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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/17/2025 has been entered.
Priority
The instant application is a U.S. National Phase of PCT/EP2020/072429 filed on 8/10/2020 and claims foreign priority to EP19195148.2 filed on 9/3/2019. Therefore, the effective filing date of the claimed invention is 9/3/2019.
DETAILED ACTION
Applicant’s amendment submitted 11/172025 is acknowledged. Claims 13-15, 17-18, 20, 22, 24-26, and 28-31 remain pending in the instant application. Claim 13 is currently amended. Claims 1-12, 16, 19, 21, 23, and 27 are canceled.
Claims 15 and 17-18 remain withdrawn pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention.
Claims 13-14, 20, 22, 24-26 and 28-31 are the subject of this office action.
Drawings
The drawings are objected to as failing to comply with 37 CFR 1.84(p)(5) because they include the following reference character not mentioned in the description: the enzyme labeled “12” in the Figure 1 is not described in the specification. Corrected drawing sheets in compliance with 37 CFR 1.121(d), or amendment to the specification to add the reference character in the description in compliance with 37 CFR 1.121(b) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
The drawings are objected to because there is only one figure and the drawing refers to it as Figure 1. However, 37 CFR 1.84(u)(1) indicates when there is only one drawing, it must not be numbered and the abbreviation FIG must not appear. Therefore, the figure must be referred to as The Figure. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Specification
The disclosure is objected to because of the following informalities:
Page 9 recites “Description of the figures” in line 1 but should recite “Description of The Figure”, since there is only one figure. All instances of “Fig. 1” on p.9, line 2, p.15, line 14, p.16, lines 22-23 and 28, p.18, line 10, p.19, lines 11-13, p.22, line 5, p.23, line 15, p.24, line 26, p.25, lines 2-3 and 5-9, and p.30, line 8, should be amended to state “The Figure” as there is only one figure.
Page 19, 3rd passage, of the originally-filed specification, comprises two distinct enzymes, i.e., N-acetylglucosamine-1-phosphate uridyltransferase and UDP-N-acetylglucosamine 2-epimerase, labeled “4” in Figure 1. It appears N-acetylglucosamine-1-phosphate uridyltransferase should be labeled “3” in Figure 1.
Appropriate correction is required.
Claim Objections
Claim 13 is objected to because of the following informalities:
Claim 13 recites “for the production of a sialylated oligosaccharide” in line 3, which is redundant and should be deleted.
Extra hyphens in the beginning of lines 3 and 15 of claim 13 are extraneous and should be removed.
In line 8 of claim 13, a comma is missing after “a heterologous sialyltransferase.”
Claim 13 recites “(CMP)-N-acetylneuraminic acid synthase” in line 8, which should be fully abbreviated to “CMP-NeuNAc synthase” as provided in line 5.
Claim 13 recites “at least one of the genes selected from the group consisting of yesZ and ganA” in line 12, and the recitation of “the group consisting of” is extraneous. Claim 13 should be amended to instead recite “at least one of the genes selected from yesZ and ganA.”
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) – Scope of Enablement
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 13-14, 20, 22, 24-26 and 28-31 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for deletion of the genes yvmC and/or cypX to abolish the formation of pulcherriminic acid during fermentation of a non-sporulating Bacillus cell, does not reasonably provide enablement for disruption of the genes yvmC and/or cypX to abolish the formation of pulcherriminic acid during fermentation of a non-sporulating Bacillus cell. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
In making a determination that a disclosure does not satisfy the enablement requirement, the factors that may be considered include: (A) the breadth of the claims, (B) the nature of the invention, (C) the state of the prior art, (D) the level of one of ordinary skill, (E) the level of predictability in the art, (F) the amount of direction provided by the inventor, (G) the existence of working examples, and (H) the quantity of experimentation needed to make or use the invention based on the content of the disclosure. While it is not essential that every factor be examined in detail, those factors deemed most relevant should be considered.
