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
The instant application is a U.S. national phase of PCT/CN2022/124823, filed on 10/12/2022. The preliminary amendment filed 01/04/2024 is entered.
Claims 3 and 7 are canceled. Claims 12-16 are new. Claims 1-2, 4-6, and 8-16 are pending and under consideration in this action.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. The instant claims are entitled to an effective filing date of 10/12/2022.
Specification
Abstract Content: Applicant is reminded of the proper content of an abstract of the disclosure. A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art. If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
The last line of the abstract filed 01/04/2024 recites “the advantages of high yield and low overall cost”, which should be deleted because the abstract should not refer to purported merits of the invention.
Abstract Language: Applicant is reminded of the proper language and format for an abstract of the disclosure. The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details. The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
The abstract filed 01/04/2024 recites “[t]he invention discloses a genetically engineered bacterium and its application in the preparation of sialyllactose” in line 1, which should be deleted because the recitation includes the implied phrase, “[t]he invention discloses”, and it includes a repetition of the invention title.
Disclosure: The specification is objected to as failing to provide proper antecedent basis for the claimed subject matter. See 37 CFR 1.75(d)(1) and MPEP § 608.01(o). Correction of the following is required: The specification recites the accession number “BAF91416.1” in line 25 on page 4 and in line 23 on page 16. It is unclear whether this accession is the same as the “BAF91416” accession number recited in instant claim 15.
The use of the terms, Novagen (p. 7 line 12), Sepax (p. 7 line 18), Tsingke (p. 7 line 28; p. 9 line 2; p. 17 line 1), which are trade names or marks used in commerce, have been noted in this application. The terms should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112(a)
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 4 and 14-15 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 enablement requirement. The claims contain subject matter which was not described in the specification in such a way as to enable one skilled in the relevant art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
It is apparent that the following GenBank accession numbers recited in claim 4 may be required to practice the claimed invention: AF305571 for the neuB gene, BAL35720 for the slr1975 gene, and NP_116637 for the Gna1 gene. Furthermore, the NCBI accession number WP_003512903.1 recited in claim 14; and the NCBI accession number BAF91616 or AJC62560.1 recited in claim 15 may be required to practice the claimed invention.
As such the biological material must be known and readily available or obtainable by a repeatable method set forth in the specification, or otherwise known and readily available to the public. If it is not so obtainable or available, the requirements of 35 USC 112(a) or pre-AIA 35 U.S.C. 112, first paragraph, may be satisfied by a deposit of: GenBank accession numbers: AF30557, BAL35720, and NP_116637; and NCBI accession numbers WP_003512903, BAF91416 and AJC62560.1.
The process disclosed in the specification does not appear to be repeatable, it is not clear that the invention will work with commonly available material and it is not apparent if the biological materials considered necessary to make and use the invention is both known and readily available to the public.
With respect to instant claim 4, the neuB gene under GenBank accession number AF305571 encodes the sialic acid synthase from Campylobacter jejuni. The slr1975 gene under GenBank accession number BAL35720 encodes Synechocystis sp. PCC6803 N-acetylglucosamine 2-epimerase. The Gna1 gene under GenBank accession number NP_116637 encodes the glucosamine-6-phosphate acetyltransferase from Saccharomyces cerevisiae. In example 2, the specification teaches ligating the neuB, slr1975, and the Gna1 gene are into the puc57 vector. The expression plasmid is related to N-acetylneuraminic acid synthesis and preparation. See the paragraph spanning pages 12-13 and see figure 10 for the Neu5Ac detection. It is unclear whether the same Neu5Ac production could be achieved without the specifically required GenBank deposited genes recited in claim 4.
