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 1-13, 16-19, 22, and 28-31 are pending.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 1-13, 16-19, 22) and the species of SEQ ID NO: 2, SEQ ID NO: 42, and E. coli in the reply filed on 1/13/2026 is acknowledged.
Claims 28-31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 1/13/2026.
In claim 13, no prior art was found for the elected species of SEQ ID NO: 42, so all SEQ ID NO: species were searched.
Claims 1-13, 16-19, and 22 are examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. See lines 11, 17, 19-22, and 27 on page 19. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 12-13 are objected to because of the following informalities:
In claim 12, “wherein at least two copies of the nucleic acid sequence encoding the enzyme is present in the genome of the cell.” The word “is” should be replaced with “are.”
In claim 13, promoter is misspelled as “promotor.”
Appropriate correction is required.
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.
Claims 1-13, 16-19, and 22 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 that “A genetically modified cell which comprises a heterologous nucleic acid sequence encoding a heterologous enzyme which on its own is capable of hydrolyzing sucrose into fructose and glucose on the extracellular side or in the periplasmic space. It is unclear whether the term “on its own” is limiting the activity of the enzyme, the location of the enzyme, or both. Furthermore, the claim recites an open transitional phrase (“comprising”), so it is unclear what additional components besides the enzyme are not permissible (the enzyme is required to have certain activity “on its own”), such as other enzymes, secretion signals, or promoters.
Claims 2-13, 16-19, and 22 are rejected for depending from a rejected base claim and not rectifying the source of indefiniteness discussed above.
Claim 6 recites “wherein a single genomically integrated copy of the heterologous nucleic acid sequence encoding the enzyme is capable of hydrolyzing sucrose into fructose and glucose.” The claim has multiple reasonable interpretations, rendering the claim indefinite. In one interpretation, the cell comprises a single genomically integrated copy of the heterologous nucleic acid sequence that encodes the enzyme that is capable of hydrolysing sucrose into fructose and glucose. In a second interpretation, the limitation is further limiting the host cell and the enzyme by requiring the nucleic acid encoding the enzyme must be capable of being integrated into the genome of the host cell.
Claim 7 recites the limitation “wherein the expression of the enzyme enables utilization of sucrose as the main and/or the sole carbon source and/or as the main and/or the sole energy source of the genetically modified cell.” The claim is indefinite because “main” is broader in scope than “sole,” leading to ambiguity in the claim scope.
Claim 9 recites that the cell does not contain a complete sucrose utilization system. However, this limitation contradicts independent claim 1, which requires that the cell comprises an enzyme that is capable of hydrolyzing sucrose into fructose and glucose. Thus, it is unclear what is meant by “complete sucrose utilization system” since claim 1 requires that the cell comprises an enzyme which is capable of sucrose hydrolysis into glucose and fructose (i.e. complete utilization of sucrose).
Claim 10 recites “wherein the cell was not capable of sustaining growth on sucrose prior to the addition of the heterologous enzyme.” There is a lack of antecedent basis for “the addition of the heterologous enzyme.” In addition, the claim language, in past tense, leads to ambiguity in the statutory category of invention, since “the addition of a heterologous enzyme” is a method step. Applicant may consider amending claim 10 to recite “wherein the cell is incapable of sustaining growth on sucrose without the heterologous enzyme.”
Claim 16 recites the limitation "the fermentation medium" in line 4. There is insufficient antecedent basis for this limitation in the claim. Claim 17 is rejected for depending from a rejected base claim and not rectifying the source of indefiniteness discussed above.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1 and 11-12 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 11 recites the genetically modified cell according to claim 1, wherein the heterologous nucleic acid sequence encodes an enzyme of SEQ ID NOs: 1 or 2 or a functional homologue of any one of SEQ ID NOs: 1 or 2 having an amino acid sequence which is more than 70% identical to any one of SEQ ID NO: 1 or 2. Claim 12 requires at least two copies of the nucleic acid sequence encoding the enzyme is present in the genome of the cell. Claims 11-12 depend from claim 1, which also requires that the enzyme is capable of hydrolyzing sucrose into fructose and glucose on the extracellular side or in the periplasmic space of the genetically modified cell.
The person of ordinary skill in the art would not have recognized that the inventors, at the time the application was filed, had possession of the claimed genus of functional homologues of SEQ ID NOs: 1 or 2 capable of hydrolyzing sucrose into fructose and glucose on the extracellular side or in the periplasmic space of a genetically modified cell.
The specification discloses only SEQ ID NOs: 1 or 2 but does not disclose any functional homologues of either protein. SEQ ID NO: 1 is 483 amino acids long and SEQ ID NO: 2 is 548 amino acids long. Thus, 70% identity to either SEQ ID NO: 1 or SEQ ID NO: 2 corresponds to up to 145 amino acid substitutions in SEQ ID NO: 1 or 164 amino acid substitutions in SEQ ID NO: 2. SEQ ID NO: 1 is a glycoside hydrolase family 32 protein from Avibacterium gallinarum (specification line 27on page 4) and SEQ ID NO: 2 is a β-fructofuranosidase protein from Arthrobacter globiformis IFO 3062 (lines 30-31 on page 4).
Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249; cited in the Restriction Requirement mailed on 12/16/2025) teaches a genetically modified E. coli cell comprising a heterologous enzyme β-fructofuranosidase from Arthrobacter (Title and Abstract). The enzyme is 100% identical to SEQ ID NO: 2 of the presently claimed invention (Isono Figure 1 and OA Appendix A), therefore the enzyme is necessarily capable of hydrolyzing sucrose into fructose and glucose. Furthermore, Isono explicitly teaches that the enzyme is capable of hydrolyzing sucrose (Abstract and page 248, left column, top paragraph).
