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 .
Election/Restrictions
Applicant’s election of Group II (claims 2-3, 5-6, 9, 24, 38, 40 and 90) in the reply filed on 5/26/2026 is acknowledged.
The Applicant also elects the following species-
a) plant species Nicotiana sp. (all Group II claims read on this elected species);
b) Enzyme (AAE1) in sub-group (i) of claim 38 and SEQ ID NO: 6 (all Group II claims read on this elected species); and
c) ALT4 and SEQ ID NO: 25 (all Group II claims read on this elected species).
Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
It is noted here that hemp (Cannabis Sativa) is re-joined.
The requirement is still deemed proper and is therefore made FINAL.
Claim Status
Claims 1-3, 5-6, 9, 24, 26, 28, 33, 37-38, 40, 50, 64, 76, 78, 80, 83-84, 86 and 90 pending.
Claims 1, 26, 28, 33, 37, 50, 64, 76, 78, 80, 83-84 and 86 are withdrawn from examination as being part of non-elected groups.
Claims 2-3, 5-6, 9, 24, 38, 40 and 90 are being examined.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 2-3, 5-6, 9, 24, 38, 40 and 90 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claims 2-3, 5-6 and 9 recites the broad recitation “a plastid localised prenyltransferase polypeptide”, and the claim also recites “preferably cannabigerolic acid synthase (CBGAS)” (for claim 2) which is the narrower statement of the range/limitation. Claim 3 recites, “…the polynucleotides are comprised within a vector”, and the claim also recites “preferably a viral vector”. Claim 5 recites, “…a high biomass plant”, and the claim also recites “preferably selected from…”. Claim 6 recites, “…the plant is Nicotiana sp.”, and the claim also recites “preferably Nicotiana benthamiana or Nicotiana tabacum”. Claim 9 recites, “…a plastid transporting peptide…”, and the claim also recites “preferably a chloroplast transit peptide (CTP)”.
The claims are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. All the dependent claims depending on any of the claims 2-3, 5-6 and 9 also inherit the indefiniteness.
Regarding claim 5, the phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 5 also recites “... high biomass plant..” in line 3. The term “high” in claim 5 is a relative term which renders the claim indefinite. The term “high biomass plant” is defined as, “High biomass plants will be understood to be plants that are capable of production of higher amounts of a cannabinoid or industrial product described herein or known in the art when compared to a lower biomass plant. High biomass plants typically have a higher mass in a given area when compared to plants of lower biomass” (spec, p.64, line 1-4). Neither “high” or “higher”, nor “low” or “lower” are defined by the claim, and the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention.
It is also not clear to the Examiner if different types/varieties/cultivars within a plant species or different species in general are compared to define the term “high biomass plant”, as recited in claim 5.
Claims 24 and 40 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 24 recites “… the plant or part thereof further comprises at least one polynucleotide selected from the group consisting of a polynucleotide encoding an acyl-lipid thioesterase…” However, there is no group of polynucleotides recited in the claim. Other polynucleotides were deleted from the claim in the amendment filed 03/21/2025. It is unclear what other polynucleotides are intended to be part of the group. Alternatively, it is unclear if polynucleotides encoding distinct acyl-lipid thioesterases are intended to be the “group”. Dependent claim 40 inherits the indefiniteness.
It is suggested to delete “at least one polynucleotide selected from the group consisting of a polynucleotide” from claim 24.
Claim 40 also recites the limitation " the polynucleotide encoding ALT4" in line 4. There is insufficient antecedent basis for this limitation in the claim.
Claim 40 depends from claim 24 which is drawn to any acyl-lipid thioesterase ecites while reciting “… at least one polynucleotide selected from the group consisting of a polynucleotide encoding an acyl-lipid thioesterase…”. Neither claim 24 nor claim 40 recite ALT4 before the said recitation in claim 40.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claims 2 and 5 are rejected under 35 U.S.C. 103 as being unpatentable over Thomas et al. (Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast, 2020, Applied Microbiology and Biotechnology, 104:9551–9563) in view of Henley et al. (AU2020357969A1), Havelka J. (What is CBGA (Cannabigerolic Acid) & what does this cannabinoid do?, 2021, Leafly; Published on 15 January 2021) and admittance by the Applicant.
Claim 2 is drawn to a genetically modified plant or part thereof comprising a polynucleotide encoding an acyl activating enzyme 1 (AAE1), a polynucleotide encoding an olivetol synthase (OLS) and a polynucleotide encoding an olivetolic acid cyclase (OAC), wherein each polynucleotide is operably linked to a promoter which is capable of directing expression of each polynucleotide in the plant or part thereof, and when expressed in the plant or part thereof in the presence of hexanoic acid (C6) and geranyl-pyrophosphate (GPP), the polypeptides encoded by the polynucleotides increase the production of cannabigerolic acid (CBGA) when compared to a wild- type plant or part thereof.
Thomas et al. describes the Tetrahydrocannabinolic acid (THCA) biosynthesis pathway in hemp (C. sativa L.) (as recited in claim 5) comprising six enzymes (AAE1, OLS, OAC, GPPS, CBGAS and THCAS) producing THCA via cannabigerolic acid (CBGA) form hexanoic acid and geranyl diphosphate via (also known as geranyl poyrosphate, GPP) (p. 9552, Fig. 1), as shown below.
Thomas et al. describes that by introducing the genes encoding olivetolic acid cyclase (OAC) and olivetol synthase (OLS) for the biosynthesis of olivetolic acid, and the integration of corresponding cannabinoid synthases like CBGAS, a new biological system is engineered for the production of natural and unnatural cannabinoids (p.9552, bridging paragraph between left column and right column) that include cannabigerolic acid (CBGA) (Fig. 1). Thomas et al. teaches that concentration of hexanoic acid is a rate limiting step (p.9556, right column, last para, line 1-2) and heterologous expression of a polynucleotide encoding the acyl-activating enzyme AAE1 from Cannabis sativa
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1065
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converts it into various short- and medium-chain fatty acids (p.9557, left column, para 1, line 5-7) including (C6) hexanoyl Co-A (Fig. 1). Isomerization of IPP to dimethylallyl diphosphate (DMAPP) to produce GPP is a slow conversion process (p.9557, right column, para 1, line 12-14) and is a rate limiting step in subsequent production of CBGA (and THCA). Thus, concentrations of hexanoic acid and GPP became two critical components in the process.
However, Thomas et al. does not explicitly teach expressing codon-optimized polynucleotide sequence(s) in a plant.
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to express the polynucleotides encoding the acyl-activating enzyme (AAE1), olivetolic acid cyclase (OAC) and olivetol synthase (OLS) in a plant including hemp (C. sativa) in presence of two rate limiting substrates comprising GPP and hexanoic acid. Expressing codon-optimized polynucleotide sequence(s) in a host organism including in a plant including in hemp (C. sativa) is well known and routine method in the art and it implies that the polynucleotide sequences are operably linked to suitable promoter(s) capable of directing its expression in the host (hemp) plant, as taught by Henley et al. (p.19, para 1, line 9-10). It is also noted here that the Applicant admits the state of prior art regarding codon optimization using GeneArt online algorithm for gene expression in N. benthamiana (instant specification, p.76, para 1, line 2-4).
Before the effective filing date of the invention, an ordinarily skilled artisan would have been motivated to increase production of economically important substance CBGA in a genetically modified plant including in hemp/cannabis by expressing the polynucleotides encoding the acyl-activating enzyme (AAE1), olivetolic acid cyclase (OAC) and olivetol synthase (OLS) in a plant including hemp (C. sativa) in presence of two rate limiting substrates comprising GPP and hexanoic acid. CBGA is a commercially important product and increasingly being used for various diseases including cardiovascular disease, metabolic disease and colon cancer, as described by Havelka J. (p.4, para 3-5). Expressing the genes involved in cannabinoids including CBGA production in a plant like hemp for production and/or use of cannabinoids is also an obvious choice especially to make commercially important hemp cultivars with higher cannabinoids including CBGA which are also economically important products.
Claims 3, 5-6, 9 and 38 are rejected under 35 U.S.C. 103 as being unpatentable over Thomas et al. in view of Henley et al. (AU2020357969A1) and Havelka J., as applied to claims 2 and 5 above, and further in view of GenBank Accession no. JN717233 (published on 1 August 2012), Deguchi et al. (Metabolic Engineering Strategies of Industrial Hemp (Cannabis sativa L.): A Brief Review of the Advances and Challenges, 2020, Front. Plant Sci., 11:580621), Norkunas et al. (Improving agroinfiltration- based transient gene expression in Nicotiana benthamiana, 2018, Plant Methods, 14:71) and Eseverri et al. (Transit Peptides From Photosynthesis-Related Proteins Mediate Import of a Marker Protein Into Different Plastid Types and Within Different Species, 2020, Front. Plant Sci., 11:56070).
Thomas et al. in view of Henley et al. and Havelka J describes a genetically modified hemp plant expressing polynucleotide sequences encoding AAE1, OLS and OAC enzymes, wherein each polynucleotide is operably linked to a promoter which is capable of directing expression of the codon optimized polynucleotides in the plant, and when expressed in the plant in the presence of hexanoic acid (C6), which is also known as caproic acid (p.9556, right column, para 2, line 12), and geranyl-pyrophosphate (GPP), increases the production of cannabigerolic acid (CBGA), as discussed above. Thomas et al. also describes heterologous expression of an acyl-activating enzyme Cannabis sativa AAE1 (CsAAE1) (p.9557, left column, para 1, line 4-6). GenBank Accession no. JN717233 encoding a Cannabis sativa acyl-activating enzyme 1 (AAE1). The polynucleotide is having at least 70% sequence identity to instant SEQ ID NO: 6, as shown below (as recited in claim 38).
