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 .
DETAILED ACTION
Applicant’s response filed on 09/30/2025 is duly acknowledged.
Claims 1, 3, 5-11, 14, 16-18, 21, 22, 24, 25, 27-29, 31-34, 36, 38, 41 and 42 (amendment dated 02/13/2023) are currently pending in this application.
Election/Restrictions
Applicant’s election without traverse of Group I (Claims 1, 3, 5-11, 14, 16-18, 21, 22, 24, 25 and 27-29; directed to “A recombinant bacterial cell…” and “A pharmaceutical composition..”) in the reply filed on 09/30/2025 (see REM, page 2) is acknowledged.
Claims 31-34, 36, 38, 41 and 42 (non-elected invention of Group II, without traverse) have been withdrawn from further considerations.
Claims 1, 3, 5-11, 14, 16-18, 21, 22, 24, 25 and 27-29 (elected invention of Group I, without traverse; directed to “A recombinant bacterial cell…” and “A pharmaceutical composition..”) have been examined on their merits in this action hereinafter.
Priority
This application is a 371 of PCT/US21/17775 (filed on 02/12/2021), which claims domestic benefits from US PRO 63/125,120 (filed on 12/14/2020), US PRO 63/043,425 (filed on 06/24/2020), and US PRO 62/975,525 (filed on 02/12/2020).
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
1. Claim 1 (as currently amended) is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites the limitation "the gene sequence encoding the methionine catabolism enzyme" in lines 3-4. There is insufficient antecedent basis for this limitation in the claim- because the earlier limitation is “a heterologous gene sequence encoding a methionine catabolism enzyme”, i.e. not any “gene sequence encoding the methionine catabolism enzyme”. Appropriate correction is required.
2. Claim 9 (as presented) is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. Claim 9 recites the following”
“9. (Original) The recombinant bacterial cell of claim 8, wherein the metNIQ has a gene sequence having at least 90% identity to SEQ ID NOs: 1004, 1005, and 1006.”
It is to be noted that as per applicant’s disclosure of record, the SEQ ID NOs: 1004, 1005 and 1006 consist of 1032bp, 654bp and 816 bp, respectively (from E. coli source). However, instant claim 9 requires “at least 90% identity” to each of said sequences of “SEQ ID NOs: 1004, 1005, and 1006”, which is confusing because all three sequences have significantly different lengths of base pairs, and “at least 90% identity” to each would not be numerically possible for a single sequence from a given source. No such example for a single methionine importer metNIQ gene sequence has been shown by the applicants (see instant specification, [0185]-[0186], [0386]-[0387], for instance) that meets such criteria, as currently required in claim 9. Thus, metes and bounds of the claimed invention does not appear to be properly defined. Appropriate correction and/or explanation is required.
If applicants intend to recite “wherein the metNIQ has a gene sequence having at least 90% identity to SEQ ID NOs: 1004, 1005, or 1006” (as interpreted hereinafter for the prior art purposes), such correction should be made appropriately.
NOTE: 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.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
1. Claims 1, 17, 21, 24 and 29 (as recited) are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Schroder et al (1995; previously made of record by examiner) as evidenced by SnapGene (2025; NPL cited as ref. [U] on PTO 892 form).
“Claim 1. (Currently Amended) A recombinant bacterial cell comprising a heterologous gene sequence encoding a methionine catabolism enzyme operably linked to a first promoter that is not associated with the gene encoding the amino acid catabolism enzyme in nature. wherein the gene sequence encoding the methionine catabolism enzyme is a methionine decarboxylase (MDC) gene sequence.”
See also limitations of instant dependent claims 17, 21, 24 and 29, as presented.
Schroder et al (1995) disclose (regarding instant claim 1) a recombinant bacterium, Escherichia coli (see page 75, left column, section “Expression in E. coli and site-directed mutagenesis”, and cited references therein) that comprises the heterologous gene sequence encoding a unmodified and mutant S-adenosyl-L-methionine decarboxylase (AdoMetDC, from Cantharanthus roseus, a Madagaskar periwinkle) gene sequence that is operably linked to a plasmid pQE-6 promoter (which has T5 constitutive promoter, that is not associated with the gene encoding the amino acid catabolism enzyme in nature), wherein the construct retained the optimal promoter-translation start configuration of the vector for expression in E. coli of the heterologous proteins pQE/AdoMetDC (unmodified proenzyme) and pQE/AdoMetDCmu (mutated proenzyme); wherein the first promoter is T5 constitutive promoter (i.e. a T5 promoter in pQE-6 expression vector; see instant claim 17; and the evidentiary disclosure by SnapGene 2025, page 2); wherein the heterologous gene encoding the methionine catabolism enzyme is located on a plasmid pQE-6 for expression in E. coli host (instant claim 21; see Figure 2, lanes 1-3 on page 76); wherein the recombinant bacterial cell is a recombinant probiotic bacterial cell, such as Escherichia coli cell culture (instant claims 24 and 29; see applicant’s definition for term “probiotic” on page 17, full paragraph [070], in particular that includes E. coli as an example of a “probiotic”, non-pathogenic microorganism; taken as a pharmaceutical composition comprising said recombinant E. coli cells with pQE-6 plasmid for expression of the heterologous AdoMetDC gene in a carrier such as culture medium; see Schroder et al, section “E. coli extracts and enzyme activity” on page 75, left column, for instance and cited reference therein; see also applicant’s broad definition for “pharmaceutically acceptable carrier” in specification, page 24, paragraphs [070]-[071]).
As per MPEP 2111.01, during examination, the claims must be interpreted as broadly as their terms reasonably allow. In re American Academy of Science Tech Center, F.3d, 2004 WL 1067528 (Fed. Cir. May 13, 2004)(The USPTO uses a different standard for construing claims than that used by district courts; during examination the USPTO must give claims their broadest reasonable interpretation.). This means that the words of the claim must be given their plain meaning unless applicant has provided a clear definition in the specification. In re Zletz, 893 F.2d 319, 321, 13 USPQ2d 1320, 1322 (Fed. Cir. 1989).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
1. Claims 1, 3, 7, 8, 10, 11, 14, 17, 18, 21, 22, 24, 25, and 27-29 (as currently amended) are rejected under 35 U.S.C. 103 as being unpatentable over Falb et al (WO 2017/123676 A1; FOR cited in IDS dated 10/01/2025) taken with Feng et al (2013; NPL cited in IDS dated 10/01/2025) and Hayashi et al (2017; NPL cited in IDS dated 10/01/2025).