With respect to instant claims 14-15, the NCBI accession number WP003512903.1 is the N-acylneuraminate cytidylyltransferase CSS, and BAF91416.1 is the α-2,6- 2,6-sialytransferase. In example 3, both genes are synthesized and ligated into pET28a. The pET28a is transformed as part of a plasmid combination into SLIS026 competent cells for the production of 6’-salyllactose (6’-SL). See lines 20-24 on page 16 and the paragraph spanning pages 17-18. The 6’-SL content (g/L) is disclosed in table 7. It is unclear whether the same 6’SL production could be achieved without the specific genes deposited under NCBI accession numbers WP003512903.1 and BAF91416.1. In example 4, the gene encoding α-2,3-sialytransferase with accession number AJC62560.1 is ligated into the pET28a vector, which is transformed into SLIS026 competent cells for 3’SL production. See lines 5-6 on page 20, table 8 and lines 3-6 on page 21. Figure 15 shows the detection profile of 3’SL fermentation broth. See lines 30-31 on page 21. It is unclear whether the same detection could be achieved without the specific gene deposited under NCBI AJC62560.1.
In the instant case, there is no indication in the specification as to public availability of the biological material. Therefore, a deposit at a recognized depository may be made to obviate this rejection.
If the deposit is made under the terms of the Budapest Treaty, then a statement, affidavit or declaration by Applicants, or by an attorney of record over his or her signature and registration number, or by someone in a position to corroborate the facts of the deposit, that the instant invention will be irrevocably and without restriction released to the public upon the issuance of a patent, would satisfy the deposit requirement made herein.
If the deposit is a non-Budapest Treaty deposit, then in order to certify that the deposit meets the requirements set forth in 37 CFR 1.801-1.809 and MPEP 2402-2411.05, a statement, affidavit or declaration by Applicant or by an attorney of record over his or her signature and registration number, or by someone in a position to corroborate the facts of the deposit would satisfy the requirements herein by stating and providing that:
(a) During the pendency of the application, access to the invention will be afforded to the Commissioner upon request;
(b) All restrictions upon availability to the public will be irrevocably removed upon granting of the patent;
(c) The deposit will be maintained in a public depository for a period of 30 years, or 5 years after the last request or for the enforceable life of the patent, whichever is longer; and
(d) Provide evidence of the test of the viability of the biological material at the time of deposit (see 37 CFR 1.807).
In the instant case, since WP_003512903.1 appears to be the protein sequence encoded by instant SEQ ID NO: 61, to obviate this rejection of claim 14, claim 14 can be amended to replace “has the NCBI accession number WP_003512903.1, or comprises the nucleic acid sequence set forth in SEQ ID NO:61” in lines 1-3 with the limitation of “has the protein sequence encoded by SEQ ID NO: 61”. Since BAF91416 and AJC6256.1 appear to be the protein sequences encoded by instant SEQ ID NOs: 62 and 63 respectively, to obviate this rejection of claim 15, the claim can be amended to replace “an enzyme with NCBI accession number BAF91416, or comprises the nucleic acid sequence set forth in SEQ ID NO:62” in lines 1-3 with the limitation of “an enzyme encoded by SEQ ID NO:62”, and replace “an enzyme with NCBI accession number AJC62560.1, or comprises the nucleic acid sequence set forth in SEQ ID NO:63” in lines 3-4 with the limitation of “an enzyme encoded by SEQ ID NO: 63”.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2, 4-6, and 8-16 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 is indefinite for three reasons. First, claim 1 recites “the genetically engineered bacterium contains multiple copies of a gene neuB and the gene neuB is initiated for expression by a Tet promoter” (lines 2-4), which is indefinite because it is unclear whether all of the multiple copies of neuB are required to be initiated for expression by a Tet promoter. Second, claim 1 recites “wherein the genetically engineered bacterium further comprises a gene neuB” (line 4), which is indefinite because it is unclear how this wherein clause further modifies a genetically engineered bacterium required to have multiple copies of neuB. For example, this wherein clause may require the minimum of neuB genes to be three because the minimum for “multiple copies” is 2 plus the one neuB gene to account for the wherein clause. However, this wherein clause may also reasonably be interpreted as encompassing genetically engineered bacteria with only one neuB gene. Third, claim 1 recites “the multiple copies” (lines 10-11), which is indefinite because it is unclear whether “the multiple copies” refer to the multiple copies of the neuB gene.
