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 .
Restriction/Election
In response to the communication received on June 3rd, 2026, from James S. Keddie, the election of Group I, claims 1-2 and 4-6, with traversal, is acknowledged. Applicant is respectfully reminded that the assertion of a search burden in a lack of unity of invention practice under PCT Rule 13.1 and 13.2 is irrelevant, therefore demonstration of a search burden is not required.
The requirement is still deemed proper for reasons cited in the Requirement for Restriction filed April 10th, 2026 and is therefore made FINAL.
Priority
Applicant’s claim for the benefit of a prior-filed application no. GB2209501.2 filed June 29th, 2022, and PCT/EP2023/067395 filed June 27th, 2023, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is June 29th, 2022.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on March 7th, 2025, was considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
The listing of references on page 67 of 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.
Status of Claims
Claims 1-2, 4-8, 10-14, 16-18, 23, and 25-28 filed October 3rd, 2025 are pending.
Claims 7-8, 10-14, 16-18, 23, and 25-28 are withdrawn.
Claims 1-2, and 4-6 are examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (see pg. 43, lns. 31-35, for example). 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.
Claim Objections
In claim 1, “OS”, “SoC28”, “SoC28C16”, “QA-GlcA”, “QA-GlcA-Gal”, “QA-Tri”, “QA-TriF”, “QA-TriFRX”, “QA-TriFRXX”, and “QA-TriF(Q)RXX” are used as abbreviation. Although these terms are later recited in dependent claims, it is suggested to insert a definition for these terms without bringing in new matter, immediately before the first appearance in claim 1; and to enclose the appearance of the terms in parentheses (in claim 1 only).
Claim 1 is further objected to for the following informality: in line 5 of claim 1, “either” should have a colon instead of a semi-colon.
Claims 2 and 4 are objected to for the following informalities: Claim 2, section (iii) recites “having at least 50% sequence identity.” This should be corrected to “has at least 50% sequence identity.” The same informality is present in claim 4, section (iv). Section (b) of claim 2 appears to be lacking “; and” at the end of the claim to make the claim inclusive of sections (i) and (iii). Further, the sections of claim 2 appear to be improperly number (e.g., (i) and (iii) only). Line 2 of claim 4 recites “covalent” which should be corrected to “covalently”.
Claim 5 is objected to for the following informality: Claim 5 currently recites “the 28-O position QA-Tri”. Examiner suggest correcting to recite “the 28-O position of QA-Tri”. Additionally, claim 5 recites the abbreviation “QATriFuT” in line 6. It is suggested to insert a definition for these terms without bringing in new matter, immediately before the first appearance in claim 5; and to enclose the appearance of the terms in parentheses (in claim 5 only).
Claims 2, and 4-6 are objected to for the recitation of “a method according to claim…” in line one of each claim. It is suggested to amend to “the method according to claim…” for proper antecedent basis.
Appropriate correction is requested.
Claim Interpretation
Claim 1, and 2 and 4-5 depending therefrom, are drawn to “a method for the production of a triterpenoid.” Under broadest reasonable interpretation, a biosynthetic pathway is a primary method of production referring to the step-by-step, enzyme catalyzed series of reactions that living organisms use to build complex molecules from precursors. Thus, a plant’s natural ability to produce a triterpenoid along a biosynthetic pathway is taken to read on a method for the production of a triterpenoid.
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 1-2 and 4-6 are rejected under 35 U.S.C. 101 because the claimed invention is directed to non-statutory subject matter. The claim(s) does/do not fall within at least one of the four categories of patent eligible subject matter because the claimed invention is directed to a natural phenomenon or product of nature without significantly more. The claims recite a method for the production of a triterpenoid.
Broadest Reasonable Interpretation:
Claim 1 recites a method for the production of a triterpenoid comprising several enzymes responsible for the synthesis of the resulting saponarioside B triterpenoid. The Examiner has interpreted “a method for the production of a triterpenoid” to be process carried out naturally by the plant from which the enzymes were isolated, Saponaria officinalis. Since claims 1-2 and 4-6 do not recite characteristics that amount to a difference between naturally occurring saponarioside B biosynthesis within the plant, according to the instant disclosure, the method for the production of a triterpenoid as claimed in claims 1-2 and 4-6 has been interpreted as a process that occurs in nature.
Step 1. Determining if the claims are under a statutory category:
Under Step 1 of the subject matter eligibility test for products and processes, it must be determined if the claim is to a process, machine, manufacture or a composition of matter. In the instant case, the claims are directed to a biological process, reciting a series of steps, and are therefore directed to a statutory category.
Step 2A. ‘Judicial exception’ analysis:
Prong One: Does the claim recite an abstract idea law of nature or natural phenomenon?
Markedly different characteristics can be expressed as the product's structure, function, and/or other properties. Non-limiting examples of characteristics that can determine the presence of a marked difference include biological or pharmacological functions or activities; chemical and physical properties; phenotype, including functional and structural characteristics; and structure and form, whether chemical, genetic, or physical. The claims recite a method for the production of a triterpenoid, reciting enzymatic steps to ultimately synthesize saponarioside B.
The Applicant has not provided evidence of a markedly different characteristic between the instantly recited biosynthetic process for the production of saponarioside B within S. officinalis, providing examples for the identification of candidate genes utilizing a publicly available S. officinalis transcriptome and RNA-seq analysis for a new soapwort transcriptome [pg. 46, lns. 15-19]. Further, the Applicant states that the major saponin found in S. officinalis are saponariosides A and B [pg. 1, lns. 18-20]. Although the characterization was done through expression in N. benthamiana, the claimed saponarioside pathway genes themselves do not have any markedly different characteristics as they were identified in naturally occurring S. officinalis. Thus, claims 1-2 and 4-6 recite a judicial exception. The judicial exception is a natural phenomenon as saponarioside B biosynthesis occurs naturally without human intervention.
Prong 2: Does the claim recite additional elements that integrate the judicial exception into a practical application?
Under Step 2A, prong 2 of the analysis, it must be determined whether the claim recites additional elements that integrate the judicial exception into a practical application. In the instant case, the claims fail to recite any additional elements that integrate the judicial into a practical application, and therefore the claims remain directed to a judicial exception invoking further analysis under step 2B.
Step 2B: ‘Significantly more’ analysis:
Under Step 2B, it must be determined if the claim recites additional elements that amount to significantly more than the judicial exception. In the instant case, claims 1-2 and 4-6 fail to recite any additional elements that amount to significantly more than the judicial exception since the claims are recited as product-by-process without reciting any structural features. Therefore, the claims as a whole do not amount to significantly more than the exception.
Therefore, claims 1-2 and 4-6 are directed to subject matter that is not patent-eligible and are, as a result, rejected under 35 U.S.C. 101.
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.
Scope of Enablement
Claims 1-2 and 4-6 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 the production of triterpenoids along the pathway to saponarioside B, does not reasonably provide enablement for all triterpenoids. Further, while the specification is enabling for the full pathway to reach saponarioside B, it is not enabling for selecting only specific steps of the pathway and still reaching saponarioside B biosynthesis. 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 or use the invention commensurate in scope with these claims.
In re Wands lists a number of factors for determining whether or not undue experimentation
would be required by one skilled in the art to make and/or use the invention. These factors are: (1) the quantity of experimentation necessary; (2) the amount of direction or guidance presented; (3) the
presence or absence of working examples of the invention; (4) the nature of the invention; (5) the state
of the prior art; (6) the relative skill of those in the art; (7) the predictability or unpredictability of the
art; (8) the breadth of the claim. In re Wands, 858 F.2d 731, 8 USPQ2d 1400 (Fed. Cir. 1988).
The Applicant teaches:
Characterization of the saponarioside B biosynthesis pathway in Saponaria officinalis using the S. officinalis transcriptome.
The Applicant does not teach:
That any triterpenoid can be produced by the claimed method, particularly that a single step (i)-(xii) of claim 1 will result in the production of any triterpenoid.
That step (xii) of claim 1 can occur without steps (i)-(xi).
The claims are broadly drawn to a method for the production of any triterpenoid. Claim 1 is broadly drawn to a method for the production of any triterpenoid comprising any one of steps (i)-(xii). Claims 2 and 4-6 require further enzymes along the biosynthesis pathway, but do not require each of the steps of claim 1. For example, this indicates that one could produce any triterpenoid using just steps (iv)-(vi) of claim 4, which would read on claim 1 as claimed.
The Applicant teaches that the first committed step of triterpene biosynthesis is predicted to be the production of β-amyrin catalyzed by an oxidosqualene cyclase, β-amyrin synthase [pg. 45, lns. 35-37]. The Applicant then follows predicted steps in the saponarioside biosynthetic pathway [pg. 46, lns. 15-40] and using the genes the Applicant had characterized to characterize candidate genes for the next steps in the pathway [pg. 48, lns. 4-42]. The Applicant does not provide sufficient working examples to show saponarioside biosynthesis, or the large possible scope of triterpenoids, from merely one predicted step (i)-(xii). The Applicant teaches each of the steps in succession, providing little to no guidance for any individual step to produce a triterpenoid alone.
