltransferase ” in line 1. Therefore, claims 1-7, and 18 are directed to compositions of matter, which is one of the statutory categories (Step 1:Yes).
Claim 1 recites “said methyltransferase can methylate more than two positions in a benzylisoquinoline alkaloid (BIA) molecule”. As an evidentiary reference, Wang (Int J Biol Macromol. 2026 Apr; 356:151603) characterizes the O-methyltransferases gene family in Stephania yunnanensis and identifies 72 SyOMT members in the BIA-producing plant. See the abstract. Wang teaches that SyOMT59 methylates monobenzylisoquinoline at 6-O/7-O/3’-O positions. See p. 11 section 5. Furthermore, the SyOMT59 performs methylation at C6, C7, C3’, C9 and C2 positions across both alkaloid classes. See p. 9 the sentence bridging the left to right columns. Compared to closest naturally occurring counterpart (i.e. SyOMT59), there is no structural difference between the natural product (i.e. SyOMT59) and the instantly claimed methyltransferase. Because there is no indication in the record that the instantly disclosed methyltransferase has a markedly different characteristic in structure, function, or other properties as compared to its natural counterpart (i.e. SyOMT59) claim 1 and dependent claims are directed to a natural-product, which is a judicial exception (Step 2A Prong 1: Yes).
The additional claimed elements separately and accumulatively fail to integrate the product of nature into practical application. Besides the natural product, claim 1 recites a “non-naturally occurring methyltransferase”. This recitation does not structurally limit the methyltransferase, nor does the recitation constitute as an additional element. Rather, the recitation does no more than generally link the natural product to a particular technological environment or field of use. Claim 2 recites: wherein the methyltransferase can methylate more than three positions in a BIA molecule. This additional element of claim 2 is recited with a high level of generality because the methyltransferase structure is not limited in anyway, nor is the BIA molecule structurally limited. As such, the limitation of claim 2 merely serves to generally link the natural product to its field of use. Claim 3 requires the BIA to be norlaudanosoline (NOR). However, claim 3 does not limit the structure of the methyltransferase that must methylate more than two positions in the NOR. As such, the limitation provides insufficient specificity to add any element of significance to the judicial exception because, at best, claim 3 implies that the methyltransferase can be used for the intended purpose of methylating NOR. Claims 4 and 18 require the methyltransferase to be represented by a protein with 90% or more sequence identity to SEQ ID NO: 6. Claim 5 requires the methyltransferase to have 95% or more identity to SEQ ID NO: 6. The instant specification indicates that SEQ ID NO: 1 is a naturally occurring GfOMT1 [i.e. O-methyltransferase 1 from Glaucium flavum]. See p. 13 line 16-17. SEQ ID NO: 6 is 98.3% identical to the instantly disclosed naturally occurring SEQ ID NO: 1. See the office action appendix. Therefore, claims 4-5 and 18 cannot integrate the natural product judicial exception into a practical application because the claims themselves encompass separate natural product judicial exceptions. Claim 6 is drawn to a nucleic acid encoding the methyltransferase of claim 1. As such, claim 6 does not integrate the judicial exception into a practical application because the limitation of claim 6 merely serves to generally link the judicial exception to a particular technological environment or field of use, i.e. genetic engineering. Claim 7 is drawn to a host cell comprising the nucleic acid of claim 6. The limitation of claim 7 is recited with a high level of generality, because the host cell is not limited in anyway. As such, claim 7 does not integrate the judicial exception into a practical application because claim 7 merely describes the natural product as being from a cell. Therefore, the claim as a whole, does not integrate the judicial exception into a practical application (Step 2A Prong 2: No).