Isono teaches mutants of SEQ ID NO: 2 with the amino acid substitutions D130A, E392A, and D306A (page 248, left column, first full paragraph). The mutants with the amino acid substitutions D130A and E392A are inactive but the enzyme with the amino acid substitution D306A does not show any considerable decrease in the activity (page 248, left column, first full paragraph). Isono Fig. 3 illustrates some conserved residues compared to other enzymes, including β-fructofuranosidase and levansucrases, but Isono also teaches that β-fructofuranosidase and levansucrase are different from each other in substrate recognition and active site formation (page 248, left column, bottom paragraph).
UniProt A0A379AWT7_AVIGA, which is identical to the instant SEQ ID NO: 1 (OA Appendix C), teaches that the protein enables the bacterium to metabolize sucrose as a sole carbon source (Function). UniProt predicts two different glycosyl hydrolase family 32 domains (see Features). However, the structure-function correlation between the assigned domains and the function of sucrose hydrolysis is not tested. Furthermore, UniProt does not teach any functional homologues of A0A379AWT7_AVIGA.
Based on the above analysis, the person of ordinary skill in the art would not have recognized that the inventors had possession, at the time the application was filed, of the claimed genus of functional homologues of SEQ ID NO: 1 and 2 capable of hydrolyzing sucrose into fructose and glucose on the extracellular side or in the periplasmic space of a genetically modified cell.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claims 1-2, 7, 11, 16, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249) in view of Chen et al. (Bioengineered 11.1 (2020): 416-427). Both references are cited in the Restriction Requirement mailed on 12/16/2025.
Regarding claims 1-2, 11, and 22, Isono teaches a genetically modified E. coli cell comprising a heterologous enzyme β-fructofuranosidase from Arthrobacter (Title and Abstract). The enzyme is 100% identical to the instant SEQ ID NO: 2 (Isono Figure 1 and OA Appendix A), therefore, the enzyme is necessarily capable of hydrolyzing sucrose into fructose and glucose. Furthermore, Isono explicitly teaches that the enzyme is capable of hydrolyzing sucrose (Abstract and page 248, left column, top paragraph). Isono identifies the native signal peptide of the enzyme (Isono Figure 1 caption).
Isono teaches that the enzyme is found in the soluble fraction of the E. coli cell pellet (page 246, right column, top paragraph), so Isono does not teach that the enzyme is located on the extracellular side of the cell (the soluble fraction of the cell pellet includes proteins found in both the cytoplasmic or the periplasmic portions of the cell).
Chen teaches the heterologous expression of an Arthrobacter β-fructofuranosidase in E. coli (Title and page 417, left column, bottom paragraph). Chen optimizes the signal peptide for extracellular expression of the enzyme (Graphical Abstract, Figure 3a). Chen teaches that the signal peptide torT results in the highest extracellular activity (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the native secretion signal of Isono’s enzyme with Chen’s torT signal peptide in order to express the enzyme in the extracellular space of the genetically modified cell. The person of ordinary skill in the art would have been motivated to recover the enzyme from the supernatant rather than the cell pellet, thus avoiding the steps of cell lysis. The person of ordinary skill in the art would have had a reasonable expectation of success in secreting the protein by replacing the native signal peptide with Chen’s torT signal peptide given that Chen is successful in the extracellular secretion of an Arthrobacter β-fructofuranosidase, which is an enzyme with the same activity from the same bacteria.
Regarding claim 7, since Isono explicitly teaches that the enzyme is capable of hydrolyzing sucrose (Abstract and page 248, left column, top paragraph), then the genetically modified cell of Isono modified by Chen is necessarily capable of utilizing sucrose as the main carbon source.
Regarding claim 16, Chen’s torT signal peptide (“secretion signal”) would have been capable of enhancing the continuous secretion of Isono’s Arthrobacter β-fructofuranosidase into the extracellular space (fermentation medium).
Claims 4-5 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249) in view of Chen et al. (Bioengineered 11.1 (2020): 416-427) as evidenced by MetaCyc (2024, website).
The teachings of Isono and Chen are incorporated into this rejection as well.
Regarding claim 4, β-fructofuranosidase hydrolyzes non-phosphorylated sucrose as evidenced by MeatCyc (Unofficial Reactions).
Regarding claim 5, β-fructofuranosidase is an invertase as evidenced by MetaCyc (Synonyms).
Regarding claim 8, β-fructofuranosidase is an enzyme as evidenced by MetaCyc (Reactions), so β-fructofuranosidase is not a sucrose transporter.
Claims 9-10 are rejected under 35 U.S.C. 103 as being unpatentable over Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249) in view of Chen et al. (Bioengineered 11.1 (2020): 416-427) as evidenced by Jahreis (J Bacteriol. 2002 Oct;184(19):5307-16).
The teachings of Isono and Chen are incorporated into this rejection as well.
Regarding claims 9-10, Isono’s E. coli strain is JM109 before heterologous expression of the Arthrobacter enzyme (page 244, right column, bottom paragraph). JM109 is incapable of growth on sucrose as evidenced by Jahreis (page 5309, right column, Results, Cloning and sequencing of the csc locus and adjacent genes). Thus, Isono’s E. coli strain JM109 does not contain a complete sucrose utilization system and was not capable of sustaining growth on sucrose prior to the heterologous expression of the Arthrobacter enzyme.
Claims 1-3, 6, 12, 18-19, and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Parschat et al. (ACS Synthetic Biology 9.10 (2020): 2784-2796) in view Vasquez-Bahena (Enzyme and Microbial Technology 40 (2006) 61–66) as evidenced by MetaCyc.
Regarding claims 1, 3, and 22, Parschat teaches a genetically modified E. coli that produces the human milk oligosaccharide (HMO) 2’-fucosyllactose using sucrose as the sole carbon source (Abstract). Parschat integrates the cscABKR gene cluster from E. coli strain W into strain B conferring the ability to grow on sucrose (page 2787, left column, bottom paragraph). The gene cluster contains a sucrose importer as well as the invertase CscA that converts sucrose into fructose and glucose (Figure 1, page 2788, right column, bottom paragraph). The expression of the invertase is sufficient to enable utilization of sucrose as a carbon or energy source of the cell because the invertase generates glucose and Parschat’s cell metabolizes glucose (Parschat Fig. 1).