Claim 1 (as amended) is directed to “A recombinant bacterial cell comprising a heterologous gene sequence encoding a methionine catabolism enzyme operably linked to a first promoter that is not associated with the gene encoding the amino acid catabolism enzyme in nature. wherein the gene sequence encoding the methionine catabolism enzyme is a methionine decarboxylase (MDC) gene sequence.”
See also limitations of dependent claims 3, 7, 8, 10, 11, 14, 17, 18, 21, 22, 24, 25, and 27-29, as currently presented.
Falb et al (2017) disclose (regarding instant claim 1) a recombinant bacterial cell comprising a heterologous gene sequence encoding a methionine catabolism enzyme operably linked to a first promoter that is not associated with the gene encoding the amino acid catabolism enzyme in nature (see Falb et al, Title, Abstract, Summary, and claims, for instance; see page 447, Claim 1-“A recombinant bacterial cell comprising a heterologous gene sequence encoding an amino acid catabolism enzyme operably linked to a first promoter that is not associated with the gene encoding the amino acid catabolism enzyme in nature”; see also page 52, [068]-“"amino acid catabolism" refers to the processing, breakdown, and/or degradation of methionine into S-adenosyl-L-homocysteine”; and page 62, [094] – “…the amino acid catabolism enzyme catabolizes methionine”); wherein (regarding instant claim 5) the bacterial cell further comprise genetic modification that reduces export of an amino acid such as methionine from the bacterial cells (see Falb et al, claim 16, and page 81, [0150]- “the exporter of an amino acid exports an amino acid out of the bacterial cell ....the exporter of an amino acid is an exporter of methionine"; and page 159, [0411]- “the bacterial cell comprises a heterologous gene encoding a methionine catabolism enzyme and a genetic modification that reduces export of methionine”); wherein (regarding instant claims 17, 18) the first promoter is an inducible promoter, or a constitutive promoter (see Falb et al, page 447, claims 2-7, for instance- “wherein the first promoter is directly or indirectly induced by environmental conditions specific to the gut of a mammal”, “…induced by low oxygen or anaerobic conditions”, “…wherein the first promoter is an FNR responsive promoter”; in addition, Falb et al also disclose the use of a promoter inducible by anhydrotetracycline, ATC for conditional expression of methionine catabolism enzymes such as PAL1-PAL3 in suitable expression vectors; see page 18, [040], and Example 3, for instance); and wherein (regarding instant claims 7, 10) the recombinant bacterial cell further comprises a heterologous gene encoding an amino acid transporter linked to a second promoter that is not associated with the amino acid transporter gene in nature (see Falb et al, page 160, section “B. Transporters of Methionine”, [0412], that are useful in treating a disease associated with methionine, such as cancer; see also pages 447-448, claims 8-15, for instance). Regarding instant claim 8, Falb et al also disclose methionine importers (see Falb et al, page 153, [0386], for instance)- “In some embodiments, methionine transporters may also be expressed or modified in the recombinant bacteria to enhance methionine import into the cell in order to increase the catabolism of methionine by the methionine catabolism enzyme” (see also Falb et al, page 160, section “B. Transporters of Methionine”, in particular); and wherein “the at least one gene encoding a transporter of methionine is a metP gene, a metN gene, a metI gene, or a metQ gene from Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis..” (see page 160, [0414], for instance); wherein (regarding instant claim 14) the cell may additionally comprise the methionine catabolism enzyme that comprises methionine gamma lyase (MGL) gene sequence (see Falb et al, page 158, [0403]- “In one embodiment, the methionine catabolism enzyme comprises a methionine gamma lyase (E.C. 4.4.1.11). In one embodiment, the methionine gamma lyase gene is a methionine gamma lyase gene from Bacillus halodurans. In one embodiment, the methionine gamma lyase is an Entamoeba histolytica methionine gamma lyase gene”); wherein (regarding instant claims 21 and 22) the heterologous gene encoding the amino acid catabolism enzyme and/or the heterologous gene encoding the amino acid transporter is located on a plasmid or on a chromosome in the bacterial cell (see Falb et al, pages 448-449, claims 18-21, for instance); wherein (regarding instant claims 24 and 25) the recombinant bacterial cell is a recombinant probiotic bacterial cell such as E. coli strain Nissle (see Falb et al, page 449, claims 24-27); wherein (regarding instant claim 27) the recombinant bacterial cell is an auxotroph in a gene that is complemented when the recombinant bacterial cell is present in a mammalian gut such as diaminopimelic acid (DAP), or an enzyme in the thymine biosynthesis pathway (see Falb et al, page 449, claims 29-30, for instance); wherein (regarding instant claim 29) the recombinant bacterial cell is comprised in a pharmaceutical composition with a pharmaceutically acceptable carrier (see Falb et al, page 450, claim 31, for instance); and wherein (regarding instant claim 28) Falb et al disclose measurement of amino acid degradation rates in recombinant probiotic bacterial cells such as E. coli Nissle strain, in terms of amino acid degraded per hour/1e9 cells (see Falb et al, Example 31, Table on pages 366-367; see also page 185, [0536]-[0539], for instance), albeit using phenylalanine degradation by the heterologous gene expressing a phenylalanine ammonia lyase enzyme (PAL3) with chromosomal insertions at various sites.
However, the recombinant bacterial cell wherein- the heterologous gene sequence encodes a methionine decarboxylase (MDC) gene sequence having “at least 90% identity” to SEQ ID NO: 1003 or 1018 (instant claim 3) has not been explicitly exemplified by the disclosure from Falb et al, discussed above.