The state of the art suggests that the first step in triterpene biosynthesis involves the cyclization of the linear precursor 2,3-oxidosqualene to a range of diverse scaffolds by a family of enzymes known as oxidosqualene cyclases (OSCs) (Jo, S., et al. 2025. “Unlocking saponin biosynthesis in soapwort.” Nat Chem Biol 21, 215–226. https://doi.org/10.1038/s41589-024-01681-7) [pg. 217, col. 1, ¶2]. Further, the aglycone core of SpA and SpB is QA, which is derived from one of the most common plant triterpenoid scaffolds, β-amyrin. The art further teaches that triterpenoids are one of the largest subclasses of specialized plant metabolites, with more than 14,000 known structures (Cárdenas, PD. 2019. “Evolution of Structural Diversity of Triterpenoids.” Front Plant Sci. 10:1523. doi: 10.3389/fpls.2019.01523) [Abstract]. Triterpenoids are cyclized from oxidized squalene precursors, creating more than 100 different cyclical triterpene scaffolds. Biosynthesis has evolved recurrently in evolution and thus the organization of the genes may not be conserved, showing great unpredictability among triterpenoid production [pg. 5, col. 2, ¶3].
The recitation of (i)-(xii) using “and/or”, suggests that each individual step does not need another to result in a triterpenoid. As the first step (i) of instant claim 1 is converting 2,3-oxidosqualene into β-amyrin, it is not clear that the method for the production of a triterpenoid would function without (i). Having merely one step of (i)-(xii) is not likely to result in the production of a triterpenoid. Particularly in the case of (xii), which would likely require the specifically claimed QA-TriF(Q)RXX of (xi) to be converted into saponarioside B. Undue experimentation would be required for one of ordinary skill in the art to see which, if any other, triterpenoids may be produced by the claimed method or individual method steps of the claimed invention.
Thus, given the quantity of experimentation necessary; the amount of direction or guidance presented; (3) the presence or absence of working examples of the invention; the nature of the invention; the state of the prior art; the predictability or unpredictability of the art; and the breadth of the claim, the instant disclosure does not sufficiently enable one of ordinary skill in the art to make or use the invention as claimed.
Written Description
Claims 1-2 and 4-6 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The instant disclosure describes:
Identification of saponarioside pathway genes based on phylogeny and by co-expression analysis.
Characterization of candidate genes by transient expression in N. benthamiana with the genes along the pathway with full identity to the SEQ ID NOs as claimed.
The instant disclosure does not describe:
That any triterpenoid can be produced by the claimed method, particularly that a single step (i)-(xii) of claim 1 will result in the production of any triterpenoid.
That step (xii) of claim 1 can occur without steps (i)-(xi).
That a sequence identity of 50-80% to the genes along the biosynthetic pathway will lead to any triterpenoid production or lead to saponarioside B production.
The claims are broadly drawn to a method for the production of any triterpenoid comprising genes with sequence identity varying from 50% to 80% to the claimed genes. Thus, the breadth of the claims is such that any triterpenoid could be produced with as low as 50% sequence identity to the genes characterized along the biosynthetic pathway. Additionally, claim 1 more broadly recites that a singular step may result in the production of any triterpenoid with relatively low sequence identity to the claimed gene.
The instant disclosure teaches characterization of the genes along the pathway to the triterpenoid saponarioside B without reducing to practice any species other than the genes with full identity to the claimed sequences. Thus, the Applicant has not reduced to practice the variability possible with the relatively low sequence identity.
For example, the Applicant claims that a method for the production of any triterpenoid comprises contacting oleanolic acid with a SoC28C16 oxidase polypeptide comprising an amino acid sequence having at least 50% identity to SEQ ID NO: 4, such that the C16 position of said oleanolic acid is oxidized to an alcohol and the C28 position of said β-amyrin is oxidized to a carboxylic acid, thereby producing echinocystic acid. SEQ ID NO: 4 is 502 amino acids long. 50% identity to SEQ ID NO: 4 allows for 251 possible substitutions, deletions or additions. Assuming only substitutions,
For just substitutions with any of the other 19 standard amino acids, there are
N
K
x
19
K
PNG
media_image1.png
28
57
media_image1.png
Greyscale
ways to choose which of the 251 positions are changes with 19 possible alternative amino acids, wherein N is the total sequence length and K is the number of changed nucleotides. This results in around 3.04 x 10469 possibilities for substitution of amino acids in SEQ ID NO: 4 with 50% sequence identity. Applicants fail to describe structural features common to the members of the broad genus. Undue experimentation would be required to ensure that another sequence falling within the broad scope of the invention may perform the function as claimed. In the instant case, undue experimentation would be required to ensure that a sequence with 50% idneity to SEQ ID NO: 4 is capable of oxidizing the C16 position of oleanolic acid and the C28 position of β-amyrin. A similar reasoning applies to SEQ ID NOs: 2, 6, 8, 12, 14, 16, 18, 20, 22, 34, and 36.
Additionally, the art teaches that triterpenoids are one of the largest subclasses of specialized plant metabolites, with more than 14,000 known structures (Cárdenas, PD. 2019. “Evolution of Structural Diversity of Triterpenoids.” Front Plant Sci. 10:1523. doi: 10.3389/fpls.2019.01523) [Abstract]. Triterpenoids are cyclized from oxidized squalene precursors, creating more than 100 different cyclical triterpene scaffolds. Biosynthesis has evolved recurrently in evolution and thus the organization of the genes may not be conserved, showing great unpredictability among triterpenoid production [pg. 5, col. 2, ¶3]. It is not clear that any sequence with only 50% identity to the claimed sequences would be sufficient to produce any triterpenoid from the large genus of triterpenoids.
The instant disclosure has not reduced to practice any species of the claimed genus other than sequences with full length identity to the claimed SEQ ID NOs. Since the genus has not been adequately described by structural features or had sufficient species reduced to practice, claim 1-2, and 4-6 do not meet the written description requirement.
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Meesapyodsuk, D. et al. (2007). “Saponin Biosynthesis in Saponaria vaccaria. cDNAs Encoding b-Amyrin Synthase and a Triterpene Carboxylic Acid Glucosyltransferase.” Plant Physiology. 143:959-969 (see IDS filed 03/07/2025), in view of Jia, Z. et al. (1998). “Major Triterpenoid Saponins from Saponaria officinalis.” J. Nat. Prod. 61:1368-1373 (see IDS filed 03/07/2025).
Claim 1 recites a method for the production of a triterpenoid comprising;
(i) contacting OS with a Saponaria officinalis p-amyrin synthase (SobAS) comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO 8, such that said OS is converted into p-amyrin.
Biosynthesis pathways for triterpenoid production are well established in the prior art, as is methodology for determining genes involved in biosynthesis. For example, Meesapyodsuk teaches saponin biosynthesis in Saponaria vaccaria, with the main saponin being vaccaroside (i.e., a method for the production of a triterpenoid; see claim interpretation) [Abstract]. Saponaria vaccaria, a soapwort, contains bioactive oleanane-type saponins similar to those found in soapbark tree (Quillaja saponaria; Rosaceae). Saponins such as vaccaroside have potentially useful pharmacological activities, including immunogenic, anticholesterolemic, and anticancer activities [pg. 959, col. 1, ¶2]. Thus, Meesapyodsuk teaches a combined polymerase chain reaction and expressed sequence tag approach to identify genes involved in saponin biosynthesis [Abstract].
Meesapyodsuk discloses identification and isolation of β-amyrin synthase, teaching that β-amyrin synthase (SvBS) provides the first step in triterpenoid biosynthesis by converting 2,3-oxidosqualene to β-amyrin (i.e., contacting OS with a β-amyrin synthase, such that said OS is converted into β-amyrin). The sequence of the SvBS cDNA was deposited in GenBank as accession number DQ915167 [pg. 961, col. 2, ¶2], with protein sequence under GenBank: ABK76265.1. The identity of the enzyme was confirmed by expression in yeast, wherein gas chromatography-mass spectrometry analysis of the yeast strain showed a single additional compound as compared to a control. The SvBS amino acid sequence revealed 81%, 80% and 72% identity with the BAS of Glycyrrhiza glabra (GenBank accession no. AB037203), Medicago truncatula (GenBank accession no. AJ430607), and Arabidopsis (Arabidopsis thaliana; GenBank accession no. NM106544), respectively [pg. 961, col. 2, ¶1], while also having 94% identity to SEQ ID NO: 8 of the instant invention (i.e., β-amyrin synthase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8; see alignment below).
Meesapyodsuk teaches that the next steps in the pathway presumably include: (1) oxidation of β-amyrin at positions 16, 23, and/or 28; (2) glycosylation at position 28 and for the major bisdesmosides, position 3; and (3) the acylation of sugars with acetyl and 2-hydroxy 2-methylglutaryl moieties [pg. 960, col. 1, ¶1]. The enzymes involved in oxidation may include cytochrome P450s and other hydroxylases, and alcohol and aldehyde dehydrogenases [pg. 61, col. 1, ¶1]. The first transfer would be expected to be Glc to the carboxyl at C-28, such as UDP-Glc.
