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 .
Claim Status
Claims 28, 34, 52, 54-57, 60, 65-66, 124, and 164 are pending.
Claims 54, 60, 66, 124, and 164 are withdrawn from the examination as being part of non-elected groups.
Claims 28, 34, 52, and 55-57 and 65 are currently being examined.
All previous objections and rejections not set forth below have been withdrawn in view
of applicant’s amendments to the claims. However, the claim amendments by the Applicant by adding new issues, which was not present in any of the claims before, necessitated new prior art references and new grounds of rejections, as discussed below.
Informalities
Claim 65 recites the limitation "the at least one modification." in line 2, while referencing preceding claim 28. It is potentially unclear if the claim requires just one of the recited targeted modifications, or all three (i)-(iii). In the interest of compact prosecution, the claim is interpreted as requiring all three (i.e., (i) –(iii)) modification recited in claim 28; which is why no 35 USC § 112(b) nor 35 USC § 102 rejection has been made over only one of (i), (ii), or (iii).
Appropriate correction is required.
Claim Rejections - 35 USC § 102(a)(1) and 112(a)
Response to Applicant’s arguments: Amendments made to the claims filed in Applicant’s
response submitted on 08/28/2025 overcame the rejections of record under 35 USC § 112(a) and 35 USC § 102(a)(1).
Claim Rejections - 35 USC § 103
Claims 28, 52, and 65 are rejected under 35 U.S.C. 103 as being unpatentable over Amir R.(1) (US 7323338 B2), in view of Tsubokura et al. (The β-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes, 2012, Plant mol. Biol., Vol.78:301-309) and Hefford MA. (Engineering Nutritious Proteins, 1997, Biotech. and Genet. Eng. Rev., 14:191-210). Due to applicant’s amendments invoking new issues, comprising the recitation of “… SEQ ID NO: 43 or 44 …”, which were not present in any claim before, this is a new rejection with new prior art references.
Claim 28 is drawn to a soybean seed or a soybean plant comprising at least about 10% increase in one or more essential amino acids wherein the seed/plant comprises- (i) a mutated glycinin protein comprising an insertion or substitution with one or more methionine, threonine, tryptophan, or lysine residues, or any combination thereof; (ii) a mutated beta-conglycinin gene with reduced expression of the gene encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 61, and (iii) a mutated Cystathionine-Gamma-Synthase (CGS) gene with increased expression and encoding a polypeptide having is at least 95% identity to SEQ ID NO: 43 or 44.
Amir R.(1) teaches a method of increasing methionine and/or methionine related metabolites in a plant (abstract). The method comprises overexpressing a mutated cystathionine γ-synthase encoded by a mutated polynucleotide lacking a region encoding an N-terminal portion of the gene, wherein the said region downregulates cystathionine γ-synthase in the plant (abstract). It describes at least about 10% increase in methionine (Met) and S-methylmethionin (SMM) concentration in several transgenic lines (e.g. plant N46) expressing mutated CGS proteins (Table 2). Levels of Met were found to be higher in all three transgenic lines at moderate to high levels than in the wild-type plants (Fig. 7a, b). The highest levels observed (11.2%, plant N46) represent a 56-fold (i.e. more than 10%) increase relative to wild-type (column, 20, line 61-66).
It also teaches a soybean cystathionine γ-synthase polypeptide having at least 95% (99.7%) sequence identity to instant SEQ ID NO: 43, as shown below.