Feng et al (2013) discloses a methionine decarboxylase sequence that can be recombinantly expressed in a bacterial host cell (see Feng et al, Abstract – “BlsE, a predicted radical S-adenosyl-L-methionine (SAM) protein, was anaerobically purified and reconstituted in vitro”; page 2, Figure 1- “General reaction scheme for radical SAM enzymes that causes SAM (10) cleavage into 5'-deoxyadenosine (11) and methionine (12).”; page 9, col. 2, paragraph 2 – “For BlsE overexpression, the blsE gene was amplified…The insert of the recombinant plasmid was sequenced and confirmed, and the correct plasmids were then transformed into E. coli BL21 (DE3).”; page 11, col. 1, paragraph 1- “Site-directed mutational analysis of BlsE ....were used to transform into E. coli BL21 (DE3) cells for subsequent expression.”; wherein the “BlsE” represents a radical S-Adenosyl-L-methionine decarboxylase enzyme).
Hayashi et al (2017) disclose gene cloning, recombinant expression, purification and characterization of L-methionine decarboxylase (MetDC) from Streptomyces sp. 590 strain (see Title, Abstract on page 389, col. 1, for instance), wherein the MDC gene was cloned and recombinant MetDC was heterologously expressed in E. coli host cells, which was purified and further characterized for its activity, both in vivo and in vitro (see Hayashi et al, section “Bacterial strains, plasmid and cultural conditions”, and Figure 1 on page 390, for instance); wherein the nucleotide sequence of the MetDC gene and corresponding amino acid sequence was depicted in Figure 1 (see Hayashi et al, page 392, Figure 2; and page 396, col. 2, section “Acknowledgements”), and was deposited in DNA databank of Japan with an accession number AB970471 (also cited in applicant’s IDS dated 10/01/2025), which has 100% identity to currently recited SEQ ID NO: 1018 of instant claim 3 (see also nucleotide sequence homology below).
SEQ ID NO: 1018 (GenEmbl database)
RESULT 1
AB970471
LOCUS AB970471 1674 bp DNA linear BCT 28-MAY-2015
DEFINITION Streptomyces sp. 590 KI-2014 gene for L-methionine decarboxylase,
complete cds.
ACCESSION AB970471
VERSION AB970471.1
KEYWORDS .
SOURCE Streptomyces sp. 590 KI-2014
ORGANISM Streptomyces sp. 590 KI-2014
Bacteria; Actinomycetota; Actinomycetes; Kitasatosporales;
Streptomycetaceae; Streptomyces.
REFERENCE 1
AUTHORS Masaya,H., Okada,A., Tamura,T. and Inagaki,K.
TITLE Gene cloning, recombinant expression, and purification of
L-methionine decarboxylase from Streptomyces sp. 590
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 1674)
AUTHORS Inagaki,K. and Tamura,T.
TITLE Direct Submission
JOURNAL Submitted (11-JUN-2014) Contact:Kenji Inagaki Okayama University,
Graduate School of Environmental and Life Science; 1-1-1
Tsushimanaka Kita-ku, Okayama, Okayama 700-8530, Japan URL
:http://www.gels.okayama-u.ac.jp/index_e.html
FEATURES Location/Qualifiers
source 1..1674
/organism="Streptomyces sp. 590 KI-2014"
/mol_type="genomic DNA"
/strain="590"
/db_xref="taxon:1510823"
/geo_loc_name="Japan:Tokyo"
/collected_by="Kyowa Hakko Kogyo Co., Ltd."
CDS 1..1674
/codon_start=1
/transl_table=11
/product="L-methionine decarboxylase"
/protein_id="BAR88117.1"
/translation="MSPTAFPAAETATAPATAVDPGPELDGGDFALPEGGLDDDRRLR
ALDAVDEYLTRKRKHLVGYQATQDMQGTALDLARFMPNNINNLGDPFQSGGYKPNTKV
VERAVLDYYAKLWHAERPHDPADPESYWGYMLSMGSTEGNMYALWNARDYLSGKALIQ
PPTAPFDAVRYVKADPDRRNPNAHHPVAFYSEDTHYSFAKAVAVLGVETFHAVGLEKY
ADECPLVDPVTGLRTWPTEVPSRPGPSGLSWDGPGEIDVDALAVLVEFFAAKGHPVFV
NLNLGSTFKGAHDDVRAVCERLLPIFERHGLVQREVVYGSCPQTGRPLVDVRRGFWIH
VDGALGAGYAPFLRLAAEDPEGYGWTPEAELPEFDFGLRLPTAGHGEVDMVSSIAMSG
HKWAGAPWPCGIYMTKVKYQISPPSQPDYIGAPDTTFAGSRNGFSPLILWDHLSRYSY
RDQVERIREAQELAAYLERRLTAMERELGVELWPARTPGAVTVRFRKPSAELVAKWSL
SSQDVLMVPGDETTRRSYVHVFVMPSVDRAKLDALLAELAEDPVILGAP
Query Match 100.0%; Score 1674; Length 1674;
Best Local Similarity 100.0%;
Matches 1674; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ATGAGCCCGACCGCCTTCCCCGCCGCCGAGACCGCGACCGCGCCCGCGACCGCCGTCGAT 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 ATGAGCCCGACCGCCTTCCCCGCCGCCGAGACCGCGACCGCGCCCGCGACCGCCGTCGAT 60
Qy 61 CCCGGTCCGGAGCTGGACGGCGGTGACTTCGCCCTCCCCGAGGGCGGCCTGGACGACGAC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 CCCGGTCCGGAGCTGGACGGCGGTGACTTCGCCCTCCCCGAGGGCGGCCTGGACGACGAC 120
Qy 121 CGGCGGCTGCGCGCGCTCGACGCCGTGGACGAGTACCTGACCCGCAAGCGCAAGCACCTG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 CGGCGGCTGCGCGCGCTCGACGCCGTGGACGAGTACCTGACCCGCAAGCGCAAGCACCTG 180
Qy 181 GTCGGCTACCAGGCCACCCAGGACATGCAGGGCACCGCACTGGACCTCGCCCGGTTCATG 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GTCGGCTACCAGGCCACCCAGGACATGCAGGGCACCGCACTGGACCTCGCCCGGTTCATG 240
Qy 241 CCGAACAACATCAACAACCTCGGCGACCCGTTCCAGAGCGGCGGATACAAGCCCAACACC 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CCGAACAACATCAACAACCTCGGCGACCCGTTCCAGAGCGGCGGATACAAGCCCAACACC 300