Title: US-18-722-386-6
Perfect score: 2201
Sequence: 1 cactctgtggtctcaaggta..........ggctttgagaccacgaagtg 2201
Searched: 1 seqs, 2533 residues
Database : NASEQ2_06242026_122839.fasta:*
RESULT 1
NASEQ2_06242026_122839
Best Local Similarity 76.0%; Query Match 60.5%; Score 1330.6; Length 2533;
Matches 1642; Conservative 0; Mismatches 519; Indels 0; Gaps 0;
Qy 20 ATGGGCAAGAACTACAAGAGCCTGGATTCTGTGGTGGCCAGCGATTTCATTGCTCTGGGT 79
||||| ||||| |||||| |||||| ||||| |||||| || ||||| || || |||
Db 96 ATGGGTAAGAATTACAAGTCCCTGGACTCTGTTGTGGCCTCTGACTTCATAGCCCTAGGT 155
Qy 80 ATTACTTCTGAGGTGGCCGAGACTCTTCATGGTAGACTTGCTGAGATCGTGTGCAACTAC 139
|| || ||||| || || ||||| || ||||||||||| || ||||||||||| || ||
Db 156 ATCACCTCTGAAGTTGCTGAGACACTCCATGGTAGACTGGCCGAGATCGTGTGTAATTAT 215
Qy 140 GGTGCTGCTACTCCTCAGACCTGGATCAACATTGCTAACCATATCCTGTCTCCGGACCTG 199
|| ||||| ||||| || || |||||||| ||||| |||||||| ||||| || |||||
Db 216 GGCGCTGCCACTCCCCAAACATGGATCAATATTGCCAACCATATTCTGTCGCCTGACCTC 275
Qy 200 CCTTTCTCACTTCACCAGATGCTTTTCTACGGCTGCTACAAGGATTTCGGTCCTGCTCCT 259
|| ||||| || ||||||||||| ||||| || ||||| || || || || ||||| |||
Db 276 CCCTTCTCCCTGCACCAGATGCTCTTCTATGGTTGCTATAAAGACTTTGGACCTGCCCCT 335
Qy 260 CCTGCTTGGATTCCTGATCCTGAGAAGGTGAAGTCTACTAACCTTGGCGCTCTGCTTGAG 319
||||||||||| || || || ||||| || ||||| || || || ||||| || | |||
Db 336 CCTGCTTGGATACCCGACCCGGAGAAAGTAAAGTCCACCAATCTGGGCGCACTTTTGGAG 395
Qy 320 AAGCGGGGTAAAGAGTTTCTGGGCGTCAAGTACAAGGACCCGATCAGCTCATTCAGCCAC 379
||||| || ||||||||| |||| |||||||| ||||| || || || ||
Db 396 AAGCGAGGAAAAGAGTTTTTGGGAGTCAAGTATAAGGATCCCATTTCAAGCTTTTCTCAT 455
Qy 380 TTCCAAGAGTTCAGCGTGAGGAACCCTGAAGTGTATTGGAGGACTGTGCTGATGGACGAG 439
|||||||| || || || |||||||| ||||||||||| || || || ||||| |||
Db 456 TTCCAAGAATTTTCTGTAAGAAACCCTGAGGTGTATTGGAGAACAGTACTAATGGATGAG 515
Qy 440 ATGAAGATCAGCTTCAGCAAGGACCCTGAGTGCATTCTGAGAAGGGACGACATTAACAAC 499
|||||||| || || ||||| || || || || || | || || || ||||| ||
Db 516 ATGAAGATAAGTTTTTCAAAGGATCCAGAATGTATATTGCGTAGAGATGATATTAATAAT 575
Qy 500 CCTGGTGGTTCTGAATGGCTTCCTGGTGGCTACCTTAACAGCGCTAAGAACTGCCTGAAC 559
|| || ||| |||||||||||| || || || |||||| || ||||| ||| ||||
Db 576 CCAGGGGGTAGTGAATGGCTTCCAGGAGGTTATCTTAACTCAGCAAAGAATTGCTTGAAT 635
Qy 560 GTGAACAGCAACAAGAAACTGAACGACACCATGATCGTGTGGCGTGATGAGGGTAACGAT 619
|| || || ||||||||| |||| || || ||||| || ||||||||||| || || |||
Db 636 GTAAATAGTAACAAGAAATTGAATGATACAATGATTGTATGGCGTGATGAAGGAAATGAT 695
Qy 620 GATCTGCCTCTTAACAAGCTGACCCTGGATCAGCTGAGGAAGAGAGTTTGGCTTGTTGGC 679
||| ||||||| ||||| |||| || || || || | || | |||||| | |||||
Db 696 GATTTGCCTCTAAACAAATTGACACTTGACCAATTGCGTAAACGTGTTTGGTTAGTTGGT 755
Qy 680 TACGCTCTGGAAGAGATGGGTCTTGAAAAGGGTTGCGCTATCGCTATCGACATGCCTATG 739
|| || || ||||| |||||| | || |||||||| || || || || || ||||| |||
Db 756 TATGCACTTGAAGAAATGGGTTTGGAGAAGGGTTGTGCAATTGCAATTGATATGCCAATG 815
Qy 740 CATGTTGACGCCGTGGTGATCTACCTTGCTATTGTGCTTGCTGGTTACGTGGTGGTGTCT 799
||||| || || ||||| ||||| || |||||||| ||||| || || || || || |||
Db 816 CATGTGGATGCTGTGGTTATCTATCTAGCTATTGTTCTTGCGGGATATGTAGTTGTTTCT 875
Qy 800 ATCGCTGACTCTTTCAGCGCTCCTGAGATTTCTACCAGGCTGAGGCTTTCTAAGGCCAAG 859
|| ||||| ||| |||||||| || || || || || | || || || || ||
Db 876 ATTGCTGATAGTTTTTCTGCTCCTGAAATATCAACAAGACTTCGACTATCAAAAGCAAAA 935
Qy 860 GCTATTTTCACCCAGGACCACATCATCAGGGGCAAGAAGAGGATTCCTCTGTACAGCAGA 919
|| ||||| || ||||| || || || | || |||||| | ||||| | ||||| |||
Db 936 GCCATTTTTACACAGGATCATATTATTCGTGGGAAGAAGCGTATTCCCTTATACAGTAGA 995
Qy 920 GTGGTCGAGGCTAAGTCTCCTATGGCTATTGTGATCCCTTGCAGCGGTTCTAACATTGGG 979
|| || || || |||||||| ||||| ||||| || ||||| || || ||||| |||||
Db 996 GTTGTGGAAGCCAAGTCTCCCATGGCCATTGTTATTCCTTGTAGTGGCTCTAATATTGGT 1055
Qy 980 GCTGAGCTTAGAGATGGGGACATCAGCTGGGACTACTTTCTCGAGAGGGCCAAAGAGTTC 1039
|| || | | ||||| || || ||||| |||||||| || || || ||||||||
Db 1056 GCAGAATTGCGTGATGGCGATATTTCTTGGGATTACTTTCTAGAAAGAGCAAAAGAGTTT 1115
Qy 1040 AAGAACTGCGAGTTCACTGCTCGTGAGCAGCCTGTTGATGCTTACACCAACATCCTGTTC 1099
|| || || || || |||||| | || || || |||||||| || || |||||||| |||
Db 1116 AAAAATTGTGAATTTACTGCTAGAGAACAACCAGTTGATGCCTATACAAACATCCTCTTC 1175
Qy 1100 AGCTCTGGTACTACCGGTGAGCCTAAGGCTATTCCTTGGACTCAAGCTACCCCTCTTAAG 1159
||||| || || || ||||| ||||| ||||| ||||||||||| || ||| | ||
Db 1176 TCATCTGGAACAACAGGGGAGCCAAAGGCAATTCCATGGACTCAAGCAACTCCTTTAAAA 1235
Qy 1160 GCTGCTGCTGATGGTTGGAGCCACCTGGATATTAGAAAGGGTGACGTGATCGTCTGGCCG 1219
|| ||||| ||||| |||||||| |||| ||||| || ||||| || || || |||||
Db 1236 GCAGCTGCAGATGGGTGGAGCCATTTGGACATTAGGAAAGGTGATGTCATTGTTTGGCCC 1295
Qy 1220 ACCAATCTTGGTTGGATGATGGGACCTTGGCTGGTGTACGCTTCTCTTCTTAACGGTGCT 1279
|| |||||||||||||||||||| ||||||||||| || ||||| || ||||| || |||
Db 1296 ACTAATCTTGGTTGGATGATGGGTCCTTGGCTGGTCTATGCTTCACTCCTTAATGGGGCT 1355
Qy 1280 TCTATCGCCCTGTACAACGGTTCTCCTCTTGTGTCTGGTTTCGCCAAGTTCGTGCAGGAT 1339
||||| ||| |||| || || || || ||||| ||||| || ||||| || |||||||||
Db 1356 TCTATTGCCTTGTATAATGGATCACCACTTGTTTCTGGCTTTGCCAAATTTGTGCAGGAT 1415
Qy 1340 GCTAAGGTTACCATGCTTGGTGTGGTGCCATCTATCGTGAGGTCATGGAAGTCCACTAAC 1399
||||| || || ||||| |||||||| || ||| || | |||||||| || ||
Db 1416 GCTAAAGTAACAATGCTAGGTGTGGTCCCTAGTATTGTTCGATCATGGAAAAGTACCAAT 1475
Qy 1400 TGCGTGTCAGGCTACGATTGGAGCACTATCCGTTGCTTCTCATCCTCTGGCGAGGCTTCT 1459
|| || ||||| |||||| ||| ||||||||||| || || ||||| || || |||
Db 1476 TGTGTTAGTGGCTATGATTGGTCCACCATCCGTTGCTTTTCCTCTTCTGGTGAAGCATCT 1535
Qy 1460 AATGTGGATGAGTACCTTTGGCTGATGGGCCGTGCTAATTACAAGCCTGTGATTGAGATG 1519
||||| ||||| ||||| ||| ||||||| | || || ||||||||||| || || |||
Db 1536 AATGTAGATGAATACCTATGGTTGATGGGGAGAGCAAACTACAAGCCTGTTATCGAAATG 1595
Qy 1520 TGCGGTGGCACTGAAATTGGTGGCGCTTTTTCTGCTGGATCCTTCTTGCAAGCTCAGAGC 1579
|| |||||||| ||||||||||| || ||||||||||| || ||||| ||||||||
Db 1596 TGTGGTGGCACAGAAATTGGTGGTGCATTTTCTGCTGGCTCTTTCTTACAAGCTCAATCA 1655
Qy 1580 CTGTCCTCATTCTCCTCACAGTGTATGGGTTGCACCCTGTACATCCTGGATAAGAACGGT 1639
| || ||||| ||||| |||||||||||||| | ||||| || || ||||| |||
Db 1656 TTATCTTCATTTAGTTCACAATGTATGGGTTGCACTTTATACATACTTGACAAGAATGGT 1715
Qy 1640 TACCCGATGCCGAAGAACAAGCCTGGTATTGGTGAACTGGCTCTGGGTCCTGTTATGTTC 1699
|| || ||||| || ||||| || || ||||||||| | || || ||||| || |||||
Db 1716 TATCCAATGCCTAAAAACAAACCAGGAATTGGTGAATTAGCGCTTGGTCCAGTCATGTTT 1775
Qy 1700 GGAGCTTCTAAGACCCTGCTGAACGGTAACCACCACGACGTTTACTTCAAGGGCATGCCT 1759
||||| || ||||| ||| |||| ||||| ||||| || ||||| || ||||| ||||||
Db 1776 GGAGCATCGAAGACTCTGTTGAATGGTAATCACCATGATGTTTATTTTAAGGGAATGCCT 1835
Qy 1760 ACCTTGAACGGTGAGGTGTTGAGAAGGCACGGTGATATTTTCGAGCTGACCAGCAACGGT 1819
|| ||||| || ||||| || || ||||| || || ||||| ||||| || || |||
Db 1836 ACATTGAATGGAGAGGTTTTAAGGAGGCATGGGGACATTTTTGAGCTTACATCTAATGGT 1895
Qy 1820 TACTACCATGCTCATGGAAGGGCTGACGATACCATGAACATCGGTGGCATCAAGATCAGC 1879