RESULT 2
US-10-475-852A-9
Sequence 9, US/10475852A
Patent No. 7323338
GENERAL INFORMATION
APPLICANT: Amir, Rachel
TITLE OF INVENTION: PLANTS CHARACTERIZED BY AN INCREASED CONTENT OF METHIONINE AND
TITLE OF INVENTION: RELATED METABOLITES AND METHODS OF GENERATING SAME
FILE REFERENCE: 27093
CURRENT APPLICATION NUMBER: US/10/475,852A
CURRENT FILING DATE: 2003-11-03
NUMBER OF SEQ ID NOS: 12
SEQ ID NO 9
LENGTH: 536
TYPE: PRT
ORGANISM: Glycine max
Query Match 99.7%; Score 2749; Length 536; Best Local Similarity 99.8%;
Matches 535; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 MAVSSSHMRFTFECRSDPDFSPPPPSFDNLRRRNFRSSAGSGAAFHGISSLILRFPPNFQ 60
||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||
Db 1 MAVSSSHMRFTFECRSDPDFSPPPPSFDNLRRRNFRSSADSGAAFHGISSLILRFPPNFQ 60
Qy 61 RQLSTKARRNCSNIGVAQIVAASWSNNSDNSPAAGAPAPPAATATDAATVPLPVVVAANE 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 RQLSTKARRNCSNIGVAQIVAASWSNNSDNSPAAGAPAPPAATATDAATVPLPVVVAANE 120
Qy 121 DVVVSAAADENGAVQLNSSSYSSFLKSDASKTIHAAERLGRGIETDGITTPVVNTSAYFF 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DVVVSAAADENGAVQLNSSSYSSFLKSDASKTIHAAERLGRGIETDGITTPVVNTSAYFF 180
Qy 181 KKTADLIDFKENRQVSYEYGRYGNPTTVVLEEKISALEGAESTVIMASGMCASVVLFMAL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 KKTADLIDFKENRQVSYEYGRYGNPTTVVLEEKISALEGAESTVIMASGMCASVVLFMAL 240
Qy 241 VPAGGHLVTTTDCYRKTRIFIETFLPKMGITTTVIDPADVGALESALEQHNVSLFFTESP 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 VPAGGHLVTTTDCYRKTRIFIETFLPKMGITTTVIDPADVGALESALEQHNVSLFFTESP 300
Qy 301 TNPFLRCVDIKLVSELCHKKGTLLCIDGTFATPLNQKALALGADLILHSLTKYMGGHHDV 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 TNPFLRCVDIKLVSELCHKKGTLLCIDGTFATPLNQKALALGADLILHSLTKYMGGHHDV 360
Qy 361 LGGCISGSIKVVSQIRTLHHVLGGTLNPNAAYLFIRGMKTLHLRVQQQNSTGMRMAKLLE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LGGCISGSIKVVSQIRTLHHVLGGTLNPNAAYLFIRGMKTLHLRVQQQNSTGMRMAKLLE 420
Qy 421 AHPKVKRVYYPGLPSHPEHELAKRQMTGFGGVVSFEIDGDLHTTIKFIDSLKIPYIAASF 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 AHPKVKRVYYPGLPSHPEHELAKRQMTGFGGVVSFEIDGDLHTTIKFIDSLKIPYIAASF 480
Qy 481 GGCESIVDQPAILSYWDLPQSERAKYKIYDNLVRFSFGVEDFEDLKADVLQALEAI 536
||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 GGCESIVDQPAILSYWDLPQSERAKYKIYDNLVRFSFGVEDFEDLKADVLQALEAI 536
However, Amir R.(1) does not describe any glycinin or beta-conglycinin.
Tsubokura et al. describes that glycinin contains a higher concentration of sulfur-containing essential amino acids (methionine and cysteine) compared to β-conglycinin (page 301, right column, para 4, line 8-9). It also describes a method to improve amino acid score (i.e., content of essential amino acids, comparing the ratio of essential amino acids in a food to a reference standard) of soybean protein by decreasing β-conglycinin, which results in an increases in glycinin content (page 301, right column, last para, and page 302, left column, first pare). Tsubokura et al. also teaches knock-out or null mutant lines for several β-conglycinin subunits in soybean (page 308, left column, para 1). It also teaches the α’ subunit of β-conglycinin in soybean (CG-α’-1, GenBank Accession Number: AB610665) with at least 90% (100%) sequence identity to SEQ ID NO: 61, as discussed in previous Office action dated 4/30/2025.
Hefford MA., teaches different approaches to make more nutritious proteins. It describes an insertion mutation in soybean glycinin protein inserting four contiguous methionine residues and expressing the methionine enriched mutated soybean glycinin protein successfully in transgenic tobacco plants/seeds (page 197, para 5).
Before the effective filing, it would have been obvious to one of ordinary skill in the art to produce a soybean plant and a seed thereof (as recited in claim 65) to improve nutritional quality/score of soybean seeds by expressing a modified CGS gene encoding a CGS polypeptide having at least 95% sequence identity to SEQ ID NO: 43, as taught by Amir R.(1), at the same time decreasing the expression of β-conglycinin comprising at least 90% sequence identity to SEQ ID NO: 61, as taught by Tsubokura et al., and as recited in claim 52. There are known standard techniques including CRISPR-Cas to reduce/knock-out/knock-down almost any gene in a plant including soybean1. The nutritional quality of the glycinin protein itself would have been further improved by expressing a mutated glycinin gene encoding a modified glycinin polypeptide comprising four or more essential amino acids like methionine, as described by Hefford MA. It is implied that a transgenic plant, as recited in claim 65, produced by the transgenic seed would be having the same genetic makeup comprising the same mutations including “the at least one modification”.
Before the date of filing of this invention, an ordinarily skilled artisan would have been motivated to enhance the nutritional value of soybean seeds/plants by- (i) expressing a mutated glycinin protein containing more methionine and comprising at least 95% sequence identity to SEQ ID NO: 61 and, (ii) reducing expression of β-conglycinin, and iii) expressing a modified Met-insensitive CGS gene, with the realistic expectation to increase methionine content in soybean plants/seeds.