Claims 2, 4-6, and 8-16 depend from claim 1 and are rejected for the reasons set forth above.
Claim 6 is indefinite for two reasons. First, claim 6 recites “the starting bacterium” in line 2. There is insufficient antecedent basis for this limitation in the claim. Claim 6 depends from claim 1, which does not require a starting bacterium. Therefore, it is unclear whether claim 6 intends to limit the genetically engineered bacterium of claim 1 or a separate starting bacterium. To obviate this rejection, “the starting bacterium” can be replaced with “the genetically engineered bacterium”. Second, claim 6 recites the parenthetical phrase “(DE3)”, which renders the claim indefinite because it is unclear whether the limitation within the parentheses is part of the claimed invention. In other words, it is unclear whether the E. coli BL21 strain is required to contain the DE3 prophage or not because the DE3 limitation is recited in parentheses.
Claim 8 is indefinite for two reasons. First, claim 8 recites “the fermentation broth” in lines 3-4, which is indefinite because there is no earlier recitation of a fermentation broth. Therefore, it is unclear whether claim 8 intends to limit the fermentation medium to a broth, or whether the claim is referring to a separate fermentation broth. Second, claim 8 recites “; and optionally, obtaining 3’-sialyllactose when the genetically engineered bacterium comprises α-2,3-sialyltransferase gene; obtaining 6’-sialyllactose when the genetically engineered bacterium comprises α-2,6-sialyltransferase gene”, which is indefinite because it is unclear whether obtaining 6’-sialyllactose is an optional or required. In other words, it is unclear whether every limitation recited after the term “optionally” is optional, or whether only obtaining 3’-sialyllactose is optional.
Claims 9-11 depend from claim 8 and are rejected for the reason set forth above.
Claim 9 recites “TB medium comprises 12 g/L trypsin”, which renders the claim indefinite because it is unclear whether the TB stands for terrific broth. The specification does not define the TB acronym as terrific broth. Although terrific broth is known in the art as TB medium, the TB medium known in the art does not include the enzyme trypsin. Rather, the known TB medium includes tryptone. Therefore, it is unclear whether the claimed TB medium is terrific broth or a separate medium.
Claim 10 is indefinite for three reasons. First, claim 10 recites “[t]he method of claim 8, comprising”, and not ‘further comprising’. Therefore, it is unclear whether claim 10 intends to further limit the method of claim 8. Second, claim 10 recites “the induction culture” in line 4, which is indefinite because it is unclear whether “the induction culture” refers to the IPTG induced culture at an OD value of 0.6 to 0.8, or a separate culture induced by any means and at any OD value. To clarify, claim 10 recites inducing culture with IPTG when cultured to OD value of 0.6 to 0.8; “and/or” supplementing…after the induction culture. Therefore, one could reasonably interpret the claim as requiring either the recited IPTG culture induction step, or the recited supplementation step. However, the claim specifically refers to supplementing after “the induction culture”, which may refer to the IPTG induced culture. Third, it is unclear whether the “OD” recited in line 2 refers to optical density because there is no wavelength associated with the claimed OD. The specification teaches an OD600 of 0.6-0.8. See page 15 line 31. However, it is unclear whether the claimed OD is limited to a 600nm wavelength described in the specification.
Regarding claims 10 and 12, the term "preferably" renders the claims indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claims 12, 13, 14, 15 and 16 refer to the methods of claims 2, 4, 5, 5, and 5 respectively, however claims 2, 4, and 5 are not method claims. Therefore, it is unclear whether claims 12-16 intend to limit the genetically engineered bacterium of claims 2, 4 and 5 or a separate method.
Claim Interpretation
Claim 1 requires a genetically engineered bacterium comprising plasmid vector 1, which encodes sialic acid synthase neuB, N-acetylglucosamine 2-epimerase slr1975, N-acetylglucosamine-6-phosphate phosphatase YqaB, glucosamine-6-phosphate acetyltransferase Gna1, L-glutamine-D-fructose-6-phosphate transaminase glmS, and phosphoenolpyruvate synthase ppsA; and the bacterium further comprises a gene encoding N-acylneuramic acid cytidylyltransferase and a gene encoding α-2,3-sialytransferase; the bacterium includes multiple, i.e. at least 2, neuB genes, and at least one of the neuB genes is regulated by the Tet promoter. The “N-acylneuramic acid cytidylyltransferase” recited in line 12 of instant claim 1 is synonymous with N-acylneuraminate cytidylyltransferase. See page 4 line 19 of the instant specification.
