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
Claims 3-4, 6-7, 13, and 16 are canceled. Claims 17-19 are new. Claims 1-2, 5, 8-12, 14-15 and 17-19 are pending and under consideration in this action.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The instant claims are entitled to an effective filing date of 10/12/2022.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2, 5, 8-12, 14-15 and 17-19 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites “a gene glmS encoding L-glutamine-D-fructose-6-phosphate transaminase comprising the amino acid sequence of SEQ ID NO: 66” see lines 10-11, which renders the claim indefinite because SEQ ID NO: 66 is a nucleic acid sequence. Therefore, it is unclear whether the claim intends to refer to a different amino acid sequence or the nucleic acid sequence of SEQ ID NO: 66.
Claims 2, 5, 8-12, 14-15 and 17-19 depend from claim 1 and are rejected for the reason set forth above.
Claim 10 recites “trace element stock solution” in line 3, which renders the claim indefinite because it is unclear which solutions constitute as trace element stock solution. The instant specification discloses that the trace element stock solution preferably comprises 54.4 g/L ferric ammonium citrate, 9.8 g/L MnCl2-4H2O, 1.6 g/L CoCl2-6H2O, 1 g/L CuCl2-2H2O, 1.9 g/L H3BO3, 9 g/L ZnSO4-7H2O, 1.1 g/L Na2MoO4-2H2O, 1.5 g/L Na2SeO3 and 1.5 g/L NiSO4-6H2O. See p. 5 lines 20-24. It is unclear whether the claimed trace element stock solution is limited to the preferred embodiment disclosed in the specification.
Claim 18 depends from claim 10 and is rejected for the reason set forth above.
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.
Claims 1-2, 5, 8-12, 15, 17 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Jennewein (WO 2019/020707, as provided with the IDS filed 04/08/2024), in view of Aesaert (WO 2022/034080) and Mahour (WO 2021/089251), with evidence from Guerry (NCBI, accession number AF305571, submitted 09/14/2002), Kanesaki (NCBI, accession number BAL35720, submitted 08/31/2011), and Lucas (accession number NC_012947, submitted 07/06/2009).
Regarding claim 1, Jennewein teaches modifying strain #942 modified for the production of sialic acid by the genomic integration of the expression cassettes <Ptet glmSm-gna1-FRT-aacC1-FRT> (SEQ ID NO: 69), <Ptet -slr1975-FRT-cat-FRT>, < Ptet -neuBC-FRT-kan-FRT> and < Ptet -ppsA-FRT-aad1-FRT>. See p. 25 lines 30-33. Jennewein teaches neuB (acc. No. AF305571). See p. 26 line 7. Accession number AF305571 is 100% identical to instant SEQ ID NO: 85, as evidenced by Guerry. See pgs. 2-3 of Guerry and the alignment in the office action appendix. Jennewein teaches slr1975 (acc. No. BAL35720). See p. 26 lines 11-12. Accession number BAL35720 is 100% identical to instant SEQ ID NO: 86, as evidenced by Kanesaki. See p. 2 of Kanesaki and the office action appendix for the alignment. Jennewein teaches gna1 encodes. See p. 26 lines 3-4. Jennewein teaches the expression cassette containing glmS (SEQ ID NO:69), which includes a subsequence that is 100% identical to instant SEQ ID NO:66. See p. 25 line 31 of Jennewein, and the office action appendix for the alignment. Jennewein teaches ppsA encoding the phosphoenolpyruvate synthase of E. coli BL21 DE3. See p. 26 lines 15-16. Jennewein teaches E. coli BL21 (DE3) strain #942 (i.e. the genetically engineered bacterium E. coli BL21 containing the DE3 prophage). See p. 25 lines 26-27. Furthermore, Jennewein teaches subcloning neuB encoding a sialic acid synthase as an operon behind the promoter Ptet fused to a FRT-site flanked kanamycin resistance gene. See p. 26 lines 7-11.