Parschat does not teach that the genetically modified cell comprises a heterologous nucleic acid sequence encoding a heterologous enzyme which on its own is capable of hydrolyzing sucrose into fructose and glucose on the extracellular side of the cell, wherein the expression of the enzyme is sufficient to enable utilization of sucrose as a carbon or energy source of the cell.
Vasquez-Bahena teaches the expression of an invertase from Zymomonas mobilis in the soluble extracellular fraction of E. coli (Title and page 62, right column, 3.2. Expression of the INVB recombinant protein right column, bottom paragraph). The invertase hydrolyzes sucrose into fructose and glucose (page 61, right column, bottom paragraph). Vasquez-Bahena teaches both the volumetric activity and specific activity of the extracellular invertase (Table 1). The culture activity is 58.29 µmol sucrose/min ml (Table 1).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the cscABKR gene cluster in the cell of Parschat with the extracellular invertase from Zymomonas mobilis. The person of ordinary skill in the art would have been motivated to eliminate the need for sucrose import into the cell by secreting the invertase instead. The person of ordinary skill in the art would have recognized the replacement as an improvement because expressing a single extracellular enzyme would have been simpler than a gene cluster comprising multiple genes. The person of ordinary skill in the art would have had a reasonable expectation of success given that Vasquez-Bahena is capable of expressing the invertase from Zymomonas mobilis extracellularly.
Regarding claim 2, Vasquez-Bahena is capable of expressing the invertase from Zymomonas mobilis extracellularly in E. coli (Title and page 62, right column, 3.2. Expression of the INVB recombinant protein right column, bottom paragraph).
Regarding claims 6 and 12, Parschat does not teach that the invertase gene is genomically integrated into E. coli in either one (claim 6) or two (claim 12) copies.
However, Parschat teaches integrating numerous heterologous genes into the genome of E. coli: see Fig. 1 caption, any blue enzyme name is a chromosomally integrated, overexpressed gene.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate the invertase gene of Vasquez-Bahena into the genome of Parschat’s E. coli in order to avoid the need for selection markers such as antibiotics. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating the invertase gene into the genome of E. coli given that Parschat integrates many different heterologous genes into the E. coli genome.
Regarding the number of gene copies, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate two copies of the invertase gene in order to increase the number of transcripts of the invertase, thus increasing the amount of translated protein. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating two copies of the invertase gene into the E. coli genome.
Regarding claims 18 and 19, Parschat teaches deleting the ptsG gene from the E. coli genome in order to slow (limit) glucose consumption such that the glucose is not converted into glucose-6-phosphate through the PTS system, thus limiting the flux of glucose to central metabolism (page 2789, left column, bottom paragraph).
Claims 4-5 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Parschat et al. (ACS Synthetic Biology 9.10 (2020): 2784-2796) in view Vasquez-Bahena (Enzyme and Microbial Technology 40 (2006) 61–66), as applied to claims 1-3, 6, 12, 18-19, and 22 above, and as evidenced by MetaCyc.
See discussion of Parschat and Vasquez-Bahena above, which is incorporated into this rejection as well.
Regarding claim 4, invertase hydrolyzes non-phosphorylated sucrose as evidenced by MetaCyc (Unofficial Reactions).
Regarding claim 5, β-fructofuranosidase is an invertase as evidenced by MetaCyc (Synonyms).
Regarding claim 8, the invertase is not a sucrose transporter. Rather, it is an enzyme that hydrolyzes sucrose as evidenced by MetaCyc (see Reactions).
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Parschat et al. (ACS Synthetic Biology 9.10 (2020): 2784-2796) in view Vasquez-Bahena (Enzyme and Microbial Technology 40 (2006) 61–66), as applied to claims 1-3, 6, 12, 18-19, and 22 above, further in view of Baumgärtner et al. (ChemBioChem 15.13 (2014): 1896-1900) and Perdersen et al. (WO 2019/123324 A1).
The teachings of Parschat and Vasquez-Bahena are incorporated into this rejection as well.
Regarding claim 13, Parschat does not teach that the heterologous nucleic acid sequence further comprises the promoter SEQ ID NO: 51 (wild-type Plac).
Baumgärtner teaches the synthesis of the human milk oligosaccharide, lacto-N-tetraose, in metabolically engineered, plasmid-free E. coli (Title). Baumgärtner teaches constructing a cassette of the heterologous N. meningitidis gene lgtA encoding β-1,3-N-acetylglucosaminyltransferase under the control of the IPTG-inducible Plac promoter and chromosomally integrating the cassette (paragraph bridging left and right columns on page 1897).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to add the IPTG-inducible Plac promoter of Baumgärtner to the heterologous invertase gene in the genetically modified cell of Parschat modified by Vasquez-Bahena. The person of ordinary skill in the art would have been motivated by the teaching of Baumgärtner, who suggests that the Plac promoter is an inducible promoter suitable for expression of heterologous enzymes in E. coli. The person of ordinary skill in the art would have had a reasonable expectation of success in this modification.
Baumgärtner does not teach the sequence of Plac.
Pedersen teaches the sequence of Plac as SEQ ID NO: 11 (line 23 on page 68).
SEQ ID NO: 11 is 100% identical to the instant SEQ ID NO: 51 (OA Appendix B).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to specifically insert the Plac sequence of Pedersen in the genetically modified cell of Parschat, Vasquez-Bahena, and Baumgärtner. The person of ordinary skill in the art would have been motivated to turn to Pedersen because Baumgärtner, although providing a strong rationale for using the Plac promoter, did not actually disclose the sequence of the promoter. The person of ordinary skill in the art would have had a reasonable expectation of success in adding SEQ ID NO: 11 of Pedersen (identical to the instant SEQ ID NO: 51) to the heterologous invertase.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-3, 6-7, 9-12, 16, and 22 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5, 7, 15-17, and 19 of copending Application No. 19/191,943 (reference application; hereafter ‘943) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025).