Claim 2 requires the N-acetylneuraminic acid catabolic pathway in the engineered bacterium to be disabled. According to the instant specification, the pathway can be disabled by knocking out all or part of the genes within the pathway, which includes nanK (N-acetylmannosamine kinase), nanE (N-acetylmannosamine-6-phosphate epimerase), and nanA (N-acetylneuraminic acid aldolase). See page 3 lines 8-18.
Claim 10 is interpreted as requiring the method of claim 8 to further comprise adding IPTG to the fermentation of claim 8 when cultured to an OD value of 0.6 to 0.8. However the wavelength of the optical density is not limited. After the induction, the fermentation may further be supplemented, but the supplementation is not required.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 4-6, 8, 10-12, and 15 are rejected under 35 U.S.C. 103 as being unpatentable over Aesaert (WO 2022/034080 published 02/17/2022, and effectively filed 08/10/2021), in view of Jennewein (WO 2019/020707, as provided with the IDS filed 04/08/2024).
Regarding claim 1, Aesaert teaches an engineered cell comprising at least one protein chosen from a list that includes: sialic acid synthase (i.e. NeuB), N-acetylglucosamine-6-phosphate phosphatase, UDP-N-acetylgucosamine 2-epimerase, glucosamine 6-phosphate N-acetyltransferase (i.e. Gna1), and L-glutamine-D-fructose-6-phosphate amino transferase (also referred to as glmS). See claims 1-3 and 11 and page 29 lines 13-18. The cell is selected from a list that includes bacterium and yeast. See claim 27 of Aesaert. Aesaert teaches a cell comprising at least one DNA sequence encoding NeuB. See claim 19 of Aesaert. Aesaert teaches presenting DNA sequences to the cell in expression modules regulated by one or more regulatory sequences, wherein the expression modules are integrated into the cell’s genome or presented in a plasmid. See claims 7-8 of Aesaert. In example 18, Aesaert teaches a modified S. cerevisiae (yeast) strain with an expression plasmid comprising constitutive transcriptional units for glmS, a phosphatase selected from a group that includes YqaB, N-acetylglucosamine 2-epimerase from B. ovatus, NeuB, N-acylneuraminate cytidylyltransferase and a beta-galactoside alpha-2,3-sialytransferase. See page 104 lines 15-27. Aesaert teaches upregulating phosphoenolpyruvate synthase gene (ppsA). See page 2 line 36 and claim 18 of Aesaert. Aesaert teaches N-acetylglucosamine 2-epimerase, an enzyme that catalyses the reaction N-acyl-D-glucosamine = N- acyl-D-mannosamine. Alternative names for this enzyme comprise N-acetylglucosamine 2-epimerase, N- acetyl-D-glucosamine 2-epimerase, GIcNAc 2-epimerase and N-acyl-D-glucosamine 2-epimerase. See page 16, lines 6-8.
Aesaert does not teach neuB, slr1975, YqaB, Gna1, GlmS and ppsA genes linked in tandem in a plasmid vector 1; however, Aesaert teaches neuB, YqaB, Gna1, and GlmS linked in a plasmid, and separately teaches upregulating ppsA. Aesaert does not teach a Tet promoter for neuB gene expression.
Jennewein teaches subcloning neuB in an operon behind the constitutive promoter Ptet. Jennewein teaches genomic integrations that lead to a Neu5Ac producing strain #1363. See the paragraph spanning pages 25-26. Jennewein teaches an E. coli BL21(DE3) strain #1363 harboring genomic integrations of: glmS, gna1, slr1975, neuB and ppsA. See table 1 on page 27.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to: (1) replace the S. cerevisiae yeast in example 18 of Aesaert with a bacterium; (2) replace the N-acetylglucosamine 2-epimerase coding gene in the plasmid of Aesaert with the slr1975 of Jennewein; (3) add ppsA as suggested by Aesaert; and (4) further add the Ptet of Jennewein to the plasmid of Aesaert.