Claims 34 and 57 are rejected under 35 U.S.C. 103 as being unpatentable over Amir R. (1), in view of Tsubokura et al. and Hefford MA. as applied to claims 28, 52, and 65 above, and further in view of Amir et al. (2) (Revisiting the attempts to fortify methionine content in plant seeds, 2019, Journal of Experimental Botany, 70:4105–4114).
Claims 34 and 57 are directly or indirectly depend from claim 28, and are drawn to the soybean seed/plant comprising knockout mutant(s) of methionine-γ-lyase (MGL) gene(s) having at least 95% identity to SEQ ID NO: 53 and/or SEQ ID NO: 54.
Amir R.(1) in view of Tsubokura et al. and Hefford MA. describe a soybean plant/seed comprising at least about 10% increase in one or more essential amino acids wherein the seed/plant comprises- (i) a mutated glycinin protein comprising an insertion of at least four methionine residues; (ii) mutated β-conglycinin gene with reduced expression of the gene encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 61, and (iii) a mutated Cystathionine-γ-Synthase (CGS) gene with increased expression and encoding a polypeptide having is at least 95% identity to SEQ ID NO: 43, as discussed above.
However, Amir R.(1) in view of Tsubokura et al. and Hefford MA. do not describe a soybean plant/seed comprising a knockout of a methionine-γ-lyase (MGL) gene encoding an MGL polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 53.
Amir et al.(2) teaches that knocking down or silencing the MGL gene, using RNAi, leads to decreasing production of MGL, resulting in 55% more methionine in potato tubers (page 4108, left column, para 4, line 10-11). It also teaches potato tubers overexpressing the Arabidopsis cystathionine γ-synthase (AtCGS), together with the RNAi::MGL construct, accumulated 2.2-fold (i.e. 220%) more methionine than the wild-type potato tubers (page 4108, left column, para 4, line 11-14), which is a 5.5-fold (or 550%) more methionine compared with transgenic potato tubers overexpressing the AtCGS alone (page 4108, right column, para 1, line 1-2).
Before the effective filing date, it would have been prima facie obvious to one of ordinary skill in the art to produce a soybean plant and a seed thereof with improved nutritional quality by knocking out or silencing the MGL gene, as taught by Amir et al.(2), while overexpressing CGS gene along with reduced expression of β conglycinin; as described by Amir R.(1), Tsubokura et al., and Hefford MA.; with a realistic expectation to further increase methionine content in soybean seeds/plants. All the methods are directed toward achieving the same result with the realistic expectation of achieving synergistic effect, as described by Amir et al.(2). There is nothing in the art to indicate that any of the genes and/or the method is incompatible and/or would ultimately reduce methionine content in the modified seeds/plants.
Knocking out a single specific gene or more than one gene is a well-known standard practice in the art using techniques like CRISPR-Cas which is equally effective in soybean1. Using known standard methods in the art, an ordinarily skilled artisan also would have been able to identify methionine-γ-lyase (MGL) gene(s) including SEQ ID NO: 53 and/or SEQ ID NO: 54 (as shown below) using fully sequenced genomes of different commercially important soybean cultivars, which was first published in 20092 and subsequently by several other groups.
Sequence identity of at least 95% to instant SEQ ID NO: 53:
RESULT 1
Title: US-17-996-639-53
Perfect score: 2250
Sequence: 1 MAESAVTNRKRSSGDDAVSE..........QKWSQMEMALERFQEKEPLV 440
A0A445LLE8_GLYSO
ID A0A445LLE8_GLYSO Unreviewed; 440 AA.
AC A0A445LLE8;
DT 08-MAY-2019, integrated into UniProtKB/TrEMBL.
DT 08-MAY-2019, sequence version 1.
DT 27-NOV-2024, entry version 23.
DE SubName: Full=Methionine gamma-lyase {ECO:0000313|EMBL:RZC24043.1};
GN ORFNames=D0Y65_003373 {ECO:0000313|EMBL:RZC24043.1};
OS Glycine soja.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; fabids; Fabales; Fabaceae; Papilionoideae; 50 kb inversion clade;
OC NPAAA clade; indigoferoid/millettioid clade; Phaseoleae; Glycine;
OC Glycine subgen. Soja.