Qy 301 AAGGTCGTCGAGCGGGCCGTGCTCGACTACTACGCGAAGCTCTGGCACGCCGAGCGCCCG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 AAGGTCGTCGAGCGGGCCGTGCTCGACTACTACGCGAAGCTCTGGCACGCCGAGCGCCCG 360
Qy 361 CACGACCCGGCCGACCCGGAGTCGTACTGGGGCTACATGCTGTCCATGGGCTCGACCGAG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 CACGACCCGGCCGACCCGGAGTCGTACTGGGGCTACATGCTGTCCATGGGCTCGACCGAG 420
Qy 421 GGCAACATGTACGCCCTCTGGAACGCCAGGGACTACCTGAGCGGCAAGGCGCTGATCCAG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GGCAACATGTACGCCCTCTGGAACGCCAGGGACTACCTGAGCGGCAAGGCGCTGATCCAG 480
Qy 481 CCGCCGACCGCCCCCTTCGACGCGGTGCGCTACGTCAAGGCCGACCCCGACCGACGGAAC 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 CCGCCGACCGCCCCCTTCGACGCGGTGCGCTACGTCAAGGCCGACCCCGACCGACGGAAC 540
Qy 541 CCGAACGCCCACCACCCGGTGGCCTTCTACTCCGAGGACACCCACTACTCCTTCGCCAAG 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 CCGAACGCCCACCACCCGGTGGCCTTCTACTCCGAGGACACCCACTACTCCTTCGCCAAG 600
Qy 601 GCCGTGGCCGTCCTCGGCGTGGAGACCTTCCACGCCGTCGGCCTGGAGAAGTACGCCGAC 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 GCCGTGGCCGTCCTCGGCGTGGAGACCTTCCACGCCGTCGGCCTGGAGAAGTACGCCGAC 660
Qy 661 GAGTGCCCGCTGGTCGACCCGGTGACCGGGCTGCGCACCTGGCCCACCGAGGTGCCCTCC 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 GAGTGCCCGCTGGTCGACCCGGTGACCGGGCTGCGCACCTGGCCCACCGAGGTGCCCTCC 720
Qy 721 CGCCCGGGTCCGTCCGGCCTGTCCTGGGACGGCCCCGGCGAGATAGACGTCGACGCCCTC 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 CGCCCGGGTCCGTCCGGCCTGTCCTGGGACGGCCCCGGCGAGATAGACGTCGACGCCCTC 780
Qy 781 GCCGTACTCGTCGAGTTCTTCGCCGCCAAGGGTCACCCGGTCTTCGTCAACCTCAACCTC 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 GCCGTACTCGTCGAGTTCTTCGCCGCCAAGGGTCACCCGGTCTTCGTCAACCTCAACCTC 840
Qy 841 GGCAGCACCTTCAAGGGCGCCCACGACGACGTCCGCGCCGTCTGCGAGCGCTTGCTGCCG 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 GGCAGCACCTTCAAGGGCGCCCACGACGACGTCCGCGCCGTCTGCGAGCGCTTGCTGCCG 900
Qy 901 ATCTTCGAGCGGCACGGGCTCGTCCAGCGCGAGGTGGTCTACGGCAGCTGCCCGCAGACC 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 ATCTTCGAGCGGCACGGGCTCGTCCAGCGCGAGGTGGTCTACGGCAGCTGCCCGCAGACC 960
Qy 961 GGCCGGCCGCTGGTGGACGTGCGCCGCGGCTTCTGGATCCACGTGGACGGCGCGCTCGGC 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 GGCCGGCCGCTGGTGGACGTGCGCCGCGGCTTCTGGATCCACGTGGACGGCGCGCTCGGC 1020
Qy 1021 GCCGGCTACGCGCCGTTCCTGCGGCTGGCCGCCGAGGACCCGGAAGGGTACGGCTGGACG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 GCCGGCTACGCGCCGTTCCTGCGGCTGGCCGCCGAGGACCCGGAAGGGTACGGCTGGACG 1080
Qy 1081 CCCGAGGCGGAGCTGCCCGAGTTCGACTTCGGCCTGCGGCTGCCCACCGCCGGGCACGGC 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 CCCGAGGCGGAGCTGCCCGAGTTCGACTTCGGCCTGCGGCTGCCCACCGCCGGGCACGGC 1140
Qy 1141 GAGGTGGACATGGTCTCCTCGATCGCGATGAGCGGCCACAAGTGGGCCGGCGCGCCGTGG 1200
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1141 GAGGTGGACATGGTCTCCTCGATCGCGATGAGCGGCCACAAGTGGGCCGGCGCGCCGTGG 1200
Qy 1201 CCGTGCGGCATCTACATGACCAAGGTGAAGTACCAGATCTCGCCGCCGTCCCAGCCGGAC 1260
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1201 CCGTGCGGCATCTACATGACCAAGGTGAAGTACCAGATCTCGCCGCCGTCCCAGCCGGAC 1260
Qy 1261 TACATCGGCGCCCCGGACACCACCTTCGCCGGCTCCCGCAACGGCTTCTCCCCGCTGATC 1320
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1261 TACATCGGCGCCCCGGACACCACCTTCGCCGGCTCCCGCAACGGCTTCTCCCCGCTGATC 1320
Qy 1321 CTCTGGGACCACCTGTCCCGGTACTCCTACCGGGACCAGGTGGAGCGGATCCGCGAGGCC 1380
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1321 CTCTGGGACCACCTGTCCCGGTACTCCTACCGGGACCAGGTGGAGCGGATCCGCGAGGCC 1380
Qy 1381 CAGGAGCTGGCCGCCTACCTGGAGCGGCGGCTGACCGCCATGGAGCGCGAACTCGGCGTC 1440
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1381 CAGGAGCTGGCCGCCTACCTGGAGCGGCGGCTGACCGCCATGGAGCGCGAACTCGGCGTC 1440
Qy 1441 GAGCTCTGGCCGGCCCGTACCCCGGGCGCCGTCACCGTACGGTTCCGCAAGCCGAGCGCC 1500
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1441 GAGCTCTGGCCGGCCCGTACCCCGGGCGCCGTCACCGTACGGTTCCGCAAGCCGAGCGCC 1500
Qy 1501 GAGCTGGTGGCCAAGTGGTCGCTGTCCTCCCAGGACGTGCTGATGGTCCCGGGCGACGAG 1560
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1501 GAGCTGGTGGCCAAGTGGTCGCTGTCCTCCCAGGACGTGCTGATGGTCCCGGGCGACGAG 1560
Qy 1561 ACCACCCGGCGCAGCTACGTGCACGTCTTCGTGATGCCCTCGGTCGACCGGGCCAAGCTG 1620
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1561 ACCACCCGGCGCAGCTACGTGCACGTCTTCGTGATGCCCTCGGTCGACCGGGCCAAGCTG 1620
Qy 1621 GACGCGCTGCTCGCCGAACTCGCCGAGGACCCGGTGATCCTGGGCGCACCGTAG 1674
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1621 GACGCGCTGCTCGCCGAACTCGCCGAGGACCCGGTGATCCTGGGCGCACCGTAG 1674