Claim 5 of ‘943 is drawn to a method for producing one or more human milk oligosaccharides comprising culturing the genetically engineered cell in a suitable medium with added lactose and harvesting one or more HMOs.
Claim 7 of ‘943 is drawn to a genetically engineered cell capable of producing LNT, LnnT, or LNT and LNnT, wherein the cell further expresses a sucrose utilization system, wherein the sucrose utilization system comprises a polypeptide capable of hydrolyzing sucrose into glucose and fructose, selected from the group consisting of SEQ ID NOs: 86 and 87.
Claim 19 of ‘943 recites that the genetically engineered cell is Escherichia coli.
SEQ ID NO: 87 of ‘943 is identical to the instant SEQ ID NO: 2.
Claim 7 of ‘943 does not recite that the polypeptide is capable of hydrolyzing sucrose into fructose and glucose on the extracellular side of the cell.
Sabri teaches that most commercial E. coli strains lack the ability to utilize sucrose (Abstract). Sucrose metabolism in certain E. coli requires the expression of both an intracellular invertase and a sucrose transporter gene (Abstract).
Chen teaches the heterologous expression of an Arthrobacter β-fructofuranosidase in E. coli (Title and page 417, left column, bottom paragraph). Chen optimizes the signal peptide for extracellular expression of the enzyme (Graphical Abstract, Figure 3a). Chen teaches that the signal peptide torT results in the highest extracellular activity (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the native secretion signal of SEQ ID NO: 87 in the genetically modified cell of claims 7 and 19 of ‘943 with Chen’s torT signal peptide in order to express the enzyme in the extracellular space of the genetically modified cell. The person of ordinary skill in the art would have been motivated to hydrolyze sucrose externally because commercial E. coli strains lack a sucrose importer, so secreting the enzyme avoids having to heterologously express both an invertase and a sucrose importer. The person of ordinary skill in the art would have had a reasonable expectation of success in secreting the protein by replacing the native signal peptide with Chen’s torT signal peptide given that Chen is successful in the extracellular secretion of an Arthrobacter β-fructofuranosidase.
Instant claims 1-2, 7, 11, and 22 are obvious over claims 7 and 19 of ‘943 in view of Sabri and Chen.
Instant claim 3 is obvious over claims 5 and 7 of ‘943 in view of Sabri and Chen.
Instant claims 6 and 12 are obvious over claims 7 and 15-17 of ‘943 in view of Sabri and Chen. Although claim 7 of ‘943 does not recite integrating one or more copies of the nucleic acid encoding SEQ ID NO: 87 into the genome of the genetically engineered cell, claims 15-17 of ‘943 recite genomically integrating other nucleic acid sequences into the cell, thus it would have also been obvious to integrate the nucleic acid sequence encoding SEQ ID NO: 87 into the genome of the cell.
Regarding the number of gene copies, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate two copies of the gene encoding SEQ ID NO: 87 in order to increase the number of transcripts of the enzyme, thus increasing the amount of translated protein. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating two copies of the nucleic acid encoding SEQ ID NO: 87 into the E. coli genome.
Instant claims 9-10 are obvious over claim 7 of ‘943 because claim 7 of ‘943 does not recite any additional sucrose utilization system components besides SEQ ID NO: 87 itself.
Regarding claim 16, Chen’s torT signal peptide (“secretion signal”) would have been capable of enhancing the continuous secretion of SEQ ID NO: 87 into the extracellular space (fermentation medium).
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 4-5 and 8 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 7 and 19 of copending Application No. 19/191,943 (reference application; hereafter ‘943) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025), as applied to claims 1-3, 6-7, 9-12, 16, and 22 above, and as evidenced by Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249; cited in the Restriction Requirement mailed on 12/16/2025) and MetaCyc (2024, website).
See discussion of claims 7 and 19 of ‘943, Sabri, and Chen above, which is incorporated into this rejection as well.
Regarding claim 4, SEQ ID NO: 87 is a β-fructofuranosidase as evidenced by Fig. 1 of Isono, which is identical to SEQ ID NO: 87 of ‘943 and the instant SEQ ID NO: 2, thus SEQ ID NO: 87 is necessarily capable of hydrolyzing non-phosphorylated sucrose as evidenced by MetaCyc (Unofficial Reactions).
Regarding claim 5, β-fructofuranosidase is an invertase as evidenced by MetaCyc (Synonyms).
Regarding claim 8, β-fructofuranosidase is an enzyme as evidenced by MetaCyc (Reactions), so β-fructofuranosidase is not a sucrose transporter.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claim 13 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 7 and 14 of copending Application No. 19/191,943 (reference application; hereafter ‘943) in view of Sabri, Chen, and Perdersen et al. (WO 2019/123324 A1).
See discussion of claim 7 of ‘943, Sabri, and Chen above, which is incorporated into this rejection as well.
Claim 14 of ‘943 recites that at least one of the nucleic acid sequences encoding the lactose permease is regulated by Plac.
Claims 7 and 14 of ‘943 do not recite the sequence of Plac or that SEQ ID NO: 87 is also regulated by Plac.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to add the Plac promoter of claim 14 of ‘943 to the nucleic acid encoding SEQ ID NO: 87 in the genetically modified cell of claim 7 of ‘943 modified by Sabri and Chen. Claim 14 of ‘943 already recites that other nucleic acid sequences in the cell are under the control of Plac, so the person of ordinary skill in the art would have been motivated to use the same promoter such that all the genes in the pathway are expressed at the same time. The person of ordinary skill in the art would have had a reasonable expectation of success in this modification.
Pedersen teaches the sequence of Plac as SEQ ID NO: 11 (line 23 on page 68).