Meesapyodsuk teaches that the EST collection, in combination with heterologous expression and other experiments, provides an effective basis with which to uncover the enzymes that catalyze the oxidation, glycosylation, and acyl transfer steps involved [pg. 965, col. 2, ¶1]. Meesapyodsuk teaches that the oxidation of β-amyrin to various sapogenins is of particular interest and relevance to a number of saponin-producing taxa.
Alignment statistics for match #1
NW Score
Identities
Positives
Gaps
4025
713/760(94%)
747/760(98%)
0/760(0%)
Query 1 MWRLKIAEGANDPYLYSTNNFVGRQTWEFDTDYGTPEAIKEVEEARQDFYKNRFQVKPCG 60
MWRLKIAEG NDPYLYSTNNFVGRQTWEFD++YGTPEAIKEVEEARQ FYKNRFQVKPCG
Sbjct 1 MWRLKIAEGGNDPYLYSTNNFVGRQTWEFDSEYGTPEAIKEVEEARQIFYKNRFQVKPCG 60
Query 61 DLLWRFQFLREKNFKQTIPQVKLGDGEEVTYEAATATVKRAVNYLAAIQAEDGHWPAEIA 120
DLLWRFQFLREKNFKQTIPQVK+GDGEEVTYEAA+ T+KR+VN L A+QA+DGHWPAEIA
Sbjct 61 DLLWRFQFLREKNFKQTIPQVKVGDGEEVTYEAASTTLKRSVNLLTALQADDGHWPAEIA 120
Query 121 GPQFFLPPLVFCLYITGHLNSVFNVHHREEILRSIYYHQNEDGGWGLHIEGHSTMFCTAL 180
GPQFFLPPLVFCLYITGHLN VFNVHHREEILRSIYYHQNEDGGWGLHIEGHSTMFCTAL
Sbjct 121 GPQFFLPPLVFCLYITGHLNVVFNVHHREEILRSIYYHQNEDGGWGLHIEGHSTMFCTAL 180
Query 181 NYICLRMLGVGPDEGDDNACPRARKWILDHGSVTHIPSWGKTWLSILGLFDWSGSNPMPP 240
NYICLRMLGVGPDEGDDNACPRARKWILDHGSVTHIPSWGKTWLSILGLFDWSGSNPMPP
Sbjct 181 NYICLRMLGVGPDEGDDNACPRARKWILDHGSVTHIPSWGKTWLSILGLFDWSGSNPMPP 240
Query 241 EFWILPSFMPMYPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIKQLREELFNEPYEDIKW 300
EFWILP+FMPMYPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIKQLREELF+EP+E+IKW
Sbjct 241 EFWILPTFMPMYPAKMWCYCRMVYMPMSYLYGKRFVGPITPLIKQLREELFSEPFEEIKW 300
Query 301 KKVRHFCAQEDLYYPHPLIQDLMWDSLYLFTEPLLTRWPFNGLIRKKALQVTMDHIHYED 360
KKVRH CA EDLYYPHPLIQDLMWDSLYLFTEPLLTRWPFN LIR+KALQVTMDHIHYED
Sbjct 301 KKVRHLCAPEDLYYPHPLIQDLMWDSLYLFTEPLLTRWPFNNLIRQKALQVTMDHIHYED 360
Query 361 ENSRYLTIGCVEKVLCMLACWVEDPNGVCYKKHLARVPDYVWIAEDGLKMQSFGSQQWDC 420
ENSRY+TIGCVEKVLCMLACWVEDPNGVCYKKHLARVPDY+WIAEDGLKMQSFGSQQWDC
Sbjct 361 ENSRYITIGCVEKVLCMLACWVEDPNGVCYKKHLARVPDYIWIAEDGLKMQSFGSQQWDC 420
Query 421 GFAVQALLASNLSLDEIGPALKKGHYFIKESQVKDNPSGDFKSMHRHISKGSWTFSDQDH 480
GFAVQALLASN+SLDEIGPALKKGH+FIKESQVKDNPSGDFKSMHRHISKGSWTFSDQDH
Sbjct 421 GFAVQALLASNMSLDEIGPALKKGHFFIKESQVKDNPSGDFKSMHRHISKGSWTFSDQDH 480
Query 481 GWQVSDCTAEGLKCCLVLSTMPPEIVGEKMDPERLYDSVNILLSLQSENGGLSAWEPAGA 540
GWQVSDCTAEGLKCCL+LSTMPPEIVGEKMDPERLYDSVN+LLSLQSENGGLSAWEPAGA
Sbjct 481 GWQVSDCTAEGLKCCLILSTMPPEIVGEKMDPERLYDSVNVLLSLQSENGGLSAWEPAGA 540
Query 541 QAWLELLNPTEFFADIVIEHEYVECTGSAIQALVLFKKLYPGHRKKEIENFILKASKYLE 600
QAWLELLNPTEFFADIVIEHEYVECTG++IQALVLFKK+YPGHRKKEIENFI KA+KYLE
Sbjct 541 QAWLELLNPTEFFADIVIEHEYVECTGASIQALVLFKKMYPGHRKKEIENFIAKAAKYLE 600
Query 601 DTQYPNGSWYGNWGVCFTYGTWFALGGLTAAGRTFSNCAAIRKGVEFLLKSQKEDGGWGE 660
DTQYPNGSWYGNWGVCFTYGTWFALGGL AAG+T++NCAA+RKGVEFLLKSQKEDGGWGE
Sbjct 601 DTQYPNGSWYGNWGVCFTYGTWFALGGLAAAGKTYANCAAMRKGVEFLLKSQKEDGGWGE 660
Query 661 SYISCPKKDFVPLEGPSNLTQTAWALMGLIYTRQMERDPTPLHRAAKLLINSQLESGDFP 720
SY+SCPKKDFVPLEGPSNLTQTAWALMGLIY RQMERDPTPLH+AAKLLINSQLE+GDFP
Sbjct 661 SYVSCPKKDFVPLEGPSNLTQTAWALMGLIYARQMERDPTPLHQAAKLLINSQLENGDFP 720
Query 721 QQEITGVFMKNCMLHYPMYRSIYPMWALAEYRKHVPLRLN 760
QQEITGVFMKNCMLHYPMYR+IYP+WA+AEYR HVPLRL+
Sbjct 721 QQEITGVFMKNCMLHYPMYRTIYPLWAIA EYRTHVPLRLS 760
Although Meesapyodsuk teaches the genes involved the production of a triterpenoid, including contacting OS with a β-amyrin synthase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8, such that said OS is converted into β-amyrin, Meesapyodsuk does not explicitly teach that the β-amyrin synthase is a Saponaria officinalis β-amyrin synthase. However, saponins and saponin biosynthesis are documented in Saponaria officinalis. Jia teaches that S. officinalis is well known for its detersive property and has been used for soap in the ancient times [pg. 1368, col. 1, ¶1]. Medicinally, it is still used as an expectorant in bronchitis, skin irritation, and rheumatic disorders. Two main triterpenoid saponins were previously established in the 1970s, saponasids A and D, and Jia teaches the isolation of saponariosides A and B from S. officinalis. Jia teaches that chemical investigations of S. officinalis have been carried out due its medicinal and commercial importance and that triterpenoid saponins with similar oligosaccharide structures have been isolated in other plants, such as the Gypsophyla paniculata, for the commercial Merck saponin extract [pg. 1372, col. 1, ¶3].
Given that Meesapyodsuk teaches the genes involved the production of a triterpenoid, including contacting OS with a β-amyrin synthase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8, such that said OS is converted into β-amyrin; and given that Jia teaches identification of saponins in S. officinalis, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to isolate the genes specific to the production of a saponin in S. officinalis, such as β-amyrin synthase, because of its medicinal and commercial importance as taught by Jia. One would have reasonable expectation of success as the methodology of isolation from S. officinalis and confirmation of identity of the β-amyrin synthase gene by expression in yeast, as well as comparable β-amyrin synthase amino acid sequences of other plant species, such as Glycyrrhiza glabra, Medicago truncatula, and Arabidopsis, was already established by Meesapyodsuk. One would further expect success as the methods as taught by Meesapyodsuk were successful in another plant from the Saponaria genus. One would be motivated to use such a method to characterize saponin biosynthesis in S. officinalis, and particularly β-amyrin synthase, given the medicinal importance of saponins and given that Meesapyodsuk teaches that the saponins of the Caryophyllaceae family, such as S. vaccaria and S. officinalis, are almost completely based on β-amyrin.
Thus, Meesapyodsuk and Jia combined teach the method of instant claim 1 (a method for the production of a triterpenoid comprising contacting OS with a Saponaria officinalis β-amyrin synthase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8, such that OS is converted into β-amyrin) to the extent that instant claim 1 requires the limitations (i)-(xii) in the alternative.
Claim 2, and 4-5 are rejected under 35 U.S.C. 103 as being unpatentable over Meesapyodsuk and Jia as applied to claim 1 above, and further in view of Aharoni, A. et al. International Publication No. WO 2020/049572 A1. “Cellulose-Synthase-Like Enzymes and Uses Thereof.” International Publication Date: 03/12/2020 (see IDS filed 03/07/2025), and Osbourn, A. et al. International Publication No. WO 2020/260475 A1. “Transferase Enzymes.” International Publication Date 12/30/2020 (see IDS filed 03/07/2025).
