DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 06/26/2025 has been entered.
Priority
This application is a 371 of PCT/US2020/034417 filed 05/22/2020 which claims benefit of provisional application 62/851 ,772 filed 05/23/2019. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 06/26/2025 complies with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Status of the Claims
Claim 13, 114 and 115 were amended. Claims 2, 4-6, 9-11, 17-28, 30, 31, 36-110 are cancelled.
Claims 1, 3, 7, 8, 12-16, 29, 32, 35 and 111-119 are pending (claim set filed 06/26/2025) and are examined on the merits herein.
Withdrawal of Rejections
The response and amendment filed on 06/26/2025 are acknowledged. The amendment to Specification is acknowledged. All of the amendment and arguments have been thoroughly reviewed and considered.
For the purposes of clarity of the record, the reasons for the Examiner's withdrawal and/or maintaining if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner's response to arguments section.
The previous objection to Specification has been withdrawn necessitated by amendment of the specification.
The previous claim 13 objection has been withdrawn necessitated by amendment of the claim 13.
The previous claim 114 rejection under 35 U.S.C. 112(b) has been withdrawn necessitated by amendment of claim 114.
New Objections
Claim Objections
Claim 32 is objected to because of the following informalities:
Claim 32 recites: “… one or more rebaudioside D_1.07, …”. Applicant is suggested to replace recitation with: “… one or more of rebaudioside D_1.07, …”.
Appropriate correction is required.
New Rejections
Claim Rejections - 35 USC § 112
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.
Claim 29 is 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 29 recites: "in a composition comprising the rebaudioside A or the rebaudioside D, the sugar moiety is transferred …”. There is insufficient antecedent basis for this limitation in the claim because neither “composition”, nor “sugar moiety” are mentioned in claim 1 from which claim 29 depends. It is also not clear whether sugar moiety is referred to glucose, which is the only sugar moiety recited to be transferred to a substrate in claim 1, since for instance sucrose has glucose and fructose sugar moieties. It is not clear what sugar moieties are encompassed by claim 29. The scope and boundaries of claim 29 are not certain making claim 29 indefinite.
Maintained Rejection
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 12-16, 29, 32, 35 and 111-119 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1 and 114 recite the limitation for glycosyltransferase polypeptides to comprise amino acid sequence that is at least 98% identical to amino acid sequence selected from the recited amino acid sequences. Claims 1 and 114 are interpreted as directed a method for transferring a sugar moiety to a substrate steviol glycoside comprising glycosyltransferase polypeptides having sequence 2% or less of which vary from listed SEQ ID Nos and can include additional sequences due to transitional phrase “comprises”.
Thus, claims 1 and 114 broadly encompass a genus of glycosyltransferases having 98% or more sequence identity to listed SEQ ID NOs. This would represent a large pools of variant protein sequences for glycosyltransferase that are functional. At the same time glycosyltransferase can have 2% or less of sequences that can differ from listed SEQ ID Nos and can include additional sequences. The Specification does not provide structure function correlation for glycosyltransferase and does not describe domains and/or amino acid residues essential for glycosyltransferase function and domain and/or amino acid residues which can be modified without loss of function. Further, applicants have not shown possession of a representative number of species for glycosyltransferase variants with sequences having less than 100% identity to listed SEQ ID Nos. The Specification provides only examples of production and validation of glycosyltransferases with sequences 100% identical to selected sequences. (Examples 1- 12).
The prior art of Monchois (Monchois et al. FEMS Microbiol. Rev., 1999, 23, 131-151) teaches structure-function relationship of glucansucrases and in particular glycosyltransferases of Streptococcus species (Abstract, p. 131, left column, 1st paragraph). Monchois describes that substitution of several amino acid residues inhibits or completely suppresses catalytic activity of glycosyltransferase indicating involvement of these residues in the catalytic mechanism of glycosyltransferase. For instance, substitution of Glu475, Asp547 and Asp437 resulted in inhibition of enzyme activity and substitution of Trp491 led to enzyme devoid of activity (p. 144, left column, 2nd paragraph and right column). Thus, the prior art demonstrates significant effect of single amino acid residues modification on glycosyltransferase function.