The additional elements fail to amount to an inventive concept. Facchini (US 2017/0058305) teaches incubating an E. coli strain harboring pGFLOMT2 with norlaudanosoline (i.e. BIA) to generate a product with m/z 344 at 3.42, which corresponds to tetrahydropapverine (i.e. methylated BIA). See [0222]. The abbreviation GFLOMT2 refers to Glaucum flavium O-methyltransferase OMT 2. See [0046]. Based on the norlaudanosoline structure provided in figure 9A and the tetrahydropapverine structure in figure 9E, Facchini suggests that the O-methyltransferase, GFLOMT2, methylates the norlaudanosoline at three positions in order to generate tetrahydropapaverine. Furthermore, Facchini teaches GFLOMT1 (SEQ ID NO: 1592). See [0132]. Facchini’s SEQ ID NO: 1592 is a 98.7% identity match to instant SEQ ID NO: 6. See the office action appendix. Thus, the additional limitations in claims 1-7, and 18 fail to amount to an inventive concept (Step 2B: No).
For all of these reasons, claims 1-7, and 18 are not patent eligible.
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.
Claims 1-18 are rejected under 35 U.S.C. 103 as being unpatentable over Facchini (US 2017/0058305).
Regarding claim 1, Facchini teaches contacting an alkaloid compound with an O-methyltransferase and/or and N-methyltransferase under reaction conditions that permit methylation of the alkaloid compound to form a second alkaloid compound; wherein the first alkaloid compound has the chemical formula (I), wherein R1, R2, R3, and R4 can each independently or simultaneously represent a hydroxyl group, and wherein R5 represents a hydrogen atom or a methyl group; wherein the second alkaloid compound has the chemical formula (II), and wherein R1, R2, R3, and R4, each independently or simultaneously represent a hydroxyl group or a methoxy group, with the proviso that at least one of R1, R2, R3, and R4 represents a methoxy group, and wherein R5 represents a hydrogen atom or a methyl group. See [0027] and claim 17 of Facchini. Furthermore, Facchini suggests that alkaloid compounds that are particularly preferred are benzylisoquinoline alkaloids. See [0096]. Facchini teaches O-methyltransferase obtained from Glaucum flavium. See claim 19 of Facchini.
[AltContent: textbox (From left to right: Facchini’s formulas (I) and (II), which are both benzylisoquinoline alkaloids)]
Facchini does not explicitly teach a non-naturally occurring methlytransferase, wherein said methyltransferase can methylate more than two positions in a benzylisoquinoline alkaloid molecule.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention that Facchini’s O-methyltransferase can methylate up to four positions in a benzylisoquinoline alkaloid molecule, because Facchini teaches that upon contact with the O-methyltransferase the hydoxyl groups at positions R1, R2, R3, and R4 of the benzylisoquinoline alkaloid of formula (I) form methoxy groups, such that formula (II) includes methoxy groups at R1, R2, R3, and R4.
Regarding claim 2, Facchini teaches contacting an alkaloid compound with an O-methyltransferase under reaction conditions that permit methylation of the alkaloid compound to form a second alkaloid compound; wherein the first alkaloid compound has the chemical formula (I), wherein R1, R2, R3, and R4 can each independently or simultaneously represent a hydroxyl group, and wherein the second alkaloid compound has the chemical formula (II), and wherein R1, R2, R3, and R4, can each independently or simultaneously represent a methoxy group. See claim 17 of Facchini. Furthermore, Facchini suggests that alkaloid compounds that are particularly preferred are benzylisoquinoline alkaloids. See [0096].
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention that Facchini’s O-methyltransferase methylates up to four positions, which is more than three positions as instantly required.
Regarding claim 3, Facchini, in example 8, discloses that incubation of an E. coli strain harboring pGFLOMT2 with norlaudanosoline (i.e. BIA) generates a product with m/z 344 at 3.42. The m/z 344 at 3.42 corresponds to tetrahydropapverine (i.e. methylated BIA). See [0222] and see figure 8C for the retention time of pGFLOMT2. The abbreviation GFLOMT2 refers to Glaucum flavium O-methyltransferase OMT 2. See [0046]. The chemical structure of norlaudanosoline (i.e. BIA) is shown in figure 9A.