SEQ ID NO: 11 is 100% identical to the instant SEQ ID NO: 51 (OA Appendix B).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to specifically use the Plac sequence of Pedersen in the genetically modified cell of claim 7 of ‘943. The person of ordinary skill in the art would have been motivated to turn to Pedersen because claims 7 and 14 of ‘943 do not recite the sequence of the Plac promoter. The person of ordinary skill in the art would have had a reasonable expectation of success in adding SEQ ID NO: 11 of Pedersen (identical to the instant SEQ ID NO: 51) to regulate the expression of SEQ ID NO: 87 in the genetically modified cell of claim 7 of ‘943.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 18-19 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5, 7, and 19 of copending Application No. 19/191,943 (reference application; hereafter ‘943) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025), as applied to claims 1-3, 6-7, 9-12, 16, and 22 above, further in view of Parschat et al. (ACS Synthetic Biology 9.10 (2020): 2784-2796)
See discussion of claims 5, 7, and 19 of ‘943, Sabri, and Chen above, which is incorporated into this rejection as well.
Claims 5, 7, and 19 of ‘943 do not recite and Sabri and Chen do not teach limiting the glucose uptake of the cell or that the cell does not contain a functional ptsG gene.
Regarding claims 18 and 19, Parschat teaches deleting the ptsG gene from the E. coli genome in order to slow (limit) glucose consumption such that the glucose is not converted into glucose-6-phosphate through the PTS system, thus limiting the flux of glucose to central metabolism (page 2789, left column, bottom paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to delete the ptsG gene from the genetically modified cell of claims 5, 7, and 19 of ‘943 in order to limit glucose consumption such that glucose is not converted into glucose-6-phosphate through the PTS system. The person of ordinary skill in the art would have been motivated to drive the flux to the desired HMOs rather than central metabolism. The person of ordinary skill in the art would have had a reasonable expectation of success in deleting the ptsG gene from the genetically modified cell of claims 5, 7, and 19 of ‘943, which is also E. coli.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-3, 7, 9-11, 16, and 22 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5, 9, and 16-17 of U.S. Patent No. 12,416,030 (hereafter ‘030) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025).
SEQ ID NO: 14 of ‘030 is identical to the instant SEQ ID NO: 2.
Claim 9 of ‘030 recites “The genetically engineered cell according to claim 8, wherein the genetically engineered cell utilizes sucrose by expressing a polypeptide capable of hydrolyzing sucrose into fructose and glucose selected from the group consisting of the SEQ ID NOs: 13 or 14, or a functional homologue thereof having an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 13 or 14.
Claim 16 of ‘030 requires a genetically engineered cell comprising a heterologous nucleotide sequence encoding a heterologous polypeptide capable of hydrolyzing sucrose into fructose and glucose which enables utilization of sucrose as sole carbon and energy source of said genetically engineered cell, and wherein the polypeptide capable of hydrolyzing sucrose into fructose and glucose is SacC Agal comprising SEQ ID NO: 13 or Bff of SEQ ID NO: 14, or a functional homologue thereof having an amino acid sequence which is at least 90% identical to any one of SEQ ID NOs: 13 or 14.
Claim 17 of ‘030 recites that the genetically engineered cell is cultured in sucrose as sole carbon and energy source.
Claim 5 of ‘030 recites that the genetically engineered cell is E. coli.
Claims 9 and 16-17 of ‘030 do not recite that the polypeptide is capable of hydrolyzing sucrose into fructose and glucose on the extracellular side of the cell.
Regarding claims 1-2 and 11, SEQ ID NO: 14 of claim 9 of ‘030 is identical to the instant SEQ ID NO: 2.
Sabri teaches that most commercial E. coli strains lack the ability to utilize sucrose (Abstract). Sucrose metabolism in certain E. coli requires the expression of an intracellular invertase and a sucrose transporter gene (Abstract).
Chen teaches the heterologous expression of an Arthrobacter β-fructofuranosidase in E. coli (Title and page 417, left column, bottom paragraph). Chen optimizes the signal peptide for extracellular expression of the enzyme (Graphical Abstract, Figure 3a). Chen teaches that the signal peptide torT results in the highest extracellular activity (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the native secretion signal of SEQ ID NO: 14 with Chen’s torT signal peptide in order to express the enzyme in the extracellular space of the genetically modified cell. The person of ordinary skill in the art would have been motivated to hydrolyze sucrose externally because Sabri teaches that many commercial E. coli lack a sucrose transporter, so secreting the enzyme avoids having to heterologously express both an invertase and a sucrose transporter. The person of ordinary skill in the art would have had a reasonable expectation of success in secreting the protein by replacing the native signal peptide with Chen’s torT signal peptide given that Chen is successful in the extracellular secretion of an Arthrobacter β-fructofuranosidase, which is an enzyme with the same activity from the same bacteria as SEQ ID NO: 14 of ‘030.
Instant claim 3 is obvious over claim 9 of ‘030 in view of Sabri and Chen because bot LNFP-1 is an HMO.
Instant claim 7 is obvious over claim 16 of ‘030 in view of Sabri and Chen.
Instant claims 9-10 are obvious over claims 9 and 16 of ‘030 because claims 9 and 16 of ‘030 do not recite any additional sucrose utilization system components besides SEQ ID NO: 13 or 14.
Regarding instant claim 16, Chen’s torT signal peptide (“secretion signal”) would have been capable of enhancing the continuous secretion of SEQ ID NO: 14 of ‘030 into the extracellular space (fermentation medium).
Instant claim 22 is obvious over claim 5 of ‘030 in view of Sabri and Chen.
Claims 4-5 and 8 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5, 9, and 16-17 of U.S. Patent No. 12,416,030 (hereafter ‘030) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025), as applied to claims 1-3, 7, 9-11, 16, and 22 above, as evidenced by Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249; cited in the Restriction Requirement mailed on 12/16/2025) and MetaCyc (2024, website).
See discussion of claims 5, 9, and 16-17 of ‘030, Sabri, and Chen above, which is incorporated into this rejection as well.
Regarding claim 4, SEQ ID NO: 14 is a β-fructofuranosidase as evidenced by Fig. 1 of Isono, which is identical to SEQ ID NO: 14 of ‘030 and the instant SEQ ID NO: 2, thus SEQ ID NO: 14 is necessarily is capable of hydrolyzing non-phosphorylated sucrose as evidenced by MetaCyc (Unofficial Reactions).