Thus, one of ordinary skill in the art would not be able to identify without further testing which amino acid sequences that have at least 98% identity to listed SEQ ID NOs encode for functional glycosyltransferase. One of ordinary skill in the art would conclude based on the lack of representative number of species and the lack of describing the domains or amino acid residues of listed SEQ ID Nos critical for the function of glycosyltransferase that the Applicant was not in possession of the claimed genus of glycosyltransferases and the specification fails to satisfy the requirements of written description under 35 U.S.C. 112 (a).
Claims 12-16, 29, 32, 35 and 111-113, dependent on claim 1, and claims 115-119, dependent on claim 114, do not resolve the issue mentioned above and are rejected.
Response to Arguments
Applicant's arguments filed 06/26/2025 have been fully considered but they are not persuasive.
In response to Applicant’s arguments (addressing p. 8 of the Remarks) that: “the Examiner has not met this initial burden. First, the Examiner has not provided evidence or a technical rationale showing that minor variations encompassed within the claimed genus would materially affect glycosyltransferase function. The Office Action does not cite any data, references, or specific mutations that would suggest loss of function in the claimed polypeptides at 98% identity. Moreover, a 2% sequence variation (i.e., 1-10 amino acids in a ~500-residue enzyme) is well within the expected tolerance for functional conservation in proteins. The skilled artisan would reasonably expect variants with 98% identity to the claimed sequences to retain the same catalytic function, absent specific contraindications.”, these arguments are not persuasive because:
The MPEP 2163 states: “[A] patentee of a biotechnological invention cannot necessarily claim a genus after only describing a limited number of species because there may be unpredictability in the results obtained from species other than those specifically enumerated.” In instant case, the description of the glycosyltransferase genera encompassed by claims 1 and 114 and having at least 98% sequence identity to listed SEQ ID NOs is not provided. The specification does not describe structure function correlation for glycosyltransferases and does not provide examples of a representative number of species which will be functional or not functional, except examples of single species for each protein with 100% identity to corresponding SEQ ID NOs. The prior art of Monchois teaches that single mutations of glycosyltransferase sequences can result in inhibition or complete elimination of glycosyltransferase activity as described above. At the same time the sequence variation of 2% represents for instance for SEQ ID NO:42, having 1422 amino acid residues, variation in 28 residues.
Thus, without description of glycosyltransferase structure-function relationship one of ordinary skill in the art will not be able to identify which amino acid residues can be modified within each genus without affecting glycosyltransferase function. Therefore, the specification does not provide sufficient written description of glycosyltransferase genera and artisan in the field would conclude that Applicant was not in possession of the claimed genera of glycosyltransferases.
Maintained/Modified Rejections
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1, 3, 7, 12-16, 29, 32, 35, 112-116, 118 and 119 are rejected under 35 U.S.C. 103 as being unpatentable over Markosyan (US 20180100175 A1 on record in IDS) in view of UniProt GTFC_STRMU (UniProt GTFC_STRMU [retrieved on 02/12/2025]. Retrieved from the Internet: <gtfC - Glucosyltransferase-SI - Streptococcus mutans serotype c (strain ATCC 700610 / UA159) | UniProtKB | UniProt>) as evidenced by Argimon (Argimon et al., PLOS One, 2013, 8, e56305, 1-11) and Gerwig (Gerwig et al. Carbohydrate Research, 2017, 440-441, 51-62 on record in IDS).
