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 .
Status of Claims
Claims 1, 5-7, 10-12, 16-17, 19 & 21 are under examination on the merits.
Claim 15 is withdrawn without traverse.
The objections to the specification are withdrawn in light of Applicant’s amendments.
The objections to claims 5, 13 & 18 are withdrawn in light of Applicant’s amendments.
The rejection of claims 12-14 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5-7, 12-14, & 16-19 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, is withdrawn in light of Applicant’s amendments.
The rejection of claim(s) 16-18 under 35 U.S.C. 102(a)(1) as being anticipated by Evans et al WO 0011196 A1 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5, 10, 11, & 16-19 under 35 U.S.C. 103 as being unpatentable over Evans et al WO 0011196 A1 in view of UniProtKB reference DEF_PENBA is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5-7, 10-13 & 16-19 under 35 U.S.C. 103 as being unpatentable over Evans and UniProtKB reference DEF_PENBA in view of Yin et al (2009) Hereditas. 31(6): 663―667 and in view of NCBI GenBank reference AY262035.1 taken with the evidence of Nunez-Palenius et al (2008) Critical Reviews in Biotechnology. 28: 13-55. is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5-7, 10-14 & 16-19 under 35 U.S.C. 103 as being unpatentable over Evans, UniProtKB reference DEF_PENBA, Yin, and NCBI GenBank reference AY262035.1 and further in view of Wolf et al (US 2007/0220636 A1) is withdrawn in light of Applicant’s amendments.
The rejection of claim(s) 1, 5-7, 10-14 & 16-19 under 35 U.S.C. 103 as being unpatentable over Evans, UniProtKB reference DEF_PENBA, Yin, and NCBI GenBank reference AY262035.1, and further in view of Huang et al US 2023/0263121 A1 is withdrawn in light of Applicant’s amendments.
Claim Objections
Claims 1, 12, 16 & 19 are objected to because of the following informalities:
Claim 1 (line 4): "n-terminal" should read --N-terminal--.
Claim 12 (line 1): "according claim" should read --according to claim".
Claim 16 (line 5): "n-terminal" should read --N-terminal--.
Claim 19 (lines 2-3): "at at least" should read --at least--.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Improper Dependency
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 12 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Due to Applicant' s amendment of the claims, the rejection is modified from the rejection as set forth in the Office action mailed 11/14/2025, as applied to claim 18. Applicant' s arguments filed 5/14/2026 have been fully considered but they are not persuasive.
Claim 12 is drawn to a plant according to claim 7 wherein a progeny or an ancestor thereof produces the sweet protein. Limitations describing the progeny or ancestors of the plant does not further limit the plant itself.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Applicant urges that claim 18 has been canceled and requests withdrawal of the rejection (Remarks, page 6, paragraphs 5-6).
This argument is unpersuasive, because the amendments to claim 12 raise additional issues under 35 U.S.C. 112(d).
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.
Claim(s) 1, 5, 10-12, 16-17, 19 & 21 are rejected under 35 U.S.C. 103 as being unpatentable over Evans et al WO 0011196 A1 (published 3/2/2000, hereafter Evans) in view of UniProtKB reference DEF_PENBA (available 7/15/1998), Wolf et al (US 2007/0220636 A1, published 9/20/2007, hereafter Wolf), and Lamphear et al (2005) Plant Biotechnology Journal. 3: 103-114. (published 11/2/2004, hereafter Lamphear).
This is a new rejection necessitated by Applicant’s amendments. Applicant’s remarks filed 5/14/2026 have been considered fully as they pertain to the instant rejection, but they are not persuasive.
Claims 1, 5, 10-12, 16-17, 19 & 21 are drawn to a watermelon plant comprising a genomic transformation event which enables the plant to produce a non-native expression or concentration of a sweet protein and a biosynthetic method for producing a non-native sweet protein comprising combining a plant with a genomic transformation event.
Evans teaches a method of expressing brazzein in tomato by preparing vector constructs comprising a brazzein gene, fused with an upstream Dahlia merckii antimicrobial protein signal peptide or not, under control of a polyubiquitin promoter or polygalacturonase promoter and cloned into a plasmid comprising a terminator (page 19, lines 3-20, figures 9-11).
The codons were optimized for tomato expression (page 19, line 17). Evans teaches that the vectors were transferred to Agrobacterium tumefaciens LBA4404, which was used to transform tomato following standard protocols, which were regenerated up to 30 individual plants and grown to maturity (page 19, lines 22-30).
Presence of the transgenic construct in plants was confirmed by PCR and expression of Brazzein was confirmed in one plant with Northern blot (page 19, lines 27-30). Brazzein production in fruits was measured by ELISA and Western blot on total protein extract, which was extracted from a sample of the pericarp of each of the fruit (page 20, lines 1-14).