OX NCBI_TaxID=3848 {ECO:0000313|EMBL:RZC24043.1, ECO:0000313|Proteomes:UP000289340};
RN [1] {ECO:0000313|EMBL:RZC24043.1, ECO:0000313|Proteomes:UP000289340}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. W05 {ECO:0000313|Proteomes:UP000289340};
RC TISSUE=Hypocotyl of etiolated seedlings {ECO:0000313|EMBL:RZC24043.1};
RA Xie M., Chung C.Y.L., Li M.-W., Wong F.-L., Chan T.-F., Lam H.-M.;
RT "A high-quality reference genome of wild soybean provides a powerful tool
RT to mine soybean genomes.";
RL Submitted (SEP-2018) to the EMBL/GenBank/DDBJ databases.
CC -!- COFACTOR:
CC Name=pyridoxal 5'-phosphate; Xref=ChEBI:CHEBI:597326;
CC Evidence={ECO:0000256|ARBA:ARBA00001933,
CC ECO:0000256|RuleBase:RU362118};
CC -!- SIMILARITY: Belongs to the trans-sulfuration enzymes family.
CC {ECO:0000256|RuleBase:RU362118}.
DR EMBL; QZWG01000002; RZC24043.1; -; Genomic_DNA.
DR EnsemblPlants; XM_028344611.1; XP_028200412.1; LOC114384810.
DR Gramene; XM_028344611.1; XP_028200412.1; LOC114384810.
DR OrthoDB; 2454808at2759; -.
DR Proteomes; UP000289340; Chromosome 2.
DR GO; GO:0005737; C:cytoplasm; IEA:TreeGrafter.
DR GO; GO:0016846; F:carbon-sulfur lyase activity; IEA:TreeGrafter.
DR GO; GO:0030170; F:pyridoxal phosphate binding; IEA:InterPro.
DR GO; GO:0019346; P:transsulfuration; IEA:InterPro.
DR FunFam; 3.40.640.10:FF:000046; Cystathionine gamma-lyase; 1.
DR FunFam; 3.90.1150.10:FF:000087; Putative methionine gamma-lyase; 1.
DR Gene3D; 3.90.1150.10; Aspartate Aminotransferase, domain 1; 1.
DR Gene3D; 3.40.640.10; Type I PLP-dependent aspartate aminotransferase-like (Major domain); 1.
DR InterPro; IPR000277; Cys/Met-Metab_PyrdxlP-dep_enz.
DR InterPro; IPR015424; PyrdxlP-dep_Trfase.
DR InterPro; IPR015421; PyrdxlP-dep_Trfase_major.
DR InterPro; IPR015422; PyrdxlP-dep_Trfase_small.
DR PANTHER; PTHR11808:SF15; CYSTATHIONINE GAMMA-LYASE; 1.
DR PANTHER; PTHR11808; TRANS-SULFURATION ENZYME FAMILY MEMBER; 1.
DR Pfam; PF01053; Cys_Met_Meta_PP; 1.
DR PIRSF; PIRSF001434; CGS; 1.
DR SUPFAM; SSF53383; PLP-dependent transferases; 1.
PE 3: Inferred from homology;
KW Lyase {ECO:0000313|EMBL:RZC24043.1};
KW Pyridoxal phosphate {ECO:0000256|ARBA:ARBA00022898,
KW ECO:0000256|PIRSR:PIRSR001434-2};
KW Reference proteome {ECO:0000313|Proteomes:UP000289340}.
FT REGION 1..32
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 9..31
FT /note="Basic and acidic residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT MOD_RES 240
FT /note="N6-(pyridoxal phosphate)lysine"
FT /evidence="ECO:0000256|PIRSR:PIRSR001434-2"
SQ SEQUENCE 440 AA; 47582 MW; FC725D83F75EDA86 CRC64;
Query Match 100.0%; Score 2250; Length 440; Best Local Similarity 100.0%;
Matches 440; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAESAVTNRKRSSGDDAVSENDRKRKNMVARNMDPAEALALTRHEFGEHGGVNMSIEASA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAESAVTNRKRSSGDDAVSENDRKRKNMVARNMDPAEALALTRHEFGEHGGVNMSIEASA 60
Qy 61 TFTVMEPETLGRMFAGELGPDRDFFIYSRHFNPTVVNLGRHMAALEGTEAAYCTASGMAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 TFTVMEPETLGRMFAGELGPDRDFFIYSRHFNPTVVNLGRHMAALEGTEAAYCTASGMAA 120
Qy 121 ISSVLLQLCSTGGHVVASTTLYGGTHALLTHFLPGKCNITTTFVDIADLESVESAIVEGK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ISSVLLQLCSTGGHVVASTTLYGGTHALLTHFLPGKCNITTTFVDIADLESVESAIVEGK 180