Nature of the invention. The claims are drawn to a method for the production of a sialylated oligosaccharide in a non-sporulating Bacillus cell wherein said Bacillus cell has been genetically engineered to possess a heterologous lactose permease, a cytidine monophosphate N-acetylneuraminic acid (CMP-NeuNAc) biosynthesis pathway that uses glucosamine-1-phosphate (GlcN-1P) as an intermediate by expressing a heterologous (CMP)-N-acetylneuraminic acid synthase, and a heterologous sialyltransferase wherein the sporulation capability of the Bacillus cell has been impaired by deletion or functional inactivation of the gene encoding sigma F; wherein the Bacillus cell has been genetically engineered by deletion or functional inactivation of at least one of the genes selected from the group consisting of yesZ and ganA; wherein formation of the pulcherriminic acid during fermentation is abolished by deletion or disruption of the genes yvmC and/or cypX; and cultivating said Bacillus cell in a fermentation broth and under conditions that are permissive for the production of a sialylated oligosaccharide.
Breadth of the claims. The breadth of the disruption of the genes yvmC and/or cypX to abolish formation of pulcherriminic acid during fermentation is exceedingly large. The claims place no limit on what the disruption to the genes yvmC and/or cypX must be as long as it results in abolished formation of pulcherriminic acid during fermentation.
State of the prior art and predictability of the art. The state of the art at the time of the invention reveals that deletion of the genes yvmC or cypX or both yvmC and cypX abolishes the formation of pulcherriminic acid during fermentation; however, no functional disruptions have been demonstrated.
Tang et al. (US2004/0096944) teaches that pulcherrimins are reddish pigments resulting from chelation of ferric ions by pulcherriminic acid (see paragraph [0005]). Tang et al. further teaches pulcherriminic acid is synthesized by Bacillus and forms pulcherrimins in presence of ferric ions, indicated by red pigment in fermentation medium (see paragraphs [0006], [0026], and [0091]). Tang et al. demonstrated that the deletion of the cypX or yvmC gene in Bacillus subtilis is necessary for elimination of the red pigment in culture medium with ferric ions present (see paragraph [0098] and Table 1). While Tang et al. postulate general modifications and disruptions to the genes yvmC and cypX to prevent formation of red pigments in paragraph [0027], Tang et al. do not disclose any specific genetic modifications that result in the abolished formation of pulcherriminic acid besides deletion of the genes yvmC and/or cypX.
Randazzo et al. (BMC Microbiol., 2016, Vol. 16(190), pp.1-16) teaches that in Bacillus subtilis, the enzymes YvmC and CypX are known to be involved in pulcherriminic acid biosynthesis; however, the mechanisms controlling the transcription of the yvmC-cypX operon were previously unknown (see Abstract: Background and p.2, paragraph bridging left and right columns). Randazzo et al. discovered that B. subtilis YvmB MarR-like regulator is the major transcription factor controlling yvmC-cypX expression (see Abstract: Results, p.2, right column, last paragraph, passage bridging pp.3-4, p.4, last passage, p.5, last passage, and Figs. 1-2 and 4-5). Transcription of yvmB was discovered to be induced by iron starvation, while it was shown that iron starvation does not induce yvmC-cypX expression (see p.3, 2nd passage, and p.11, paragraph bridging left and right columns). Randazzo et al. demonstrates that yvmB and iron starvation play a role in yvmC-cypX expression.
Therefore, the prior art only recognized deletion of at least one of yvmC and cypX to abolish the formation of pulcherriminic acid during fermentation. At the time of the invention, there were no recognized disruptions to the genes yvmC and cypX other than deletion that resulted in the abolished production of pulcherriminic acid.
Guidance in the specification. The specification does not provide any guidance for the disruption of at least one of the genes yvmC and cypX that abolishes the formation of pulcherriminic acid during fermentation. Only general discussion of gene disruption in yvmC and cypX is provided in passages 2-3 on p.41.
Amount of experimentation necessary. In view of the foregoing analysis, one of skill in the art would not be able to make or use the full scope of the claimed invention without performing experimentation on a case by case basis for each possible disruption in the genes yvmC and cypX in a Bacillus cell to discover which disruptions abolish pulcherriminic acid synthesis during fermentation. The person of skill in the art would have no reasonable expectation of success across the breadth of disruptions to the genes yvmC and cypX, besides deletion of these genes, to abolish formation of pulcherriminic acid in Bacillus.
Taking these factors into account, undue experimentation would be required by one of ordinary skill in the art to practice the full scope of the claimed invention. Thus, the claims are not fully enabled by the disclosure.