Evans envisions transformed plants of various species, including melon, and the progeny and seeds of such plants comprising a polynucleotide of the invention stably incorporated (page 6, lines 20-30).
Although Evans teaches that the gene encoding brazzein may be codon optimized, Evans does not teach the expression cassette comprises a nucleotide sequence encoding the sweet protein having a sequence identity of at least 96% sequence identity to SEQ ID NO: 30. Although Evans suggests the application in melon, Evans does not specifically teach the method in watermelon. Finally, Evans does not teach a signal peptide which is an N-terminal secretion signal operatively linked to a nucleotide sequence encoding the sweet protein.
UniProtKB reference DEF_PENBA teaches that the protein sequence of brazzein comprises a sequence that can be encoded by a sequence with 100% identity to instant SEQ ID NO: 30 and has 100% sequence identity to instant SEQ ID NO: 7. See alignments below.
DEF_PENBA
ID DEF_PENBA Reviewed; 54 AA.
AC P56552;
DT 15-JUL-1998, integrated into UniProtKB/Swiss-Prot.
DT 15-JUL-1998, sequence version 1.
DT 09-APR-2025, entry version 90.
DE RecName: Full=Defensin-like protein;
DE AltName: Full=Brazzein {ECO:0000303|PubMed:7957951};
OS Pentadiplandra brazzeana.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Pentadiplandraceae; Pentadiplandra.
OX NCBI_TaxID=43545;
RN [1]
RP PROTEIN SEQUENCE, AND PYROGLUTAMATE FORMATION AT GLN-1.
RC TISSUE=Fruit;
RX PubMed=7957951; DOI=10.1016/0014-5793(94)01184-2;
RA Ming D., Hellekant G.;
RT "Brazzein, a new high-potency thermostable sweet protein from
RT Pentadiplandra brazzeana B.";
RL FEBS Lett. 355:106-108(1994).
RN [2]
RP FUNCTION.
RX PubMed=15118082; DOI=10.1073/pnas.0401567101;
RA Yount N.Y., Yeaman M.R.;
RT "Multidimensional signatures in antimicrobial peptides.";
RL Proc. Natl. Acad. Sci. U.S.A. 101:7363-7368(2004).
RN [3]
RP STRUCTURE BY NMR.
RX PubMed=9628478; DOI=10.1038/nsb0698-427;
RA Caldwell J.E., Abildgaard F., Dzakula Z., Ming D., Hellekant G.,
RA Markley J.L.;
RT "Solution structure of the thermostable sweet-tasting protein brazzein.";
RL Nat. Struct. Biol. 5:427-431(1998).
CC -!- FUNCTION: Taste-modifying protein; sweet-tasting. It is 2000 sweeter
CC than sucrose on a molar basis. {ECO:0000269|PubMed:15118082}.
CC -!- FUNCTION: Has a pH-specific antimicrobial activity against bacteria
CC (B.subtilis, E.coli and S.aureus) and the fungus C.albicans.
CC {ECO:0000269|PubMed:15118082}.
CC -!- SUBUNIT: Monomer.
CC -!- SUBCELLULAR LOCATION: Secreted.
CC -!- SIMILARITY: Belongs to the DEFL family. {ECO:0000305}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
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DR PIR; S51208; S51208.
DR PDB; 1BRZ; NMR; -; A=2-54.
DR PDB; 2BRZ; NMR; -; A=2-54.
DR PDB; 2KGQ; NMR; -; A=2-54.
DR PDB; 2KYQ; NMR; -; A=3-54.
DR PDB; 2LY5; NMR; -; A=2-54.
DR PDB; 2LY6; NMR; -; A=2-54.
DR PDB; 2N66; NMR; -; A=1-54.
DR PDB; 2N69; NMR; -; A=1-54.
DR PDB; 4HE7; X-ray; 1.80 A; A=1-54.
DR PDB; 7W8E; X-ray; 1.34 A; A=1-54.
DR PDB; 7W8H; X-ray; 1.50 A; A/B/C/D/E/F/G/H=1-54.
DR PDBsum; 1BRZ; -.
DR PDBsum; 2BRZ; -.
DR PDBsum; 2KGQ; -.
DR PDBsum; 2KYQ; -.
DR PDBsum; 2LY5; -.
DR PDBsum; 2LY6; -.
DR PDBsum; 2N66; -.
DR PDBsum; 2N69; -.
DR PDBsum; 4HE7; -.
DR PDBsum; 7W8E; -.
DR PDBsum; 7W8H; -.
DR AlphaFoldDB; P56552; -.
DR BMRB; P56552; -.
DR SMR; P56552; -.
DR TCDB; 1.C.45.4.2; the plant defensin (plant defensin) family.
DR EvolutionaryTrace; P56552; -.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0042742; P:defense response to bacterium; IEA:UniProtKB-KW.