Jennewein does not teach (1) YqaB encoding N-acetylglucosamine-6-phosphate phosphatase, wherein the gene YqaB is derived from the E. coli BL21 genome.
Jennewein does not teach (1) Gna1 encoding glucosamine-6-phosphate acetyltransferase comprising the amino acid sequence of SEQ ID NO:87.
Jennewein does not teach (2) a plasmid vector 2 having a pET28a plasmid as the backbone, wherein the plasmid vector 2 comprises a gene encoding N-acylneuraminic acid cytidylytransferase and a gene encoding a sialytransferase, wherein the sialytransferase is alpha-2,6-sialyltransferase or alpha-2,3-sialyltransferase.
Aesaert teaches a pRS420-plasmid series containing constitutive transcriptional units for one or more copies of mutant glmS*54, a phosphatase like the E. coli gene YqaB, an N-acetylglucosamine 2-epimerase, one or more copies of N-acetylneuraminate synthase like NeuB, and one or more copies of N-acylneuraminate cytidylyltransferase. Optionally, a constitutive transcriptional unit comprising one or more copies of GNA1 with SEQ ID NO: 7, which is 100% identical to instant SEQ ID NO: 87 (see the alignment provided in the office action appendix). To produce sialyated oligosaccharides, Aesaert teaches that the plasmid further comprises constitutive transcriptional units of a alpha-2,3-sialyltransferase like, and/or a alpha 2,6-sialytransferase. See p. 93 lines 9-28. Evidentiary reference Lucas discloses that YqaB is present in the E. coli BL21 genome. See p. 1-2 of Lucas. Aesaert teaches presenting DNA sequences to the cell in expression modules regulated by one or more regulatory sequences, wherein the expression modules are integrated into the cell’s genome or presented in a plasmid. See claims 7-8 of Aesaert. In example 4, Aesaert teaches modifying an E. coli for sialic acid and 6' -siayllactose production. See p. 95 line 6. Aesaert teaches a mutant E. coli modified with genomic knock-ins and/or expression plasmids with constitutive transcriptional units to express N-acylneuraminate cytidylyltransferase enzyme and beta-galactoside alpha-2,6-sialytransferase. See p. 95 lines 15-18.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Aesaert's plasmid for Jennewein’s expression cassettes such that Aesaert’s plasmid carries Jennewein’s neuB, slr1975, glms, and ppsA genes; to further add Aesaert’s YqaB and Gna1 genes to that plasmid; and to further modify the E. coli BL21 (DE3) strain #942 with Aesaert’s separate plasmid for expression of N-acylneuraminate cytidylyltransferase enzyme and alpha-2,6-sialytransferase. One of ordinary skill in the art would have been motivated to use Aesaert’s plasmid, because Aesaert suggests plasmids can be used as an alternative to genome integration. There would have been a reasonable expectation of success because Jennewein demonstrates integrating neuB, slr1975, glmS, a gna1 and ppsA into the genome of E. coli BL21 (DE3) strain #942; and Aesaert teaches using a plasmid for the gene integration of neuB, N-acetylglucosamine 2-epimerase (i.e. same type of enzyme as slr1975), glmS, YqaB and gna1. One of ordinary skill in the art would have been further motivated to add YqaB and gna1 of Aesaert to the modified plasmid, because Aesaert suggests that YqaB and GNA1 SEQ ID NO: 7 (i.e. 100% identical to instant SEQ ID NO: 87) are useful for the production of sialic acid (see p. 93 lines 9, 14, and 21-22). There would have been a reasonable expectation of success because Aesaert suggests combining the YqaB and GNA1 SEQ ID NO: 7 with NeuB, glmS and N-acetylglucosamine 2-epimerase (i.e. same type of enzyme as slr1975) (see p. 93 lines 9-22) and Jennewein teaches introducing neuB, slr1975, glmS, and a gna1 into E. coli BL21 (DE3). In the process, one of ordinary skill in the art would arrive at a first plasmid comprising neuB, slr1975, YqaB, Gna1, glmS and ppsA linked in tandem. One of ordinary skill in the art would have been further motivated to insert Aesaert’s separate plasmid for expressing N-acylneuraminate cytidylyltransferase and alpha-2,6-sialytransferase, because Jennewein teaches producing sialylated oligosaccharides (see claim 1 of Jennewein) selected from a group including 6’-sialyllactose (see claim 2 of Jennewein); and Aesaert suggests using the plasmid for 6' -siayllactose production (see p. 95 line 6). There would have been a reasonable expectation of success because Aesaert demonstrates integrating the plasmid into an E. coli cell for 6’-sialyllactose production, and Jennewein teaches E. coli cells and 6’siallyactose production.