Examiner notes that the Osbourn reference shares common inventors with the instant application but constitutes prior art given the disclosure with prior public availability date. This reference does not qualify for an exception under 102(b)(1)(A) because it falls outside of the one-year grace period disclosure by inventor.
Claim 2 recites a method according to claim 1 comprising;
(i) contacting p-amyrin with a Saponaria officinalis C28C16 oxidase (SoC28C16 oxidase) to oxidise the C28 position of the p-amyrin to a carboxylic acid and the C16 position to an alcohol to form echinocystic acid, wherein the amino acid sequence of the C28C16 oxidase has at least 50% sequence identity to SEQ ID NO: 4; and
(ii) contacting echinocystic acid with a Saponaria officinalis C-23 oxidase (SoC23 oxidase) to oxidise the C-23 position of echinocystic acid to an aldehyde to form quillaic acid (QA), wherein the amino acid sequence of the SoC23 oxidase having at least 50% sequence identity to SEQ ID NO: 6.
Claim 4 recites a method according to claim 2 or claim 3 further comprising;
(iv) contacting QA with a Saponaria officinalis QA 3-0 glucuronosyl transferase ("SoCSL") to covalent attach D-glucuronic acid ("GIcA") to the 3-O position of quillaic acid to form "QA-GIcA"; wherein the amino acid sequence of the SoCSL having at least 60% sequence identity to SEQ ID NO: 10;
(v) contacting QA-GIcA with Saponaria officinalis QA-GIcA galactosyl transferase ("SoC3Gal") to covalently attach D-Galactose ("Gal") via a 1->2 linkage to QA-GIcA to form “QA-GIcA- Gal"; wherein the amino acid sequence of the QA-GIcA-Gal has at least 50% sequence identity to SEQ ID NO: 12; and
(vi) contacting QA-GIcA-Gal with a Saponaria officinalis QA-GIcA-Gal xylosyl transferase ("SoC3Xyl") to covalently attach D-Xylose ("Xyl") via a 1,3 linkage to QA-GIcA-Gal to form "QA-GIcA-[Gal]-Xyl" QA-Tri; wherein the amino acid sequence of SoC3Xyl has at least 50% sequence identity to SEQ ID NO: 14.
Claim 5 recites a method according to claim 4 further comprising;
(vii) contacting QA-Tri with a Saponaria officinalis QA-Tri fucosyl transferase ("SoC28Fu") to attach fucose to the 28-0 position QA-Tri to form QA-TriF; wherein the amino acid sequence of QATriFuT has at least 60% sequence identity to SEQ ID NO: 16;
(viii) contacting QA-TriF with a Saponaria officinalis QA-TriF rhamnosyl transferase ("SoC28Rha") to covalently attach rhamnose via a 1, 2 linkage to QA-TriF to form QA-TriFR; wherein the amino acid sequence of SoC28Rha has at least 50% sequence identity to SEQ ID NO: 18;
(ix) contacting QA-TriFR with a Saponaria officinalis QA-TriFR xylosyl transferase ("SoC28Xyl1 ") to covalently attach xylose via a 1,4 linkage to QA-TriFR to form QA- TriFRX; wherein the amino acid sequence of SoC28Xy1 has at least 50% sequence identity to SEQ ID NO: 20; and
(x) contacting QA-TriFRX with a Saponaria officinalis QA-TriFRX-xylosyl transferase ("SoC28Xyl2 ") to covalently attach xylose via a 1,3 linkage to QA-TriFRX to form QA-TriFRXX; wherein the amino acid sequence of SoC28Xyl2 has at least 50% sequence identity to SEQ ID NO: 22.
Regarding claim 2, Meesapyodsuk teaches the genes involved the production of a triterpenoid, including contacting OS with a β-amyrin synthase comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8, such that said OS is converted into β-amyrin, and Jia teaches identification of saponins in S. officinalis. Meesapyodsuk and Jia combined render obvious the method for the production of a triterpenoid comprising a S. officinalis β-amyrin synthase with an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 8 as they provide the rationale that S. officinalis and saponins, and thus the biosynthetic pathway, have medicinal value and provide the methodology for isolation. Meesapyodsuk teaches that the next steps in the pathway beyond β-amyrin synthesis presumably include: (1) oxidation of β-amyrin at positions 16, 23, and/or 28; (2) glycosylation at position 28 and for the major bisdesmosides, position 3; and (3) the acylation of sugars with acetyl and 2-hydroxy 2-methylglutaryl moieties [pg. 960, col. 1, ¶1]. Further, Meesapyodsuk teaches that the enzymes involved in oxidation may include cytochrome P450s and other hydroxylases, and alcohol and aldehyde dehydrogenases [pg. 61, col. 1, ¶1] and that the first transfer would be expected to be Glc to the carboxyl at C-28, such as UDP-Glc.
Meesapyodsuk and Jia do not explicitly teach that the amino acid sequence of the C28C16 oxidase has at least 50% sequence identity to SEQ ID NO: 4, or that the amino acid sequence of the SoC23 oxidase having at least 50% sequence identity to SEQ ID NO: 6. However, Aharoni teaches a method of producing a triterpenoid saponin and teaches the key genes and enzymes in the biosynthesis pathway of triterpenoid saponins [Abstract]. Aharoni teaches that there are plentiful reports underlying saponins’ benefits to human health and medical appplications [¶06]. Fig. 20A shows the biosynthetic pathway of spinach saponins comprising co-expressed saponin β-amyrin synthase genes (see Fig. 20A below). Aharoni further teaches identification of key biosynthesis genes in a number of other plant species, including Glycyrrhiza uralensis, Beta vulgaris, Chenopodium quinoa, Medicago sativa, and Glycine max [Table 11] and teaches that the method of producing a triterpenoid saponin includes when the plant cell comprises a cell from a plant in the Caryophyllales order, the plant selected from the Saponaria genus [¶112].
Aharoni teaches the nucleotide and amino acid sequences for triterpenoid biosynthetic pathway in spinach including saponin β-amyrin synthase (SEQ ID NO: 48), cytochrome P450s (SEQ ID NOs: 49 and 52), cytochrome P450 (C-23 oxidase) (SEQ ID NO: 54), glycosyl transferases (UDP-glycosyltransferase; Fucosyl transferase) (SEQ ID NO: 56), glycosyl transferases (UDP-glycosyltransferase) (SEQ ID NO: 58 and 60), glycosyl transferases (UDP-glycosyltransferase; Xylosyl transferase) (SEQ ID NO: 62), acyl transferase (BAHD acyl transferase) (SEQ ID NO: 64), and glucuronic acid transferase (SEQ ID NO: 66) [Table 16]. Aharoni teaches that the biosynthetic intermediate of a triterpenoid saponin comprises oleanolic acid [claim 31]. Fig. 42C shows that β-amyrin is oxidized by cytochromes P450 giving rise to multiple aglycons (bayogenin; serjanic acid; oleanolic acid; medicagenic acid; glycyrrhetinic acid; soyasapogenol A; soyasapogenol B) that are decorated by CSLGs and other glycosyltransferases giving rise to triterpenoid saponins (8 - bayogenin -hexA-hex-hex (M. sativa); 9 - serjanic acid -hexA-hex (Chenopodium quinoa (C. quinoa)); 10 - betavulgaroside IV (B. vulgaris); 11 - yossoside V (S. oleracea); 12 - glycyrrhizin (G. uralensis); 13 - soyasapogenol A-hexA-hex-pent (L. japonicus);14 - soyasaponin VI (G. max)) [¶71].
SEQ ID NO: 2 of the instant application shares 84.4% identity with SEQ ID NO: 48. Examiner notes that SEQ ID NO: 48 was published to NCBI in 2017 as a beta-amyrin 28-oxidase-like (NCBI Reference Sequence: XP_021834918.1), and is thus taken to read on the C28 oxidase (i.e., wherein the amino acid sequence of the C28 oxidase has at least 80% identity to SEQ ID NO: 2).