DR GO; GO:0050832; P:defense response to fungus; IEA:UniProtKB-KW.
DR GO; GO:0031640; P:killing of cells of another organism; IEA:UniProtKB-KW.
DR Gene3D; 3.30.30.10; Knottin, scorpion toxin-like; 1.
DR InterPro; IPR036574; Scorpion_toxin-like_sf.
DR SUPFAM; SSF57095; Scorpion toxin-like; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antibiotic; Antimicrobial; Direct protein sequencing;
KW Disulfide bond; Fungicide; Pyrrolidone carboxylic acid; Secreted;
KW Taste-modifying protein.
FT CHAIN 1..54
FT /note="Defensin-like protein"
FT /id="PRO_0000221427"
FT MOD_RES 1
FT /note="Pyrrolidone carboxylic acid"
FT /evidence="ECO:0000269|PubMed:7957951"
FT DISULFID 4..52
FT DISULFID 16..37
FT DISULFID 22..47
FT DISULFID 26..49
FT STRAND 4..7
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 13..17
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 21..30
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 33..39
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 41..43
FT /evidence="ECO:0007829|PDB:2BRZ"
FT STRAND 45..50
FT /evidence="ECO:0007829|PDB:7W8E"
SQ SEQUENCE 54 AA; 6498 MW; 5BAB4D7215291252 CRC64;
Length: 54
Score: 315.00 Matches: 53
Percent Similarity: 100.0% Conservative: 0
Best Local Similarity: 100.0% Mismatches: 0
Query Match: 100.0% Indels: 0
Gaps: 0
US-18-565-025A-30 (1-159) x DEF_PENBA (1-54)
Qy 1 GATAAGTGTAAGAAGGTTTATGAAAATTATCCTGTTTCTAAGTGTCAACTTGCTAATCAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 AspLysCysLysLysValTyrGluAsnTyrProValSerLysCysGlnLeuAlaAsnGln 21
Qy 61 TGTAATTATGATTGTAAGCTTGATAAGCATGCTAGATCTGGAGAATGTTTTTATGATGAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 22 CysAsnTyrAspCysLysLeuAspLysHisAlaArgSerGlyGluCysPheTyrAspGlu 41
Qy 121 AAGAGAAATCTTCAATGTATTTGTGATTATTGTGAATAT 159
|||||||||||||||||||||||||||||||||||||||
Db 42 LysArgAsnLeuGlnCysIleCysAspTyrCysGluTyr 54
DEF_PENBA
ID DEF_PENBA Reviewed; 54 AA.
AC P56552;
DT 15-JUL-1998, integrated into UniProtKB/Swiss-Prot.
DT 15-JUL-1998, sequence version 1.
DT 09-APR-2025, entry version 90.
DE RecName: Full=Defensin-like protein;
DE AltName: Full=Brazzein {ECO:0000303|PubMed:7957951};
OS Pentadiplandra brazzeana.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Pentadiplandraceae; Pentadiplandra.
OX NCBI_TaxID=43545;
RN [1]
RP PROTEIN SEQUENCE, AND PYROGLUTAMATE FORMATION AT GLN-1.
RC TISSUE=Fruit;
RX PubMed=7957951; DOI=10.1016/0014-5793(94)01184-2;
RA Ming D., Hellekant G.;
RT "Brazzein, a new high-potency thermostable sweet protein from
RT Pentadiplandra brazzeana B.";
RL FEBS Lett. 355:106-108(1994).
RN [2]
RP FUNCTION.
RX PubMed=15118082; DOI=10.1073/pnas.0401567101;
RA Yount N.Y., Yeaman M.R.;
RT "Multidimensional signatures in antimicrobial peptides.";
RL Proc. Natl. Acad. Sci. U.S.A. 101:7363-7368(2004).
RN [3]
RP STRUCTURE BY NMR.
RX PubMed=9628478; DOI=10.1038/nsb0698-427;
RA Caldwell J.E., Abildgaard F., Dzakula Z., Ming D., Hellekant G.,
RA Markley J.L.;
RT "Solution structure of the thermostable sweet-tasting protein brazzein.";
RL Nat. Struct. Biol. 5:427-431(1998).
CC -!- FUNCTION: Taste-modifying protein; sweet-tasting. It is 2000 sweeter
CC than sucrose on a molar basis. {ECO:0000269|PubMed:15118082}.
CC -!- FUNCTION: Has a pH-specific antimicrobial activity against bacteria
CC (B.subtilis, E.coli and S.aureus) and the fungus C.albicans.
CC {ECO:0000269|PubMed:15118082}.
CC -!- SUBUNIT: Monomer.
CC -!- SUBCELLULAR LOCATION: Secreted.
CC -!- SIMILARITY: Belongs to the DEFL family. {ECO:0000305}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR PIR; S51208; S51208.