Applicant may overcome this rejection by amending claim 13 to remove recitation of disruption to limit claim 13 to only deletion of the genes yvmC and/or cypX.
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 13-14, 20, 22, 24-26 and 28-31 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 13 recites “providing a non-sporulating Bacillus cell” in line 3. It is not clear if the step of providing intends to include active method steps for encoding the heterologous genes that are recited in claim 13 in the non-sporulating Bacillus cell and deleting or inactivating the gene encoding sigma F, at least one of the genes yesZ and ganA, and at least one of the genes yvmC and cypX or if the step of providing is intended to be fulfilled by means such as purchasing a Bacillus cell with the aforementioned genetic requirements. Thus, the metes and bounds of claim 13 are uncertain. Applicant may overcome this rejection by amending claim 13 to:
genetically engineering a nonsporulating Bacillus cell:
to express a heterologous lactose permease,
to catalyze a cytidine monophosphate N-acetylneuraminic acid (CMP-NeuNAc) biosynthesis pathway that uses glucosamine-1-phosphate (GlcN-1P) as an intermediate by expressing a heterologous UDP N-acetylglucosamine 2-epimerase, a heterologous sialic acid synthase, a heterologous CMP-NeuNAc synthase, and a heterologous sialyltransferase,
to impair the sporulation capability of the Bacillus cell by deleting or functionally inactivating the gene encoding sigma F;
to abolish or reduce the YesZ-mediated β-galactosidase activity by deleting or functionally inactivating the genes yesZ and/or ganA; and
to abolish formation of pulcherriminic acid during fermentation by deleting the genes yvmC and/or cypX; and
cultivating said Bacillus cell in a fermentation broth under conditions that are permissive for the production of a sialylated oligosaccharide.
Claims 14, 20, 22, 24-26 and 28-31 are also rejected for being dependent on a rejected base claim and failing to remedy the issues set forth above.
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 24, 26, and 29-30 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which they depend, or for failing to include all the limitations of the claim upon which they depend.
Regarding claim 24, the limitation “wherein the CMP-NeuNAc biosynthesis pathway uses a sialic acid salvage pathway” does not further limit claim 13. Claim 13 recites that the Bacillus cell expresses genes that catalyze a cytidine monophosphate N-acetylneuraminic acid (CMP-NeuNAc) biosynthesis pathway. On page 15, lines 13-22, of the specification, a NeuNAc salvage pathway is described as a conversion of internalized sialic acid to CMP-NeuNAc (see also the figure). Since claim 13 recites a CMP-NeuNAc biosynthesis pathway that results in the production of CMP-NeuNAc, sialic acid salvaging is already implied and required by claim 13.
Regarding claim 26, the limitation “wherein the Bacillus cell lacks any β-galactosidase activity or possesses reduced β-galactosidase activity as compared to a wild-type progenitor Bacillus cell of the same species” does not further limit claim 13. Claim 13 requires deletion or functional inactivation of a yesZ and/or ganA gene, whichnd and 4th-6th passages). Thus, since claim 13 already requires deletion or functional inactivation of at least one gene encoding β-galactosidase, claim 26 does not further limit claim 13.
Claims 29 and 30 recite the limitation, “wherein the CMP-NeuNAc biosynthesis pathway uses GlcN-1P as an intermediate,” which repeats a limitation that is already recited in claim 13. Claim 13 specifically recites “a cytidine monophosphate N-acetylneuraminic acid (CMP-NeuNAc) biosynthesis pathway that uses glucosamine-1-phosphate (GlcN-1P) as an intermediate” in lines 5-6.
Applicant may cancel the claims, amend the claims to place the claims in proper dependent form, rewrite the claims in independent form, or present a sufficient showing that the dependent claims complies with the statutory requirements.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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 13-14, 20, 22, 24-26, and 28-31 are rejected under 35 U.S.C. 103 as being unpatentable over Beauprez et al. (WO2018/122225 A1; Of Record in IDS filed 8/11/2022) in view of Overkamp et al. (PLoS ONE, 2015, Vol. 10(10), pp.1-12; Of Record), Koski et al. (U.S. Patent No. 7,319,030; Of Record), Priem et al. 2002 (Glycobiol., Vol. 12(4), p.235-240; Of Record), Carneiro et al. 2018 (Intern. J. Biolog. Macromol., Vol. 120, p.279-287; Of Record in IDS filed 8/30/2024), and Tang et al. (US2004/0096944).