Thus, given the detailed disclosure for heterologously expressed MDC in probiotic microbial strains from Feng et al and Hayashi et al, and in addition, since Falb et al already disclose the recombinant bacterial cell comprises any of various exogenous methionine catabolism enzymes (see Falb et al, page 153, [0384]-[0390], section “8. Methionine” - "A. Methionine Catabolism Enzymes"; paragraph [068] – “"amino acid catabolism" refers to the processing, breakdown, and/or degradation of methionine”; paragraph [094] - "the amino acid catabolism enzyme catabolizes methionine"), including a decarboxylase enzyme (page 159, paragraph [0407]- "In one embodiment, the at least one methionine catabolism enzyme comprises a 2-oxo acid decarboxylase"), and methods comprising E. coli host expression (paragraph [07]; Claim 27- "…wherein the recombinant bacterial cell is of the species Escherichia coli strain Nissle."; paragraph [057] - "FIG. 84 depicts a schematic of the E. coli…"), it would have been obvious to an artisan of ordinary skill in the art to successfully include the methionine decarboxylase sequence of Feng et al in the E coli expression system taught by Falb et al, at least in order to improve the methionine catabolism enzyme efficiency in a desired probiotic microbial host cell.
In addition, since Feng et al teach the recombinant expression of a methionine decarboxylase (MDC) gene sequence (see detailed teachings above), and Falb et al already disclose recombinant bacterial cell expressing recombinant methionine catabolism enzymes (see detailed teachings above), it would have been obvious to an artisan of ordinary skill in the art to include the methionine decarboxylase gene sequence taught by Hayashi et al (i.e. MDC with gene sequence as shown in AB970471) in the E coli expression system of Falb et al (taken with Feng et al), in order to improve the desired methionine catabolism enzyme efficiency for the recombinant probiotic bacterial cells. Also, given the detailed disclosure for the genetic cloning and related gene/expression vector manipulations disclosed in the cited prior art references of Falb et al taken with Feng et al and Hayashi et al, an artisan of ordinary skill in the art would have had a reasonable expectation of success in modifying the recombinant probiotic bacteria disclosed by Falb et al in order to incorporate one or more suitable methionine catabolism enzymes including MGL under suitable inducible and/or constitutive promoters depending on the desired outcome and/or downstream applications, as already suggested by the disclosure of Falb et al (see teachings and/or advantages for use in cancer treatment, discussed above for instance). Additionally, the limitations for the specific amounts and/or range of the methionine degradation activity for the recombinant probiotic cells comprising such combination(s) of one or more methionine catabolism enzymes as disclosed in the cited prior art would have been obvious as all the cited prior art references, in particular Falb et al disclose such activity measures for amino acid degradation rate in micromoles/hour/109 cells (see Falb et al, activity of strains in Table disclosed on page 367), albeit for degradation of phenylalanine, and since the specific assays for methionine degrading enzymes were already known in the art (see teachings from Feng et al, page 10, col. 2, 2nd and 3rd paragraphs; and Hayashi et al, page 390, col. 1, section “Enzyme assay”, for instances), such determinations for methionine degradation rates for the recombinant probiotic cells would have been obvious and/or fully contemplated by an artisan of ordinary skill in the art. Thus, the invention as generically claimed fails to distinguish itself over the combined teachings and/or suggestions in the cited prior art (Falb et al taken with Feng et al and Hayashi et al) of record, as discussed above.
2. Claims 5 and 6 (as amended) are rejected under 35 U.S.C. 103 as being unpatentable over Falb et al (WO 2017/123676 A1; FOR cited in IDS dated 10/01/2025) taken with Feng et al (2013; NPL cited in IDS dated 10/01/2025) and Hayashi et al (2017; NPL cited in IDS dated 10/01/2025) as applied to claims 1, 3, 7, 8, 10, 11, 14, 17, 18, 21, 22, 24, 25, and 27-29 above, and further in view of Liu et al (2015; NPL cited in IDS dated 10/01/2025) and Reeves et al (2011; NPL cited as ref. [V] on PTO 892 form).
Claim 5 is directed to “The recombinant bacterial cell of claim 1[[4]], wherein the recombinant bacterial cell further comprises knock-out of an endogenous methionine efflux pump.”
Claim 6 is directed to “The recombinant bacterial cell of claim 5, wherein the endogenous methionine efflux pump is yjeH, and wherein the yjeH comprises a sequence having at least 90% identity with SEQ ID NO:1014.”
The combined teachings and suggestions from the cited prior art of Falb et al taken with Feng et al and Hayashi et al have been discussed above in details, and are further relied upon in the same manner hereinafter.
Liu et al (2015) disclose a methionine efflux pump, yjeH (see Abstract - "we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of L-methionine ... suggesting that YjeH functions as an exporter of L-methionine"; page 7754 col. 1, paragraph 3) and further discloses yjeH knock out experiments in E. coli (page 7754 col. 1, paragraph 4 - "Gene knockout was introduced into E. coli"; Table 1 "yjeH deletion").