Regarding claims 4-5 and 18, Facchini teaches the O-methyltransferase GFLOMT1 (SEQ.ID NO: 1592). See [0153]. Facchini teaches O-methyltransferase obtained from G. flavium and selected from a group that includes SEQ ID NO: 1592. See claim 19 of Facchini. Facchini’s SEQ ID NO: 1592 is a 98.7% identity match to instant SEQ ID NO: 6. See the office action appendix.
Regarding claim 6, Facchini teaches a first polynucleotide obtainable from a first plant capable of producing an alkaloid compound encoding a first enzyme capable of catalyzing a chemical reaction that converts a first alkaloid compound into a second alkaloid compound. See claim 11 of Facchini. Facchini teaches a first alkaloid compound that has the chemical formula (I), wherein R1, R2, R3, and R4 can each independently or simultaneously represent a hydroxyl group; and a second alkaloid compound has the chemical formula (II), and wherein R1, R2, R3, and R4, can each independently or simultaneously represent a methoxy group, with the proviso that at least one of R1, R2, R3, and R4 represents a methoxy group. See claim 17 of Facchini. Furthermore, Facchini suggests that alkaloid compounds that are particularly preferred are benzylisoquinoline alkaloids. See [0096].
Regarding claim 7, Facchini teaches a method of producing an alkaloid compound in a host cell, the method comprising: (a)(i) a first polynucleotide obtainable from a first plant capable of producing an alkaloid compound encoding a first enzyme capable of catalyzing a chemical reaction that converts a first alkaloid compound into a second alkaloid compound; and (b)(i) providing a second chimeric nucleic acid sequence; and (c) introducing the first and second chimeric nucleic acid sequence into a cell; and wherein the first and second polynucleotide are non-homologous. See claim 11 of Facchini. In example 8, Facchini teaches an E. coli strain (i.e. host cell) harboring different combinations and permutations of pGFLOMT1, pGFLOMT2, pGFLOMT6 and pCNMT (i.e. nucleic acids encoding methyltransferases). See [0221].
Regarding claim 8, Facchini teaches a method of producing an alkaloid compound in a host cell, the method comprising: introducing the first and second chimeric nucleic acid sequence into a cell; and wherein the first and second polynucleotide are non-homologous (i.e. from a different organism than the host cell). See claim 11 of Facchini.
Regarding claim 9, Facchini teaches a method of producing an alkaloid compound in a host cell, the method comprising: introducing the first and second chimeric nucleic acid sequence into a cell; and wherein the first and second polynucleotide are non-homologous (i.e. from a different organism than the host cell). See claim 11 of Facchini. Furthermore, Facchini teaches marker genes that may be employed to identify transformance through visional inspection including green fluorescent protein. See [0130].
Facchini does not explicitly teach a host cell that comprises a third nucleic acid encoding a protein from a different organism than the host cell.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Facchini’s marker gene (i.e. a third nucleic acid) encoding a GFP protein (i.e. a protein from a different organism than the host cell) with the host cell of Facchini comprising first and second nucleic acid sequences. One of ordinary skill in the art would have been motivated to do so because Facchini suggests that marker genes allow for the distinction of transformed cells from non-transformed cells. There would have been a reasonable expectation of success because Facchini suggests that cloning vectors typically contain a marker allowing for the selection of transformed cells (see [0131]).