Query Match 84.8%; Score 2202; Length 483;
Best Local Similarity 82.5%;
Matches 400; Conservative 39; Mismatches 44; Indels 2; Gaps 1;
Qy 1 MELFFICGLVLFSTLSLISLFLLHNHSSARGYRLPPGRMGWPFIGESYEFLANGWKGYPE 60
|||||:|||||| :||| | || :|| |||:||||:|||| :|||:|| |||||||
Db 1 MELFFMCGLVLFLSLSLASFFLFYNHHRTRGYKLPPGKMGWPVVGESFEFFQTGWKGYPE 60
Qy 61 KFIFSRLAKYKPNQVFKTSILGEKVAVMCGATCNKFLFSNEGKLVNAWWPNSVNKIFPSS 120
|||| || || |:|||||||:||||||:||| ||||:||| ||| ||||:||:||||||
Db 61 KFIFDRLNKYTPSQVFKTSIVGEKVAVLCGAAGNKFLYSNENKLVQAWWPSSVDKIFPSS 120
Qy 121 TQTSSKEEAKKMRKLLPTFFKPEALQRYIPIMDEIAIRHMEDEWEGKSKIEVFPLAKRYT 180
||||||||||||||||| | ||||| ||||||| ||||||| |||| |:||||||| ||
Db 121 TQTSSKEEAKKMRKLLPNFLKPEALHRYIPIMDSIAIRHMESGWEGKDKVEVFPLAKNYT 180
Qy 181 FWLACRLFLSIDDPVHVAKFADPFNDIASGIISIPIDLPGTPFNRGIKASNVVRQELKTI 240
||||||||||::|| |||||::||||||:||||:||||||||||||||:|||||:||: |
Db 181 FWLACRLFLSVEDPAHVAKFSEPFNDIAAGIISMPIDLPGTPFNRGIKSSNVVRKELRAI 240
Qy 241 IKQRKLDLSDNKASPTQDILSHMLLTPDEDGRYMNELDIADKILGLLIGGHDTASAACTF 300
||||||||:| ||||||||||||||| |||::|:|:|||||||||||||||||||:|||
Db 241 IKQRKLDLADGKASPTQDILSHMLLTCTEDGKFMSEMDIADKILGLLIGGHDTASASCTF 300
Qy 301 VVKFLAELPHIYDGVYKEQMEIAKSKKEGERLNWEDIQKMKYSWNVACEVMRLAPPLQGA 360
||||||||||||:|||||||||| ||| || ||||||||||||||||||||||||||||
Db 301 VVKFLAELPHIYEGVYKEQMEIANSKKAGELLNWEDIQKMKYSWNVACEVMRLAPPLQGG 360
Qy 361 FREALSDFMYAGFQIPKGWKLYWSANSTHRNPECFPEPEKFDPARFDGSGPAPYTYVPFG 420
|||||||||| |||||||||||||||||| ||||||||: |||:||||:|||||||||||
Db 361 FREALSDFMYNGFQIPKGWKLYWSANSTHMNPECFPEPKTFDPSRFDGTGPAPYTYVPFG 420
Qy 421 GGPRMCPGKEYARLEILVFMHNIVKRFKWEKLIPDETIVVNPMPTPAKGLPVRLRPHSKP 480
||||||||||||||||||||||:||||||||::||| ::||||| | |||||| || |
Db 421 GGPRMCPGKEYARLEILVFMHNVVKRFKWEKMLPDEKVIVNPMPIPEHGLPVRLFPH--P 478
Qy 481 VTVSA 485
||:|
Db 479 RTVAA 483
SEQ ID NO: 6 of the instant application is a 63.1% match with SEQ ID NO: 54, both annotated as C23 oxidases (i.e., wherein the amino acid sequence of the C-23 oxidase ha(see alignment below).
Query Match 63.1%; Score 1724; Length 531;
Best Local Similarity 61.2%;
Matches 319; Conservative 94; Mismatches 96; Indels 12; Gaps 6;
Qy 10 SIACIVILRWALNMMQWLWFEPRRLEKLLRKQGLQGNSYKFLFGDMKESSMLRNEALAKP 69
|: ||:|| | ::: || |::||| |::||| ||||||| ||||||| |||||| |
Db 13 SLGCIIILYWMWKLLKGLWLTPKKLEKCLKQQGLVGNSYKFLIGDMKESSKLRNEALQK- 71
Qy 70 MPMPFDNDYFPRINPFVDQLLNK--YGMNCFLWMGPVPAIQIGEPELVREAFNRMHEFQK 127
|:|| :||: || ||: |:|| | | : |:|||| | | :|||:::|||||: |||
Db 72 -PIPFTHDYYNRIQPFIHQILNNSGAGKNIYTWLGPVPTILITQPELIKDAFNRMNNFQK 130
Qy 128 PKTNPLSALLATGLVSYEGDKWAKHRRLINPSFHVEKLKLMIPAFRESIVEVVNQWEKKV 187
|: || : :|:||| :||| ||||||:|:||:| ::|||||| | : : :|:||| |
Db 131 PRLNPYTQMLSTGLPNYEGQKWAKHRKLLNPAFQLDKLKLMIHTFETCVTDTLNKWEKLV 190
Qy 188 PENGSAEIDVWPSLTSLTGDVISRAAFGSVYGDGRRIFELLAVQKELVLSLLKFSYIPGY 247
: ||:|:|:|| ||:|||| |:|||||| : ||||||||| :||::|:||||:|||||:
Db 191 CKTGSSEVDIWPYLTTLTGDGIARAAFGSSFEDGRRIFELLTLQKDIVISLLKYSYIPGF 250
Qy 248 TYLPTEGNKKMKAVNNEIQRLLENVIQNRKKAMEAGEAAKDDLLGLLMDSNYKESML--- 304
|:| :||:||| :|||: || |:| |:|||||||| ||||||:|::|| |:
Db 251 KYMPIKGNRKMKEADNEIKPLLTNIINRRRKAMEAGEAPKDDLLGMLLESNANEARQVNE 310
Qy 305 -EGGGKNKK--LIMSFQDLIDECKLFFLAGHETTAVLLVWTLILLCKHQDWQTKAREEVL 361
| | :| | ||| ::|| || ||||| |||:| | ||::|| |||||||:||:|||
Db 311 NESGSSKRKSDLTMSFPEMIDACKQFFLAGQETTSVALTWTMLLLAKHQDWQTRARQEVL 370
Qy 362 ATFGMSEPTDYDAL-NRLKIVTMILNEVLRLYPPVVSTNRKLFKGETKLGNLVIPPGVGI 420
|||||: | |:| : |||||||||| ||||||||| :|:|:: |||||:|||| |||:
Db 371 ATFGMNTP-DFDGIHNRLKIVTMILYEVLRLYPPVPATSRRVHDRETKLGDLVIPQGVGV 429
Qy 421 SLLTIQANRDPKVWGEDASEFRPDRFAEGLVKATKGNVAFFPFGWGPRICIGQNFALTES 480
| : |: :|::||:|| ||:|||||||: |||||| ::||||||||||||||||| |:
Db 430 SFSILHAHLNPEIWGDDAKEFKPDRFAEGIAKATKGNNSYFPFGWGPRICIGQNFALVEA 489
Qy 481 KMAVAMILQRFTFDLSPSYTHAPSGLITLNPQYGAPLMFRR 521
|||: ||||||:||||||| |||: ||:| ||:|| :: |
Db 490 KMALCMILQRFSFDLSPSYIHAPTSLISLQPQHGAHIILHR 530
Though Aharoni teaches cytochrome P450s involved in oleanolic acid and C-23 oxidase involved in triterpenoid biosynthesis, Aharoni does not explicitly teach a C28C16 oxidase to oxidize the C16 position of the oleanic acid to an alcohol to form echinocystic acid to eventually form quillaic acid. However, the biosynthetic process and enzymatic steps involved in the process are known in the art. For example, Osbourn teaches genes and polypeptides for glycosylating quillaic acid in host cells, including the triterpenoid biosynthesis pathway (i.e., a method for the production of a triterpenoid).
The triterpenoid QS-21 consists of a C-30 triterpenoid backbone known as quillaic acid [pg. 1, ln. 28]. Biosynthesis of quillaic acid proceeds from β-Amyrin which is synthesised through cyclisation of the universal linear precursor 2,3-oxidosqualene (OS) by oxidosqualene cyclases (OSCs) (β-amyrin synthase) (i.e., contacting OS with a β-amyrin synthase) [Fig. 2; pg. 1, lns. 34-46]. The β-amyrin scaffold is further oxidized with a carboxylic acid, alcohol and aldehyde at the C-28, C-16a and C-23 positions, respectively, to form oleanolic acid, echinocystic acid, and lastly quillaic acid (QA) (i.e., contacting β-amyrin with a C28 oxidase to oxidize the C28 position of the β-amyrin to a carboxylic acid to form oleanolic acid; contacting oleanolic acid with an oxidase to oxidize the C16 position of the oleanolic acid to an alcohol to form echinocystic acid; contacting echinocystic acid with a C23 oxidase to oxidize the C-23 position of echinocystic acid to an aldehyde to form quillaic acid) (see Fig. 2 below).
PNG
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496
1137
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Greyscale
Osbourn teaches that other patent applications, such as WO 2019/122259 describe identification of enzymes participating in QA production. Candidate enzymes were cloned from leaf cDNA. Enzymes were tested by transient co-expression in Nicotiana benthamiana, allowing for the identification of one OSC and three cytochrome P450s (P450s) required for synthesis of β-amyrin and oxidation to quillaic acid.
Osbourn teaches the requirement of an enzyme capable of oxidizing β-amyrin or an oxidized derivative thereof, such as oleanolic acid, at the C-16 position to an alcohol and further teaches SEQ ID NO: 16, a QsCYP716-C-16α capable of oxidizing the C16 position of the oleanolic acid with 50.41% identity to SEQ ID NO: 4 (i.e., wherein the amino acid sequence of the C16 oxidase has at least 50% sequence identity to SEQ ID NO: 4) (see alignment below) [pg. 7, lns. 29-30].
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
512 bits(1319)
0.0
Compositional matrix adjust.