Regarding claim 13, Beauprez teaches a method of producing sialylated compounds by fermentative growth of microorganisms (see p.4, lines 26-27). The sialylated compounds include sialylated oligosaccharides such as 3’-sialyllactose and 6’-sialyllactose (see p.8, lines 19-44 and p.9, lines 1-11). The producer microorganisms are engineered such that they express lactose permease, sialyltransferase and catalyze a pathway as shown in Figs. 1A-1B, which corresponds to the CMP-NeuNAc biosynthesis pathway of the present invention (see p.7, lines 31-37, p.8, lines 3-7, p.10, lines 17-30, and p.21, lines 34-38). Beauprez performs the method with a Bacillus subtilis 168 strain, and cultivates it in Luria Broth (LB) medium (see Example 1, p.33, line 24; p.37, line 24). Example 7 of Beauprez describes the production of sialyllactoses and other sialylated compounds by cultivation of the strains described in Example 1, which includes Bacillus subtilis 168 as previously described, using the construct of Example 3 (see p.41, lines 1-8). Beauprez further describes different sialyllactoses (i.e., 6’-sialyllactose and 3’-sialyllactose) and mixtures can be obtained by substituting the sialyltransferase for one with a separate activity (see p.41, lines 1-7). Example 3 describes a daughter strain of the strain used in Example 2 (see p.38, lines 14-16). Example 2 describes an E. coli strain that overexpresses a glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (ScGNA1), a N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE), and a sialic acid synthase from Campylobacter jejuni (CjneuB) (see p.37, line 25,-p.38, line 1). Example 3 describes that the daughter strain is further modified by overexpressing the lactose permease EclacY from E. coli, a CMP-sialic acid synthetase from Neisseria meningitides (NmneuA), and a sialyltransferase from Photobacterium damselae (Pdbst) (see p.38, lines 13-20). Therefore, according to Example 7, a Bacillus subtilis 168 strain that overexpresses a glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (ScGNA1), a N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE), a sialic acid synthase from Campylobacter jejuni (CjneuB), the lactose permease EclacY from E. coli, a CMP-sialic acid synthetase from Neisseria meningitides (NmneuA), and a sialyltransferase from Photobacterium damselae (Pdbst) is produced for the production of sialyllactoses, specifically 6’-sialyllactose. The EclacY reads on a heterologous lactose permease, the BoAGE reads on a heterologous UDP-N-acetylglucosamine 2-epimerase, the CjneuB reads on a heterologous sialic acid synthase, the NmneuA reads on a (CMP)-N-acetylneuraminic acid synthase, and the Pdbst reads on a heterologous sialyltransferase as recited in claim 13. Furthermore, the Bacillus subtilis 168 strain described above is expected to use glucosamine-1-phosphate as an intermediate in the CMP-NeuNAc biosynthesis pathway because it expresses the required enzymes as recited in claim 13.
Beauprez does not teach that the Bacillus subtilis 168 strain is non-sporulating wherein the sporulation capability of the Bacillus cell has been impaired by deletion or functional inactivation of the gene encoding sigma F, deletion or functional inactivation at least one of the genes yesZ and ganA, or wherein formation of pulcherriminic acid during fermentation is abolished by deletion or disruption of the genes yvmC and/or cypX.