Regarding claim 5, β-fructofuranosidase is an invertase as evidenced by MetaCyc (Synonyms).
Regarding claim 8, β-fructofuranosidase is an enzyme as evidenced by MetaCyc (Reactions), so β-fructofuranosidase is not a sucrose transporter.
Claims 6, 12, and 18-19 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5, 9, and 16-17 of U.S. Patent No. 12,416,030 (hereafter ‘030) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025), as applied to claims 1-3, 7, 9-11, 16, and 22 above, further in view of Parschat et al. (ACS Synthetic Biology 9.10 (2020): 2784-2796).
See discussion of claims 5, 9, and 16-17 of ‘030, Sabri, and Chen above, which is incorporated into this rejection as well.
Regarding claims 6 and 12, claims 5, 9, and 16-17 of ‘030 do not teach that the invertase gene is genomically integrated into E. coli in either one (claim 6) or two (claim 12) copies.
Parschat teaches a genetically modified E. coli that produces the human milk oligosaccharide (HMO) 2’-fucosyllactose using sucrose as the sole carbon source (Abstract). Parschat integrates the cscABKR gene cluster from E. coli strain W into strain B conferring the ability to grow on sucrose (page 2787, left column, bottom paragraph). The gene cluster contains a sucrose importer as well as the invertase CscA the converts sucrose into fructose and glucose (Figure 1, page 2788, right column, bottom paragraph). Parschat teaches integrating numerous heterologous genes into the genome of E. coli: see Fig. 1 caption, any blue enzyme name is a chromosomally integrated, overexpressed gene.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate the gene encoding SEQ ID NO: 14 of claims 5, 9, and 16-17 of ‘030 into the genome of the genetically modified cell per the teaching of Parschat in order to avoid the need for selection markers such as antibiotics. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating the gene encoding SEQ ID NO; 14 into the genome of the genetically modified cell of claims 5, 9, and 16-17 of ‘030 given that Parschat integrates many different heterologous genes into the E. coli genome.
Regarding the number of gene copies, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate two copies of the invertase gene in order to increase the number of transcripts of the invertase, thus increasing the amount of translated protein. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating two copies of the invertase gene into the E. coli genome.
Regarding claims 18 and 19, claims 5, 9, and 16-17 of ‘030 do not recite and Chen does not teach limiting glucose uptake of the cell or that the cell does not contain a functional ptsG gene.
Parschat teaches deleting the ptsG gene from the E. coli genome in order to slow (limit) glucose consumption such that the glucose is not converted into glucose-6-phosphate through the PTS system, thus limiting the flux of glucose to central metabolism (page 2789, left column, bottom paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to delete the ptsG gene from the genetically modified cell of claims 5, 9, and 16-17 of ‘030 in order to limit glucose consumption such that glucose is not converted into glucose-6-phosphate through the PTS system. The person of ordinary skill in the art would have been motivated to drive the flux to the desired HMOs rather than central metabolism. The person of ordinary skill in the art would have had a reasonable expectation of success in deleting the ptsG gene from the genetically modified cell of claims 5, 9, and 16-17 of ‘030, which is also E. coli in one embodiment (see claim 5 of ‘030).
Claim 13 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 6 and 9 of U.S. Patent No. 12,416,030 (hereafter ‘030) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487), Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025) and Perdersen et al. (WO 2019/123324 A1).
See discussion of claim 9 of ‘030, Sabri, and Chen above, which is incorporated into this rejection as well.
Claim 6 of ‘030 recites the genetically engineered cell according to claim 1, wherein the expression of the recombinant nucleic acid is regulated by a promoter sequence selected from the group consisting of SEQ ID NO: 16-39.
Claim 9 of ‘030 does not recite that SEQ ID NO: 14 (identical to the instant SEQ ID NO: 2) are under the control of the instant SEQ ID NO; 51.
Pedersen teaches the sequence of Plac as SEQ ID NO: 11 (line 23 on page 68), which is identical to SEQ ID NO: 36 of claim 6 of ‘030.
SEQ ID NO: 11 is 100% identical to the instant SEQ ID NO: 51 (OA Appendix B).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to specifically use the Plac promoter of claim 6 of ‘030 to control expression of SEQ ID NO: 14 in the genetically modified cell of claim 9 of ‘030. Claim 6 of ‘030 already recites that other nucleic acid sequences in the cell are under the control of Plac, so the person of ordinary skill in the art would have been motivated to use the same promoter such that all the genes in the pathway are expressed at the same time. The person of ordinary skill in the art would have had a reasonable expectation of success in this modification.
Claims 1-10, 12, 16, 18-19, and 22 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 of U.S. Patent No. 12,534,701 (hereafter ‘701). Although the claims at issue are not identical, they are not patentably distinct from each other because claims 1-5 and 11 are anticipated by claim 5 of ‘701 and claims 6-10, 12, 16, 18-19, and 22 are obvious over claims 2-10 and 15 of ‘701.
Claim 1 of ‘701 recites “A genetically modified cell capable of producing one or more Human Milk Oligosaccharides (HMOs), which comprises a heterologous nucleic acid sequence encoding an invertase, wherein the invertase is SacC Agal comprising the amino acid sequence of SEQ ID NO: 1 or a functional homologue thereof, having an amino acid sequence which is at least 90% identical to SEQ ID NO: 1, wherein the cell further comprises at least one additional heterologous nucleic acid sequence encoding one or more functional enzymes with glycosyltransferase activity.
Claim 4 of ‘701 recites that the heterologous nucleic acid sequence encoding the invertase comprises a nucleic acid sequence encoding a signal peptide capable of enhancing the continuous secretion of the invertase into the periplasm of the genetically modified cell or into the extracellular medium, wherein the signal peptide is selected from SEQ ID NO: 5-27 and 29.
Claim 5 of ‘701 recites the invertase is capable of hydrolyzing non-phosphorylated sucrose into fructose and glucose in the periplasmic space or on the extracellular side of the cell.