245/486(50%)
335/486(68%)
19/486(3%)
Query 29 GLLLLALF-LSVSFLLYLSRRAYASLPNPPPGKLGFPVVGESLEFLSTRRKGVPEKFVFD 87
LL+LA+ LS F+LY + PPG++G+P +GES +F + KG PE F+FD
Sbjct 9 ALLVLAIVSLSTFFVLYYNTPTKDG-KTLPPGRMGWPFIGESYDFFAAGWKGKPESFIFD 67
Query 88 RMAKYCRD----VFKTSILGATTAVMCGTAGNKFLFSNEKKHVTGWWPKSVELIFPTSLE 143
R+ K+ + F+TS+ G + V+ G A NK LFSNEKK VT WWP S++ FP++ +
Sbjct 68 RLKKFAKGNLNGQFRTSLFGNKSIVVAGAAANKLLFSNEKKLVTMWWPPSIDKAFPSTAQ 127
Query 144 KSSNEESIMMKQFLPNFL-KPEPLQKYIPVMDIITQRHFNT-SWEGRNVVKVFPTAAEFT 201
S+NEE+++M++F P+FL + E LQ+YIP+MD T+RHF T +W + ++ F ++T
Sbjct 128 LSANEEALLMRKFFPSFLIRREALQRYIPIMDDCTRRHFATGAWGPSDKIEAFNVTQDYT 187
Query 202 TLLACRVFLSV---EDPIEVAKISEPFEILAAGFLSIPINLPGTKLNKAVKAADQIRDAI 258
+ACRVF+S+ EDP V + F +L AG S+ I+LP T + A+KA+ IR A+
Sbjct 188 FWVACRVFMSIDAQEDPETVDSLFRHFNVLKAGIYSMHIDLPWTNFHHAMKASHAIRSAV 247
Query 259 VQILKRRRVEIAENKANGMQDIASMLLTTPTNAG--------FYMTEAHISEKILGMIVG 310
QI K+RR E+AE KA QD+ S +L TP + Y+ +A I KILG++VG
Sbjct 248 EQIAKKRRAELAEGKAFPTQDMLSYMLETPITSAEDSKDGKAKYLNDADIGTKILGLLVG 307
Query 311 GRDTASTVITFIIKYLAENPEIYNKVYEEQMEVVKSKKPGELLNWEDVQKMKYSWCVACE 370
G DT+STVI F K++AENP +Y +Y+EQMEV +K PGELLNW+D+QKMKYSWC CE
Sbjct 308 GHDTSSTVIAFFFKFMAENPHVYEAIYKEQMEVAATKAPGELLNWDDLQKMKYSWCAICE 367
Query 371 AMRLAPPVQGGFKVAINDFVYSGFNIRKGWKLYWSAIA THMNPEYFPEPEKFNPSRFEGK 430
MRL PPVQG F+ AI DF ++G+ I KGWK+YWS +TH NPE FP+PEKF+P+RFEG
Sbjct 368 VMRLTPPVQGAFRQAITDFTHNGYLIPKGWKIYWSTHSTHRNPEIFPQPEKFDPTRFEGN 427
Query 431 GPVPYSFVPFGGGPRMCPGKEYSRLETLVFMHHLVTRYNWEKVYPTEKITVDPMPFPVNG 490
GP +SFVPFGGGPRMCPGKEY+RL+ L F+HH+VT++ WE++ P EKI V PMP+P
Sbjct 428 GPPAFSFVPFGGGPRMCPGKEYARLQVLTFVHHIVTKFKWEQILPNEKIIVSPMPYPEKN 487
Query 491 LPIRLI 496
LP+R+I
Sbjct 488 LPLRMI 493
Meesapyodsuk, Aharoni and Osbourn teach characterization, identification, and confirmation of key genes in saponin triterpenoid biosynthesis pathways. In particular, Aharoni, Osbourn, and Meesapyodsuk teach β-amyrin synthase for β-amyrin synthesis from OS and Aharoni and Osbourn further teach oxidation at positions C-28, C-16, and C-23 to form quillaic acid. As detailed above, the references teach key genes in saponin triterpenoid biosynthesis, but within other plant species. The instant claims are generally directed to the identification of enzymes forming the biosynthetic pathway producing saponarioside in S. officinalis. Existence of this major saponin in Saponaria was generally known in the art, as taught by Jia. The steps of the pathway are similar to other saponin triterpenoids and taught in both Aharoni and Osbourn. Meesapydsuk additionally teaches that soapwort contains saponins similar to those found in Quillaja Saponaria [Abstract]. Further, the methods for identifying the genes involved in the pathway were known at the time of filing. As taught in Aharoni and Osbourn, the methods rely on next-gen sequencing, transcriptome analysis and exploitation of knowledge from similar pathways, such as Saponaria vaccaria and Quillaja saponaria. Insofar as the claim limitations rely on known principles of gene identification in the art, identification of the genes involved in the saponarioside biosynthetic pathway would have been obvious in view of Aharoni, Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis. Thus, the teachings of Meesapyodsuk, Aharoni, Osbourn, and Jia render obvious claim 2.
Regarding claim 4, Osbourn teaches that, as depicted in Figure 3, the branched trisaccharide chain at the C-3 position in QS-21 is initiated with a β-0-glucuronic acid (GlcpA) residue attached at the 3-0 position of QA [pg. 2, lns. 29-32]. The GlcpA residue is then linked to a o-Galactose (Galp) via a 13-1->2 linkage and to a o-Xylose (Xylp) via a 13-1,3 linkage. Osbourn teaches a QA 3-0 glucuronosyl transferase ("QA-GlcA T") capable of transferring o-glucuronic acid ("GlcpA") at the 3-0 position of quillaic acid to form 3[3-{[13-D-glucopyranosiduronic acid]oxy}-quillaic acid ("QA-GlcpA") (i.e., contacting QA with a QA 3-O glucuronosyl transferase to covalently attach D-glucuronic acid to the 3-O position of quillaic acid to form QA-GlcA); QA-GlcpA galactosyl transferase ("QA-GalT") capable of transferring o-Galactose ("Galp") via a 13-1->2 linkage to QA-GlcpA to form 3β-{[β-D-galactopyranosyl-(1->2)-β-Doglucopyranosiduronic acid]oxy}-quillaic acid ("QA-GlcpA-Galp'') (i.e., contacting QA-GlcA with QA-GlcA galactosyl transferase to covalently attach Gal via a β-1->2 linkage to QA-GlcA to form QA-GlcA-Gal); and ("Xylp'') via a 1->3 linkage to QA-GlcpA-Galp to form 3β-{[β - D-xylopyranosyl-( 1->3)-[[3-o-galactopyranosyl-( 1->2) H3-oglucopyranosiduronic acid]oxy}-quillaic acid ("QA-GlcpA-[Galp]-Xylp") (i.e., contacting QA-GlcA-Gal with a QA-GlcA-Gal xylosyl transferase to covalently attach d-Xylose (“Xyl”) via a 1 ,3 linkage to QA-GlcA-Gal to form QA-GlcA-[Gal]-Xyl or QA-Tri) [pg. 4, lns. 5-18].
Osbourn teaches characterization of two glucuronosyl transferases, a galactosyl transferase, and Rhamnosyl, Xylosyl, and dual Rhamnosyl/Xylosyl transferases which permit glycosylation of the 3-0 position of QA with the respective saccharide within the 3-0 branched trisaccharide from Quillaja Saponaria [pg. 2, lns. 40-43], and additionally teaches that the polypeptides or nucleotide sequences may be derived from other plant species [pg. 4, lns. 28-29]
Similarly, Aharoni teaches several glucuronosyltransferases, galactosyltransferases, and xylosyltransferases, and characterization thereof [¶1004; 1018; 812; Table 5]. Aharoni teaches Beta vulgaris CSL with SEQ ID NO: 94. SEQ ID NO: 94 has 74.4% identity to instant SEQ ID NO: 10 (i.e., wherein the amino acid sequence of the CSL has at least 60% sequence identity to SEQ ID NO: 10) (see alignment below). Examiner notes that Aharoni teaches CSLs from other species with similar sequence identity to that of SEQ ID NO: 10, including Chenopodium quinoa (CqCSL; SEQ ID NO: 96) and spinach CSLG (SEQ ID NO: 66).