Qy 181 TNVLYFESMANPSLKVADIPELCRVGHAKGVTVVVDNTFAPMVISPARLGADVVVHSISK 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 TNVLYFESMANPSLKVADIPELCRVGHAKGVTVVVDNTFAPMVISPARLGADVVVHSISK 240
Qy 241 FISGGADIIAGAVCGPASLVNSMMDLHDGSLMLLGPTMNAKVAFELSERIPHLSLRMKEH 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 FISGGADIIAGAVCGPASLVNSMMDLHDGSLMLLGPTMNAKVAFELSERIPHLSLRMKEH 300
Qy 301 CHRALVFATRLKKLGLKVIYPGLEDHPQHKLLKSIANREYGYGGLLCIDMGTVERANLLM 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 CHRALVFATRLKKLGLKVIYPGLEDHPQHKLLKSIANREYGYGGLLCIDMGTVERANLLM 360
Qy 361 AHLQNDAQFGFMAVSLGYYETLMSCSGSSTSSEMTEEEQKLAGISPGLVRMSIGYIGTLE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 AHLQNDAQFGFMAVSLGYYETLMSCSGSSTSSEMTEEEQKLAGISPGLVRMSIGYIGTLE 420
Qy 421 QKWSQMEMALERFQEKEPLV 440
||||||||||||||||||||
Db 421 QKWSQMEMALERFQEKEPLV 440
Sequence identity of at least 95% to instant SEQ ID NO: 54:
RESULT 1
Title: US-17-996-639-54
Perfect score: 2254
Sequence: 1 MAESAVVNRKRSGTDDVVSE..........QKWSQMEIALDRFQEKQPLV 440
A0A445H9T1_GLYSO
ID A0A445H9T1_GLYSO Unreviewed; 440 AA.
AC A0A445H9T1;
DT 08-MAY-2019, integrated into UniProtKB/TrEMBL.
DT 08-MAY-2019, sequence version 1.
DT 27-NOV-2024, entry version 26.
DE SubName: Full=Methionine gamma-lyase isoform A {ECO:0000313|EMBL:RZB70378.1};
DE SubName: Full=Methionine gamma-lyase isoform B {ECO:0000313|EMBL:RZB70379.1};
GN ORFNames=D0Y65_035388 {ECO:0000313|EMBL:RZB70378.1};
OS Glycine soja
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; fabids; Fabales; Fabaceae; Papilionoideae; 50 kb inversion clade;
OC NPAAA clade; indigoferoid/millettioid clade; Phaseoleae; Glycine;
OC Glycine subgen. Soja.
OX NCBI_TaxID=3848 {ECO:0000313|EMBL:RZB70378.1, ECO:0000313|Proteomes:UP000289340};
RN [1] {ECO:0000313|EMBL:RZB70378.1, ECO:0000313|Proteomes:UP000289340}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. W05 {ECO:0000313|Proteomes:UP000289340};
RC TISSUE=Hypocotyl of etiolated seedlings {ECO:0000313|EMBL:RZB70378.1};
RA Xie M., Chung C.Y.L., Li M.-W., Wong F.-L., Chan T.-F., Lam H.-M.;
RT "A high-quality reference genome of wild soybean provides a powerful tool
RT to mine soybean genomes.";
RL Submitted (SEP-2018) to the EMBL/GenBank/DDBJ databases.
CC -!- COFACTOR:
CC Name=pyridoxal 5'-phosphate; Xref=ChEBI:CHEBI:597326;
CC Evidence={ECO:0000256|ARBA:ARBA00001933,
CC ECO:0000256|RuleBase:RU362118};
CC -!- SIMILARITY: Belongs to the trans-sulfuration enzymes family.
CC {ECO:0000256|RuleBase:RU362118}.
CC {ECO:0000313|EMBL:RZB70378.1}.
DR EMBL; QZWG01000013; RZB70378.1; -; Genomic_DNA.
DR EMBL; QZWG01000013; RZB70379.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A445H9T1; -.
DR SMR; A0A445H9T1; -.
DR EnsemblPlants; XM_028342274.1; XP_028198075.1; LOC114382697.
DR Gramene; XM_028342274.1; XP_028198075.1; LOC114382697.
DR OrthoDB; 2454808at2759; -.
DR Proteomes; UP000289340; Chromosome 13.
DR GO; GO:0005737; C:cytoplasm; IEA:TreeGrafter.
DR GO; GO:0016846; F:carbon-sulfur lyase activity; IEA:TreeGrafter.
DR GO; GO:0030170; F:pyridoxal phosphate binding; IEA:InterPro.
DR GO; GO:0019346; P:transsulfuration; IEA:InterPro.
DR FunFam; 3.40.640.10:FF:000046; Cystathionine gamma-lyase; 1.
DR FunFam; 3.90.1150.10:FF:000087; Putative methionine gamma-lyase; 1.