Jennewein and Aesaert do not teach (1) a plasmid vector 1 having pACYCDuet plasmid as the backbone. Therefore, Jennewein and Aesaert do not teach (1) the genes neuB, slr1975, YqaB, Gna1, glmS and ppsA linked in tandem in the plasmid 1 vector. However, Jennewein teaches neuB, slr1975, glmS, and ppsA as claimed; and Aesaert teaches a pRS420 plasmid, and Aesaert teaches YqaB and Gna1 as claimed.
Jennewein and Aesaert do not teach (2) a plasmid vector 2 having a pET28a plasmid as the backbone. However, Aesaert teaches an expression plasmid with constitutive transcriptional units to express N-acylneuraminate cytidylyltransferase enzyme and alpha-2,6-sialytransferase.
Mahour teaches a process that can be adapted for producing human milk oligosaccharides. See p. 1 lines 9-11. Mahour teaches E. coli BL21 Gold (DE3) (i.e. E. coli BL21 containing the DE3 prophage) carrying the plasmid pET28a with kanamycin resistance with gene sequences encoding enzymes. See p. 76 lines 9 and 15-16. Mahour teaches growing cells carrying three vectors. See p. 79 lines 5-6. The vectors include pACYCDuet with chloramphenicol resistance. See table 3 on p. 78.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Mahour’s pACYCDuet template for Aesaert’s pRS420-plasmid template as to arrive at a pACYCduet containing Jennewein’s neuB, slr1975, glmS, and ppsA, and Aesaert’ YqaB and Gna1 (i.e. plasmid 1); and to further substitute Mahour’s pET28a for Aesart’s plasmid template used for expressing N-acylneuraminate cytidylyltransferase and alpha-2,6-sialytransferase (i.e. plasmid 2). One of ordinary skill in the art would have been motivated to use Mahour’s pACYCDuet template because Mahour discloses that the template has chloramphenicol resistance. There would have been a reasonable expectation of success because Mahour demonstrates transforming an E. coli BL21 (DE3) cell with an pACYCDuet encoding enzymes; and Jennewein teaches integrating enzyme genes into E. coli BL21 (DE3) cells. One of ordinary skill in the art would have been further motivated to use Mahour’s pET28a template, because Mahour suggests that plasmid pET28a has kanamycin resistance. There would have been a reasonable expectation of success because Mahour demonstrates transforming an E. coli BL21 (DE3) cell with an pET28a encoding enzymes; and Jennewein teaches integrating enzyme genes into E. coli BL21 (DE3) cells.
Jennewein, Aesaert and Mahour do not teach (3) multiple copies of a plasmid vector 3, wherein the plasmid vector 3 comprises a gene neuB encoding sialic acid synthase, and the gene neuB is initiated for expression by a Tet promoter. However, Jennewein teaches subcloning neuB encoding a sialic acid synthase behind the promoter Ptet (see p. 26 lines 7-11).