Query Match 74.4%; Score 2734; Length 704;
Best Local Similarity 70.8%;
Matches 498; Conservative 97; Mismatches 102; Indels 6; Gaps 2;
Qy 1 MSPHNTCTLQITRALLSRLHILFHSALVASVFYYRFSNFSSGPA----WALMTFAELTLA 56
|| : | :| |||:||| |||||| : ::|||||::||: : | |:| ||: |
Db 1 MSSLHICKVQTTRAILSRFHILFHSLAILALFYYRFTSFSTTKSGILPWTLLTTAEVVLG 60
Qy 57 FIWALTQAFRWRPVVRAVFGPEEIDPAQLPGLDVFICTADPRKEPVMEVMNSVVSALALD 116
|:||||||||||||:| | | : | ||||:||||||||| ||||:||||:|:||:|||
Db 61 FVWALTQAFRWRPVLRDVAGWDSIKEEQLPGVDVFICTADPIKEPVLEVMNTVLSAMALD 120
Qy 117 YPAEKLAVYLSDDGGSPLTREVIREAAVFGKYWVGFCGKYNVKTRCPEAYFSSFCDGERV 176
|||||| |||||||||||||| |:||: | | |: || || :|||||:|:|||||||||:
Db 121 YPAEKLGVYLSDDGGSPLTREAIKEASKFAKVWLPFCSKYGIKTRCPQAFFSSFCDGERL 180
Qy 177 DHNQDYLNDELSVKSKFEAFKKYVQKASEDATKCIVVNDRPSCVEIIHDSKQNGEGEVKM 236
| |||: ||| :|||:|||| ||:||||| :|| : :|| |||||||:||||||||||
Db 181 DWNQDFKADELVLKSKYEAFKNYVEKASEDESKCTMAHDRSPCVEIIHDNKQNGEGEVKM 240
Qy 237 PLLVYVAREKRPGFNHHAKAGAINTLLRVSGLLSNSPFFLVLDCDMYCNDPTSARQAMCF 296
||||||:||||| | ||||:| ||||||:||| |: |||||||||||||||||:|||
Db 241 PLLVYVSREKRPNRPHRFKAGALNALLRVSGVLSNGPYLLVLDCDMYCNDPTSARQSMCF 300
Qy 297 HLDPKLAPSLAFVQYPQIFYNTSKNDIYDGQARAAFKTKYQGMDGLRGPVMSGTGYFLKR 356
|||||||||||||||||||||||||||||||||:|:|||:|||||:||||::||||:|||
Db 301 HLDPKLAPSLAFVQYPQIFYNTSKNDIYDGQARSAYKTKWQGMDGIRGPVLTGTGYYLKR 360
Qy 357 KALYGKPHDQDELLREQPTKAFGSSKIFIASLGENT--CVALKGLSKDELLQETQKLAAC 414
|||||:||::|| | || |||||| ||||: |: :||| :::|:||:| : || |
Db 361 KALYGQPHNEDEFLINQPEKAFGSSTKFIASVSSNSKQNMALKEMTRDDLLEEAKNLATC 420
Qy 415 TYESNTLWGSEVGYSYDCLLESTYCGYLLHCKGWISVYLYPKKPCFLGCATVDMNDAMLQ 474
||||| ||:::||||:||||||: |||||||||||||||||:|||||| |:|| |||:|
Db 421 AYESNTEWGNKIGYSYECLLESTFTGYLLHCKGWISVYLYPKRPCFLGCTTIDMKDAMVQ 480
Qy 475 IMKWTSGLIGVGISKFSPFTYAMSRISIMQSLCYAYFAFSGLFAVFFLIYGVVLPYSLLQ 534
:|||||||:||||||||| ||| ||:||:||:|| || || || | |||||:||| ||:
Db 481 LMKWTSGLLGVGISKFSPLTYAFSRMSILQSMCYGYFTFSALFGVSFLIYGIVLPVCLLK 540
Qy 535 GVPLFPKAGDPWLLAFAGVFISSLLQHLYEVLSSGETVKAWWNEQRIWIIKSITACLFGL 594
|||:||| |||: | || ||||||||||||| :::| |||| |||||||:|| |||
Db 541 GVPVFPKVSDPWIGVFVVVFASSLLQHLYEVLSSDDSIKTWWNEIRIWIIKSVTASLFGT 600
Qy 595 LDAMLNKIGVLKASFRLTNKAVDKQKLDKYEKGRFDFQGAQMFMVPLMILVVFNLVSFFG 654
:||:: |||: ||||||||| |||:||:|||||:|||||| :|||||:|||| |:||| |
Db 601 MDAIMKKIGIQKASFRLTNKVVDKEKLEKYEKGKFDFQGAAVFMVPLIILVVLNMVSFVG 660
Qy 655 GLRRTVIHKNYEDMFAQLFLSLFILALSYPIMEEIVRKARKGR 697
|||| :|:|| ::|| ||||| |:| ||||::| || | ||||
Db 661 GLRRAIINKNCDEMFGQLFLSFFLLVLSYPVLEGIVTKVRKGR 703
Although the references do not explicitly teach that the CSL, C3Gal, and C3Xyl are from Saponaria officinalis, or have the claimed sequence identity as claimed in the instant application, as detailed above, the references explicitly teach glucuronosyl transferase, galactosyl transferase, and xylosyl transferase within other plant species. The instant claims are generally directed to the identification of enzymes forming the biosynthetic pathway producing saponarioside in S. officinalis, particularly transferases that covalently attach sugars to quillaic acid. Existence of this major saponin in Saponaria was generally known in the art, as taught by Jia. The steps of the covalent attachments are similar to other saponin triterpenoids and taught in both Aharoni and Osbourn. Meesapyodsuk teaches that sugars are transferred by the characterized enzymes, forming bonds to the saponin carbon skeleton such as an ester linkage (i.e., covalently attached). Further, the methods for identifying the genes involved in the pathway were known at the time of filing, as detailed above. The lack of sequences with high similarity or from S. officinalis does not render the claims novel because identification of the genes involved in the saponarioside biosynthetic pathway would have been obvious in view of Aharoni, Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis. Thus, the teachings of Meesapyodsuk, Aharoni, Osbourn, and Jia combined make obvious claim 4.
Regarding claim 5, Aharoni teaches that the most abundant spinach saponin, Yossoside V, has acetyl-fucose, rhamnose and glucose linked to the carboxyl at C-28, as revealed by mass spectrometry [¶948]. Aharoni teaches that a spinach fucosyltransferase (SEQ ID NO: 56) works directly on MA-GlcA to link fucose at position C-28 [¶976]. Next, a glycosyltransferase (SEQ ID NO: 58) transfers a rhamnose onto the fucose [¶977].
SEQ ID NO: 56 of Aharoni has 62% identity to SEQ ID NO: 16 of the instant application across two ranges (see alignment below) (i.e., wherein the amino acid sequence of QATriFuT has at least 60% identity to SEQ ID NO: 16).
Range 1: 347 to 467
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
163 bits(413)
1e-50
Compositional matrix adjust.
75/121(62%)
92/121(76%)
0/121(0%)
Query 242 QLDVLAHEAVGCFITHCGWNSIIEATNFGVPMLGMPQFMDQFLDAHFMEKVWGVGIRAKA 301
QLDVL HE++ CF+THCGWNSI EA +FGVPML +PQF+DQ +DAHF+E+VWG GI K
Sbjct 347 QLDVLVHESISCFVTHCGWNSITEALSFGVPMLSVPQFLDQPVDAHFVEQVWGAGITVKR 406
Query 302 DEKNFVTCDEIKCGVNEIMYGDKANMIKENAAKWKDLAKEAVGEGGSSDKNIDEIINWLA 361
E VT DEI + + G+KA IK N A+WK LAKEA+ EGGSSDK+IDEII W++
Sbjct 407 SEDGLVTRDEIVRCLEVLNNGEKAEEIKANVARWKVLAKEALDEGGSSDKHIDEIIEWVS 466
Query 362 S 362
S
Sbjct 467 S 467
Range 2: 7 to 230
Alignment statistics for match #2
Score
Expect
Method
Identities
Positives
Gaps
158 bits(399)
2e-48
Compositional matrix adjust.
88/242(36%)
132/242(54%)
19/242(7%)
Query 6 RTMEVIMMPFHHQGHLTPMLQFAKRFAWKGAGSIRITLATTLSTAQNMTNSKNNNNNNDY 65
+ +E+I+ P+H QGH+ MLQFAKR AWK A ++T+ATTLST M + N
Sbjct 7 KKVEIIVFPYHGQGHMNTMLQFAKRIAWKNA---KVTIATTLSTTNKMKSKVENAWGTS- 62
Query 66 DFLTVESIYDDTDDSQLKFMGRMGKFKSEASLQLGRLITTKSID-NNKCMLVYDAYLPWA 124
+T++SIYDD+D+SQ+KFM RM +F++ A+ L +L+ K + +NK +LVYD LPWA
Sbjct 63 --ITLDSIYDDSDESQIKFMDRMARFEAAAASSLSKLLVQKKEEADNKVLLVYDGNLPWA 120
Query 125 LDVGKDHNIQAAAFFVQACAYMASFYPMFLEEFGSDDQHPVVAAAKAESVPSLSVELPSR 184
LD+ +H ++ AAFF Q+CA +A++Y ++ E G + + + A P
Sbjct 121 LDIAHEHGVRGAAFFPQSCATVATYYSLYQETQGKELETELPAV------------FPPL 168
Query 185 EEMERYAPKCAQSPSSDDKPNTVKKSLHPVYRMVVSSITTLHLADFVLINSFDHLEHQLD 244
E ++R P + K P V+ L AD +L N FD L +
Sbjct 169 ELIQRNVPNVFGLKFPEAVVAKNGKEYSPFVLFVLRQCINLEKADLLLFNQFDKLVEPGE 228
Query 245 VL 246
VL
Sbjct 229 VL 230
SEQ ID NO: 58 of Aharoni has 50% identity to SEQ ID NO: 18 of the instant application (i.e., wherein the amino acid sequence of C28Rha has at least 50% sequence identity to SEQ ID NO: 18).
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
449 bits(1155)
2e-160
Compositional matrix adjust.