Regarding claim 1, Markosyan teaches methods of preparing steviol glycosides: “The present invention provides a biocatalytic process for preparing a composition comprising a target steviol glycoside by contacting a starting composition comprising an organic substrate with a microorganism and/or biocatalyst, thereby producing a composition comprising a target steviol glycoside.” (paragraph 0007). Markosyan describes different non-UDP-sugar sugars that can be used with the invention including maltose and sucrose: “Examples of carbohydrates which may be used in accordance with this invention include, but are not limited to, tagatose, trehalose, galactose, rhamnose, various cyclodextrins, cyclic oligosaccharides, various types of maltodextrins, dextran, sucrose, glucose, … maltose …” (paragraph 0123). Although Markosyan provides working examples with UDP-glucose as a sugar donor (for instance Example 4, paragraph 0294), Markosyan mentions embodiment wherein “the starting composition is sucrose” (paragraph 0127) and discloses that: “The organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein” (paragraph 0130). Therefore, Markosyan does not exclude that sucrose can serve as a substrate for glycosylation of steviol glycosides. Besides, sucrose can serve as glycosyl donor for reaction of glycosylation of rebaudioside A catalyzed by glucansucrase (which is glycosyltransferase) as evidenced by Gerwig (Abstract).
Markosyan teaches different target steviol glycosides including rebaudioside D, its isomer, rebaudioside D2, rebaudioside M and its isomer, rebaudioside M2 (paragraph 0009). Markosyan discloses the use of glycosyltransferase: “The UDP-glucosyltransferase can be any UDP-glucosyltransferase capable of adding at least one glucose unit to the steviol and/or steviol glycoside substrate to provide the target steviol glycoside.” (paragraph 0023). Markosyan describes that glycosyltransferase is capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D (paragraph 0029) and adding at least one glucose unit to rebaudioside D to form rebaudioside M (paragraph 0030).
Markosyan does not teach glycosyltransferases comprising amino acid sequences that are at least 98% identical to amino acid sequences with recited SEQ ID Nos.
UniProt GTFC_STRMU teaches glycosyltransferase from Streptococcus mutans serotype (P13470) that can transfer glucose moiety from sucrose (p. 1). The amino acid sequence of S. mutans glycosyltransferase P13470 comprises the amino acid sequence with more than 98% identity to claimed SEQ ID NO:42 (p. 7, last updated 11/28/2002) according to BLAST analysis. The amino acid residues 35- 1455 of UniProt GTFC_STRMU teaching are identical to amino acid residues 2-1422 of SEQ ID NO:42. Thus, UniProt GTFC_STRMU teaches the first and the second glycosyltransferase of claim 1.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Markosyan and UniProt GTFC_STRMU and use S. mutans glycosyltransferase P13470 from UniProt teaching for production of rebaudiosides as described by Markosyan teaching. One would have been motivated to do so since UniProt GTFC_STRMU provides annotated sequence for glycosyltransferase that can transfer glucose moiety from sucrose as a substrate, Markosyan teaches sucrose as a substrate for glycosyltransferase for production of rebaudiosides and glycosyltransferase can glycosylate steviol glycosides as evidenced by Gerwig. Besides, pursuant to MPEP 2112.01(II), “[p]roducts of identical chemical composition cannot have mutually exclusive properties.” In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Therefore, given glycosyltransferase with SEQ ID NO: 42 is taught by UniProt GTFC_STRMU, it is necessarily capable of transferring glycosyl from sucrose or maltose to rebaudioside A as taught in claim 1 for the first and second glycosyltransferase. A skilled artisan would have reasonably expected success in the combination because Markosyan, UniProt GTFC_STRMU and Gerwig describe various glycosyltransferases. Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claim 1 obvious.