DR Gene3D; 3.90.1150.10; Aspartate Aminotransferase, domain 1; 1.
DR Gene3D; 3.40.640.10; Type I PLP-dependent aspartate aminotransferase-like (Major domain); 1.
DR InterPro; IPR000277; Cys/Met-Metab_PyrdxlP-dep_enz.
DR InterPro; IPR015424; PyrdxlP-dep_Trfase.
DR InterPro; IPR015421; PyrdxlP-dep_Trfase_major.
DR InterPro; IPR015422; PyrdxlP-dep_Trfase_small.
DR PANTHER; PTHR11808:SF15; CYSTATHIONINE GAMMA-LYASE; 1.
DR PANTHER; PTHR11808; TRANS-SULFURATION ENZYME FAMILY MEMBER; 1.
DR Pfam; PF01053; Cys_Met_Meta_PP; 1.
DR PIRSF; PIRSF001434; CGS; 1.
DR SUPFAM; SSF53383; PLP-dependent transferases; 1.
PE 3: Inferred from homology;
KW Lyase {ECO:0000313|EMBL:RZB70378.1};
KW Pyridoxal phosphate {ECO:0000256|ARBA:ARBA00022898,
KW ECO:0000256|PIRSR:PIRSR001434-2};
KW Reference proteome {ECO:0000313|Proteomes:UP000289340}.
FT REGION 1..27
FT /note="Disordered"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT COMPBIAS 9..27
FT /note="Basic and acidic residues"
FT /evidence="ECO:0000256|SAM:MobiDB-lite"
FT MOD_RES 240
FT /note="N6-(pyridoxal phosphate)lysine"
FT /evidence="ECO:0000256|PIRSR:PIRSR001434-2"
SQ SEQUENCE 440 AA; 47421 MW; EE365CC70E0CE582 CRC64;
Query Match 100.0%; Score 2254; Length 440; Best Local Similarity 100.0%;
Matches 440; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 MAESAVVNRKRSGTDDVVSENDGKRKNVVAPDMDPAAALALTRHEFGEHGGVNMSIEASA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAESAVVNRKRSGTDDVVSENDGKRKNVVAPDMDPAAALALTRHEFGEHGGVNMSIEASA 60
Qy 61 TFTVMEPETLCRMFTGELGPDRDFFIYSRHFNPTVLNLGRHMAALEGTEAAYCTASGMAA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 TFTVMEPETLCRMFTGELGPDRDFFIYSRHFNPTVLNLGRHMAALEGTEAAYCTASGMAA 120
Qy 121 ISSVLLQLCSSGGHVVASTTLYGGTHALLTHFLPGKCNISTTFVDISDLESVESAIVEGK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ISSVLLQLCSSGGHVVASTTLYGGTHALLTHFLPGKCNISTTFVDISDLESVESAIVEGK 180
Qy 181 TNVLYFESMANPSLKVANIPELCRVGHAKGVTVVTDNTFAPMVISPARLGADVVVHSISK 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 TNVLYFESMANPSLKVANIPELCRVGHAKGVTVVTDNTFAPMVISPARLGADVVVHSISK 240
Qy 241 FISGGADIIAGAVCGPASLVNSMMDLHDGSLMLLGPTMNAKVAFELSERIPHLSLRMKEH 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 FISGGADIIAGAVCGPASLVNSMMDLHDGSLMLLGPTMNAKVAFELSERIPHLSLRMKEH 300
Qy 301 CHRALVFATRLKKLGLKVIYPGLEDHPQHELLKSIANREYGYGGLLCIDMETVERANLLM 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 CHRALVFATRLKKLGLKVIYPGLEDHPQHELLKSIANREYGYGGLLCIDMETVERANLLM 360
Qy 361 AHLQNDAQFGFMAVSLGYYETLMSCSGSSTSSGMTEEEQKLAGISPGLVRMSIGYIGTLE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 AHLQNDAQFGFMAVSLGYYETLMSCSGSSTSSGMTEEEQKLAGISPGLVRMSIGYIGTLE 420
Qy 421 QKWSQMEIALDRFQEKQPLV 440
||||||||||||||||||||
Db 421 QKWSQMEIALDRFQEKQPLV 440
Before the effective filing date of the invention, an ordinarily skilled artisan would have been motivated to decrease expression of MGL polypeptide in soybean to increase one or more essential amino acid(s) including methionine.