Regarding claim 10, Facchini teaches a method of producing an alkaloid compound in a host cell, the method comprising: (a) providing a first chimeric nucleic acid sequence comprising as operably linked components: (i) a first polynucleotide obtainable from a first plant capable of producing an alkaloid compound encoding a first enzyme capable of catalyzing a chemical reaction that converts a first alkaloid compound into a second alkaloid compound; and (ii) one or more polynucleotides capable of controlling expression in a cell; (b) providing a second chimeric nucleic acid sequence comprising as operably linked components: (i) a second polynucleotide obtainable from a second plant capable of producing an alkaloid compound encoding a second enzyme capable of catalyzing a chemical reaction that converts the second alkaloid compound into a third alkaloid compound (i.e. composition of interest); and (ii) one or more polynucleotides capable of controlling expression in a cell; (c) introducing the first and second chimeric nucleic acid sequence into a cell (i.e. host cell encoding a pathway); and (d) growing the cell to produce the first and second enzyme and the second and third alkaloid compound (i.e. compound of interest); and wherein the first and second polynucleotide are non-homologous and wherein the third alkaloid compound is produced in the cell at a level that is in excess of the level of alkaloid compound produced when a homologous first and second polynucleotide are used. See claim 11 of Facchini.
Regarding claim 11, Facchini teaches a first polynucleotide encoding a first enzyme capable of catalyzing a chemical reaction that converts a first alkaloid compound into a second alkaloid compound (i.e. precursor for the composition of interest). See claim 11 of Facchini. Facchini teaches an O-methyltransferase enzyme that permits methylation of a first alkaloid compound to form a second alkaloid compound. See claim 17 of Facchini. Facchini teaches a second polynucleotide encoding a second enzyme capable of catalyzing a second alkaloid compound into a third alkaloid compound (i.e. composition of interest). See claim 11 of Facchini.
[AltContent: textbox ((left) figure 9A and (right) figure 9E)]Regarding claim 12, Facchini, in example 8, teaches cultures of E. coli harboring different permutations of pGFLOMT1, pGFLOMT2, pGFLOMT6, and pCNMT. See [0221]. Facchini teaches incubating an E. coli strain harboring pGFLOMT2 with norlaudanosoline (i.e. precursor of the BIA composition) to generate products including a product with m/z 344 at 3.42. The m/z 344 at 3.42 corresponds to tetrahydropapverine (i.e. methylated BIA). See [0222] and figure 8C. As shown in figure 9A provided below, norlaudanosoline (i.e. precursor of the BIA composition) does not include any methyl groups; and the structure of tetrahydropapverine in figure 9E has three methylated positions.
Facchini does not explicitly teach c. allowing the methyltransferase to methylate the precursor to the BIA composition, wherein the methyltransferase methylates more than two positions in the precursor of the BIA composition, thereby producing a methylated composition of interest.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention that Facchini’s GFLOMT2 O-methyltransferase methylates three positions of a norlaudanosoline precursor of the BIA composition. For clarity, the tetrahydropapaverine of Facchini is being interpreted in view of the structure provided in figure 9E; however, tetrahydropapaverine is understood to include 4 methoxy groups rather than three shown in Facchini’s figure 9E.
Regarding claim 13, Facchini teaches a method of producing an alkaloid compound in a host cell, the method comprising: introducing the first and second chimeric nucleic acid sequence into a cell; and wherein the first and second polynucleotide are non-homologous (i.e. from a different organism than the host cell). See claim 11 of Facchini. In example 8, Facchini teaches E. coli harboring different combinations and permutations of pGFLOMT1, pGFLOMT2, pGFLOMT6 and pCNMT (i.e. nucleic acids encoding methyltransferase proteins from a different organism than the host). See [0221].
Regarding claim 14, Facchini teaches a method of producing an alkaloid compound in a host cell, the method comprising: introducing the first and second chimeric nucleic acid sequence into a cell; and wherein the first and second polynucleotide are non-homologous (i.e. from a different organism than the host cell). See claim 11 of Facchini. Furthermore, Facchini teaches marker genes that may be employed to identify transformance through visional inspection including green fluorescent protein. See [0130].