Regarding claims 3 and 7, UniProt GTFC_STRMU teaches Streptococcus mutans to be a secreted protein (p. 3, Subcellular Location) the sequence of which contains amino acid sequence for the signal peptide, i.e. amino acid residues 1-34 (p. 5) and the mature polypeptide with amino acid residues 35- 1455. The amino acid residues 35-1455 are identical to instant amino acid residues 2-1422 of SEQ ID NO:42. The mature polypeptide is an active form of glycosyltransferase as evidenced by Argimon teaching that Streptococcus mutans glycosyltransferases are extracellular proteins that synthesize extracellular glucans from dietary sucrose (p. 1, left column, 1st and 2nd paragraphs).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the sequence of glycosyltransferase from UniProt GTFC_STRMU teaching by removing signal peptide and substituting it with the start codon coding for methionine thus arriving to the amino acid sequence 100% identical to instant sequence with SEQ ID NO: 42 and use it as the first and the second glycosyltransferase for steviol glycoside production. One would have been motivated to do so since UniProt GTFC_STRMU teaches glycosyltransferase to be a secreted protein and processed mature glycosyltransferase is active in the extracellular space as evidenced by Argimon and the active form of glycosyltransferase (without signal peptide) can be expressed in a host cell as described in instant application. A skilled artisan would have reasonably expected success in the modification because it is within the skill of artisan in the art to modify sequence of the recombinant polypeptide. Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig and Argimon render claims 3 and 7 obvious.
Regarding claims 12-13, Markosyan mentions that glucosyltransferase can be expressed in a microorganism and provides examples of such microorganisms including those in instant claim 13: “In one embodiment, steviol biosynthesis enzymes and UDP-glucosyltransferases are produced in a microorganism. The microorganism may be, for example, E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Bacillus sp., Yarrowia sp. etc” (paragraph 0024). Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claims 12 and 13 obvious.
Regarding claims 14-16, Markosyan teaches that enzyme (glycosyltransferase) “can be in free form or immobilized to a solid support made from inorganic or organic materials (paragraph 0018). Markosyan discloses that the enzyme can be in a recombinant microorganism and the microorganism can be immobilized on a solid support comprising materials of instant application and immobilization is achieved by the same methods as in instant application: “In another embodiment, the microorganism is immobilized. For example, the microorganism may be immobilized to a solid support made from inorganic or organic materials. Non-limiting examples of solid supports suitable to immobilize the microorganism include derivatized cellulose or glass, ceramics, metal oxides or membranes. The microorganism may be immobilized to the solid support, for example, by covalent attachment, adsorption, cross-linking, entrapment or encapsulation.” (paragraph 0158). Even though Markosyan does not explicitly describes details of enzyme immobilization, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention that Markosyan will apply same methods for enzyme immobilization as was described by him for cell immobilization. One would be motivated to assume so with reasonable expectation of success since Markosyan describes several materials for immobilization and several methods for immobilization and these methods are known in the art. Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claims 14-16 obvious.
Regarding claim 29, Markosyan provided example of 80% conversion of rebaudioside D to rebaudioside M: “HPLC indicated 80% conversion of rebaudioside D to rebaudioside M after 120 h as seen in FIG. 41” (paragraph 0297). Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claim 29 obvious.
Regarding claims 32 and 35, the specification defines rebaudioside isomers described in claims 32 and 35 by their relative retention time: “Rebaudioside isomers were classified by calculating relative retention times for all rebaudioside isomers detected by the method described in Example 4 (the 4- minute method) and the method described in Example 16 (the 20-minute method).” (paragraph 0081).
Regarding claims 32 and 35, Markosyan teaches presence of several isomers of rebaudioside D and rebaudioside M. Rebaudioside D2 and rebaudioside M2 isomers were identified (paragraph 0039) and their structures were confirmed by NMR (paragraphs 0506 and 0539). Besides rebaudioside D2 and rebaudioside M2 isomers, several other isomers of rebaudioside D and rebaudioside M were detected in Markosyan teaching by LC-MS system (paragraph 0437). Note that Markosyan does not determine retention time relative to standard and additionally retention time can vary depending on conditions of glycosyltransferase catalytic reaction (particular enzyme and sugar substrate used) and LC-MS parameters. Therefore, isomers found by Markosyan can be similar to instant rebaudioside D and rebaudioside M isomers in claims 32 and 35 or obvious variants of those.