|| || ||||| ||||| | || || ||||| ||||| || || ||||||||||| ||
Db 1896 TATTATCATGCACATGGTCGTGCAGATGATACAATGAATATTGGAGGCATCAAGATTAGT 1955
Qy 1880 AGTATCGAGATTGAGCGGGTGTGCAATGAGGTGGACGATAGGGTTTTCGAGACTACCGCT 1939
|| |||||||| || || || ||||| || || || || ||||||||||| || |||
Db 1956 TCCATAGAGATTGAACGAGTTTGTAATGAAGTTGATGACAGAGTTTTCGAGACAACTGCT 2015
Qy 1940 ATTGGTGTGCCTCCTCTTGGTGGTGGTCCTGAGCAACTTGTGATTTTCTTCGTGCTGAAG 1999
||||| ||||| ||| | || ||||| ||||||||| | || |||||||| || | ||
Db 2016 ATTGGAGTGCCACCTTTGGGCGGTGGACCTGAGCAATTAGTAATTTTCTTTGTATTAAAA 2075
Qy 2000 GACAGCAACGATACCACCATCGATCTGAACCAGCTCCGGCTTTCTTTCAACCTTGGTCTG 2059
|| || ||||| || || || | || || | || | ||||||||| | ||| |
Db 2076 GATTCAAATGATACAACTATTGACTTAAATCAATTGAGGTTATCTTTCAACTTGGGTTTA 2135
Qy 2060 CAGAAGAAGCTGAACCCGCTTTTCAAGGTGACAAGGGTTGTGCCTCTTAGCTCTTTGCCT 2119
|||||||| || || || || |||||||| || | |||||||||||| || | ||
Db 2136 CAGAAGAAACTAAATCCTCTGTTCAAGGTCACTCGTGTTGTGCCTCTTTCATCACTTCCG 2195
Qy 2120 AGGACTGCCACCAACAAGATTATGAGAAGGGTGCTGAGGCAGCAGTTCTCCCATTTTGAA 2179
|| || || ||||||||||| ||||||||||| || | ||||| || || || ||||||
Db 2196 AGAACAGCAACCAACAAGATCATGAGAAGGGTTTTGCGCCAGCAATTTTCTCACTTTGAA 2255
Qy 2180 T 2180
|
Db 2256 T 2256
Moreover, the polypeptide encoded by the polynucleotide of instant SEQ ID NO: 6 is having 100% sequence identity to the protein encoded by the mRNA set forth by GenBank Accession no. JN717233, as shown below.
Title: AASEQ1_06242026_123900
Perfect score: 3801
Sequence: 1 MGKNYKSLDSVVASDFIALG..........RTATNKIMRRVLRQQFSHFE 720
Searched: 1 seqs, 720 residues
Database : AASEQ2_06242026_123915.fasta:*
RESULT 1
AASEQ2_06242026_123915
Best Local Similarity 100.0%; Query Match 100.0%; Score 3801; Length 720;
Matches 720; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MGKNYKSLDSVVASDFIALGITSEVAETLHGRLAEIVCNYGAATPQTWINIANHILSPDL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MGKNYKSLDSVVASDFIALGITSEVAETLHGRLAEIVCNYGAATPQTWINIANHILSPDL 60
Qy 61 PFSLHQMLFYGCYKDFGPAPPAWIPDPEKVKSTNLGALLEKRGKEFLGVKYKDPISSFSH 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 PFSLHQMLFYGCYKDFGPAPPAWIPDPEKVKSTNLGALLEKRGKEFLGVKYKDPISSFSH 120
Qy 121 FQEFSVRNPEVYWRTVLMDEMKISFSKDPECILRRDDINNPGGSEWLPGGYLNSAKNCLN 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 FQEFSVRNPEVYWRTVLMDEMKISFSKDPECILRRDDINNPGGSEWLPGGYLNSAKNCLN 180
Qy 181 VNSNKKLNDTMIVWRDEGNDDLPLNKLTLDQLRKRVWLVGYALEEMGLEKGCAIA IDMPM 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VNSNKKLNDTMIVWRDEGNDDLPLNKLTLDQLRKRVWLVGYALEEMGLEKGCAIA IDMPM 240
Qy 241 HVDAVVIYLAIVLAGYVVVSIADSFSAPEISTRLRLSKAKAIFTQDHIIRGKKRIPLYSR 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 HVDAVVIYLAIVLAGYVVVSIADSFSAPEISTRLRLSKAKAIFTQDHIIRGKKRIPLYSR 300
Qy 301 VVEAKSPMAIVIPCSGSNIGAELRDGDISWDYFLERAKEFKNCEFTAREQPVDAYTNILF 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 VVEAKSPMAIVIPCSGSNIGAELRDGDISWDYFLERAKEFKNCEFTAREQPVDAYTNILF 360
Qy 361 SSGTTGEPKAIPWTQATPLKAAADGWSHLDIRKGDVIVWPTNLGWMMGPWLVYASLLNGA 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 SSGTTGEPKAIPWTQATPLKAAADGWSHLDIRKGDVIVWPTNLGWMMGPWLVYASLLNGA 420
Qy 421 SIALYNGSPLVSGFAKFVQDAKVTMLGVVPSIVRSWKSTNCVSGYDWSTIRCFSSSGEAS 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 SIALYNGSPLVSGFAKFVQDAKVTMLGVVPSIVRSWKSTNCVSGYDWSTIRCFSSSGEAS 480
Qy 481 NVDEYLWLMGRANYKPVIEMCGGTEIGGAFSAGSFLQAQSLSSFSSQCMGCTLYILDKNG 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 NVDEYLWLMGRANYKPVIEMCGGTEIGGAFSAGSFLQAQSLSSFSSQCMGCTLYILDKNG 540
Qy 541 YPMPKNKPGIGELALGPVMFGASKTLLNGNHHDVYFKGMPTLNGEVLRRHGDIFELTSNG 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 YPMPKNKPGIGELALGPVMFGASKTLLNGNHHDVYFKGMPTLNGEVLRRHGDIFELTSNG 600
Qy 601 YYHAHGRADDTMNIGGIKISSIEIERVCNEVDDRVFETTAIGVPPLGGGPEQLVIFFVLK 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 YYHAHGRADDTMNIGGIKISSIEIERVCNEVDDRVFETTAIGVPPLGGGPEQLVIFFVLK 660
Qy 661 DSNDTTIDLNQLRLSFNLGLQKKLNPLFKVTRVVPLSSLPRTATNKIMRRVLRQQFSHFE 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 DSNDTTIDLNQLRLSFNLGLQKKLNPLFKVTRVVPLSSLPRTATNKIMRRVLRQQFSHFE 720
However, Thomas et al. does not describe any polynucleotide encoding a fusion polypeptide comprising a plastid transporting (transit) peptide.
Deguchi et al. describes different metabolic engineering strategies for industrial hemp to increase production of commercially important compounds including cannabinoids (title and abstract) by overexpressing various cannabinoid synthesis genes. Deguchi et al. teaches that high amount of GPP, which is an essential substrate to synthesize CBGA, is produced in plastid, thus the overexpression of GPP synthase or activating MEP-pathway genes in the plastid is essential or useful (p.3, left column, para 2, line 9-15). Deguchi et al. also teaches that in hemp, olivetolic acid synthesized in cytosol is transferred to plastid, where olivetolic acid and GPP are converted into CBGA (p.3, right column, para 2, line 23-25). Deguchi et al. describes introducing a gene (encoding tetrahydrocannabinolic acid synthase, THCAS) in tobacco (Nicotiana sp.), as recited in claim 6, to produce THCA from cannabigerolic acid (CBGA) (p.2, right column, last para, line 5-7). Deguchi et al. also describes transient gene expression via agroinfiltration (p.7, left column, last para, line 2-7) which often use tobacco (Nicotiana sp.) plants, as described in detail by Norkunas et al. (title and abstract).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to express the polynucleotide sequences encoding acyl-activating enzyme (AAE1), olivetolic acid cyclase (OAC) and olivetol synthase (OLS) enzymes in a plant including hemp (C. sativa), as described by Thomas et al, and direct the proteins encoded by nuclear genes (as transgeneses introduced via agrobacterium infiltration or other methods are mostly integrated in the nuclear genome) to plastid using a plastid transit/transporting peptide fused to the proteins because the substrates of the enzymes are accumulated in plastids, as taught by Deguchi et al. Expressing fusion proteins using a transit/transporting peptide(s) (as recited in claims 3 and 9) to target the fusion protein(s) to plastids/chloroplasts in a plant is well known and a routine method in the art, as described by Eseverri et al. (Title and abstract). Eseverri et al. describes transit peptides (TPs) of three Arabidopsis proteins related to photosynthetic and photo-protection processes direct highly efficient import of a recombinant reporter protein to different types of plastids including chloroplasts and leucoplasts (p. 2, para 3, line 5-8).