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Jennewein’s Tet promoter with the plasmid 1 of Jennewein as modified by Aesaert and Mahour, and to further duplicate that plasmid 1 as to arrive at multiple copies of a plasmid vector 3 that comprises Jennewein’s neuB (100% identical to instant SEQ ID NO: 85) under Jennewein’s Tet promoter. One of ordinary skill in the art would be motivated to add the Tet promoter to the plasmid 1, because Jennewein suggests promoters that provide a site for initiation of transcription (see p. 12 lines 18-20). There would have been a reasonable expectation of success because Jennewein demonstrates constructing expression cassette with neuB under the Ptet promoter. One of ordinary skill in the art would have been further motivated to duplicate the plasmid, because Aesaert teaches a cell that comprises at least one DNA sequence encoding neuB (see claim 19 of Aesaert). Thus, Aesaert reasonably suggests cells containing more than one sequence encoding neuB. There would have been a reasonable expectation of success because Mahour demonstrates transforming E. coli BL21 (DE3) cells carrying three vectors.
Regarding claim 2, Jennewein teaches deleting nanA (N-acetylneuraminate lyase), nanK (N-acetylneuraminate kinase) and nanE from E. coli. See example 1 spanning p. 10-11. These gene knockouts or deletions would have disabled an N-acetylneuraminic acid catabolic pathway.
Aesaert teaches modifying an E. coli strain by knocking out nanA, nanE and nanK. See p. 95 lines 6-7, p. 97 line 4, and p. 98 lines 11-12.
Regarding claim 5, Aesaert teaches a mutant E. coli modified with genomic knock-ins and/or expression plasmids with constitutive transcriptional units to express N-acylneuraminate cytidylyltransferase enzyme and beta-galactoside alpha-2,6-sialytransferase. See p. 95 lines 15-18. Therefore, Aesaert suggests that the genes are linked in tandem within a plasmid vector.
Jennewein and Aesaert do not teach plasma vector 2 having a pET28a backbone, as required in amended claim 1 from which claim 5 depends.
Mahour teaches E. coli BL21 Gold (DE3) (i.e. E. coli BL21 containing the DE3 prophage) carrying the plasmid pET28a with kanamycin resistance with gene sequences encoding enzymes. See p. 76 lines 9 and 15-16.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Mahour’s pET28a for Aesart’s plasmid template used for expressing N-acylneuraminate cytidylyltransferase and alpha-2,6-sialytransferase (i.e. plasmid 2) as discussed above; and to further arrange the genes encoding N-acylneuraminate cytidylyltransferase and alpha-2,6-sialytransferase in any order including in tandem. A person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would have been a reasonable expectation of success because Aesaert demonstrates arranging the N-acylneuraminate cytidylyltransferase and alpha-2,6-sialytransferase in one plasmid.
Regarding claim 8, Jennewein teaches a method for producing a sialylated oligosaccharide that is selected from a group that includes 3’-sialyllactose and 6’-sialyllactose. See claims 1 and 2 of Jennewein. The cell is cultivated in the presence of lactose. See claim 6 of Jennewein. The sialylated oligosaccharide can be recovered from the fermentation broth (i.e. extracting from the fermentation broth). See p. 17 lines 25-26. In example 4, Jennewein teaches growing E. coli BL21 (DE3) #1363. Lactose is added after one hour of incubation. See the paragraph spanning pgs. 36-37. As a result, 3’-siallylactose (3’-SL) and 6’-siallylactose (6’-SL) are produced. See p. 37 lines 11-12.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply siallylactose production method of Jennewein to the modified E. coli BL21 (DE3) of Jennewein, Aesaert and Mahour.
Regarding claim 9, Jennewein teaches growing E. coli BL21 (DE3) #1363 harboring plasmids in a supplemented mineral salts medium. See p. 36 lines 12-14.
Jennewein does not teach a fermentation medium that comprises 2.31 g/L KH2PO4 and 12.54 g/L K2HPO4.