Although Markosyan discloses formation of isomers of rebaudioside D and rebaudioside M, they are not defined by the relative retention time. However, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to look at the guidance provided by Markosyan for the presence of several isomers of rebaudioside D and rebaudioside M produced by glycosyltransferase action and identify isomers produced. One would be motivated to do so to find novel steviol glycosides for application in food industry since Markosyan teaches steviol glycosides to be high intensity sweeteners not causing glycemic response (paragraph 0002). Therefore, a skilled artisan would have reasonably expected success in identification of produced rebaudioside D and rebaudioside M isomers following the guidance of the prior art.
Additionally and/or alternatively, MPEP 2112-2112.02 states that when a reference teaches all the limitations of a claim except for a property or function, and the examiner cannot determine whether or not the reference inherently possesses properties which anticipate or render obvious the claimed invention but has basis for shifting the burden of proof to applicant as in In re Fitzgerald, 619 F.2d 67, 205 USPQ 594 (CCPA 1980). In the instant case, as discussed supra, Markosyan teaches presence of several isomers of rebaudioside D and rebaudioside M. Plus, Markosyan and UniProt GTFC_STRMU teach the identical method of making the steviol glycosides as instantly claimed in instant claim 1.
The Patent and Trademark Office is not equipped to conduct experimentation in order to determine whether or not Applicants’ rebaudioside D and rebaudioside M isomers differ, and if so to what extent, from the rebaudioside D and rebaudioside M isomers taught in Markosyan. The cited art taken as a whole demonstrates a reasonable probability that the rebaudioside D and rebaudioside M isomers of Markosyan are either identical or sufficiently similar to the claimed rebaudioside D and rebaudioside M isomers and whatever differences exist are not patentably significant. Therefore, with the showing of the reference, the burden of establishing non-obviousness by objective evidence is shifted to the Applicants.
Merely because a property (retention time) of instant isomers of rebaudioside D and rebaudioside M is not expressly taught in a reference does not make the known method patentable. The steviol glycosides preparation method possesses property to produce instant rebaudiosides necessarily present which might not be displayed in the tests used in Markosyan. Accordingly, the disclosures of Markosyan and UniProt GTFC_STRMU is a sufficient basis that the method of steviol glycoside preparation produces the instant rebaudioside D and rebaudioside M isomers.
In the alternative, even if the claimed rebaudioside D and rebaudioside M isomers are not identical to the Markosyan rebaudioside D and rebaudioside M isomers with regard to some unidentified properties, the differences between that which is taught and that which is claimed are considered to be so slight that the Markosyan and UniProt GTFC_STRMU method of steviol preparation is likely to inherently possess the same property of producing instant rebaudioside D and rebaudioside M isomers particularly in view of the similar method steps, substrates and enzyme, glycosyltransferase. Thus, the claimed rebaudioside D and rebaudioside M isomers would have been obvious to those of ordinary skill in the art under the meaning of USC 103. Accordingly, the claimed invention as a whole was at least prima facie obvious, especially in the absence of sufficient, clear, and convincing evidence to the contrary.
Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claims 32 and 35 obvious.
Regarding claim 114, Markosyan teaches methods of preparing steviol glycosides (paragraph 0007). Markosyan describes different non-UDP-sugar sugars that can be used with the invention (paragraph 0123). As described above for claim 1, although Markosyan provides working examples with UDP-glucose as a sugar donor (for instance Example 4, paragraph 0294), Markosyan mentions embodiment wherein “the starting composition is sucrose” (paragraph 0127) and discloses that: “The organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein” (paragraph 0130). Therefore, Markosyan does not exclude that sucrose can serve as a substrate for glycosylation of steviol glycosides. Besides, sucrose can serve as glycosyl donor for reaction of glycosylation of rebaudioside A catalyzed by glucansucrase (which is glycosyltransferase) as evidenced by Gerwig (Abstract).