Overkamp teaches an asporogenous (non-sporulating) strain can be constructed by replacing bp 2443565 to 2444182 of the Bacillus subtilis 168 chromosome, which corresponds to the majority of the sigma F gene, with a spectinomycin resistance cassette (see Abstract and p.5, 2nd passage). This is considered to read on functionally inactivating the gene encoding sigma F, as claimed. Koski teaches that industrial processes generate significant amounts of bacterial mass that needs to be inactivated prior to its discharge into the environment, especially if the bacterium has been genetically engineered (see Col. 1, lines 19-23). Koski further teaches that the spores of Bacillus endure heat much better than vegetative cells and therefore, destroying them by heating requires high temperatures and longer treatments, resulting in increasing costs and equipment needs for production (See Col. 1, lines 24-29). Koski teaches it is then more desirable to use a non-sporulating Bacillus strain for production (see Col. 1, lines 29-30). Koski teaches the non-sporulating B. subtilis bacterium, from which a sigG gene has been substantially deleted, is suitable for a production strain of various biologically prepared products (see Col. 7, lines 21-23).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to inactivate the sigma F gene in order to create a non-sporulating B. subtilis 168, as taught by Overkamp, in the B. subtilis 168 strain used in the methods of Beauprez to arrive at the claimed invention. One of ordinary skill would have been motivated to remove the sporulation ability of the B. subtilis 168 of Beauprez because Koski teaches non-sporulating B. subtilis are more desirable to use in industrial production of biologically prepared products because they are more susceptible to inactivation procedures, and Beauprez teaches production of sialylated compounds in genetically engineered B. subtilis, yielding predictable results. One of ordinary skill in the art would have further been motivated to prevent sporulation of the B. subtilis 168 of Beauprez because Koski further teaches inactivation of the bacterial mass created in industrial process is especially important before discharge into the environment when dealing with genetically engineered bacteria, which the bacterium of Beauprez is engineered.
Overkamp and Koski do not teach deletion or functional inactivation at least one of the genes yesZ and ganA, or wherein formation of pulcherriminic acid during fermentation is abolished by deletion or disruption of the genes yvmC and/or cypX.
Priem teaches a fermentation process that allows large-scale production of human milk oligosaccharides by a β-galactosidase-negative E. coli strain still expressing lactose permease gene lacY and also expressing a glycosyltransferase that accepts lactose (see Abstract and p.236, left column, 2nd passage, and p.238, right column, 2nd passage). Priem teaches that lactose transported into the cell by lactose permease LacY cannot be degraded since the cell is devoid of β-galactosidase activity, and is glycosylated (see Fig. 1). Priem teaches engineering the E. coli strain to include a CMP-NeuAc biosynthesis pathway and α-2,3-sialyltransferase for the high-yield production of 3’-sialyllactose (see p.237, left and right columns, p.238, left column, 1st passage, and Fig. 5).
Carneiro teaches a β-galactosidase gene yesZ for expression in Bacillus subtilis as a heterologous protein (see Abstract, and p.280, left column, 1st passage).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have deactivated the β-galactosidase gene, as taught by Priem, and specifically deactivated the β-galactosidase gene yesZ, as disclosed by Carneiro, in the non-sporulating Bacillus subtilis engineered for the production of sialylated oligosaccharides, as taught by Beauprez in view of Overkamp and Koski, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to do so because Priem teaches deactivating β-galactosidase allows for the accumulation of lactose intracellularly, which is critical for the production of sialyllactose oligosaccharides, yielding predictable results. One of ordinary skill in the art would have had a reasonable expectation of success because the non-sporulating B. subtilis for producing 6’-sialyllactose as taught by Beauprez in view of Overkamp and Koski is engineered to include the lactose permease LacY, the CMP-NeuNAc biosynthesis pathway, and a sialyltransferase as was done in Priem.
Priem and Carneiro do not teach wherein formation of pulcherriminic acid during fermentation is abolished by deletion or disruption of the genes yvmC and/or cypX.
Tang teaches Bacillus cells are well-established hosts for the production of heterologous biological substances; however, these Bacillus cells also produce undesirable pigment byproducts that must be removed during purification of the heterologous biological substance of interest (see paragraphs [0005]-[0007]). Tang teaches Bacillus subtilis produces pulcherriminic acid which becomes pulcherrimins, a reddish pigment, in the presence of ferric ions in the culture medium (see paragraphs [0005]-[0006] and [0026]). Tang further teaches that the synthesis of pulcherriminic acid by Bacillus subtilis can be prevented if at least one of the genes yvmC and cypX are deleted (see paragraphs [0024]-[0025], [0098], and Table 1).
Therefore, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have further deleted at least one of the genes yvmC and cypX, as taught by Tang, in the non-sporulating Bacillus subtilis engineered for the production of sialylated oligosaccharides, as taught by Beauprez in view of Overkamp, Koski, Priem, and Carneiro, to arrive at the claimed invention. One of ordinary skill in the art would have been motivated to prevent the formation of red pigment in the culture medium to simplify purification of the sialylated oligosaccharides, yielding predictable results. One of ordinary skill in the art would have had a reasonable expectation of success since each of Beauprez and Tang are directed to Bacillus subtilis.