Since Falb et al already suggest reducing the activity of a methionine exporter (see paragraph [0150] -"the exporter of an amino acid exports an amino acid out of the bacterial cell ... the exporter of an amino acid is an exporter of methionine"; paragraph [0411] - "the bacterial cell comprises a heterologous gene encoding a methionine catabolism enzyme and a genetic modification that reduces export of methionine"), and discloses the recombinant bacterial cell further comprising a gene knockout of an endogenous essential/metabolic gene (paragraph [0716]-[0717]- "In some embodiments, the genetically engineered bacteria comprise a deletion or mutation in two or more genes required for cell survival and/or growth ... examples of essential genes include . . . YjeE"), it would have been obvious to one of ordinary skill in the art to include the methionine efflux pump, yjeH, and genetic knockout compositions and methods of Liu et al in the E. coli expression system of Falb et al (when taken with the disclosure from Feng et al and Hayashi et al), in order to help improve methionine processing, and for other desired downstream applications thereof.
Although, Liu et al do not disclose “wherein the yjeH comprises a sequence having at least 90% identity with SEQ ID NO:1014” (instant claim 6), such would have been obvious to an artisan of ordinary skill in the art as an E. coli strain with an endogenous amino acid transporter having the sequence 100% identical to SEQ ID NO:1014 was already known and disclosed by Reeves et al (2011; see page 8, section “Data deposition”) as accession number CP002211 (see nucleotide sequence homology below):
SEQ ID NO: 1014 (GenEmbl-database)
RESULT 29
CP002211/c
LOCUS CP002211 5038386 bp DNA circular BCT 31-JAN-2014
DEFINITION Escherichia coli str. 'clone D i2', complete genome.
ACCESSION CP002211
VERSION CP002211.1
DBLINK BioProject: PRJNA52021
BioSample: SAMN02603902
KEYWORDS .
SOURCE Escherichia coli str. 'clone D i2'
ORGANISM Escherichia coli str. 'clone D i2'
Bacteria; Pseudomonadota; Gammaproteobacteria; Enterobacterales;
Enterobacteriaceae; Escherichia.
REFERENCE 1 (bases 1 to 5038386)
AUTHORS Reeves,P.R., Liu,B., Zhou,Z., Li,D., Guo,D., Ren,Y., Clabots,C.,
Lan,R., Johnson,J.R. and Wang,L.
TITLE Rates of Mutation and Host Transmission for an Escherichia coli
Clone over 3 Years
JOURNAL PLoS ONE 6 (10), E26907 (2011)
PUBMED 22046404
REMARK Publication Status: Online-Only
REFERENCE 2 (bases 1 to 5038386)
AUTHORS Reeves,P.R., Liu,B., Zhou,Z., Li,D., Guo,D., Ren,Y., Clabots,C.,
Lan,R., Johnson,J.R. and Wang,L.
TITLE Direct Submission
JOURNAL Submitted (10-SEP-2010) TEDA School of Biological Sciences and
Biotechnology, Nankai University, 23 Hongda Street, Tianjin
Economic-Technological Development Area (TEDA), Tianjin, Tianjin
300457, P. R. China
COMMENT Bacteria available from Prof. James. Johnson's lab in Veterans
Affairs Medical Center and Department of Medicine, University of
Minnesota, Minneapolis, Minnesota, USA. Email address:
johns007\@umn.edu.
FEATURES Location/Qualifiers
source 1..5038386
/organism="Escherichia coli str. 'clone D i2'"
/mol_type="genomic DNA"
/strain="clone D i2"
/db_xref="taxon:885276"
gene 190..255
/gene="thrL"
/locus_tag="i02_5004"
CDS 190..255
/gene="thrL"
/locus_tag="i02_5004"
/codon_start=1
/transl_table=11
/product="thr operon leader peptide homoserine kinase"
/protein_id="AER82599.1"
/translation="MKRISTTITTTITITTGNGAG"
gene 336..2798
/gene="thrA"
/locus_tag="i02_0001"
CDS 336..2798
/gene="thrA"
/locus_tag="i02_0001"
/codon_start=1
/transl_table=11
/product="bifunctional aspartokinase I/homoserine"
/protein_id="AER82600.1"
/translation="MRVLKFGGTSVANAERFLRVADILESNARQGQVATVLSAPAKIT
NHLVAMIEKTISGQDALPNISDAERIFAELLTGLAAAQPGFPLAQLKTFVDQEFAQIK
HVLHGISLLGQCPDSINAALICRGEKMSIAIMAGVLEARGHNVTVIDPVEKLLAVGHY
LESTVDIAESTRRIAASRIPADHMVLMAGFTAGNEKGELVVLGRNGSDYSAAVLAACL
RADCCEIWTDVDGVYTCDPRQVPDARLLKSMSYQEAMELSYFGAKVLHPRTITPIAQF
QIPCLIKNTGNPQAPGTLIGASRDEDELPVKGISNLNNMAMFSVSGPGMKGMVGMAAR
VFAAMSRARISVVLITQSSSEYSISFCVPQSDCVRAERAMQEEFYLELKEGLLEPLAV