Before the effective filing date of the invention, an ordinarily skilled artisan would have been motivated to use plastid transporting peptide fused to AAE1, OAC and OLS proteins to plastids with a realistic objective to increase production of commercially important CBGA in a genetically modified plant including in hemp/cannabis.
Regarding claims 3 and 5- 6, use of tobacco (Nicotiana sp.) plants to transiently express various proteins including heterologous fusion proteins is a well-known, routine and a preferred method in the art for rapid and efficient production of recombinant proteins while checking its expression and/or efficacy of the proteins in a plant expression system, as described by Norkunas et al. (title; abstract line 1-3). Transient transformation using Agrobacterium tumefaciens is by far the preferred method of protein production as it provides safe, high-level and very rapid transgene expression in comparison to transgenic plants (Norkunas et al., p.1, bridging paragraph between left and right column). Norkunas et al. describes using tobacco (N. benthamiana) for agroinfiltration (p.4, right column, para 3). Transient expression of the codon optimized genes as encompassed by claim 3 (which depends from claim 2) in Nicotiana, satisfies the definition of “genetically modified” plant (instant specification, p.65, para 3, line 1-5).
Claims 24, 40 and 90 are rejected under 35 U.S.C. 103 as being unpatentable over unpatentable over Thomas et al. in view of Henley et al. and Havelka J. as applied to claims 2 and 5 above, and further in view of Kalinger et al. (Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa, 2018, Plant Physiology and Biochemistry, 127:104–118), Wang et al. (A Plastid Phosphatidylglycerol Lipase Contributes to the Export of Acyl Groups from Plastids for Seed Oil Biosynthesis, 2018, The Plant Cell, 29:1678–1696) and Shen et al. (A 13-lipoxygenase, TomloxC, is essential for synthesis of C5 flavour volatiles in tomato, 2014, Journal of Experimental Botany, 65:419–428), Patel et al. (Volatile Fatty Acids (VFAs) Generated by Anaerobic Digestion Serve as Feedstock for Freshwater and Marine Oleaginous Microorganisms to Produce Biodiesel and Added-Value Compounds, 2021, Front. Microbiol., 12:614612) (published on 28 January 2021) and Pulsifer et al. (Acyl‑lipid thioesterase1–4 from Arabidopsis thaliana form a novel family of fatty acyl–acyl carrier protein thioesterases with divergent expression patterns and substrate specificities, 2014, Plant Mol. Biol., 84:549–563).
Claim 40 is directed to the plant or part thereof of claim 24, wherein:
i) the polynucleotide encoding ALT4 comprises a nucleotide sequence which is at least 70% to a sequence set forth as SEQ IDNO: 25;
ii) the polynucleotide encoding PLIP1 comprises a nucleotide sequence which is at least 70 to the sequence set forth as SEQ ID NO:26; and
iii) the polynucleotide encoding TomLoxC comprises a nucleotide sequence which is at least 70% to the sequence set forth as SEQ ID NO:27.
Thomas et al. in view of Henley et al. and Havelka J. describes a genetically modified plant expressing polynucleotide sequences encoding AAE1, OLS and OAC enzymes, wherein each polynucleotide is operably linked to a promoter which is capable of directing expression of the codon optimized polynucleotides in the plant, and when expressed in the plant in the presence of hexanoic acid (C6), which is also known as caproic acid (p.9556, right column, para 2, line 12), and geranyl-pyrophosphate (GPP), increases the production of cannabigerolic acid (CBGA), as discussed above.
However, Thomas et al. in view of Henley et al. and Havelka J. does not describe any acyl-lipid thioesterase, Plastid Lipase 1 (PLIP1), or Tomato 13-lipoxygenase (LOX), TomloxC.
Kalinger et al. describes that in Arabidopsis thaliana, a family of plastid-localized acyl-lipid thioesterases (AtALT1-4) (as recited in claim 24) generate medium-chain (C6-C14) fatty acids and β–keto fatty acids (abstract, left column, line 2-4), which is also known as MK precursors. The volatile fatty acids attract insect pollinators or deter predatory insects, and its homologues are present in all plant taxa (abstract, line 4-5). In addition to biofuels, medium-chain FAs and MKs are industrially valuable as components of insecticides, flavourings, fragrances, and pharmaceutical precursors (p.117, left column, para 4, line 1-3).
Kalinger et al. also describes that AtALT4 protein (as recited in claim 40) generates 6:0 (C6) and 8:0 (C8) fatty acids. It is known in the art that hexanoic acid is a C6 fatty acid. With the reference to Pulsifer et al., Kalinger et al. describes the sequence of AtALT4 (p.105, left column, para 2m line 2-4; p.108, left column, para 1, line 9-10) (GenBank Accession No. At1g68280 or F4HX80) (Pulsifer et al., p.553, right column, last para, line 4-5) which is having 100% sequence identity to the polypeptide (SEQ ID NO: 21) encoded by instant SEQ ID NO: 25 (spec, p.22, para: “Key to Sequence Listing”), as recited by claim 40, as shown below.
RESULT 1
ALT4_ARATH
ID ALT4_ARATH Reviewed; 188 AA.
AC F4HX80; Q9C9G3;
DT 20-JAN-2016, integrated into UniProtKB/Swiss-Prot.
DT 28-JUN-2011, sequence version 1.
DT 18-JUN-2025, entry version 80.
DE RecName: Full=Acyl-acyl carrier protein thioesterase ATL4, chloroplastic {ECO:0000305};
DE EC=3.1.2.- {ECO:0000305};
DE AltName: Full=Acyl-ACP thioesterase ATL4 {ECO:0000305};
DE AltName: Full=Acyl-lipid thioesterase 4 {ECO:0000303|PubMed:24214063};
DE Flags: Precursor;
GN Name=ALT4 {ECO:0000303|PubMed:24214063};
GN OrderedLocusNames=At1g68280 {ECO:0000312|Araport:AT1G68280};
GN ORFNames=T22E19.9 {ECO:0000312|EMBL:AAG52600.1};
OS Arabidopsis thaliana (Mouse-ear cress).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Brassicaceae; Camelineae; Arabidopsis.
OX NCBI_TaxID=3702;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Columbia;
RX PubMed=11130712; DOI=10.1038/35048500;
RA Theologis A., Ecker J.R., Palm C.J., Federspiel N.A., Kaul S., White O.,
RA Alonso J., Altafi H., Araujo R., Bowman C.L., Brooks S.Y., Buehler E.,
RA Chan A., Chao Q., Chen H., Cheuk R.F., Chin C.W., Chung M.K., Conn L.,
RA Conway A.B., Conway A.R., Creasy T.H., Dewar K., Dunn P., Etgu P.,
RA Feldblyum T.V., Feng J.-D., Fong B., Fujii C.Y., Gill J.E., Goldsmith A.D.,
RA Haas B., Hansen N.F., Hughes B., Huizar L., Hunter J.L., Jenkins J.,
RA Johnson-Hopson C., Khan S., Khaykin E., Kim C.J., Koo H.L.,
RA Kremenetskaia I., Kurtz D.B., Kwan A., Lam B., Langin-Hooper S., Lee A.,
RA Lee J.M., Lenz C.A., Li J.H., Li Y.-P., Lin X., Liu S.X., Liu Z.A.,
RA Luros J.S., Maiti R., Marziali A., Militscher J., Miranda M., Nguyen M.,
RA Nierman W.C., Osborne B.I., Pai G., Peterson J., Pham P.K., Rizzo M.,
RA Rooney T., Rowley D., Sakano H., Salzberg S.L., Schwartz J.R., Shinn P.,
RA Southwick A.M., Sun H., Tallon L.J., Tambunga G., Toriumi M.J., Town C.D.,
RA Utterback T., Van Aken S., Vaysberg M., Vysotskaia V.S., Walker M., Wu D.,
RA Yu G., Fraser C.M., Venter J.C., Davis R.W.;
RT "Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana.";
RL Nature 408:816-820(2000).
RP GENOME REANNOTATION.
RC STRAIN=cv. Columbia;
RX PubMed=27862469; DOI=10.1111/tpj.13415;
RA Cheng C.Y., Krishnakumar V., Chan A.P., Thibaud-Nissen F., Schobel S.,
RA Town C.D.;
RT "Araport11: a complete reannotation of the Arabidopsis thaliana reference
RT genome.";
RL Plant J. 89:789-804(2017).
RN [3]
RP FUNCTION, SUBCELLULAR LOCATION, AND TISSUE SPECIFICITY.
RX PubMed=24214063; DOI=10.1007/s11103-013-0151-z;
RA Pulsifer I.P., Lowe C., Narayaran S.A., Busuttil A.S., Vishwanath S.J.,
RA Domergue F., Rowland O.;
RT "Acyl-lipid thioesterase1-4 from Arabidopsis thaliana form a novel family
RT of fatty acyl-acyl carrier protein thioesterases with divergent expression
RT patterns and substrate specificities.";
RL Plant Mol. Biol. 84:549-563(2014).
CC -!- FUNCTION: Acyl-ACP thioesterase involved in the production of fatty
CC acids and beta-keto fatty acids. Can produce fatty acids of medium to
CC long chain (6:0, 8:0, 10:0 and 16:1) and small amounts of medium to
CC long chain beta-keto fatty acids (8:0, 14:0 and 16:1) when expressed in
CC a heterologous organism (E.coli). Possesses thioesterase activity for
CC lauroyl-ACP (12:0-ACP) in vitro. May play a role in the development of
CC floral organs by generating short chain fatty acids.