DR PDB; 1BRZ; NMR; -; A=2-54.
DR PDB; 2BRZ; NMR; -; A=2-54.
DR PDB; 2KGQ; NMR; -; A=2-54.
DR PDB; 2KYQ; NMR; -; A=3-54.
DR PDB; 2LY5; NMR; -; A=2-54.
DR PDB; 2LY6; NMR; -; A=2-54.
DR PDB; 2N66; NMR; -; A=1-54.
DR PDB; 2N69; NMR; -; A=1-54.
DR PDB; 4HE7; X-ray; 1.80 A; A=1-54.
DR PDB; 7W8E; X-ray; 1.34 A; A=1-54.
DR PDB; 7W8H; X-ray; 1.50 A; A/B/C/D/E/F/G/H=1-54.
DR PDBsum; 1BRZ; -.
DR PDBsum; 2BRZ; -.
DR PDBsum; 2KGQ; -.
DR PDBsum; 2KYQ; -.
DR PDBsum; 2LY5; -.
DR PDBsum; 2LY6; -.
DR PDBsum; 2N66; -.
DR PDBsum; 2N69; -.
DR PDBsum; 4HE7; -.
DR PDBsum; 7W8E; -.
DR PDBsum; 7W8H; -.
DR AlphaFoldDB; P56552; -.
DR BMRB; P56552; -.
DR SMR; P56552; -.
DR TCDB; 1.C.45.4.2; the plant defensin (plant defensin) family.
DR EvolutionaryTrace; P56552; -.
DR GO; GO:0005576; C:extracellular region; IEA:UniProtKB-SubCell.
DR GO; GO:0042742; P:defense response to bacterium; IEA:UniProtKB-KW.
DR GO; GO:0050832; P:defense response to fungus; IEA:UniProtKB-KW.
DR GO; GO:0031640; P:killing of cells of another organism; IEA:UniProtKB-KW.
DR Gene3D; 3.30.30.10; Knottin, scorpion toxin-like; 1.
DR InterPro; IPR036574; Scorpion_toxin-like_sf.
DR SUPFAM; SSF57095; Scorpion toxin-like; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Antibiotic; Antimicrobial; Direct protein sequencing;
KW Disulfide bond; Fungicide; Pyrrolidone carboxylic acid; Secreted;
KW Taste-modifying protein.
FT CHAIN 1..54
FT /note="Defensin-like protein"
FT /id="PRO_0000221427"
FT MOD_RES 1
FT /note="Pyrrolidone carboxylic acid"
FT /evidence="ECO:0000269|PubMed:7957951"
FT DISULFID 4..52
FT DISULFID 16..37
FT DISULFID 22..47
FT DISULFID 26..49
FT STRAND 4..7
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 13..17
FT /evidence="ECO:0007829|PDB:7W8E"
FT HELIX 21..30
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 33..39
FT /evidence="ECO:0007829|PDB:7W8E"
FT STRAND 41..43
FT /evidence="ECO:0007829|PDB:2BRZ"
FT STRAND 45..50
FT /evidence="ECO:0007829|PDB:7W8E"
SQ SEQUENCE 54 AA; 6498 MW; 5BAB4D7215291252 CRC64;
Query Match 100.0%; Score 315; Length 54;
Best Local Similarity 100.0%;
Matches 53; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY 53
|||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 2 DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY 54
Lamphear teaches that availability of foods low in sugar and high in flavor is important to millions of individuals for diabetic or dietetic purposes (abstract). Lamphear teaches that commercial production of sweet proteins is limited because natural sources for the proteins are tropical plant species that are difficult to cultivate (page 103, left column, paragraph 1).
Lamphear teaches a method of expressing brazzein polypeptides in maize (page 110, right column, paragraph 3-page 111, right column, paragraph 1). Lamphear teaches vectors for expressing brazzein comprising a promoter, a terminator, and a barley alpha-amylase signal sequence (figure 1). Only low expression was detected in lines in which brazzein expression was targeted to the cytoplasm (page 105, left column, paragraph 1), while the highest brazzein-expressing lines used a plant signal sequence fused to the amino-terminal end of the brazzein to direct the protein to the plant cell wall (page 106, right column, paragraph 2). Sequencing of the N-terminal amino acid sequence of purified products showed that the signal sequence was processed accurately to remove the signal peptide (page 107, left column, paragraph 1).
Wolf teaches a motivation to modify watermelon sweet characteristics, because watermelon fruit quality depends on sweetness (paragraph [0002]). However, elevating total sugar content in watermelon fruit is not desirable because people consume large portions of watermelon; it would be highly advantageous to have watermelon varieties producing fruits that are sweeter yet contain the same amount or fewer calories compared to current watermelon fruit (paragraphs [0007-0008]). The method of Wolf does not involve genetic modification, but Wolf does teach that transformation of watermelon for the addition or deletion of traits is encompassed (paragraph [0020, 0053]).