Claim 6 of ‘701 requires that the heterologous nuclei acid sequence encodes a genomically integrated copy of the invertase.
Instant claim 2 is anticipated by claim 5 of ‘701 since in order for the invertase to be capable of hydrolyzing non-phosphorylated sucrose into fructose and glucose in the periplasmic space or on the extracellular side of the cell, the enzyme is necessarily located in either the periplasm or on the extracellular side of the cell.
Instant claim 3-5 are anticipated by claim 5 of ’701.
Claims 2-4, 6-10 and 15 of ‘701 do not recite that the invertase is capable of hydrolyzing sucrose into fructose and glucose in the periplasmic space r on the extracellular side of the cell. However, claim 5 of ‘701 recites that the invertase is capable of hydrolyzing sucrose into fructose and glucose in the periplasmic space of the extracellular side of the cell.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply the invertase of claim 5 of ‘701 to any of the genetically modified cells of claims 2-4, 6-10 and 15 of ‘701 in order to introduce this capability into the cell. The person of ordinary skill in the art would have had a reasonable expectation of success.
Instant claim 6 is obvious over claims 5-6 of ‘701.
Instant claim 7 is obvious over claims 5 and 8 of ‘701.
Instant claim 8 is obvious over claims 5 and 9 of ‘701.
Instant claim 9 is obvious over claims 5 and 10 of ‘701.
Instant claim 10 is obvious over claims 5 and 10 of ‘701 since if the cell does not contain a complete sucrose utilization system, then the cell was not capable of sustaining growth on sucrose prior to the addition of the heterologous enzyme.
Instant claim 11 is anticipated by claim 5 of ‘701 for the embodiment in which the heterologous nucleic acid is SEQ ID NO: 1.
Instant claim 12 is obvious over claims 5 and 7 of ‘701.
Instant claim 16 is obvious over claims 4-5 of ‘701.
Instant claim 18 is obvious over claims 2 and 5 of ‘701.
Instant claim 19 is obvious over claims 3 and 5 of ‘701.
Instant claim 22 is obvious over claims 5 and 15 of ‘701.
Claim 13 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 5 and 13 of U.S. Patent No. 12,534,701 (hereafter ‘701) as evidenced by the instant specification. Although the claims at issue are not identical, they are not patentably distinct from each other because claim 13 is obvious over claims 5 and 13 of ‘701.
Claim 13 of ‘701 does not recite that the invertase is capable of hydrolyzing sucrose into fructose and glucose in the periplasmic space r on the extracellular side of the cell. However, claim 5 of ‘701 recites that the invertase is capable of hydrolyzing sucrose into fructose and glucose in the periplasmic space of the extracellular side of the cell.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to apply the invertase of claim 5 of ‘701 to the genetically modified cell of claim 13 of ‘701 in order to introduce this capability into the cell. The person of ordinary skill in the art would have had a reasonable expectation of success. Instant claim 13 is thus obvious over claims 5 and 13 of ‘701 because the list of promoter names corresponds exactly to the instant SEQ ID NO: 29-40 and 42 as evidenced by the instant specification (Table 6 on page 22).
Claims 1-2, 7, 9-11, 16, and 22 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 4 and 18 of U.S. Patent No. 12,286,658 (hereafter ‘658) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025).
Claim 2 of ‘658 is drawn to a genetically engineered cell capable of producing HMOs. Claim 4 of ‘658 recites that the genetically engineered cell further expresses a sucrose utilization system comprising a polypeptide capable of hydrolyzing sucrose into
glucose and fructose, selected from the group consisting of SEQ ID NOs: 86 and 87, or a functional homologue of any one of SEQ ID NOs: 86 and 87.
Claims 18 of ‘658 limits the cell to E. coli.
SEQ ID NOs: 87 is identical to the instant SEQ ID NO: 2.
Regarding claims 1-2, 11, and 22, claim 4 of ‘658 does not recite that SEQ ID NO: 87 is capable of hydrolyzing sucrose into fructose and glucose on the extracellular side or in the periplasmic space in the genetically modified cell or that expression of the enzyme is sufficient to enable utilization of sucrose as a carbon or energy source of the genetically modified cell.
Sabri teaches that most commercial E. coli strains lack the ability to utilize sucrose (Abstract). Sucrose metabolism in certain E. coli requires the expression of an intracellular invertase and a sucrose transporter gene (Abstract).
Chen teaches the heterologous expression of an Arthrobacter β-fructofuranosidase in E. coli (Title and page 417, left column, bottom paragraph). Chen optimizes the signal peptide for extracellular expression of the enzyme (Graphical Abstract, Figure 3a). Chen teaches that the signal peptide torT results in the highest extracellular activity (Abstract).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to replace the native secretion signal of SEQ ID NO: 87 with Chen’s torT signal peptide in order to express the enzyme in the extracellular space of the genetically modified cell. The person of ordinary skill in the art would have been motivated to hydrolyze sucrose externally because commercial E. coli lack a sucrose transporter, so secreting the enzyme avoids having to heterologously express both an invertase and a sucrose transporter. The person of ordinary skill in the art would have had a reasonable expectation of success in secreting the protein by replacing the native signal peptide with Chen’s torT signal peptide given that Chen is successful in the extracellular secretion of an Arthrobacter β-fructofuranosidase, which is an enzyme with the same activity from the same bacteria as SEQ ID NO: 87.
Instant claim 7 is obvious over claim 4 of ‘658 in view of Sabri and Chen.
Instant claims 9-10 are obvious over claim 4 of ‘658 because claim 4 of ‘658 does not recite any additional sucrose utilization system components besides SEQ ID NO: 86 or 87.
Regarding claim 16, Chen’s torT signal peptide (“secretion signal”) would have been capable of enhancing the continuous secretion of SEQ ID NO: 87 into the extracellular space (fermentation medium).
Instant claim 22 is obvious over claim 18 of ‘658 in view of Sabri and Chen.