Markosyan teaches different target steviol glycosides including rebaudioside D, its isomer, rebaudioside D2, rebaudioside M and its isomer, rebaudioside M2 (paragraph 0009, 0506 and 0539). Besides rebaudioside D2 and rebaudioside M2 isomers, several other isomers of rebaudioside D and rebaudioside M were detected in Markosyan teaching by LC-MS system (paragraph 0437). Note that Markosyan does not determine retention time relative to standard and additionally retention time can vary depending on conditions of glycosyltransferase catalytic reaction (particular enzyme and sugar substrate used) and LC-MS parameters. Therefore, isomers found by Markosyan can be similar to instant isomers defined by the retention time as described above or obvious variants of those. Markosyan discloses the use of glycosyltransferase (paragraph 0023). Markosyan describes that glycosyltransferase is capable of adding at least one glucose unit to rebaudioside A to form rebaudioside D (paragraph 0029) and adding at least one glucose unit to rebaudioside D to form rebaudioside M (paragraph 0030).
Markosyan does not teach glycosyltransferases comprising amino acid sequences that are at least 98% identical to amino acid sequences with recited SEQ ID Nos.
UniProt GTFC_STRMU teaches glycosyltransferase from Streptococcus mutans serotype (P13470) that can transfer glucose moiety from sucrose (p. 1). The amino acid sequence of S. mutans glycosyltransferase P13470 comprises the amino acid sequence with more than 98% identity to claimed SEQ ID NO:42 (p. 7, last updated 11/28/2002). The amino acid residues 35-1455 of UniProt teaching are identical to amino acid residues 2-1422 of SEQ ID NO:42.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine teachings of Markosyan and UniProt and use S. mutans glycosyltransferase P13470 from UniProt GTFC_STRMU teaching for production of rebaudiosides as described by Markosyan teaching. One would have been motivated to do so since UniProt GTFC_STRMU provides annotated sequence for glycosyltransferase that can transfer glucose moiety from sucrose as a substrate, Markosyan teaches sucrose as a substrate for glycosyltransferase for production of rebaudiosides and glycosyltransferase can glycosylate steviol glycosides as evidenced by Gerwig. Besides, pursuant to MPEP 2112.01(II), “[p]roducts of identical chemical composition cannot have mutually exclusive properties.” In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). A chemical composition and its properties are inseparable. Therefore, if the prior art teaches the identical chemical structure, the properties applicant discloses and/or claims are necessarily present. Therefore, given glycosyltransferase with SEQ ID NO: 42 is taught by UniProt GTFC_STRMU, it is necessarily capable of transferring glycosyl from sucrose or maltose to rebaudioside A as taught in claim 1 for the first and second glycosyltransferase. A skilled artisan would have reasonably expected success in the combination because Markosyan, UniProt GTFC_STRMU and Gerwig describe various glycosyltransferases.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to look at the guidance provided by Markosyan for the presence of several isomers of rebaudioside D and rebaudioside M produced by glycosyltransferase action and identify isomers produced. One would be motivated to do so to find novel steviol glycosides for application in food industry since Markosyan teaches steviol glycosides to be high intensity sweeteners not causing glycemic response (paragraph 0002). Therefore, a skilled artisan would have reasonably expected success in identification of produced rebaudioside D and rebaudioside M isomers following the guidance of the prior art. Additionally, please see discussion above for claims 32 and 35 on obviousness of rebaudioside D and rebaudioside M isomers. Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claim 114 obvious.
Regarding claims 112, 113, 118 and 119, Markosyan describes reaction of transformation of rebaudioside D to rebaudioside M by lysate containing glycosyltransferase and reaction is performed at 30°C for 90 hours (paragraph 0325). Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claims 112, 113, 118 and 119 obvious.