TERLAIISVVGDGMRTLRGISAKFFAALARANINIVAIA QGSSERSISVVVNNDDATT
GVRVTHQMLFNTDQVIEVFVIGVGGVGGALLEQLKRQQSWLKNKHIDLRVCGVANSKA
LLTNVHGLNLENWQEELAQAKEPFNLGRLIRLVKEYHLLNPVIVDCTSSQAVADQYAD
FLREGFHVVTPNKKANTSSMDYYHQLRYAAEKSRRKFLYDTNVGAGLPVIENLQNLLN
AGDELMKFSGILSGSLSYIFGKLDEGMSFSEATTLAREMGYTEPDPRDDLSGMDVARK
LLILARETGRELELADIEIEPVLPAEFNAEGDVAAFMANLSQLDNLFAARVAKARDEG
KVLRYVGNIDEDGVCRVKIAEVDSNDPLFKVKNGENALAFYSHYYQPLPLVLRGYGAG
NDVTAAGVFADLLRTLSWKLGV"
gene 2800..3732
/gene="thrB"
/locus_tag="i02_0002"
CDS 2800..3732
/gene="thrB"
/locus_tag="i02_0002"
/codon_start=1
/transl_table=11
/product="homoserine kinase"
/protein_id="AER82601.1"
/translation="MVKVYAPASSANMSVGFDVLGAAVTPVDGALLGDVVTVEAAETF
SLNNLGRFADKLPSEPRENIVYQCWERFCQELGKQIPVAMTLEKNMPIGSGLGSSACS
VVAALMAMNEHCGKPLNDTRLLALMGELEGRISGSIHYDNVAPCFLGGMQLMIEENDI
ISQQVPGFDEWLWVLAYPGIKVSTAEARAILPAQYRRQDCIAHGRHLAGFIHACYSRQ
PELAANLMKDVIAEPYRERLLPGFRQARQAVAEIGAVASGISGSGPTLFALCDKPDTA
QRVADWLGKNYLQNQEGFVHICRLDTAGARVLEN"
gene 3733..5019
/gene="thrC"
/locus_tag="i02_0003"
CDS 3733..5019
/gene="thrC"
/locus_tag="i02_0003"
/codon_start=1
/transl_table=11
/product="threonine synthase"
/protein_id="AER82602.1"
/translation="MKLYNLKDHNEQVSFAQAVTQGLGKNQGLFFPHDLPEFSLTEID
EMLKLDFVTRSAKILSAFIGDEIPQEILEERVRAAFAFPAPVANVESDVGCLELFHGP
TLAFKDFGGRFMAQILTHIAGDKPVTILTATSGDTGAAVAHAFYGLPNVKVVILYPRG
KISPLQEKLFCTLGGNIETVAIDGDFDACQALVKQAFDDEELKVALGLNSANSINISR
LLAQICYYFEAVAQLPQEARNQLVVSVPSGNFGDLTAGLLAKSLGLPVKRFIAATNVN
DTVPRFLHDGQWSPKATQATLSNAMDVSQPNNWPRVEELFRRKIWQLKELGYAAVDDE
TTQQTMRELKELGYTSEPHAAVAYRALRDQLNPGEYGLFLGTAHPAKFKESVEAILGE
TLDLPKELAERADLPLLSHNLPADFAALRKLMMNHQ"
gene 5087..5236
/locus_tag="i02_0004"
CDS 5087..5236
/locus_tag="i02_0004"
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="AER82603.1"
/translation="MEKNDREKGEILNKCGNLEIRIAENNNCRSHRVISGYRPITGND
KRSNL"
gene 5233..5529
/gene="yaaX"
/locus_tag="i02_0005"
CDS 5233..5529
/gene="yaaX"
/locus_tag="i02_0005"
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="AER82604.1"
/translation="MKKMQSIVLALSLVLVAPMAAQAAEITLVPSVKLQIGDRDNRGY
YWDGGHWRDHGWWKQHYEWRGNRWHPYGPPPSPRHNKHNDHRGDHRPGPDKHHR"
gene complement(5589..6365)
/gene="yaaA"
/locus_tag="i02_0006"
CDS complement(5589..6365)
/gene="yaaA"
/locus_tag="i02_0006"
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="AER82605.1"
/translation="MLILISPAKTLDYQSPLTTTRYTLPELLDNSQQLIHEARKLTPP
QISSLMRISDKLAGINAARFHDWQPDFTPENARQAILAFKGDVYTGLQAETFSEDDFD
FAQQHLRMLSGLYGVLRPLDLMQPYRLEMGIRLENARGKDLYQFWGDIITNKLNEALA
AQGDNVVINLASDEYFKSVKPKKLNAEIIKPVFLDEKNGKFKIISFYAKKARGLMSRF
IIENRLTKPEQLTGFNSEGYFFDEASSSNGELVFKRYEQR"
gene complement(6435..7865)
/gene="yaaJ"
/locus_tag="i02_0007"
CDS complement(6435..7865)
/gene="yaaJ"
/locus_tag="i02_0007"
/codon_start=1
/transl_table=11
/product="putative transporter yaaJ"
/protein_id="AER82606.1"
/translation="MPDFFSFINSVLWGSVMIYLLFGAGCWFTFRTGFVQFRYIRQFG
KSLKNSIHPQPGGLTSFQSLCTSLAARVGSGNLAGVALAITAGGPGAVFWMWVAAFIG
MATSFAECSLAQLYKERDANGQFRGGPAWYMARGLGMRWMGVLFAVFLLIAYAIIFSG
VQANAVARALSFSFDFPPLVTGIILAVFAMLAITRGLHGVARLMQGFVPLMAIIWVLT
CLVICVMNIGQLPHVIWSIFESAFGWQEAAGGAAGYTLSQAITNGFQRSMFSNEAGMG
STPNAAAAAASWPPHPAAQGIVQMIGIFIDTLVICTASAMLILLAGNGTTYMPLEGIQ
LIQKAMRVLMGSWGAEFVTLVVILFAFSSIVANYIYAENNFFFLRLNNPKAIWCLRIC
TFATVIGGTLLSFPLMWQLADIIMACMAITNLTAILLLSPVVHTIASDYLRQRKLGVR
PVFDPLRYPEIGRQLSPDAWDDVSQE"
gene 8144..9097
/gene="talB"
/locus_tag="i02_0008"
CDS 8144..9097
/gene="talB"
/locus_tag="i02_0008"
/codon_start=1
/transl_table=11
/product="transaldolase B"
/protein_id="AER82607.1"
/translation="MTDKLTSLRQYTTVVADTGDIAAMKLYQPQDATTNPSLILNAAQ
IPEYRKLIDDAVAWAKQQSNDRAQQIVDATDKLAVNIGLEILKLVPGRISTEVDARLS
YDTEASIAKAKRLIKLYNDAGISNDRILIKLASTWQGIRAAEQLEKEGINCNLTLLFS
FAQARACAEAGVFLISPFVGRILDWYKANTDKKEYAPAEDPGVVSVSEIYQYYKEHGY
ETVVMGASFRNIGEILELAGCDRLTIAPALLKELAESEGAIERKLSYTGEVKARPARI
TESEFLWQHNQDPMAVDKLAEGIRKFAVDQEKLEKMIGDLL"
gene 9209..9799
/gene="mogA"
/locus_tag="i02_0009"
CDS 9209..9799
/gene="mogA"
/locus_tag="i02_0009"
/codon_start=1
/transl_table=11
/product="molybdenum cofactor biosynthesis protein"
/protein_id="AER82608.1"
/translation="MMNTLRIGLVSISDRASSGVYQDKGIPALEEWLTSALTTPFELE
TRLIPDEQAIIEQTLCELVDEMSCHLVLTTGGTGPARRDVTPDATLAVADREMPGFGE