Claims 4-5 and 8 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 4 and 18 of U.S. Patent No. 12,286,658 (hereafter ‘658) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025), as applied to claims 1-2, 7, 9-11, 16, and 22 above, as evidenced by Isono et al. (Journal of bioscience and bioengineering 97.4 (2004): 244-249; cited in the Restriction Requirement mailed on 12/16/2025) and MetaCyc (2024, website).
See discussion of claims 4 and 18 of ‘658, Sabri, and Chen above, which is incorporated into this rejection as well.
Regarding claim 4, SEQ ID NO: 87 is a β-fructofuranosidase as evidenced by Fig. 1 of Isono, which is identical to SEQ ID NO: 87 of ‘658 and the instant SEQ ID NO: 2, thus SEQ ID NO: 87 is necessarily is capable of hydrolyzing non-phosphorylated sucrose as evidenced by MetaCyc (Unofficial Reactions).
Regarding claim 5, β-fructofuranosidase is an invertase as evidenced by MetaCyc (Synonyms).
Regarding claim 8, β-fructofuranosidase is an enzyme as evidenced by MetaCyc (Reactions), so β-fructofuranosidase is not a sucrose transporter.
Claims 6, 12, and 18-19 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 4 and 18 of U.S. Patent No. 12,286,658 (hereafter ‘658) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025), as applied to claims 1-2, 7, 9-11, 16, and 22 above, further in view of Parschat et al. (ACS Synthetic Biology 9.10 (2020): 2784-2796).
See discussion of claims 4 and 18 of ‘658, Sabri, and Chen above, which is incorporated into this rejection as well.
Regarding claims 6 and 12, claims 4 and 18 of ‘658 do not recite that the gene encoding SEQ ID NO: 87 is genomically integrated into E. coli in either one (claim 6) or two (claim 12) copies.
Parschat teaches a genetically modified E. coli that produces the human milk oligosaccharide (HMO) 2’-fucosyllactose using sucrose as the sole carbon source (Abstract). Parschat integrates the cscABKR gene cluster from E. coli strain W into strain B conferring the ability to grow on sucrose (page 2787, left column, bottom paragraph). The gene cluster contains a sucrose importer as well as the invertase CscA the converts sucrose into fructose and glucose (Figure 1, page 2788, right column, bottom paragraph). Parschat teaches integrating numerous heterologous genes into the genome of E. coli: see Fig. 1 caption, any blue enzyme name is a chromosomally integrated, overexpressed gene.
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate the gene encoding SEQ ID NO: 87 into the genome of the cell of claims 4 and 18 of ‘658 in order to avoid the need for selection markers such as antibiotics. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating the gene encoding SEQ ID NO: 87 into the genome of E. coli given that Parschat integrates many different heterologous genes into the E. coli gene.
Regarding the number of gene copies, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to integrate two copies of the invertase gene in order to increase the number of transcripts of the invertase, thus increasing the amount of translated protein. The person of ordinary skill in the art would have had a reasonable expectation of success in integrating two copies of the invertase gene into the E. coli genome.
Regarding claims 18 and 19, claims 4 and 18 of ‘658 do not recite and Chen does not teach limiting glucose uptake of the cell or that the cell does not contain a functional ptsG gene.
Parschat teaches deleting the ptsG gene from the E. coli genome in order to slow (limit) glucose consumption such that the glucose is not converted into glucose-6-phosphate through the PTS system, thus limiting the flux of glucose to central metabolism (page 2789, left column, bottom paragraph).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to delete the ptsG gene from the genetically modified cell of claims 4 and 18 of ‘658 in order to limit glucose consumption such that glucose is not converted into glucose-6-phosphate through the PTS system. The person of ordinary skill in the art would have been motivated to drive the flux to the desired HMOs rather than central metabolism. The person of ordinary skill in the art would have had a reasonable expectation of success in deleting the ptsG gene from the genetically modified cell of claims 4 and 18 of ‘658, which is also E. coli.
Claim 13 is rejected on the ground of nonstatutory double patenting as being unpatentable over claims 4, 13, and 18 of U.S. Patent No. 12,286,658 (hereafter ‘658) in view of Sabri et al. (Applied and environmental microbiology 79.2 (2013): 478-487) and Chen et al. (Bioengineered 11.1 (2020): 416-427; cited in the Restriction Requirement mailed on 12/16/2025) and as evidenced by the instant specification.
See discussion of claims 4 and 18 of ‘658, Sabri, and Chen above, which is incorporated into this rejection as well.
Regarding claim 13, claims 4 and 18 of ‘658 do not recite that the heterologous nucleic acid sequence further comprises the promoter SEQ ID NO: 29-54.
Claim 13 of ‘658 recites at least one of the nucleic acid sequences encoding lactose permease is regulated by one or more promoter sequences. The promoters include promoters SEQ ID NO: 34, 38 42-45, and 51 as evidenced by the instant specification (Table 6 on pages 22-23).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to add any of the promoters of claim 13 of ‘658 to regulate the expression of SEQ ID NO: 87 in the cell of claims 4 and 18 of ‘658. Claim 13 of ‘658 already recites that other nucleic acid sequences in the cell are under the control of these promoters, so the person of ordinary skill in the art would have been motivated to use the same promoter such that all the genes in the pathway are expressed at the same time. The person of ordinary skill in the art would have had a reasonable expectation of success in this modification.
Examiner’s Comment
SEQ ID NO: 28 is not taught by the prior art. De Geus et al. (WO0066751A1) teaches a fusion gene comprising the Alcaligenes faecalis secretion signal, a modified trp promoter and the E. coli penicillin G acylase (lines 932 on page 16). SEQ ID NO: 12 is the corresponding protein sequence of this fusion. Amino acid residues 1-10 and 12-26 of SEQ ID NO: 12 are identical to the instant SEQ ID NO: 28. However, the amino acid residue at position 11 is valine in De Geus’s SEQ ID NO: 12, whereas the presently claimed SEQ ID NO: 28 has alanine at position 11.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657