Claims 55-56 are rejected under 35 U.S.C. 103 as being unpatentable over Amir R.(1) in view of Tsubokura et al. and Hefford MA. as applied to claims 28, 52, and 55 above, and further in view of Hacham et al. (An in vivo internal deletion in the N-terminus region of Arabidopsis cystathionine γ-synthase results in CGS expression that is insensitive to methionine, 2006, The Plant Journal, 45:955–967),
Claim 55 depends from claim 28 and is drawn to a soybean seed/plant comprising a modified CGS gene wherein a self-regulatory domain encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 64 is removed. Claim 56 depends from claim 55 and is drawn to the soybean plant/seed wherein the modified CGS gene encodes a CGS protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 36.
Amir R.(1) in view of Tsubokura et al. and Hefford MA. describe a soybean plant/seed comprising at least about 10% increase in one or more essential amino acids wherein the seed/plant comprises- (i) a mutated glycinin protein comprising an insertion of at least four methionine residues; (ii) a mutated β-conglycinin gene with reduced expression of the gene encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 61, and (iii) a mutated Cystathionine-γ-Synthase (CGS) gene with increased expression, and encoding a polypeptide having is at least 95% identity to SEQ ID NO: 43, as discussed above.
Amir R.(1) also describes a soybean CGS protein (column 6, line 3-4). It teaches that the CGS gene is regulated at the level of mRNA stability wherein the CGS transcript encoding an specific amino acid sequence in the first exon acts in cis to destabilize its own mRNA in a process that is activated by methionine or one of its metabolites (column 1, line 56-61).
Amir R.(1) teaches a CGS polypeptide (SEQ ID NO: 9, as described above) comprising at least 95% sequence identity to instant SEQ ID NO: 64, as shown below.
Title: US-17-996-639-64
Perfect score: 233
Sequence: 1 HGISSLILRFPPNFQRQLST..........CSNIGVAQIVAAWSNNSDNS 45
Searched: 1 seqs, 536 residues
Total number of hits satisfying chosen parameters: 1
Database : AASEQ2_09302025_155022.pep:*
RESULT 1
AASEQ2_09302025_155022
Query Match 95.5%; Score 222.5; DB 1; Length 536; Best Local Similarity 97.8%;
Matches 45; Conservative 0; Mismatches 0; Indels 1; Gaps 1;
Qy 1 HGISSLILRFPPNFQRQLSTKARRNCSNIGVAQIVAA-WSNNSDNS 45
||||||||||||||||||||||||||||||||||||| ||||||||
Db 46 HGISSLILRFPPNFQRQLSTKARRNCSNIGVAQIVAASWSNNSDNS 91
However, Amir R.(1) in view of Tsubokura et al. and Hefford MA. do not describe a CGS protein comprising an amino acid sequence with at least 95% homology to SEQ ID NO: 36.
Hacham et al. teaches a method (ribonuclease protection analysis) to identify the N-terminal nucleotide sequence conferring methionine sensitive feedback regulation in cystathionine γ-synthase (CGS) enzyme in Arabidopsis (abstract). It also teaches that overexpression of the feedback-insensitive AtCGS gene increased methionine level in the transgenic plants (page 955, abstract).
Sequence identity between SEQ ID NO: 36 and the unmated wild type CGS protein, as taught by Amir R.(1), is shown below. The sequence highlighted in grey (SEQ ID NO: 64) is deleted by the Applicant to modify the endogenous CGS protein to get feedback-insensitive cystathionine γ-synthase (CGS) protein.
SEQ43: Instant SEQ ID NO: 43 (wild type soybean CGS protein)
SEQ9_Amir(1): Wild type soybean CGS protein (SEQ ID NO: 9), as taught by Amir R.(1).