Regarding claim 115 and 116, Markosyan teaches different non-UDP-sugars that can be used with the invention including maltose and sucrose (paragraph 0123). UniProt GTFC_STRMU teaches sucrose as a sugar donor for Streptococcus mutans glycosyltransferase P13470. As described above, the amino acid sequence of S. mutans glycosyltransferase P13470 comprises the amino acid sequence with more than 98% identity to claimed SEQ ID NO:42 and will necessarily possess the claimed properties including ability to use non-UDP sugar donors. Thus, Markosyan and UniProt GTFC_STRMU teachings as evidenced by Gerwig render claims 115 and 116 obvious.
Claims 111 and 117 are rejected under 35 U.S.C. 103 as being unpatentable over Markosyan (US 20180100175 A1 on record in IDS) in view of UniProt GTFC_STRMU (UniProt GTFC_STRMU [retrieved on 02/12/2025]. Retrieved from the Internet: <gtfC - Glucosyltransferase-SI - Streptococcus mutans serotype c (strain ATCC 700610 / UA159) | UniProtKB | UniProt>) as evidenced by Gerwig (Gerwig et al. Carbohydrate Research, 2017, 440-441, 51-62 on record in IDS) as applied to claims 1 and 114 above, and further in view of Holm (Holm and Sander The EMBO Journal, 1995, 14, 1287-1293).
The teachings of Markosyan and UniProt GTFC_STRMU have been set forth above.
Markosyan does not teach the reaction medium to comprise pyridoxal phosphate.
Regarding claims 111 and 117, Holm teaches unexpected similarity in three-dimensional structure between glycosyltransferases, i.e. β-glycosyltransferase from bacteriophage T4 and glycogen phosphorylase which is α-glycosyltransferase (Abstract). β-glycosyltransferase transfers glucose unit from UDP-glucose to DNA and glycogen phosphorylase transfers terminal glucose unit extracting it from glycogen in the presence of coenzyme, pyridoxal phosphate (PLP) (Abstract). The structural alignment of two enzymes showed superimposition of PLP and UDP sites: “the phosphate groups in the catalytic site of β-glucosyltransferase overlap with those of glycogen phosphorylase and the pyridoxal ring in glycogen phosphorylase occupies a position structurally equivalent to that of the ribose ring of UDP in β- glucosyltransferase.” (p. 1290, left column, Fig. 5). The glucose binding sites appeared to be structurally equivalent (p. 1290, right column, 2nd paragraph). The structural equivalency of UDP and PLP and phosphate sites can indicate that PLP can facilitate catalysis of glycosyltransferase when the non-UDP substrate is used substituting the UDP function.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to try adding pyridoxal phosphate from Holm teaching to the reaction medium of glycosyltransferase reaction with non-UDP substrate based on Markosyan and UniProt GTFC_STRMU teachings. One would have been motivated to do so since Holm showed structural superimposition of UDP and PLP for two glycosyltransferases with similar biochemical features of transferring glucose units. A skilled artisan would have reasonably expected success in that because UniProt GTFC_STRMU describes glycosyltransferase transferring glucose moiety from non-UDP sugar donor, sucrose, Markosyan teaches UDP-glucose as sugar donor for steviol glycosides glycosylation (not excluding sucrose as a substrate) and addition of PLP which may substitute the UDP function can potentially facilitate the reaction and experimenting with addition of different components to the reaction medium is routine and conventional in the art. Thus, Markosyan, UniProt GTFC_STRMU and Holm teachings as evidenced by Gerwig render claims 111 and 117 obvious.
Response to Arguments
Applicant's arguments filed 06/26/2025 have been fully considered but they are not persuasive.
In response to Applicant’s arguments (addressing p. 9-10 of the Remarks) that: “Markosyan's carbohydrates are not non-UDP-sugar sugar donors, as claimed, at least because Markosyan' s carbohydrates are not taught or suggested as a sugar donor for an enzymatic process. … But to the extent Markosyan mentions a sugar donor in the context of its UDP-glycosyltransferases, Markosyan describes only UDP-glucose. …Markosyan's carbohydrates are, instead, and contrary to the Office Action's interpretation, simply provided as composition additives alongside highly purified target steviol glycosides. … GTFC _ STRMU fails to cure the deficiencies of Markosyan”, these arguments are nor persuasive because:
Even though Markosyan does not explicitly describe non-UDP sugars as substrates for
glycosylation, Markosyan does not exclude non-UDP sugar donors, such as sucrose, as substrates since
he discloses embodiment wherein “the starting composition is sucrose” (paragraph 0127) without mentioning UDP-glucose and discloses that: “The organic compound(s) of starting composition serve as a substrate(s) for the production of the target steviol glycoside(s), as described herein” (paragraph 0130).