Aesaert teaches a minimal medium for E. coli that includes 2.993 g/L KH2PO4 and 7.315 g/L K2HPO4. See p. 82 lines 31-32.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Aesaert’s minimal medium containing 2.993 g/L KH2PO4 and 7.315 g/L K2HPO4 for Jennewein’s mineral salt medium; and to further optimize the concentrations of the KH2PO4 and K2HPO4. One of ordinary skill in the art would have been motivated to use Aesaert’s mineral medium because Aesaert discloses that the medium is for E. coli. There would be a reasonable expectation of success because Jennewein and Aesaert demonstrate culturing E. coli. One of ordinary skill in the art would have been further motivated to optimize the KH2PO4 and K2HPO4, because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would have been a reasonable expectation of success because Aesaert teaches includes 2.993 g/L KH2PO4 and 7.315 g/L K2HPO4 amounts from which one could reasonably optimize. MPEP 2144.05(II)(A) indicates that differences in concentration generally amount to “routine optimization” and will not support patentability unless there is evidence indicating the claimed feature is critical. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Aesaert and Jennewein do not teach a fermentation medium that is a TB medium; the TB medium comprises 12 g/L tryptone, 24 g/L yeast extract, 4 mL/L glycerol.
Mahour teaches growing E. coli in TB media. See p. 77 line 16-17 and table 2. The TB medium includes 12 g tryptone, 24 g yeast extract, 5 g glycerol and phosphate buffer in 1 L dH2O. See table 2.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Aesaert’s minimal medium containing KH2PO4 and K2HPO4 with Mahour’s TB medium; and to further optimize the 5 g/l glycerol. One of ordinary skill in the art would have been motivated to use the TB medium of Mahour, because Mahour suggests that the medium can be used to culture E. coli, and Mahour teaches E. coli BL21 (DE3), which is the same strain taught by Jennewein. There would have been a reasonable expectation of success because Mahour demonstrates growing E. coli in the TB medium, which includes a phosphate buffer; and Aesaert’s minimal medium includes phosphates, i.e. KH2PO4 and K2HPO4. One of ordinary skill in the art would have been further motivated to optimize the glycerol, because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would have been a reasonable expectation of success because Mahour teaches a 5g/L amount from which one could reasonably optimize. MPEP 2144.05(II)(A) indicates that differences in concentration generally amount to “routine optimization” and will not support patentability unless there is evidence indicating the claimed feature is critical. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 10, Jennewein teaches growing E. coli BL21 (DE3) cells harboring expression plasmids in a mineral salts medium. When the cultures reach OD600 of 0.1 to 0.3, gene expression is induced by the addition of IPTG. After an hour of incubation, 1.5 mM lactose is added. See p. 36 line 12 to p. 37 line 1.
Jennewein does not teach OD600 value of 0.6-0.8.
Mahour teaches culturing E. coli until OD600 -of 0.8-1.0, afterwards, induction is carried out with IPTG. See p. 85 lines 5, and 9-10.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize the OD600 of 0.1 to 0.3 taught by Jennewein based on the suggestion of Mahour. One of ordinary skill in the art would have been motivated to do so because Mahour suggests culturing E. coli until an OD600 -of 0.8-1.0 before IPTG induction, and an OD600 -of 0.8 overlaps with the instantly claimed 0.6-0.8 range. There would have been a reasonable expectation of success because Mahour and Jennewein demonstrate culturing E. coli and an induction with IPTG.
Jennewein and Mahour do not teach subsequently supplementing 2 g/L MgSO4·7H2O, 20 g/L glycerol, 1 mL/L trace element stock solution and 5 g/L lactose. However, Jennewein teaches subsequently adding 1.5 mM lactose after induction culture.