CC {ECO:0000269|PubMed:24214063}.
CC -!- SUBCELLULAR LOCATION: Plastid, chloroplast
CC {ECO:0000269|PubMed:24214063}.
CC -!- TISSUE SPECIFICITY: Expressed specifically in anther walls
CC (endothecium) and in microspores. {ECO:0000269|PubMed:24214063}.
CC -!- SIMILARITY: Belongs to the 4-hydroxybenzoyl-CoA thioesterase family.
CC {ECO:0000305}.
CC -!- SEQUENCE CAUTION:
CC Sequence=AAG52600.1; Type=Erroneous gene model prediction; Evidence={ECO:0000305};
DR EMBL; AC016447; AAG52600.1; ALT_SEQ; Genomic_DNA.
DR EMBL; CP002684; -; NOT_ANNOTATED_CDS; Genomic_DNA.
DR PIR; D96706; D96706.
DR AlphaFoldDB; F4HX80; -.
DR SMR; F4HX80; -.
DR FunCoup; F4HX80; 4.
DR STRING; 3702.F4HX80; -.
DR PaxDb; 3702-AT1G68280.1; -.
DR Araport; AT1G68280; -.
DR TAIR; AT1G68280; ALT4.
DR eggNOG; ENOG502RYRP; Eukaryota.
DR HOGENOM; CLU_101141_1_2_1; -.
DR InParanoid; F4HX80; -.
DR BioCyc; ARA:AT1G68280-MONOMER; -.
DR PRO; PR:F4HX80; -.
DR Proteomes; UP000006548; Chromosome 1.
DR ExpressionAtlas; F4HX80; baseline.
DR GO; GO:0009507; C:chloroplast; IDA:UniProtKB.
DR GO; GO:0016297; F:fatty acyl-[ACP] hydrolase activity; IDA:UniProtKB.
DR GO; GO:0006629; P:lipid metabolic process; IEA:UniProtKB-KW.
DR CDD; cd00586; 4HBT; 1.
DR FunFam; 3.10.129.10:FF:000037; acyl-acyl carrier protein thioesterase ATL3, chloroplastic; 1.
DR Gene3D; 3.10.129.10; Hotdog Thioesterase; 1.
DR InterPro; IPR050563; 4-hydroxybenzoyl-CoA_TE.
DR InterPro; IPR029069; HotDog_dom_sf.
DR PANTHER; PTHR31793; 4-HYDROXYBENZOYL-COA THIOESTERASE FAMILY MEMBER; 1.
DR PANTHER; PTHR31793:SF27; NOVEL THIOESTERASE SUPERFAMILY DOMAIN AND SAPOSIN A-TYPE DOMAIN CONTAINING PROTEIN (0610012H03RIK); 1.
DR Pfam; PF13279; 4HBT_2; 1.
DR SUPFAM; SSF54637; Thioesterase/thiol ester dehydrase-isomerase; 1.
PE 2: Evidence at transcript level;
KW Chloroplast; Hydrolase; Lipid metabolism; Plastid; Reference proteome;
KW Transit peptide.
FT TRANSIT 1..47
FT /note="Chloroplast"
FT /evidence="ECO:0000255"
FT CHAIN 48..188
FT /note="Acyl-acyl carrier protein thioesterase ATL4,
FT chloroplastic"
FT /id="PRO_0000435264"
FT ACT_SITE 64
FT /evidence="ECO:0000250|UniProtKB:P56653,
FT ECO:0000250|UniProtKB:Q9C7I5"
SQ SEQUENCE 188 AA; 21318 MW; CD993F75DAB7335F CRC64;
Best Local Similarity 100.0%; Query Match 100.0%; Score 970; Length 188;
Matches 188; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MIRVTGTAAPAMSVVFPTSWRQPVMLPLRSAKTFKPHTFLDLKGGKEMSEFHEVELKVRD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MIRVTGTAAPAMSVVFPTSWRQPVMLPLRSAKTFKPHTFLDLKGGKEMSEFHEVELKVRD 60
Qy 61 YELDQFGVVNNAVYANYCQHGMHEFLESIGINCDEVARSGEALAISELTMNFLAPLRSGD 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YELDQFGVVNNAVYANYCQHGMHEFLESIGINCDEVARSGEALAISELTMNFLAPLRSGD 120
Qy 121 KFVVKVNISRTSAARIYFDHSILKLPNQEVILEAKATVVWLDNKHRPVRIPSSIRSKFVH 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 KFVVKVNISRTSAARIYFDHSILKLPNQEVILEAKATVVWLDNKHRPVRIPSSIRSKFVH 180
Qy 181 FLRQNDTV 188
||||||||
Db 181 FLRQNDTV 188
Wang et al. describes that PLASTID LIPASE1 (PLIP1), as recited in claims 40 and 90, hydrolyzes polyunsaturated fatty acids (acyl groups) in-vivo (abstract). Insertion mutation lines in the PLIP1 gene show an approximate 10% reduction in total seed fatty acid (acyl group) content (p. 1685, right column, para 2, line 25-26) and delayed germination (abstract), while the overexpression lines had a 40 to 50% increased seed fatty acid (acyl group) content (p.1685, right column, para 2, line 26-28), which includes, inter alia, C6 hexanoic acid. Wang et al. also describes the coding sequence of PLASTID LIPASE1 (PLIP1) gene (GenBank Accession No. At3G61680 or Phytozome gene ID: At3G61680) (p.1694, left column, para 5, line 3) which is having more than 75% sequence identity to instant SEQ ID NO: 26 (as recited in claim 40), as shown below.Title: US-18-722-386-26
Perfect score: 1985
Sequence: 1 cactctgtggtctcaaatgg..........ggctttgagaccacgaagtg 1985
Searched: 1 seqs, 1950 residues
Database : NASEQ2_06242026_145105.fasta:*
RESULT 1
NASEQ2_06242026_145105
Best Local Similarity 75.3%; Query Match 59.4%; Score 1179.4; DB 1; Length 1950;
Matches 1468; Conservative 0; Mismatches 481; Indels 0; Gaps 0;
Qy 17 ATGGCTTTCAACACCGCTATGGCTTCTACTTCTCCTGCTGCTGCTAACGATGTGCTGAGA 76
||||| || || || |||||||| ||||| ||||| || || || || || || | |||
Db 1 ATGGCGTTTAATACGGCTATGGCGTCTACATCTCCAGCGGCGGCAAATGACGTTTTAAGA 60
Qy 77 GAGCACATTGGTCTTAGGCGGTCTTTGTCTGGTCAGGACCTTGTGCTTAAAGGCGGCGGT 136
|| || ||||| || | | || ||||| ||||| || || || | |||||||| |||
Db 61 GAACATATTGGCCTCCGTAGATCGTTGTCCGGTCAAGATCTCGTCTTAAAAGGCGGTGGT 120
Qy 137 ATTCGTAGGTCCTCTTCTGATAATCACCTGTGCTGCAGGTCCGGGAACAACAACAATAGG 196
|| || || || ||| || |||||| |||| || | ||||| || || || ||| |
Db 121 ATACGGAGATCGAGTTCCGACAATCACTTGTGTTGTCGCTCCGGTAATAATAATAATCGC 180
Qy 197 ATTCTGGCTGTGTCTGTGCGGCCTGGTATGAAAACTTCTAGATCTGTGGGCGTGTTCAGC 256
||||| ||||||||||| || || || |||||||| | |||||||||| ||||||
Db 181 ATTCTTGCTGTGTCTGTTCGTCCGGGGATGAAAACGAGTCGATCTGTGGGAGTGTTCTCG 240
Qy 257 TTCCAGATCTCCAGCTCTATTATCCCGAGTCCGATTAAGACCCTGCTGTTCGAGACTGAT 316
|| ||||| || || ||||| ||||| |||||||| || || |||| || || || ||
Db 241 TTTCAGATATCGAGTTCTATAATCCCAAGTCCGATAAAAACGTTGCTATTTGAAACGGAC 300
Qy 317 ACCAGCCAGGATGAACAAGAGTCCGACGAGATTGAGATCGAGACAGAGCCTAACCTGGAT 376
|| || || || |||||| ||| ||||||||||| ||||||||||| || || |||
Db 301 ACGTCTCAAGACGAGCAAGAGAGCGATGAGATTGAGATTGAGACAGAGCCAAATCTAGAT 360
Qy 377 GGCGCTAAGAAGGCTAATTGGGTTGAGCGGCTTCTTGAGATTAGGCGTCAGTGGAAGAGA 436
|| || |||||||| |||||||| ||| |||| |||||||| ||| | ||||||||||||
Db 361 GGAGCCAAGAAGGCAAATTGGGTCGAGAGGCTGCTTGAGATAAGGAGACAGTGGAAGAGA 420
Qy 437 GAGCAAAAGACCGAGAGCGGTAACTCTGATGTGGCTGAAGAGTCTGTGGATGTGACTTGT 496
|||||||| || ||||| || ||| ||| || || || || ||| || || || |||
Db 421 GAGCAAAAAACAGAGAGTGGAAACAGTGACGTTGCAGAGGAAAGTGTTGACGTTACGTGT 480
Qy 497 GGTTGCGAAGAGGAAGAGGGCTGCATTGCTAATTACGGTAGCGTGAACGGTGATTGGGGC 556
||||| ||||| ||||| || |||||||| |||||||| || || |||||||||||
Db 481 GGTTGTGAAGAAGAAGAAGGTTGCATTGCGAATTACGGATCTGTAAATGGTGATTGGGGA 540
Qy 557 CGTGAGTCTTTTTCTAGGCTTCTGGTTAAGGTGAGCTGGTCCGAGGCTAAGAAGCTTTCT 616
|| || || || ||||| | || || ||||| ||||| |||||||| |||||||||