Before the time of filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Evans to use a nucleotide sequence capable of encoding the brazzein protein taught by UniProtKB reference DEF_PENBA. One of ordinary skill in the art would have been motivated to use a nucleotide sequence codon optimized in another plant species, because although Evans used a sequence codon optimized for tomato, transformation of other species was suggested. One of ordinary skill in the art would have had reasonable expectation of success substituting one nucleic acid sequence for another, because codon optimization was routine prior to the filing of the instant application and the encoded protein would comprise an identical sequence.
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Evans to substitute watermelon for tomato. One of ordinary skill in the art would have been motivated to introduce a brazzein encoding gene into watermelon in order to create sweeter watermelon without increasing calories. One of ordinary skill in the art would have had reasonable expectation of success, because Wolf teaches that transformation of watermelon is a method to improve sweetness and Evans suggests application of the method in melon.
Finally, before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Evans to use an N-terminal secretion signal operatively linked to the nucleotide sequence encoding the sweet protein instead of a Dahlia antimicrobial protein signal peptide. One of ordinary skill in the art would have been motivated to use a N-terminal secretion signal because constructs comprising the barley alpha-amylase signal sequence had highest brazzein expression. One of ordinary skill in the art would have had reasonable expectation of success, because both tomato and maize brazzein expression systems had successfully used signal peptides fused to the brazzein peptide.
Evans’ method reads on combining a plant with a genomic transformation event that enables the plant to produce a non-native expression of the sweet protein, forming a genetically modified plant and growing and regenerating a population of the genetically modified plant (instant claim 16). Confirming brazzein production by PCR, Northern blot, ELISA and/or Western blot reads on selecting the genetically modified plants that produce the sweet protein and harvesting the sweet protein (instant claim 16). Evans’ method also reads on preparing plasmids comprising an expression cassette which expresses the sweet protein and comprises nucleotide sequences encoding the protein, transforming a host cell with the plasmids, and transfecting the plant with a plurality of the transformed host cell (instant claim 17). Codon optimizing to arrive at a nucleotide sequence with at least 97% sequence identity to instant SEQ ID NO: 30 would be obvious (instant claim 19).
Moreover, the plant comprising a genomic transformation event to produce a non-native expression of a sweet protein comprising an expression cassette comprising a sequence of at least 96% or at least 97% sequence identity to SEQ ID NO: 30, including wherein the expression cassette comprises an N-terminal secretion signal and promoter, or terminator, would be obvious because the expression cassettes of Evans comprise these regulatory sequences (instant claims 1 & 5). Such a plant would express a sweet protein comprising an amino acid sequence having at least 90% sequence identity to instant SEQ ID NO: 7 (claims 10 & 21). Plant parts of the plant, including fruits comprising the sweet protein (instant claim 11), would likewise be obvious. Claim 12 is drawn to the plant, not a progeny or an ancestor, and so is obvious as presented above.
Claims 1, 5, 10-12, 16-17, 19 & 21 are obvious over Evans, UniProtKB reference DEF_PENBA, Wolf, and Lamphear.
Applicant urges that amended claims require a nucleotide sequence encoding an N-terminal secretion signal operatively linked to a nucleotide sequence encoding the sweet protein. Applicant urges that the claims are directed to the surprising discovery that a brazzein can not only be expressed but can be secreted out of the watermelon cells. Applicant urges that one of ordinary skill in the art would not have motivation to nor reasonable expectation of success in reaching the instant claims (Remarks, page 8, paragraphs 3-4).
This argument is unpersuasive, because Lamphear teaches that an N-terminal secretion signal for transgenically expressing brazzein in maize leads to higher expression. One of ordinary skill in the art would have been motivated to use an N-terminal secretion signal for the expression of brazzein in another plant system, and one of ordinary skill in the art would have had reasonable expectation of success, because signal peptides had been used in conjunction with brazzein in other systems such as tomato. Moreover, Evans envisions the system of brazzein expression in melon, and Wolf teaches motivation to increase sweetness in watermelon specifically. The limitations of the instant inventions would therefore have been obvious over the prior art.
Claim(s) 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Evans, UniProtKB reference DEF_PENBA, Wolf, and Lamphear as applied to claims 1, 5, 10-12, 16-17, 19 & 21 above, and further in view of Yin et al (2009) Hereditas. 31(6): 663―667 (published June, 2009, hereafter Yin, uploaded with machine translation appended) and in view of NCBI GenBank reference AY262035.1, available 5/24/2003.
Claims 6-7 are drawn to the plant wherein the regulatory sequences have a sequence identity of at least 70% to SEQ ID NOs: 1-6 & 31-36.