Aesaert, in example 2, teaches materials and methods for E. coli. See. p. 82 line 18. Aesaert teaches a minimal medium containing 0.5 g/L MgSO4.7H2O, 30 g/L glycerol, 1 mL/L vitamin solution. See p. 82 lines 21-24. Aesaert discloses that 20 g/L lactose can be added to the medium as a precursor. See p. 82 lines 25-26. The vitamin solution includes MnCl2.2H2O and CuCl2.H2O (e.g. trace elements). See p. 82 lines 27-29.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine Aesaert’s minimal medium containing 0.5 g/L MgSO4.7H2O, 30 g/L glycerol, 1 mL/L vitamin solution (i.e. trace element stock solution) with the IPTG induced E. coli culture of Jennewein and Mahour; to further optimize the concentrations of the MgSO4.7H2O and glycerol components, and to optimize Jennewein’s 1.5 mM lactose concentration based on Aesaert’s suggestion. One of ordinary skill in the art would have been motivated to supplement the IPTG induced culture with the minimal medium of Aesaert, because Jennewein suggests incubating cells after the IPTG induction (see p. 37 lines 2-3); and Aesaert suggests that the minimal medium is suitable for E. coli incubation. There would have been a reasonable expectation of success because Jennewein demonstrates culturing E. coli cells, and Aesaert suggests that the minimal medium is suitable for E. coli. One of ordinary skill in the art would have been further motivated to optimize the 0.5 g/L MgSO4.7H2O and 30 g/L glycerol concentrations of Aesaert, because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would have been a reasonable expectation of success because Aesaert teaches starting concentrations from which one could optimize. One of ordinary skill in the art would have been further motivated to optimize the lactose, because Aesaert suggests that 20 g/L lactose can be added to E. coli cells. There would have been a reasonable expectation of success because Jennewein teaches a 1.5 mM lactose amount from which one could reasonably optimize. MPEP 2144.05(II)(A) states that “[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 11, Jennewein teaches growing E. coli BL21 (DE3) #1363 at 30˚C in shake flasks. See p. 36 lines 12-13.
Jennewein and Mahour do not teach 250 rpm.
Aesaert teaches growing host strains at 30˚C and shaking at 200 rpm. See p. 94 lines 31-32
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize Jennewein’s shaking based on Aesaert’s suggestion. One of ordinary skill in the art would have been motivated to do so because Aesaert suggests growing at 30˚C and shaking at 200 rpm. There would be a reasonable expectation of success because Jennewein demonstrates growing E. coli BL21 (DE3) #1363 at 30˚C in shake flasks, and Aesaert teaches a 200 rpm from which one could optimize. MPEP 2144.05(II)(A) states that “[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 12, Jennewein teaches deleting nanA, nanK and nanE from E. coli. See example 1 spanning pgs. 10-11.
Aesaert teaches modifying an E. coli strain by knocking out nanA (N-acetylneuraminate lyase), nanE and nanK (N-acetylneuraminate kinase). See, for example, p. 95 lines 6-7, p. 97 line 4, p. 98 lines 11-12.
Regarding claim 15, Jennewein teaches a sialyltransferase gene with the accession number BAF91416 SEQ ID NO: 30, which is 100% identical to instant SEQ ID NO: 62. See the table on p. 35 and the office action appendix.
Regarding claim 17, Jennewein teaches screening genes that encode α-2,3-sialyltransferase, thus producing 3’-sialyllactose. Jennewein teaches enzymes that synthesize 6’sialyllactose depicted as α-2,6-sialyltransferase. See p. 37 lines 11-12. Jennewein teaches quantifying the obtained sialylated products. See table 5 on p. 38.
Regarding claim 19, Jennewein teaches deleting nanA (N-acetylneuraminate lyase), nanK (N-acetylneuraminate kinase) and nanE from E. coli. See example 1 spanning p. 10-11. These gene knockouts or deletions would have disabled an N-acetylneuraminic acid catabolic pathway.
Aesaert teaches modifying an E. coli strain by knocking out nanA, nanE and nanK. See p. 95 lines 6-7, p. 97 line 4, and p. 98 lines 11-12.