Regarding claim 14, Beauprez teaches the LB broth contains lactose (see p.37, lines 12-15).
Regarding claim 20, Beauprez teaches isolating sialyllactose from the culture medium, which reads on claim 20 (see p.12, lines 8-16).
Regarding claim 22, Beauprez teaches the lactose permease is E. coli LacY, reading on claim 22 (see p.38, lines 16-17).
Regarding claim 24, the specification as originally-filed discloses that the N-acetylneuraminic acid (NeuNAc, a.k.a. sialic acid) salvage pathway comprises internalization of exogenous sialic acid by the Bacillus cell and conversion of the internalized sialic acid to CMP-NeuNAc (see p.15, lines 21-22). Beauprez depicts the endogenous generation of CMP-NeuNAc by sialic acid in Figs. 1-3, and thus teaches a salvage pathway of sialic acid in which CMP-NeuNAc is generated, reading on claim 24 (see Figs. 1-3, p.10, lines 26-30, and p.21, lines 34-38). Furthermore, the generation of the sialylated oligosaccharides 3’-sialyllactose and 6’-sialyllactose is done by a sialyltransferase that sialylates lactose internalized by lactose permease using CMP-NeuNAc, which is another case of sialic acid salvaging (see p.10, lines 26-30 and Figs. 1-3).
Regarding claim 25, Beauprez teaches the sialylated compounds include sialylated oligosaccharides such as 3’-sialyllactose and 6’-sialyllactose (see p.8, lines 19-44 and p.9, lines 1-11), which would imply that the sialyltransferase is a lactose-accepting sialyltransferase as described in the instant specification at p.11, lines 15-17.
Regarding claim 26, the non-sporulating Bacillus subtilis taught by Beauprez in view of Overkamp, Koski, Priem, Carneiro, and Tang has a deactivated yesZ β-galactosidase gene, and thus possesses reduced β-galactosidase activity as compared to a wild-type progenitor Bacillus cell of the same species.
Regarding claim 28, Beauprez in view of Overkamp, Koski, Priem, Carneiro, and Tang teach a non-sporulating Bacillus subtilis cell, reading on claim 28.
Regarding claims 29-31, Beauprez teaches producing 3’-sialyllactose and 6’-sialyllactose in mixture by using a combination of the sialyltransferases alpha-2,3-sialyltransferase, alpha-2,6-sialyltransferase, and alpha-2,8-sialyltransferase (see p.10, lines 26-30 and p.41, lines 1-7). Furthermore, the non-sporulating Bacillus subtilis 168 strain described in the rejection of claim 13 above is expected to use glucosamine-1-phosphate as an intermediate in the CMP-NeuNAc biosynthesis pathway because it expresses the required enzymes as recited in claim 13.
Therefore, claims 13-14, 20, 22, 24-26, and 28-31 are prima facie obvious over Beauprez in view of Overkamp, Koski, Priem, Carneiro, and Tang.
Response to Arguments
Applicant's arguments filed 11/17/2025 have been fully considered but they are not persuasive.
In Applicant’s Remarks, see p.7, 3rd paragraph, Applicant argues that Beauprez teaches away from using the methods of the claimed invention for producing sialylated compounds in the Background section. Applicant further argues pending claim 13 now requires deletion or disruption of the yvmC and/or cypX genes, a feature not taught by Beauprez nor the secondary references Overkamp and Koski (see Applicant’s Remarks – paragraph bridging pp.7-8). Applicant further argues the Priem and Carneiro references fail to remedy the issues above (see Applicant’s Remarks – p.7, 2nd paragraph). Lastly, Applicant argues that the Zhang reference fails to remedy the issues set forth above and only provides an invitation to experiment further with sialic acids and sialyllactoses (see Applicant’s Remarks – paragraph bridging pp.8-9 and p.9, 1st paragraph). This is not found persuasive.