SEQ36: Instant SEQ ID NO: 36 (part of feedback-insensitive soybean CGS
protein)
SEQ43 MAVSSSHMRFTFECRSDPDFSPPPPSFDNLRRRNFRSSAGSGAAFHGISSLILRFPPNFQ
SEQ9_Amir(1) MAVSSSHMRFTFECRSDPDFSPPPPSFDNLRRRNFRSSADSGAAFHGISSLILRFPPNFQ
SEQ36 MAVSSSHMRFTFECRSDPDFSPPPPSFDNLRRRNFRSSAGS-------------------
***************************************.*
SEQ43_17996639 RQLSTKARRNCSNIGVAQIVAASWSNNSDNSPAAGAPAPPAATATDAATVPLPVVVAANE
SEQ9_Amir RQLSTKARRNCSNIGVAQIVAASWSNNSDNSPAAGAPAPPAATATDAATVPLPVVVAANE
SEQ36 ------------------------------------------------------------
SEQ43_17996639 DVVVSAAADENGAVQLNSSSYSSFLKSDASKTIHAAERLGRGIETDGITTPVVNTSAYFF
SEQ9_Amir DVVVSAAADENGAVQLNSSSYSSFLKSDASKTIHAAERLGRGIETDGITTPVVNTSAYFF
SEQ36 --------GENGTVQLNSSSYS-FLKSDASKTIHA-------------------------
.***:********* ************
SEQ43_17996639 KKTADLIDFKENRQVSYEYGRYGNPTTVVLEEKISALEGAESTVIMASGMCASVVLFMAL
SEQ9_Amir KKTADLIDFKENRQVSYEYGRYGNPTTVVLEEKISALEGAESTVIMASGMCASVVLFMAL
SEQ36 ------------------------------------------------------------
SEQ43_17996639 VPAGGHLVTTTDCYRKTRIFIETFLPKMGITTTVIDPADVGALESALEQHNVSLFFTESP
SEQ9_Amir VPAGGHLVTTTDCYRKTRIFIETFLPKMGITTTVIDPADVGALESALEQHNVSLFFTESP
SEQ36 ------------------------------------------------------------
SEQ43_17996639 TNPFLRCVDIKLVSELCHKKGTLLCIDGTFATPLNQKALALGADLILHSLTKYMGGHHDV
SEQ9_Amir TNPFLRCVDIKLVSELCHKKGTLLCIDGTFATPLNQKALALGADLILHSLTKYMGGHHDV
SEQ36 ------------------------------------------------------------
SEQ43_17996639 LGGCISGSIKVVSQIRTLHHVLGGTLNPNAAYLFIRGMKTLHLRVQQQNSTGMRMAKLLE
SEQ9_Amir LGGCISGSIKVVSQIRTLHHVLGGTLNPNAAYLFIRGMKTLHLRVQQQNSTGMRMAKLLE
SEQ36 ------------------------------------------------------------
SEQ43_17996639 AHPKVKRVYYPGLPSHPEHELAKRQMTGFGGVVSFEIDGDLHTTIKFIDSLKIPYIAASF
SEQ9_Amir AHPKVKRVYYPGLPSHPEHELAKRQMTGFGGVVSFEIDGDLHTTIKFIDSLKIPYIAASF
SEQ36 ------------------------------------------------------------
SEQ43_17996639 GGCESIVDQPAILSYWDLPQSERAKYKIYDNLVRFSFGVEDFEDLKADVLQALEAI
SEQ9_Amir GGCESIVDQPAILSYWDLPQSERAKYKIYDNLVRFSFGVEDFEDLKADVLQALEAI
SEQ36 --------------------------------------------------------
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan to identify the N-terminal self-regulatory sequence downstream of ATG start codon in the wild type CGS gene in soybean, by following the method described by Hacham et al., and delete the self-regulatory sequence in the endogenous CGS gene (without inserting any foreign or heterologous DNA in the plant) with a realistic expectation to increase methionine and/or methionine related metabolites, as described by Amir R.(A). There are several standard methods in the art including CRISPR-Cas technique to delete specific genomic sequence in plants including soybean1. Deleting any specific sequence (functional equivalent sequences) is an experimental design choice of an ordinarily skilled artisan so long the self-regulatory domain in the N-terminal part of the cDNA is absent in the mRNA while the encoded CGS polypeptide retains its enzymatic activity. Deleting the regulatory sequence in the endogenous CGS gene without inserting any foreign or heterologous DNA in the (soybean) plant would be helpful to pass regulatory hurdles in many countries that classify transgene-free genome-edited products under the “non-genetically modified organism” (non-GMO) status.
Before the effective filing, one of ordinary skill in the art would have been motivated to express a feedback-insensitive CGS gene in soybean encoding a functional CGS enzyme by deleting the self-regulatory domain of the CGS gene with the realistic goal to improve nutritional quality of soybean seeds/plants by increasing one or more essential amino acid content including methionine and/or methionine related metabolites.
Response to Applicant’s Arguments
The argument set forth in the Applicant’s reply on 08/28/2025 has been considered but is
not found fully persuasive.
The applicant amended the claims including the independent base claim 28 by adding new issues including reciting “SEQ ID NO: 43 or 44”, which were not present in any of the claims before. Accordingly, prior art references are changed. The Applicant’s amendments necessitate new grounds of rejections and are being examined accordingly, as presented in this office action.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new grounds 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.
Communication
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JAY CHATTERJEE whose telephone number is (703)756-1329. The examiner can normally be reached (Mon - Fri) 8.30 am to 5.30 pm..
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/ Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/ Primary Examiner, Art Unit 1663
1Jacobs et al. (Targeted genome modifications in soybean with CRISPR/Cas9, 2015, BMC Biotechnology, 15:16) provide the evidence of known method of CRISPR-Cas based gene editing technique in soybean.
2Schmutz et al. (Genome Sequence of the Palaeopolyploid Soybean, 2010, Nature 463:178-183) (First published on 2009-11-02).