Db 541 CGAGAATCGTTCTCTAGATTGCTTGTGAAGGTTTCTTGGTCTGAGGCTAAAAAGCTTTCT 600
Qy 617 CAGCTTGCTTACCTGTGCAACCTGGCTTACACCATTCCTGAGATCAAGGGCGAAGATCTG 676
||| | ||||| |||| ||| |||||||||| || |||||||||||||| || ||| ||
Db 601 CAGTTAGCTTATTTGTGTAACTTGGCTTACACGATACCTGAGATCAAGGGTGAGGATTTG 660
Qy 677 CGGAGGAATTACGGTCTTAAGTTCGTGACCAGCAGCCTCGAGAAGAAAGCTAAGGCTGCT 736
| || || || || | ||||| ||||| | || ||||||||||| || ||
Db 661 AGAAGAAACTATGGGTTAAAGTTTGTGACATCTTCATTGGAAAAGAAAGCTAAAGCAGCG 720
Qy 737 ATCCTGCGTGAGAAGCTTGAACAGGATCCTACTCACGTGCCAGTGATCACCTCTCCTGAT 796
|| || | ||||| || || || ||||| || || || || || || || || || |||
Db 721 ATACTTAGAGAGAAACTAGAGCAAGATCCAACACATGTCCCTGTTATTACATCCCCGGAT 780
Qy 797 CTTGAAAGCGAGAAGCAGTCTCAGCGGAGCGCTTCTAGTTCTGCTAGCGCTTATAAGATC 856
| ||| ||||||||||||||| || ||||| ||||||| ||||| |||||
Db 781 TTAGAATCCGAGAAGCAGTCTCAACGATCAGCTTCATCTTCTGCTTCTGCTTACAAGATT 840
Qy 857 GCTGCTTCCGCTGCTAGCTACATCCACTCTTGCAAAGAGTACGATCTGAGCGAGCCGATC 916
|||||||| ||||| ||||| ||||||||||||||||| ||||| || || ||
Db 841 GCTGCTTCAGCTGCGTCTTACATTCACTCTTGCAAAGAGTATGATCTTTCAGAACCAATT 900
Qy 917 TACAAGTCTGCTGCAGCTGCTCAAGCTGCTGCTTCTACTATGACTGCTGTTGTTGCTGCT 976
|| || || ||||| |||||||| ||||| || ||||| ||||| || || ||||||||
Db 901 TATAAATCAGCTGCTGCTGCTCAGGCTGCAGCGTCTACCATGACCGCGGTGGTTGCTGCG 960
Qy 977 GGCGAGGAAGAAAAGCTTGAAGCTGCTAGAGAGCTGCAAAGCCTGCAATCTTCTCCATGC 1036
|| ||||| || ||||| ||||| || || ||| | || || ||||| ||||| ||
Db 961 GGTGAGGAGGAGAAGCTAGAAGCGGCAAGGGAGTTACAGTCGCTACAATCATCTCCTTGT 1020
Qy 1037 GAGTGGTTCGTGTGCGACGATCCTAATACTTACACCCGGTGCTTCGTGATCCAGGGCTCT 1096
|||||||| || || || ||||| || || ||||| ||||||| ||||| ||||| |||
Db 1021 GAGTGGTTTGTTTGTGATGATCCAAACACATACACTAGGTGCTTTGTGATTCAGGGATCT 1080
Qy 1097 GATTCTCTTGCTAGCTGGAAGGCTAACCTGTTCTTCGAGCCTACCAAGTTCGAGGACACT 1156
|||||| | ||| ||||| || ||||| ||||||||||| || ||||| ||||||||
Db 1081 GATTCTTTAGCTTCTTGGAAAGCAAACCTTTTCTTCGAGCCAACTAAGTTTGAGGACACA 1140
Qy 1157 GATGTGCTTGTTCACAGGGGAATCTACGAGGCTGCAAAGGGAATCTATGAGCAGTTCCTG 1216
||||| | || ||||| |||||||||||||| ||||| ||||| || || |||||| |
Db 1141 GATGTATTAGTCCACAGAGGAATCTACGAGGCAGCAAAAGGAATATACGAACAGTTCTTA 1200
Qy 1217 CCTGAGATTACCGAGCACCTTTCTAGGCATGGTGACAGGGCTAAGTTCCAGTTCACCGGT 1276
|| || || || ||||| | ||||| ||||| || || |||||||| |||||||| |||
Db 1201 CCAGAAATAACAGAGCATTTGTCTAGACATGGAGATAGAGCTAAGTTTCAGTTCACGGGT 1260
Qy 1277 CATTCTCTTGGCGGTAGCCTTTCTCTTATCGTGAACCTGATGCTGATCTCCAGGGGCCTT 1336
||||||||||| || || || || | || ||||| ||||||| ||||| || || ||
Db 1261 CATTCTCTTGGAGGCAGTCTCTCATTAATAGTGAATTTGATGCTTATCTCTAGAGGACTC 1320
Qy 1337 GTTTCTTCAGAGGCTATGAAGTCTGTGGTGACCTTCGGTTCTCCTTTCGTTTTCTGTGGT 1396
||| || || |||||||| || || || || |||||||| || || || || ||||||
Db 1321 GTTAGCTCTGAAGCTATGAAATCCGTTGTCACGTTCGGTTCACCGTTTGTGTTTTGTGGT 1380
Qy 1397 GGCGAGAAGATCCTTGCTGAGCTTGGTCTTGATGAGTCTCATGTGCACTGCGTGATGATG 1456
|| |||||||| || || |||||||||||||| ||| |||||| ||||| |||||||||
Db 1381 GGTGAGAAGATTCTAGCGGAGCTTGGTCTTGACGAGAGTCATGTTCACTGTGTGATGATG 1440
Qy 1457 CACAGGGATATTGTGCCTAGGGCCTTCTCTTGCAACTACCCTGATCATGTGGCTCTGGTG 1516
|| || ||||| || || | ||||| || || || || ||||| ||||| ||||| ||
Db 1441 CATAGAGATATCGTCCCACGAGCCTTTTCGTGTAATTATCCTGACCATGTTGCTCTCGTT 1500
Qy 1517 CTTAAGAGGCTGAACGGTTCTTTCAGGACTCACCCTTGCCTGAACAAGAACAAGCTCCTG 1576
|| ||| | |||| || || ||| | || || ||||| || ||||| || || || ||
Db 1501 CTCAAGCGTTTGAATGGCTCCTTCCGTACACATCCTTGTCTCAACAAAAATAAACTGTTG 1560
Qy 1577 TACAGCCCTATGGGCAAAGTGTACATTCTGCAGCCTAGCGAGTCTGTGTCTCCTACTCAT 1636
|| || ||||| ||||| || ||||| ||||| || ||| || || || || ||
Db 1561 TATTCACCGATGGGGAAAGTATATATTCTACAGCCGAGTGAGAGCGTCTCGCCGACGCAC 1620
Qy 1637 CCTTGGTTGCCTCCAGGTAACGCTCTGTACATCCTCGAGAATTCTAACGAGGGCTACTCT 1696
|| ||| | || || || |||||||||||||| | || ||| ||||| || ||||||
Db 1621 CCATGGCTTCCACCGGGAAACGCTCTGTACATTTTAGAAAATAGCAACGAAGGTTACTCT 1680
Qy 1697 CCTACTGCTCTGAGGGCTTTTCTTAACAGGCCTCATCCTCTCGAGACTCTGTCTCAAAGG 1756
||||| || | | || ||| | ||| | ||||| || ||||| || ||| |||| |
Db 1681 CCTACGGCGTTACGAGCATTTTTAAACCGCCCTCACCCGCTCGAAACGCTGAGTCAACGC 1740
Qy 1757 GCTGCTTATGGTTCTGAGGGTTCTGTGCTTAGGGACCACGACTCTAAGAACTACGTGAAG 1816
|| |||||||| || || ||||| || | |||||||||||||| ||||||||||| |||
Db 1741 GCAGCTTATGGCTCGGAAGGTTCAGTCTTGAGGGACCACGACTCCAAGAACTACGTTAAG 1800
Qy 1817 GCTGTGAATGGTGTGTTGAGGCAGCACACCAAGCTGATTGTGAGGAAGGCTAGGATTCAG 1876
|| ||||| || || | ||||||||||| ||||| || || ||||| || ||||| ||
Db 1801 GCCGTGAACGGAGTTCTCAGGCAGCACACGAAGCTCATAGTTAGGAAAGCCAGGATACAA 1860
Qy 1877 AGGCGTTCTGTTTGGCCTGTGCTTACTTCTGCTGGTAGGGGTCTTAACGAGTCTCTTACT 1936
||| | ||||||||| ||||| || || || || | || | |||||| || ||
Db 1861 AGGAGGAGTGTTTGGCCCGTGCTGACATCAGCAGGACGTGGATTAAACGAGAGCCTGACG 1920
Qy 1937 ACCGCCGAGGAAATCATGACCCGGGTTTA 1965
|| |||||||| |||||||| || || ||
Db 1921 ACGGCCGAGGAGATCATGACACGTGTCTA 1949
Moreover, the protein encoded by the SEQ ID NO: 26 is having 100% sequence identity to the protein encoded by the same PLIP1 gene as described by Phytozome gene ID: At3G61680 (data not shown).
Shen et al. describes the peroxidation of C18 polyunsaturated fatty acids, such as linolenic and linoleic acids, is initially catalyzed by lipoxygenases (LOXs) which act upon polyunsaturated fatty acids at either the C9 or C13 position; the enzymes are referred to as 9- and 13-LOXs respectively, depending on which position is oxygenated (p. 420, left column, para 2, line 1-7). Tomato 13-lipoxygenase (LOX), TomloxC, enzyme responsible for synthesis of C5 (derived from fatty acids) and C6 volatile (fatty acids) compounds (abstract). The most common volatile fatty acids include hexanoic acid which is also known as caproic acid (Patel et. al., p.2, right column, para 3, line 1-3). C5 volatiles is responsible for consumer liking for the taste of fresh tomato (Shen et al., abstract). Shen et al. describes the full length open reading frame of TomloxC cloned into a vector (p. 421, left column, last para, line 1-2) and also teaches the sequence (GenBank accession No. AAB65766) of TomLoxC (Supplementary Fig. 1). The TomLoxC protein is encoded by the gene taught by GenBank accession No. U37839 which is having at least 70% sequence identity to instant SEQ ID NO: 27, as recited in claim 40 and as shown below.