Assuming arguendo that glycosyltransferase of Markosyan teaching does not work with
non-UDP sugar substrates, the rejection is based on combination of references of Markosyan and UniProt GTFC_STRMU. The Streptococcus mutans glycosyltransferase comprising instant SEQ ID NO: 42 and taught by UniProt GTFC_STRMU uses sucrose as a substrate (p. 1). The same property of S. mutans glycosyltransferase encoded by gtf-C gene is confirmed by Monchois teaching glycosyl residue transfer from sucrose during glucan synthesis (p. 134, left column 3rd paragraph). Additionally, Gerwig showed that sucrose can not only serve as a sugar donor but also be used in reaction of glycosylation of steviol glycoside, rebaudioside A (Abstract). Therefore, at least some glycosyltransferases are able to use non-UDP sugar donor for glycosylation of steviol glycosides.
Finally, UniProt GTFC_STRMU teaches S. mutans glycosyltransferase comprising instant
SEQ ID NO: 42 and as described in the rejection above for claims 3 and 7, one of ordinary skill in the art would have been motivated to modify the sequence of glycosyltransferase from UniProt GTFC_STRMU for expression in the host cell by removing signal peptide and substituting it with the start codon coding for methionine thus arriving to the amino acid sequence 100% identical to instant sequence with SEQ ID NO: 42 because UniProt GTFC_STRMU teaches glycosyltransferase as a secreted protein and processed mature glycosyltransferase is active as evidenced by Argimon (p. 1, left column, 1st and 2nd paragraphs).
Pursuant to MPEP 2112.01(I), where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). Thus, since prior art teaches instant glycosyltransferase (with 100% identity), this enzyme is capable of transferring glycosyl moiety from non-UDP sugars, such as sucrose or maltose to steviol glycosides as taught in instant claims.
Therefore, the 35 U.SD.C. 103 rejection is maintained.
Allowable Subject Matter
Claim 8 is 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. Claim 8 is directed to glycosyltransferase polypeptide with amino acid sequence 100% identical to SEQ ID NO:41. It is noted that the sequence with the SEQ ID NO: 41 is novel.
The closest prior art of UniProt PBPA_PSEAE (UniProt PBPA_PSEAE [retrieved on 02/12/2025]. Retrieved from the Internet: <mrcA - Penicillin-binding protein 1A - Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) | UniProtKB | UniProt>) teaches penicillin binding protein 1A from Pseudomonas aeruginosa Q07806. The penicillin binding protein has the glycosyltranferase activity (peptidoglycan glycosyltransferase activity) and carboxypeptidase activity (p. 2) with the N-terminal section belonging to the glycosyltransferase 51 family and the C-terminal section belonging to the transpeptidase family (p. 6). The amino acid sequence of penicillin binding protein 1A of UniProt PBPA_PSEAE comprises the amino acid sequence with claimed SEQ ID NO:41 (p. 7, last updated 01/01/1998) according to BLAST analysis. However, the amino acid sequence of UniProt PBPA_PSEAE has additional 35 amino acid residues of N-terminus which are not present in SEQ ID NO:41. The additional amino acid residues comprise the cytoplasmic domain (residues 1-5) and the signal-anchor for type II membrane protein domain (residues 6-26) (p. 4).
Thus, there is not teaching or suggestion to achieve 100% identity with the same length to SEQ ID NO: 41 .
Conclusion
No claims are allowed.
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/L.G.K./Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653