227/454(50%)
299/454(65%)
15/454(3%)
Query 7 ASEQLHIVMIPWFAYGHILPYFELSNKLAEKGHKITLVVPNKVKLDLEPKIRHPSLISLH 66
+++ LH+VM PWFAYGH++P+ LSNKLAE GHK+T ++P K L+ +P+ I+
Sbjct 2 SAKMLHVVMYPWFAYGHMIPFLHLSNKLAETGHKVTYILPPKALTRLQNLNLNPTQITFR 61
Query 67 AFTVPHIEPLPPGTETCSDVP-IELQHHLAVAMDRARPEVESIISAIDDPKPDLLFYDNA 125
TVP ++ LP G E +D+P I L HLA A+DR RPE E+I+ I KPD++ YD A
Sbjct 62 TITVPRVDGLPAGAENVTDIPDITLHTHLATALDRTRPEFETIVELI---KPDVIMYDVA 118
Query 126 YWVPEIATKLGMKSVFYQIACALSITRIK-----QTPSASASASASAKLFTLPKWVLTPK 180
YWVPE+A K G KSV Y + A S++ K TP K K+ P
Sbjct 119 YWVPEVAVKYGAKSVAYSVVSAASVSLSKTVVDRMTPLEKPMTEEERK----KKFAQYPH 174
Query 181 VLGDARANYGEGITYYQRVKKALSSCDAIA LRTCREIEGESSDILAAQYNKPVFLTGPVL 240
++ +GEGIT Y R+ LS CDAIA RTCREIEG+ L+ QY K V LTGPVL
Sbjct 175 LI-QLYGPFGEGITMYDRLTGMLSKCDAIA CRTCREIEGKYCQYLSTQYEKKVTLTGPVL 233
Query 241 PEVEFLPPLDNSWAEWLAKFGPKSVVLCCFGSQYVPDKAQLQEMALALEDTGLPFLMSVK 300
PE E L+ W+EWL++F SV+ C FGSQ+ DK Q QE+ L LE T LPFLM+V+
Sbjct 234 PEPEVGATLEAPWSEWLSRFKLGSVLFCAFGSQFYLDKDQFQEIILGLEMTNLPFLMAVQ 293
Query 301 PPTECATIEEALPEGFSERVKERGVVHGGWVQQLQILAHPSVGCFICHCGYGSMWEGLLS 360
PP CATIEEA PEGF+ERVK+RGVV WVQQL ILAHP+VGCF+ HC +G+MWE LLS
Sbjct 294 PPKGCATIEEAYPEGFAERVKDRGVVTSQWVQQLVILAHPAVGCFVNHCAFGTMWEALLS 353
Query 361 DNQLVLLPQLPDQLMMAQMLAEKLKVGVMVDREEDDGWVSRKNLCQAVKSVMDPHSEFAA 420
+ QLV++PQL DQ++ +MLA++LKVGV V+R GWVS++NLC+A+KSVMD SE
Sbjct 354 EKQLVMIPQLGDQILNTKMLADELKVGVEVER-GIGGWVSKENLCKAIKSVMDEDSEIGK 412
Query 421 LLKNNHANFRDKLLTNGFMANYLEVFDQDLKRFL 454
+K +H +R L + M+ Y++ F +DL+ +
Sbjct 413 DVKQSHEKWRATLSSKDLMSTYIDSFIKDLQALV 446
Aharoni teaches that Yossoside II is further converted into Yossoside III by SEQ ID NO: 60, which accepts as a substrate only an aglycone having a fucose and rhamnose attached to the C-28 position with 63.76% identity to instant SEQ ID NO: 20 (see alignment below) and another xylosyltransferase [¶977].
Alignment of Aharoni SEQ ID NO: 60 with instant SEQ ID NO: 20:
Alignment statistics for match #1
Score
Expect
Method
Identities
Positives
Gaps
580 bits(1494)
0.0
Compositional matrix adjust.
285/447(64%)
351/447(78%)
15/447(3%)
Query 3 GEKELRIVMFPWLAFGHFIPYLHLSNKLAEKGHKITLLLPNKARLQLESLNLHPSLITFH 62
G KEL IVM+PWLAFGHFIPYLHLSNKLA+KGHKIT LLP++A+LQL+S NL+PSLIT
Sbjct 2 GTKELHIVMYPWLAFGHFIPYLHLSNKLAQKGHKITFLLPHRAKLQLDSQNLYPSLITLV 61
Query 63 SITVPPLETLPYGTETTADISLDQHGELSISMDRTRPEVESFLST--HKPDLVLYDMAHW 120
ITVP ++TLP G E+TADI L QHG+LSI+MDRTRPE+ES LS KPDL+ +DMA W
Sbjct 62 PITVPQVDTLPLGAESTADIPLSQHGDLSIAMDRTRPEIESILSKLDPKPDLIFFDMAQW 121
Query 121 VPEIAAKVGIKSVSYNVVCAIA VSHVRPSLPLPKGTAAHVPLPLSSVPKWSLNQHGSSTP 180
VP IA+K+GIKSVSYN+VCAI++ VR G S+VP W+L SS
Sbjct 122 VPVIASKLGIKSVSYNIVCAISLDLVRDWYKKDDG---------SNVPSWTLKHDKSS-- 170
Query 181 YFGEGITLLERSVISLSSADAIA IRTCREIEGVYCDRVAATFNKPVLVTSHALPDLELEL 240
+FGE I++LER++I+L + DAI IR+CREIEG YCD +A F KPVL++ LP E
Sbjct 171 HFGENISILERALIALGTPDAIGIRSCREIEGEYCDSIAERFKKPVLLSGTTLP--EPSD 228
Query 241 SPLETRWAEWLARFEPGSVIFCCLGSQHVLDAPQLQELALGLEMTGLPFLMAVKPPVGCT 300
PL+ +W +WL +FE GSVIFCCLGSQHVLD PQLQELALGLEMTGLPF +A+KPP+G
Sbjct 229 DPLDPKWVKWLGKFEEGSVIFCCLGSQHVLDKPQLQELALGLEMTGLPFFLAIKPPLGYA 288
Query 301 SLEEVLPEGFNDRVSGRGVVHGGWVQQQQIMAHPSLGCFVTLCGSSSMWEGLVSESQLVL 360
+L+EVLPEGF++RV RGV HGGWVQQ Q++AHPS+GCF+ CGSSSMWE LVS++QLVL
Sbjct 289 TLDEVLPEGFSERVRDRGVAHGGWVQQPQMLAHPSVGCFLCHCGSSSMWEALVSDTQLVL 348
Query 361 LPQLADQTLYAKLMADELKVGVKVEREENGWMTKRSLCEAIKSVMDEDSDISHVVRKNHA 420
PQ+ DQ L A LMAD+LKVGVKVERE++G ++K AIKSVMD++S+I+ V+KNH
Sbjct 349 FPQIPDQALNAVLMADKLKVGVKVEREDDGGVSKEVWSRAIKSVMDKESEIAAEVKKNHT 408
Query 421 KYRSMLISPGFISGYIDNFIKDLQALV 447
K+R MLI+ F++GYID+FIKDLQ LV
Sbjct 409 KWRDMLINEEFVNGYIDSFIKDLQDLV 435
The references explicitly teach triterpenoid biosynthesis including frucosyl transferase, rhamnosyl transferase, and xylosyl transferase within other plant species and with different sequence identity. The instant claims are generally directed to the identification of enzymes forming the biosynthetic pathway producing saponarioside in S. officinalis, particularly frucosyl transferase, rhamnosyl transferase, and xylosyl transferases resulting in the further enzymatic development of quillaic acid. Existence of this major saponin in Saponaria was generally known in the art, as taught by Jia, and the soapwort saponins found to be similar to that of Q. saponaria. The steps of the covalent attachments are similar to other saponin triterpenoids and taught in both Aharoni and Osbourn and the covalent attachment suggested by Meesapyodsuk, as detailed above. The references teach that the saponin structure can vary based on the plant species and saponin pathway at hand [Meesapydosk, pg. 965, col. 2, ¶2], thus identification of triterpenoid biosynthesis enzymes for saponarioside B would result in the specific linkage as claimed. Further, the methods for identifying the genes involved in the pathway were known at the time of filing, as detailed above. The lack of sequences with high similarity or from S. officinalis does not render the claims novel because identification of the genes involved in the saponarioside B biosynthetic pathway would have been obvious in view of Aharoni, Osbourn, which disclose the general principle for identifying the genes, as well as the rationale to do so, such as the medical and industrial importance of saponins from S. officinalis, as taught by Meesapydosuk. Thus, the teachings of Meesapyodsuk, Aharoni, Osbourn, and Jia combined make obvious claim 5.
Subject Matter Free of Art
Aharoni teaches that takes part in the last step of saponin biosynthesis in spinach by transferring the acetyl group by a member of the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) superfamily of acyltransferases reported to be involved in triterpenoid saponin biosynthesis. However, the references do not teach or reasonaby suggest the application of quinovosyl transferase from Saponaria officinalis with 50% identity to SEQ ID NO: 36, thus the subject matter of claim 6 appears to be free of the prior art due to the recitation of quinovosyl transferase from Saponaria officinalis with 50% identity to SEQ ID NO: 36.
Conclusion
No claims allowed.
Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY K. JOHNSON whose telephone number is (571)272-5761. The examiner can normally be reached Monday - Friday 7:30 am - 5:00 pm.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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