Title: US-18-722-386-27
Perfect score: 2726
Sequence: 1 cactctgtggtctcaaatgc..........ggctttgagaccacgaagtg 2726
Searched: 1 seqs, 2802 residues
Database : NASEQ2_06242026_155310.fasta:*
RESULT 1
NASEQ2_06242026_155310
Best Local Similarity 73.5%; Query Match 57.0%; Score 1554; DB 1; Length 2802;
Matches 1983; Conservative 0; Mismatches 715; Indels 0; Gaps 0;
Qy 15 AAATGCTGAAGCCTCAGTTCCAGCAGTCTACCAAGACTCTGATCCCGAGCTGGAACACCA 74
||||| |||||||||| || || || ||||| || || || || || ||||| || |
Db 22 AAATGTTGAAGCCTCAATTTCAACAATCTACAAAAACCCTAATTCCATCTTGGAATACTA 81
Qy 75 ATACCTTGTTCCTCGCTAGCTTCCCGATCAACATCCTGAACAAGAACTTCATTCTGAAGA 134
|||| || ||| | || || || || || || | || || ||||| || || || |
Db 82 ATACATTATTCTTAGCCTCTTTTCCCATAAATATTTTAAATAAAAACTTTATACTTAAAA 141
Qy 135 AGAAGAACAACTTCCGGGTCCACCACAACTACAACGGTGCTAACACTATCAAGGCCGTGC 194
| ||||| || || |||| || || || || || ||||| || || || ||||| ||||
Db 142 AAAAGAATAATTTTAGGGTTCATCATAATTATAATGGTGCAAATACCATTAAGGCTGTGC 201
Qy 195 TGAACAGCACCCAGAAGTCTATTGGAGTTAAGGCTGTGGTGACCGTGCAGAAACAGGTGA 254
| || || || || || || || ||||| ||||||||||| || || ||||| || |
Db 202 TTAATTCTACTCAAAAATCCATAGGTGTTAAAGCTGTGGTGACTGTCCAAAAACAAGTTA 261
Qy 255 ACCTTAACCTTCTGAGGGGCCTTGATGGTATCGGTGATCTGCTTGGTAAGAGCCTGATTC 314
| | || | | || || ||||||||||| |||||| | |||||||| || |||
Db 262 ATTTAAATTTATTAAGAGGACTTGATGGTATTGGTGATTTACTTGGTAAATCACTAATTT 321
Qy 315 TGTGGATTGTGGCTGCTGAGCTTGATCACAAGACCGGTTTGGAGAAGCCGAGCATTAGGT 374
| |||||||| ||||||||||||||||| ||||| || | || ||||| || |||||||
Db 322 TATGGATTGTTGCTGCTGAGCTTGATCATAAGACTGGACTTGAAAAGCCAAGTATTAGGT 381
Qy 375 CTTATGCTCACAGGGGTCTTGATGTGGATGGCGATACTTACTACGAGGCCGATTTCGAGA 434
| ||||| || | || || ||||||||||| || || || || ||||| ||||| || |
Db 382 CATATGCACATCGTGGACTAGATGTGGATGGTGACACATATTATGAGGCTGATTTTGAAA 441
Qy 435 TCCCTGAGGATTTTGGTGAGGTGGGCGCTATTCTTGTTGAGAACGAGCACCACAAAGAGA 494
| ||||| || ||||| ||||| || || ||| | || || || ||||||||||| || |
Db 442 TTCCTGAAGACTTTGGGGAGGTTGGTGCAATTTTAGTAGAAAATGAGCACCACAAGGAAA 501
Qy 495 TGTACGTCAAGAACATCGTGATCGACGGTTTCGTGCACGCCAAGGTTGAGATTACTTGCA 554
|||| || || || || || || |||||||| || || ||||| ||||| ||||| ||||
Db 502 TGTATGTGAAAAATATTGTAATTGACGGTTTTGTCCATGCCAAAGTTGAAATTACATGCA 561
Qy 555 ACTCTTGGGTGCACAGCAAGTTCGCTAACCCTGACAAGAGGATCTTCTTTACCAACAAGA 614
|||||||||| || ||| || ||||| ||||| || ||||| ||||| || || |||
Db 562 ACTCTTGGGTTCATTCCAAATTTGCTAATCCTGATAAAAGGATTTTCTTCACAAATAAGT 621
Qy 615 GCTACCTGCCTTCTCAGACCCCTTCTGGTGTGATTAGACTGAGAGAGGGTAGAACCAGGA 674
|| | || ||||| ||||| ||| || ||||| | |||||||| ||||| | |
Db 622 CATATTTACCATCTCAAACCCCAAGTGGAGTAATTAGGTTAAGAGAGGGGAGAACTCGTA 681
Qy 675 CCTTGAGAGGTGATGGTGTTGGTGAGAGGAAGGTGTTCGAGAGGATCTACGATTACGACG 734
| || ||||||||||| ||||| || || || || || |||||||| || ||||| || |
Db 682 CATTAAGAGGTGATGGAGTTGGAGAAAGAAAAGTATTTGAGAGGATTTATGATTATGATG 741
Qy 735 TGTACAACGATCTTGGCGAGGTGGTGAGCAACAACGATGATGCTAAGAGGCCTATCCTTG 794
| || || ||||| || || || || || || || ||||||||||| || || || ||||
Db 742 TTTATAATGATCTCGGAGAAGTCGTTAGTAATAATGATGATGCTAAAAGACCAATACTTG 801
Qy 795 GCGGTAAGAAGCTGCCTTATCCTAGAAGATGCAGGACTGGTAGGCAGCGGTCTAAAAAGG 854
| || || || | || ||||||||||| || || || ||| | || | |||||| |
Db 802 GTGGAAAAAAATTACCATATCCTAGAAGGTGTAGAACCGGTCGACAAAGAAGTAAAAAAG 861
Qy 855 ATCCTCTGTACGAGACTCGGAGCACCTTCGTTTATGTGCCTAGAGATGAGGCTTTCAGCG 914
|||| | || || || |||| ||||| || ||||| || |||||||| || || |
Db 862 ATCCATTATATGAAACAAGGAGTACCTTTGTGTATGTACCAAGAGATGAAGCATTTTCAG 921
Qy 915 CCGTGAAGTCTCTTACCTTCTCTGGTAACACCGTGTACTCTGCTCTGCATGCTGTTGTGC 974
| |||||| | | || ||||| || |||||||| ||||| || || || || || ||||
Db 922 CAGTGAAGAGTTTAACATTCTCCGGCAACACCGTTTACTCCGCCCTACACGCGGTGGTGC 981
Qy 975 CTGCTCTTGAGTCTGTTGTGTCTGATCCTGATCTGGGCTTCCCTCACTTCCCTGCTATTG 1034
| || | || || || || || ||||| || || || || || || || || || ||||
Db 982 CGGCATTGGAATCAGTCGTATCCGATCCCGACCTAGGGTTTCCACATTTTCCGGCCATTG 1041
Qy 1035 ACTCTCTTTTCAACGTGGGCGTTGACCTGTCTGGCCTGTCTGATAAGAAGTCCAGCCTGT 1094
|||| |||||||| ||||| || || || || || || |||||| || ||||| |
Db 1042 ACTCGCTTTTCAATGTGGGTGTCGATCTTTCAGGACTTAGTGATAAAAAAAGTAGCCTTT 1101
Qy 1095 TCAACATCGTGCCGAGGCTGATCAAGAGCATCTCTGAGACTGGTAAGGACGTGCTGCTTT 1154
| |||||||| || ||||| || ||| || ||||| || || || || || |||| |
Db 1102 TTAACATCGTACCAAGGCTTATTAAGTCTATTTCTGAAACCGGAAAAGATGTCTTGCTCT 1161
Qy 1155 TCGAGTCTCCTCAGCTTGTTCAGCGGGACAAGTTCTCATGGTTTCGGGATGTTGAGTTCG 1214
| || || ||||| | || || ||||||| || || |||||| | ||||| || || |
Db 1162 TTGAATCCCCTCAATTGGTCCAAAGGGACAAATTTTCTTGGTTTAGAGATGTGGAATTTG 1221
Qy 1215 CTAGGCAGACTCTTGCTGGTCTGAACCCTTACTCTATTAGGCTTGTGACCGAGTGGCCTC 1274
|||| || || | |||||| |||| || || ||| | | || || || |||||
Db 1222 CTAGACAAACCTTAGCTGGTTTGAATCCATATAGTATCCGATTGGTTACGGAATGGCCAT 1281
Qy 1275 TGAGGTCTAACTTGGATCCTAAGGTTTCAGGCCCTCCTGAGAGCGAGATTACCAAAGAGC 1334
||||| || | || |||||||| || || ||||| || || || || |||||||
Db 1282 TGAGGAGCAATCTAGACCCTAAGGTGTCTGGACCTCCAGAATCAGAAATCACAAAAGAGC 1341
Qy 1335 TTATCGAGAACGAGATCGGCAACAACATGACCGTTGAGCAAGCAGTGCAGCAGAAGAAGC 1394
|||| ||||| || || || ||||| ||||| ||||| || || || || || ||||||
Db 1342 TTATTGAGAATGAAATTGGAAACAATATGACTGTTGAACAGGCCGTTCAACAAAAGAAGT 1401
Qy 1395 TGTTCATCCTGGATTACCACGATCTGCTGCTGCCGTACGTTAACAAGGTGAACGAGCTTA 1454
|||||||||| ||||| || ||| || | |||| || || ||||| ||||| || || |
Db 1402 TGTTCATCCTCGATTATCATGATTTGTTATTGCCATATGTGAACAAAGTGAATGAACTCA 1461
Qy 1455 AGGGCAGCGTGTTGTACGGTTCTCGGACTATCTTCTTCCTGACTCCTCACGGAACCCTTA 1514
| || || || || || || | ||||| |||||| |||||||||| || || | |
Db 1462 AAGGGTCTGTATTATATGGATCAAGAACTATATTCTTCTTGACTCCTCATGGCACATTGA 1521
Qy 1515 AGCCTCTTGCTATTGAGCTTACCAGGCCTCCTATCGATGATAAGCCGCAGTGGAAAGAGG 1574
| ||| | || |||||||| || ||||| || || |||||||| || || ||||| || |
Db 1522 AACCTTTGGCCATTGAGCTAACTAGGCCACCAATAGATGATAAACCTCAATGGAAGGAAG 1581
Qy 1575 TGTACAGCCCTAACAATTGGAACGCTACTGGTGCTTGGCTTTGGAAGCTTGCTAAGGCTC 1634
| || ||| || |||||||| || ||||| |||||||| ||||| | ||||| ||||
Db 1582 TTTATTCCCCAAATAATTGGAATGCCACTGGGGCTTGGCTATGGAAATTGGCTAAAGCTC 1641
Qy 1635 ATGTGCTGAGCCACGATTCTGGTTACCATCAGCTTGTGTCTCACTGGCTTAGGACTCATT 1694
|||| || || || || || || ||||| || || ||||||||| || |||||||
Db 1642 ATGTTCTTTCTCATGACTCCGGATATCATCAACTAGTCAGTCACTGGCTAAGAACTCATT 1701