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Jennewein (WO 2019/020707, as provided with the IDS filed 04/08/2024), in view of Aesaert (WO 2022/034080) and Mahour (WO 2021/089251), as applied to claims 1-2, 5, 8-12, 15, 17 and 19 above, and further in view of Jennewein2016 (US 2016/0333042).
Regarding claim 18, Aesaert teaches a minimal medium containing 1 mL/L vitamin solution. See p. 82 lines 21-24. Furthermore, Aesaert teaches a trace element mix that includes 5 g/L MnCl2.4H2O, 1.6 g/L CoCl2.6H2O, 0.033 g/L CuCl2.2H2O, 11.4 g/L H3BO3, 0.06 g/L Na2MoO4, and 22 g/L ZnSO4.7H2O. See p. 107 lines 20-21. Aesaert teaches a molybdate solution containing Na2MoO4.2H2O. See p. 82 line 29.
Jennewein, Aesaert and Mahour do not teach 54.4 g/L ferric ammonium citrate, 9.8 g/L MnCl2, 1 g/L CuCl2·2H2O, 1.9 g/L H3BO3, 9 g/L ZnSO4·7H2O, 1.1 g/L Na2MoO4·2H2O, 1.5 g/L Na2SeO-3 and 1.5 g/L NiSO4.
Jennewein2016 teaches purifying neutral human milk oligosaccharides (HMOs) produced by microbial fermentation. See [0001]. Furthermore, Jennewein2016 teaches E. coli strain (E. coli BL21(DE3)). See [0054]. Jennewein2016, in example 2, teaches a microbial fermentation that uses trace elements: ammonium ferric citrate 56 mg/l, MnCl2.4H2O 9.8 mg/l, CoCl2.6H2O 1.6 mg/l, CuCl2.2H2O 1 mg/l, H3BO3 1.6 mg/l, ZnSO4.7H2O 9 mg/l, Na2MoO4.2H2O 1.2 mg/l, Na2SeO3 1.2 mg/l; and feed substances glycerol and lactose. See [0077]. Furthermore, Jennewein2016 teaches trace elements including 0.002 gl−1 NiSO4x6H2O. See [0055].
Jennewein, Aesaert, Mahour and Jennewein2016 do not teach 54.4 g/L ferric ammonium citrate, 9.8 g/L MnCl2, 1 g/L CuCl2·2H2O, 1.9 g/L H3BO3, 9 g/L ZnSO4·7H2O, 1.1 g/L Na2MoO4·2H2O, 1.5 g/L Na2SeO-3 and 1.5 g/L NiSO4.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to substitute Jennewein’s trace elements for the 1 mL/L vitamin solution in the minimal medium of Aesaert; and to further optimize the concentration of each trace element. One of ordinary skill in the art would have been motivated to use the trace elements of Jennewein2016 because Jennewein2016 suggests that the trace elements can be used with a glycerol and lactose feed during a microbial fermentation of E. coli BL21(DE3). There would have been a reasonable expectation of success because Jennewein2016 demonstrates fermenting E. coli BL21(DE3) with the trace elements, and E. coli BL21(DE3) is the same strain taught by Jennewein and Mahour. One of ordinary skill in the art would have been further motivated to optimize the concentration of each trace element taught by Jennewein2016, because Aesaert discloses that the vitamin mix is present in the minimal medium at a concentration of 1 mL/L. Thus, one would have been motivated to convert the mg/L concentrations taught by Jennewein2016 to g/L, such that the concentration that enters the minimal medium of Aesaert is in the mg/L concentrations taught by Jennewein2016. There would have been a reasonable expectation of success because Jennewein2016 teaches starting concentrations from which one could reasonably optimize. MPEP 2144.05(II)(A) indicates that differences in concentration or temperature generally amount to “routine optimization” and will not support patentability unless there is evidence indicating the claimed feature is critical. “Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Response to Arguments
Applicant's arguments filed 5/26/2026 have been fully considered but they do not apply to the new grounds of rejection set forth above.
Conclusion
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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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./
Examiner, Art Unit 1657
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