The teachings of Evans, UniProtKB reference DEF_PENBA, Wolf, and Lamphear are presented above. They do not teach a regulatory sequence having an identity of at least 70% to instant SEQ ID NOs: 1-6 or 31-36.
Yin teaches the transformation of tomato with a gene encoding brazzein under the control of the ADP-glucose pyrophosphorylase large subunit 1 promoter from watermelon (page 664, left column, paragraph 4-right column, paragraph 3). Yin teaches that the promoter is expressed in tomato fruit (page 664, left column, paragraph 3 of translation). Yin teaches a motivation to use a fruit specific promoter, because constitutive expression leads to large consumption of nutrients within the plant (page 667, left column, first paragraph of the translation).
Yin teaches that brazzein is high in sweetness, highly water soluble, and can retain sweetness after being treated, making it one of the most promising sweet protein for applications (page 664, left column, paragraph 2 of translation). Yin teaches that sweet protein is both sweet and low in calories, so expressing sweet protein genes can improve flavor (page 666, right column, paragraph 2 of translation).
NCBI GenBank reference AY262035.1 teaches a promoter region for a watermelon ADP-glucose pyrophosphorylase large subunit with 100% sequence identity to 97% of instant SEQ ID NO: 5. See alignment below.
Citrullus lanatus ADP-glucose pyrophosphorylase large subunit (wml1) gene, promoter region
Sequence ID: AY262035.1Length: 1573Number of Matches: 1
Range 1: 276 to 1573GenBankGraphicsNext MatchPrevious Match
Alignment statistics for match #1
Score
Expect
Identities
Gaps
Strand
2398 bits(1298)
0.0
1298/1298(100%)
0/1298(0%)
Plus/Plus
Query 1 GTTAATATGTAAAATCGATAATGCAAAAATATTTTAACTATTTATTTTGAGATGAAATAA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 276 GTTAATATGTAAAATCGATAATGCAAAAATATTTTAACTATTTATTTTGAGATGAAATAA 335
Query 61 AGTAGGAGCGATCTTAATCTTCCAATTGTAAAAACATACGTACACATCTAAATTTTATAC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 336 AGTAGGAGCGATCTTAATCTTCCAATTGTAAAAACATACGTACACATCTAAATTTTATAC 395
Query 121 TTAAGGAGGTGTTTGGGCGTGACTTTAAATGGGTGGGGTAAACTATCATAGCTCACTCCA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 396 TTAAGGAGGTGTTTGGGCGTGACTTTAAATGGGTGGGGTAAACTATCATAGCTCACTCCA 455
Query 181 TGTTTAAGAATGTAGTTATAGTAATTGGTGTTTCCAACTATTATAATTCACAATTAACTA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 456 TGTTTAAGAATGTAGTTATAGTAATTGGTGTTTCCAACTATTATAATTCACAATTAACTA 515
Query 241 CCGTATTACTTGTTACAATATTTACTATTTTCCACCTATCTTCTCTTCCCCTAATTGTTA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 516 CCGTATTACTTGTTACAATATTTACTATTTTCCACCTATCTTCTCTTCCCCTAATTGTTA 575
Query 301 TAGTGTTTATTATTACTTAGACTAAAATAGTATGCACCTTATACATGCAGAAGTAGAATA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 576 TAGTGTTTATTATTACTTAGACTAAAATAGTATGCACCTTATACATGCAGAAGTAGAATA 635
Query 361 GTAATATAGTTTAAAACTACATTGGTCCAGTATCAAGAAGATATCTTAATTTTATTTAGG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 636 GTAATATAGTTTAAAACTACATTGGTCCAGTATCAAGAAGATATCTTAATTTTATTTAGG 695
Query 421 AATTTAATAATTGGGTGTGGTTTTGTAAACATAAAAATAAAGAAAGCTATAAACAGAGGT 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 696 AATTTAATAATTGGGTGTGGTTTTGTAAACATAAAAATAAAGAAAGCTATAAACAGAGGT 755
Query 481 CCTAGGATGCAGATTGGTTACAGAATTCAGTGCCCCAGGAAGTAGCACCAATTTTCATCA 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 756 CCTAGGATGCAGATTGGTTACAGAATTCAGTGCCCCAGGAAGTAGCACCAATTTTCATCA 815
Query 541 TTTATTTAAAAAGTATATTTTGAAGAAATATTATTGTGAAATAGTTTTTGAATTTGTTGC 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 816 TTTATTTAAAAAGTATATTTTGAAGAAATATTATTGTGAAATAGTTTTTGAATTTGTTGC 875
Query 601 GTGCTTGGTGGAAGTGGGCAACACAAATACATGGGTGCCCATCCAAGTATTGCCTTGTTT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 876 GTGCTTGGTGGAAGTGGGCAACACAAATACATGGGTGCCCATCCAAGTATTGCCTTGTTT 935