Beauprez discloses embodiments in Example 7 that produce sialyllactoses in Bacillus cells heterologously expressing a lactose permease, a cytidine monophosphate N-acetylneuraminic acid (CMP) biosynthesis pathway that uses glucosamine 1-phosphate (GlcN-1P) as an intermediate by expressing a heterologous UDP N-acetylglucosamine 2-epimerase, a heterologous sialic acid synthase, and a heterologous (CMP)-N-acetylneuraminic acid synthase, and a heterologous sialyltransferase. Beauprez teaches a method of producing sialylated compounds by fermentative growth of microorganisms (see p.4, lines 26-27). The sialylated compounds include sialylated oligosaccharides such as 3’-sialyllactose and 6’-sialyllactose (see p.8, lines 19-44 and p.9, lines 1-11). The producer microorganisms are engineered such that they express lactose permease, sialyltransferase and catalyze a pathway as shown in Figs. 1A-1B, which corresponds to the CMP-NeuNAc biosynthesis pathway of the present invention (see p.7, lines 31-37, p.8, lines 3-7, p.10, lines 17-30, and p.21, lines 34-38). Beauprez performs the method with a Bacillus subtilis 168 strain, and cultivates it in Luria Broth (LB) medium (see Example 1, p.33, line 24,-p.37, line 24). Example 7 of Beauprez describes the production of sialyllactoses and other sialylated compounds by cultivation of the strains described in Example 1, which includes Bacillus subtilis 168 as previously described, using the construct of Example 3 (see p.41, lines 1-8). Beauprez further describes different sialyllactoses (i.e., 6’-sialyllactose and 3’-sialyllactose) and mixtures can be obtained by substituting the sialyltransferase for one with a separate activity (see p.41, lines 1-7). Example 3 describes a daughter strain of the strain used in Example 2 (see p.38, lines 14-16). Example 2 describes an E. coli strain that overexpresses a glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (ScGNA1), a N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE), and a sialic acid synthase from Campylobacter jejuni (CjneuB) (see p.37, line 25; p.38, line 1). Example 3 describes that the daughter strain is further modified by overexpressing the lactose permease EclacY from E. coli, a CMP-sialic acid synthetase from Neisseria meningitides (NmneuA), and a sialyltransferase from Photobacterium damselae (Pdbst) (see p.38, lines 13-20). Therefore, according to Example 7, a Bacillus subtilis 168 strain that overexpresses a glucosamine-6-P-aminotransferase from Saccharomyces cerevisiae (ScGNA1), a N-acetylglucosamine-2-epimerase from Bacteroides ovatus (BoAGE), a sialic acid synthase from Campylobacter jejuni (CjneuB), the lactose permease EclacY from E. coli, a CMP-sialic acid synthetase from Neisseria meningitides (NmneuA), and a sialyltransferase from Photobacterium damselae (Pdbst) is produced for the production of sialyllactoses, specifically 6’-sialyllactose. The EclacY reads on a heterologous lactose permease, the BoAGE reads on a heterologous UDP-N-acetylglucosamine 2-epimerase, the CjneuB reads on a heterologous sialic acid synthase, the NmneuA reads on a (CMP)-N-acetylneuraminic acid synthase, and the Pdbst reads on a heterologous sialyltransferase as recited in claim 13. Furthermore, the Bacillus subtilis 168 strain described above is expected to use glucosamine-1-phosphate as an intermediate in the CMP-NeuNAc biosynthesis pathway because it expresses the required enzymes as recited in claim 13. Deletion or disruption of the genes yvmC and/or cypX are taught by Tang. Tang teaches Bacillus cells are well-established hosts for the production of heterologous biological substances; however, these Bacillus cells also produce undesirable pigment byproducts that must be removed during purification of the heterologous biological substance of interest (see paragraphs [0005]-[0007]). Tang teaches Bacillus subtilis produces pulcherriminic acid which becomes pulcherrimins, a reddish pigment, in the presence of ferric ions in the culture medium (see paragraphs [0005]-[0006] and [0026]). Tang further teaches that the synthesis of pulcherriminic acid by Bacillus subtilis can be prevented if at least one of the genes yvmC and cypX are deleted (see paragraphs [0024]-[0025], [0098], and Table 1). Thus, it would have been obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to delete at least one of the genes yvmC and cypX to prevent the formation of red pigment in the Bacillus subtilis strain for production of sialyllactose biological products. One of ordinary skill in the art would have had a reasonable expectation of success since each of Beauprez and Tang are directed to Bacillus subtilis. With respect to Applicant’s arguments regarding Zhang, the rejection set forth above no longer relies on Zhang. Thus, the arguments pertaining to Zhang are moot.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/J.P.S./Examiner, Art Unit 1657