QMRQISLHFVPTAILSRQVGVIRKQALILNLPGQPKSIKETLEGVKDAEGNVVVHGIF
ASVPYCIQLLEGPYVETAPEVVAAFRPKSARREVSE"
gene complement(10004..10570)
/gene="yaaH"
/locus_tag="i02_0010"
CDS complement(10004..10570)
/gene="yaaH"
/locus_tag="i02_0010"
/codon_start=1
Query Match 100.0%; Score 1257; Length 5038386;
Best Local Similarity 100.0%;
Matches 1257; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 ATGAGTGGACTCAAACAAGAACTGGGGCTGGCCCAGGGCATCGGCCTACTATCGACGTCA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4749101 ATGAGTGGACTCAAACAAGAACTGGGGCTGGCCCAGGGCATCGGCCTACTATCGACGTCA 4749042
Qy 61 TTATTAGGCACTGGCGTGTTTGCCGTTCCTGCGTTAGCTGCGCTAGTAGCAGGCAATAAC 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4749041 TTATTAGGCACTGGCGTGTTTGCCGTTCCTGCGTTAGCTGCGCTAGTAGCAGGCAATAAC 4748982
Qy 121 AGCCTGTGGGCGTGGCCCGTTTTGATTATCTTAGTGTTCCCGATTGCGATTGTGTTTGCG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748981 AGCCTGTGGGCGTGGCCCGTTTTGATTATCTTAGTGTTCCCGATTGCGATTGTGTTTGCG 4748922
Qy 181 ATTCTGGGTCGCCACTATCCCAGCGCAGGCGGCGTCGCACACTTCGTCGGTATGGCGTTT 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748921 ATTCTGGGTCGCCACTATCCCAGCGCAGGCGGCGTCGCACACTTCGTCGGTATGGCGTTT 4748862
Qy 241 GGTTCGCGGCTTGAGCGAGTCACCGGCTGGTTGTTTTTATCGGTCATTCCCGTGGGTTTG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748861 GGTTCGCGGCTTGAGCGAGTCACCGGCTGGTTGTTTTTATCGGTCATTCCCGTGGGTTTG 4748802
Qy 301 CCTGCCGCGCTACAAATTGCTGCCGGATTCGGCCAGGCAATGTTTGGCTGGCATAGCGGG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748801 CCTGCCGCGCTACAAATTGCTGCCGGATTCGGCCAGGCAATGTTTGGCTGGCATAGCGGG 4748742
Qy 361 CAACTGTTGTTGGCAGAACTCGGTACGCTGGCGCTGGTGTGGTATATCGGTACTCGAGGT 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748741 CAACTGTTGTTGGCAGAACTCGGTACGCTGGCGCTGGTGTGGTATATCGGTACTCGAGGT 4748682
Qy 421 GCCAGTTCCAGTGCTAATCTACAAACAGTTATTGCCGGGCTTATCGTCGCACTGATTGTC 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748681 GCCAGTTCCAGTGCTAATCTACAAACAGTTATTGCCGGGCTTATCGTCGCACTGATTGTC 4748622
Qy 481 GCTATCTGGTGGGCGGGCGATATCAAACCTGCGAATATCCCCTTCCCTGCGCCAGGAAAT 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748621 GCTATCTGGTGGGCGGGCGATATCAAACCTGCGAATATCCCCTTCCCTGCGCCAGGAAAT 4748562
Qy 541 ATCGAACTTACCGGGTTATTCGCTGCGTTATCAGTGATGTTCTGGTGTTTTGTCGGTCTG 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748561 ATCGAACTTACCGGGTTATTCGCTGCGTTATCAGTGATGTTCTGGTGTTTTGTCGGTCTG 4748502
Qy 601 GAAGCATTTGCCCATCTTGCCTCGGAATTTAAAAATCCAGAGCGTGATTTTCCTCGTGCT 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748501 GAAGCATTTGCCCATCTTGCCTCGGAATTTAAAAATCCAGAGCGTGATTTTCCTCGTGCT 4748442
Qy 661 TTGATGATTGGCCTGCTGCTGGCAGGATTAGTCTATTGGGGCTGTACGGTAGTCGTCTTA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748441 TTGATGATTGGCCTGCTGCTGGCAGGATTAGTCTATTGGGGCTGTACGGTAGTCGTCTTA 4748382
Qy 721 CACTTCGACGCCTATGGTGAACAAATGGCGGCGGCAGCATCGCTTCCCAAAATTGTAGTG 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748381 CACTTCGACGCCTATGGTGAACAAATGGCGGCGGCAGCATCGCTTCCCAAAATTGTAGTG 4748322
Qy 781 CAGTTATTCGGTGTAGGAGCGTTATGGATTGCCTGCGTAATTGGCTATCTGGCCTGCTTT 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748321 CAGTTATTCGGTGTAGGAGCGTTATGGATTGCCTGCGTAATTGGCTATCTGGCCTGCTTT 4748262
Qy 841 GCCAGTCTCAACATTTATATACAGAGCTTCGCCCGCCTGGTCTGGTCGCAGGCGCAACAT 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748261 GCCAGTCTCAACATTTATATACAGAGCTTCGCCCGCCTGGTCTGGTCGCAGGCGCAACAT 4748202
Qy 901 AATCCTGACCATTACCTGGCACGCCTCTCTTCTCGCCATATTCCGAATAATGCCCTCAAT 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748201 AATCCTGACCATTACCTGGCACGCCTCTCTTCTCGCCATATTCCGAATAATGCCCTCAAT 4748142
Qy 961 GCGGTGCTCGGCTGCTGCGTGGTGAGCACGTTGGTGATTCATGCTTTAGAGATCAATCTG 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748141 GCGGTGCTCGGCTGCTGCGTGGTGAGCACGTTGGTGATTCATGCTTTAGAGATCAATCTG 4748082
Qy 1021 GACGCTCTTATTATTTATGCCAATGGCATCTTTATTATGATTTATCTGTTATGCATGCTG 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 4748081 GACGCTCTTATTATTTATGCCAATGGCATCTTTATTATGATTTATCTGTTATGCATGCTG 4748022
Qy 1081 GCAGGCTGTAAATTATTGCAAGGACGTTATCGACTACTGGCAGTGGTTGGCGGGCTATTA 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db