Facchini does not explicitly teach a host cell that comprises a third nucleic acid encoding a protein from a different organism than the host cell.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Facchini’s marker gene (i.e. a third nucleic acid) encoding a GFP protein (i.e. a protein from a different organism than the host cell) with the host cell of Facchini comprising first and second nucleic acid sequences. One of ordinary skill in the art would have been motivated to do so because Facchini suggests that marker genes allow for the distinction of transformed cells from non-transformed cells. There would have been a reasonable expectation of success because Facchini suggests that cloning vectors typically contain a marker allowing for the selection of transformed cells (see [0131]).
Regarding claim 15, Facchini teaches incubating an E. coli strain harboring pGFLOMT2 (i.e. one nucleic acid sequence encoding a O-methyltransferase protein). Facchini discloses that E. coli strain harboring pGFLOMT2 generates tetrahydropapverine (i.e. BIA composition). See [0222] and figure 8C.
Regarding claim 16, Facchini, in example 8, teaches incubating an E. coli strain harboring pGFLOMT2 with norlaudanosoline (i.e. precursor of the BIA composition) to generate products including a product with m/z 344 at 3.42. The m/z 344 at 3.42 corresponds to tetrahydropapverine (i.e. methylated BIA). See [0222] and figure 8C.
Regarding claim 17, Facchini, in example 8, teaches incubating mixed E. coli strains harboring pGFLOMT1 and pGFLOMT2 with norlaudanosoline to yield compounds including a compound with m/z 344 at 3.42, which corresponds to tetrahydropapaverine. See [0222]. GFLOMT1 is SEQ.ID NO: 1592. See [0153]. Facchini’s SEQ ID NO: 1592 is a 98.7% identity match to instant SEQ ID NO: 6. See the office action appendix.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Facchini (US 2017/0058305) in view of Smolke (US 2014/0273109).
Regarding claim 19, Facchini teaches a host cell comprising an introduced first polynucleotide that encodes a first enzyme capable of catalyzing a chemical reaction that converts a first alkaloid compound into a second alkaloid compound. See claim 11 of Facchini. Facchini teaches an O-methyltransferase enzyme that can be contacted a first alkaloid compound under reaction conditions that permit methylation of the first alkaloid compound to form a second alkaloid compound; wherein the first alkaloid compound has the chemical formula (I), wherein R1, R2, R3, and R4 can each independently or simultaneously represent a hydroxyl group; and wherein the second alkaloid compound has the chemical formula (II), and wherein R1, R2, R3, and R4, can each independently or simultaneously represent a methoxy group, with the proviso that at least one of R1, R2, R3, and R4 represents a methoxy group. See claim 17 of Facchini and the structures of formulas (I) and (II) above. Furthermore, Facchini suggests that alkaloid compounds that are particularly preferred are benzylisoquinoline alkaloids. See [0096]. Facchini teaches O-methyltransferase obtained from Glaucum flavium. See claim 19 of Facchini.
Facchini does not teach a kit.
Facchini does not explicitly teach a non-naturally occurring methyltransferase, wherein said methyltransferase can methylate more than two positions in a benzylisoquinoline alkaloid molecule.
Smolke teaches host cells engineered to produce benzylisoquinoline alkaloids (BIAs). See the abstract. Smolke teaches host cells that comprise heterologous coding sequences for one or more methyltransferases. See claim 114 of Smolke. Smolke teaches a kit that includes a host cell. The cell expresses a BIA of interest. See [0179]
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to organize Facchini’s methyltransferase into a kit based on the suggestion of Smolke; and to further recognize that Facchini’s O-methyltransferase can methylate up to four positions in a benzylisoquinoline alkaloid molecule. One of ordinary skill in the art would have been motivated to organize Facchini’s methyltransferase into a kit, because Smolke suggests kits that include host cells that encode methyltransferases for the purpose of BIA production. There would have been a reasonable expectation of success because Facchini demonstrates host cells encoding an O-methylase enzyme that methylates norlaudanosoline at three to generate tetrahydropapaverine (see [0222] and the structures in figures 9A and 9E).
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657
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