Query 661 GTTTGTTTGTATGCGGCGTAGAAATAGCGGATGATCGTTGGAAATTGAGTTTTGTAGGAA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 936 GTTTGTTTGTATGCGGCGTAGAAATAGCGGATGATCGTTGGAAATTGAGTTTTGTAGGAA 995
Query 721 TAGCaaaaaaagaaagaaagaaatatgaagaagatgaagatgtaaatgaggaagaagaag 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 996 TAGCAAAAAAAGAAAGAAAGAAATATGAAGAAGATGAAGATGTAAATGAGGAAGAAGAAG 1055
Query 781 aaCCATTTGCTGACATGAATGAACCTTTCCCACTTTCTTGTTTTTTACTATAAATCAATC 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1056 AACCATTTGCTGACATGAATGAACCTTTCCCACTTTCTTGTTTTTTACTATAAATCAATC 1115
Query 841 CTCGTGAATGAAAACGCCTTACATTCACATGCCCATTAAGCATTAATCCCCTTTCTCCAC 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1116 CTCGTGAATGAAAACGCCTTACATTCACATGCCCATTAAGCATTAATCCCCTTTCTCCAC 1175
Query 901 CGCcttcttcatcatcactcacatttgtcactctcttttcctctttctctcttcgtcttc 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1176 CGCCTTCTTCATCATCACTCACATTTGTCACTCTCTTTTCCTCTTTCTCTCTTCGTCTTC 1235
Query 961 ttcCCCCATTTCCAACGCTTCTACCTTTGATTCGTTTCCCCTTTGTAAGTTTTCGATTTC 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1236 TTCCCCCATTTCCAACGCTTCTACCTTTGATTCGTTTCCCCTTTGTAAGTTTTCGATTTC 1295
Query 1021 TTCTGCTTTCCTTTCTGGGATTTCTTATTTGCATCATTTACTTTTCTGGGTGTCTATTAT 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1296 TTCTGCTTTCCTTTCTGGGATTTCTTATTTGCATCATTTACTTTTCTGGGTGTCTATTAT 1355
Query 1081 TTTATTGTATTAGAGCTTTGTCAGATGATTTCTTGTATTTGTTTAGCTACCCCTTTTGCT 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1356 TTTATTGTATTAGAGCTTTGTCAGATGATTTCTTGTATTTGTTTAGCTACCCCTTTTGCT 1415
Query 1141 TTTTTCTGTTCTTGGTGTGATCTGTACTCTATATGGTTGCTTGGATTGGGGCTTTTGCTT 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1416 TTTTTCTGTTCTTGGTGTGATCTGTACTCTATATGGTTGCTTGGATTGGGGCTTTTGCTT 1475
Query 1201 TTTCTTATTGGGATTTGAGCTGGGGGTGGGGCTATTAGATTAGATTGTAATTTGTGCCAT 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sbjct 1476 TTTCTTATTGGGATTTGAGCTGGGGGTGGGGCTATTAGATTAGATTGTAATTTGTGCCAT 1535
Query 1261 TTCTGATGCAtttttttttttttCCAAGGGAGAGAGTT 1298
||||||||||||||||||||||||||||||||||||||
Sbjct 1536 TTCTGATGCATTTTTTTTTTTTTCCAAGGGAGAGAGTT 1573
Before the filing of the instant application, one of ordinary skill in the art would have been motivated to modify the method and plants taught by Evans to substitute the constitutive promoters with a watermelon AGPL1 promoter as taught by Yin and NCBI GenBank reference AY262035.1. One of ordinary skill in the art would have been motivated to substitute the fruit specific promoter for a constitutive promoter, because constitutive promoters can lead to large consumption of nutrients within the plant. One of ordinary skill in the art would have had reasonable expectation of success, because Evans suggests the method in melon, and the AGPL1 promoter is a watermelon promoter.
In light of Evans, UniProtKB reference DEF_PENBA, Wolf, Lamphear, Yin and NCBI GenBank reference AY262035.1, the plant comprising the expression cassette with the regulatory sequence with >70% sequence identity to instant SEQ ID NO: 5, wherein the cassette comprises a promoter operably linked with the nucleotide sequence encoding the protein would be obvious (instant claims 6-7). Claims 1, 5-7, 10-12, 16-17, 19 & 21 are thus obvious.
Claim(s) 1, 5, 10-12, 16-17, 19 & 21 are rejected under 35 U.S.C. 103 as being unpatentable over Evans et al WO 0011196 A1 (published 3/2/2000, hereafter Evans) in view of UniProtKB reference DEF_PENBA (available 7/15/1998), Huang et al US 2023/0263121 A1 (published 8/24/2023 but with an effective filing date of 3/30/2021, prior to the effective filing date of the instant application), and Lamphear et al (2005) Plant Biotechnology Journal. 3: 103-114. (published 11/2/2004, hereafter Lamphear).