Qy 1695 GCTGTACCGAGCCTTACATTATCGCCACCAACAGGCAGCTTTCTGCTATGCATCCTATCT 1754
| ||||| || || |||||||| || || || || || | ||| |||||||| || |
Db 1702 GTTGTACAGAACCATACATTATTGCAACAAATAGACAATTAAGTGCAATGCATCCAATAT 1761
Qy 1755 ACAGGCTGCTGCATCCTCACTTCAGGTACACCATGGAAATCAACGCTCTGGCTAGGGAAG 1814
| || || |||||||||| ||||| ||||| ||||| || || || | ||||| ||||
Db 1762 ATAGATTGTTGCATCCTCATTTCAGATACACAATGGAGATAAATGCCTTAGCTAGAGAAG 1821
Qy 1815 CTCTGATCAACGCCAACGGTATTATCGAGTCCTCATTCTTCCCGGGCAAGTACAGCGTTG 1874
| || || || || || ||||| || ||| ||||| ||||| |||||||| ||||
Db 1822 CACTTATTAATGCTAATGGTATCATTGAGAGTTCATTTTTCCCAGGCAAGTATTCAGTTG 1881
Qy 1875 AGCTGTCCTCTATTGCTTACGGTGCTGAGTGGCGTTTCGATCAAGAAGCTTTGCCTCAGA 1934
|| || |||||||| || |||||||| ||| | || || ||||| || | || || |
Db 1882 AGTTGAGTTCTATTGCCTATGGTGCTGAATGGAGATTTGACCAAGAGGCACTCCCACAAA 1941
Qy 1935 ACCTGATCAGCAGAGGTCTTGCTGAGGAAGATCCTAACGAGCCTCACGGTCTTAAGCTGG 1994
|||| || || || || | || ||||||||||| || || || || || | || || |
Db 1942 ACCTTATTAGTAGGGGATTGGCAGAGGAAGATCCAAATGAACCACATGGCTTGAAACTAG 2001
Qy 1995 CTATTGAGGATTACCCTTTCGCTAACGATGGTCTGGTGCTGTGGGATATTCTTAAGCAGT 2054
| || || ||||||||||| ||||| |||||| | || || ||||| || ||||| || |
Db 2002 CAATAGAAGATTACCCTTTTGCTAATGATGGTTTAGTACTTTGGGACATACTTAAACAAT 2061
Qy 2055 GGGTGACCAACTACGTGAACCACTACTACCCTCAGACCAACCTGATCGAGAGCGACAAAG 2114
|||| || || || || ||||| || || || || || || || || || || ||||
Db 2062 GGGTAACAAATTATGTAAACCATTATTATCCACAAACAAATCTCATTGAATCTGATAAAG 2121
Qy 2115 AATTGCAGGCTTGGTGGTCCGAGATCAAGAATGTTGGTCACGGCGACAAGAAAGACGAAC 2174
|| | || ||||||||||| ||||| || |||||||| || || ||||||||||| || |
Db 2122 AACTCCAAGCTTGGTGGTCAGAGATTAAAAATGTTGGACATGGTGACAAGAAAGATGAGC 2181
Qy 2175 CTTGGTGGCCTGAACTTAAGACCCCTAACGATCTGATCGGCATCATCACCACTATCGTCT 2234
| |||||||| || | || || || || || | || || || ||||| || || || |
Db 2182 CATGGTGGCCAGAGTTAAAAACTCCAAATGACTTAATTGGTATTATCACAACAATAGTTT 2241
Qy 2235 GGGTTACCTCTGGTCATCATGCTGCTGTGAACTTCGGCCAGTACTCTTACGGTGGTTACT 2294
|||| || ||||| |||||||| ||||| ||||| ||||| || || || || ||||
Db 2242 GGGTAACTTCTGGCCATCATGCAGCTGTTAACTTTGGCCAATATAGCTATGGAGGCTACT 2301
Qy 2295 TCCCTAATAGGCCTACCACCGCTAGGTCTAAGATGCCTACTGAGGATCCTACTGCTGAAG 2354
| || |||||||| || || |||||||| || ||||| ||||||||||| || ||||| |
Db 2302 TTCCAAATAGGCCAACAACTGCTAGGTCAAAAATGCCAACTGAGGATCCAACAGCTGAGG 2361
Qy 2355 AGTGGGAGTGGTTCCTTAACAAGCCTGAAGAGGCTCTGCTGCGGTGTTTCCCATCTCAAA 2414
| |||||||||||| | || || ||||| ||||| || || | || ||||| || ||||
Db 2362 AATGGGAGTGGTTCTTGAATAAACCTGAGGAGGCACTACTAAGATGCTTCCCTTCACAAA 2421
Qy 2415 TTCAGGCTACTAAGGTGATGACCATCCTCGACGTGCTGTCTAACCATTCTCCTGACGAAG 2474
|||| || || || |||||||| || | || || | || |||||||| || || ||||
Db 2422 TTCAAGCAACAAAAGTGATGACAATTTTGGATGTCTTATCAAACCATTCACCAGATGAAG 2481
Qy 2475 AGTACATCGGCGAGAAGATTGAGCCTTACTGGGCTGAAGATCCGGTGATTAACGCTGCTT 2534
| || || || || |||||||||||||| |||||||| ||||| ||||||||||| || |
Db 2482 AATATATTGGTGAAAAGATTGAGCCTTATTGGGCTGAGGATCCCGTGATTAACGCGGCGT 2541
Qy 2535 TCGAGGTGTTCTCCGGCAAGCTTAAAGAGCTTGAGGGCATCATCGACGCCCGGAACAATG 2594
|||||||||| || || || | ||||||||||| || || || || || | || ||||
Db 2542 TCGAGGTGTTTTCGGGGAAATTGAAAGAGCTTGAAGGGATTATTGATGCTAGAAATAATG 2601
Qy 2595 ATTCTAAGCTGAACAATAGGAACGGCGCTGGCGTTATGCCTTACGAGCTTCTTAAGCCTT 2654
|| |||| |||| ||||| || || ||||| ||||||||||| || | | || ||||
Db 2602 ATAGTAAGTTGAATAATAGAAATGGAGCTGGAGTTATGCCTTATGAATTGTTGAAACCTT 2661
Qy 2655 ACAGCGAGCCTGGTGTTACTGGAAAGGGTGTGCCATACAGCATCAGCATCTAGGCTTT 2712
| || ||||| ||||||||||| ||||| || || || || || | |||
Db 2662 ATTCTGAACCTGGAGTTACTGGAAAAGGTGTACCTTATAGTATTTCAATTTGATTTTT 2719
Moreover, the protein encoded by the SEQ ID NO: 27 is having 100% sequence identity to the protein encoded by the same TomLoxC gene as described by Shen et al. and GenBank accession no. U37839 (data not shown).
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to overexpress the polynucleotide sequences encoding acyl-activating enzyme (AAE1), olivetolic acid cyclase (OAC) and olivetol synthase (OLS) enzymes in a plant (as described by Thomas et al.), a polynucleotide encoding the ALT4 protein (as described by Kalinger et al.), a polynucleotide sequence encoding the PLIP1 protein (as described by Wang et al.) and a polynucleotide sequence encoding the TomLoxC protein (as described by Shen et al.).
Before the effective filing date of the invention, an ordinarily skilled artisan would have been motivated to overexpress the ALT4 gene encoding the ALT4 protein in the plant already overexpressing polynucleotide sequences encoding acyl-activating enzyme (AAE1), olivetolic acid cyclase (OAC) and olivetol synthase (OLS) enzymes to attract insect pollinators and/or to deter predatory insects, as described by Kalinger et al. It is also known in the art that ALT-type thioesterases (including ALT4) generate the ß-keto fatty acids (precursors of MKs) which are used in the manufacture of fragrances, flavorings, and diesel biofuels, and have insecticidal and antifungal properties, as described by Kalinger et al. (p.117, left column, para 4, line 1-3).
There is also a realistic expectation that overexpression of ALT4 protein would also increase the concentration of hexanoic acid (a C6 fatty acid) which is the precursor to produce CBGA in plants, as discussed above. Overexpressing the polynucleotide encoding PLASTID LIPASE1 (PLIP1) and TomLoxC enzymes would increase seed fatty acid content including (C6) hexanoic acid (as described by Wang et al. and Shen et al.) while overexpressing TomLoxC would improve the taste, as in fresh tomato as liked by consumers, as taught by Shen et al. Increased (C6) hexanoic acid content, due to overexpression of PLIP1 and TomLoxC, would have a realistic expectation to increase commercially important CBGA content in presence of overexpressing polynucleotide sequences encoding acyl-activating enzyme (AAE1), olivetolic acid cyclase (OAC) and olivetol synthase (OLS) enzymes in the plant.
Conclusion
No Claim is allowed.
Communication
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J.C.
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Supervisory Patent Examiner, Art Units 1661 & 1662
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