The applied reference Huang has a common inventor with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
This is a new rejection necessitated by Applicant’s amendments. Applicant’s remarks filed 5/14/2026 have been considered fully as they pertain to the instant rejection, but they are not persuasive.
The teachings of Evans are presented above. Although Evans teaches that the gene encoding brazzein may be codon optimized, Evans does not teach the expression cassette comprises a nucleotide sequence encoding the sweet protein having a sequence identity of at least 96% sequence identity to SEQ ID NO: 30. Although Evans suggests the application in melon, Evans does not specifically teach the method in watermelon. Finally, Evans does not teach a signal peptide which is an N-terminal secretion signal operatively linked to a nucleotide sequence encoding the sweet protein.
The teachings of UniProtKB reference DEF_PENBA and Lamphear are also presented above.
Huang teaches modification of watermelon by genome editing by introducing an expression construct that used a cassava vein mosaic virus promoter operably linked to a mogroside gene homolog to increase expression of the homologs of the mogroside synthesis pathway in watermelon (paragraphs [0289-0297]). Huang teaches that mogrosides are used as a natural sweetener in China (paragraph [0004]).
Huang teaches a motivation for increasing sweetness in watermelon, because people like sweetness but non-sugar sweeteners can avoid the risks of obesity, diabetes, metabolic syndrome and cardiovascular disease from sugar (paragraph [0004]). However, Huang also teaches that mogrosides are from monk fruit, which has high production cost and poor productivity, and so production of the mogroside sweetener in widely adapted plant systems would be beneficial (paragraph [0006]). Huang mentions brazzein as another sweetener (paragraph [0235]).
Before the filing date of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of Evans to substitute watermelon for tomato. One of ordinary skill in the art would have been motivated to transform watermelon to express the sweet protein brazzein, because Huang teaches watermelon is a widely adapted plant system beneficial for the production of an alternative sweetener. One of ordinary skill in the art would have had reasonable expectation of success, because Huang successfully introduced nucleotide sequences encoding a sweetener into watermelon.
Claims 1, 5, 10-12, 16-17, 19 & 21 are obvious over Evans, UniProtKB reference DEF_PENBA, Huang, and Lamphear.
Applicant urges that amended claims require a nucleotide sequence encoding an N-terminal secretion signal operatively linked to a nucleotide sequence encoding the sweet protein. Applicant urges that the claims are directed to the surprising discovery that a brazzein can not only be expressed but can be secreted out of the watermelon cells. Applicant urges that one of ordinary skill in the art would not have motivation to nor reasonable expectation of success in reaching the instant claims (Remarks, page 8, paragraphs 3-4).
This argument is unpersuasive, because Lamphear teaches that an N-terminal secretion signal for transgenically expressing brazzein in maize leads to higher expression. One of ordinary skill in the art would have been motivated to use an N-terminal secretion signal for the expression of brazzein in another plant system, and one of ordinary skill in the art would have had reasonable expectation of success, because signal peptides had be used in conjunction with brazzein in other systems such as tomato. Moreover, Evans envisions the system of brazzein expression in melon, and Huang teaches methods of increasing sweetness in watermelon specifically. The limitations of the instant inventions would therefore have been obvious over the prior art.
Claim(s) 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Evans, UniProtKB reference DEF_PENBA, Huang, and Lamphear as applied to claims 1, 5, 10-12, 16-17, 19 & 21 above, and further in view of Yin et al (2009) Hereditas. 31(6): 663―667 (published June, 2009, hereafter Yin, uploaded with machine translation appended) and in view of NCBI GenBank reference AY262035.1, available 5/24/2003.
The teachings of Evans, UniProtKB reference DEF_PENBA, Huang, and Lamphear are presented above. They do not teach a regulatory sequence having an identity of at least 70% to instant SEQ ID NOs: 1-6 or 31-36.
The teachings of Yin and NCBI GenBank reference AY262035.1 are presented above.
Before the filing of the instant application, one of ordinary skill in the art would have been motivated to modify the method and plants taught by Evans to substitute the constitutive promoters with a watermelon AGPL1 promoter as taught by Yin and NCBI GenBank reference AY262035.1. One of ordinary skill in the art would have been motivated to substitute the fruit specific promoter for a constitutive promoter, because constitutive promoters can lead to large consumption of nutrients within the plant. One of ordinary skill in the art would have had reasonable expectation of success, because Evans suggests the method in melon, and the AGPL1 promoter is a watermelon promoter.
In light of Evans, UniProtKB reference DEF_PENBA, Hang, Lamphear, Yin and NCBI GenBank reference AY262035.1, claims 1, 5-7, 10-12, 16-17, 19 & 21 are obvious.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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