(1) One would be motivated to replace the S. cerevisiae yeast in example 18 of Aesaert with a bacterium, because Aesaert suggests that yeast cells are interchangeable with bacteria cells (see claim 27 of Aesaert). There would be a reasonable expectation of success because Aesaert demonstrates integrating the following genes into the genome of a bacterium: gna1, glmS, a phosphatase chosen from a group that includes YqaB, N-acetylglucosamine 2-epimerase, NeuB, N-acylneuraminate cytidylyltransferases, and beta-galactosidase alpha-2,3-sialytransferase (see example 26 spanning pages 110-111); and Aesaert further suggests that genes can be introduced into a host’s genome or presented on a vector plasmid (see claims 7-8 of Aesaert). In the process, one would arrive at a bacterium comprising the plasmid described in example 18 of Aesaert.
(2) One would be motivated to replace the N-acetylglucosamine 2-epimerase coding gene in the plasmid of Aesaert with the slr1975 gene of Jennewein, because this is simple substitution of one gene for another coding the same enzyme. Jennewein suggests that the enzyme may be part of a simple a cost-efficient method for the purification of a sialyllactose (see page 4 lines 3-5 of Jennewein). There would be a reasonable expectation of success because Aesaert demonstrates constructing a plasmid comprising a N-acetylglucosamine 2-epimerase, which is the same type of enzyme as the slr1975 of Jennewein.
(3) One would be motivated to add the ppsA of Aesaert to the plasmid because Aesaert suggests modifying cells for enhanced synthesis and/or supply of phosphoenolpyruvate. There would be a reasonable expectation of success because Aesaert teaches upregulating the ppsA gene.
(4) One would be motivated to add the Ptet of Jennewein to the plasmid because Aesaert suggests introducing expression modules into cells under regulatory sequences. There would be a reasonable expectation of success because Jennewein demonstrates integrating NeuB, Slr1975, Gna1 and GlmS genes into a bacterium and initiating integrated genes with the Ptet promoter.
Regarding claim 2, Aesaert teaches modifying an E. coli strain by knocking out nanA (N-acetylneuraminate lyase), nanE and nanK (N-acetylneuraminate kinase). See page 95 lines 6-7, page 97 line 4, and page 98 lines 11-12. Jennewein teaches deleting nanA, nanK and nanE from E. coli. See example 1 spanning pages 10-11. These gene knockouts or deletions would have disabled an N-acetylneuraminic acid catabolic pathway.
Regarding claim 4, Jennewein teaches neuB (acc. no. AF305571 as disclosed in the instant specification), slr1975 (acc. No. BAL35720 as disclosed in the instant specification), and the ppsA encoding the phosphoenolpyruvate synthase of E. coli BL21. See the paragraph spanning pages 25-26.
Regarding claim 5, in example 18, Aesaert teaches an expression plasmid comprising constitutive transcriptional units for glmS, a phosphatase selected from a group that includes YqaB, N-acetylglucosamine 2-epimerase, NeuB, N-acylneuraminate cytidylyltransferase and a beta-galactoside alpha-2,3-sialytransferase. See page 104 lines 15-27. Aesaert teaches presenting DNA coding sequences to the cells in one or more expression modules that are integrated into the host’s genome or on a plasmid. See claims 7-8 of Aesaert. Aesaert teaches DNA sequences encoding N-acylneuraminate cytidylyltransferase and alpha-2,3-sialytransferase, oralpha-2,6-sialytransferase. See claim 19 of Aesaert.
Aesaert and Jennewein do not teach N-acylneuraminate cytidylyltransferase and sialyltransferase tandemly linked in a plasma vector 2
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to combine the N-acylneuraminate cytidylyltransferase and sialyltransferases of Aesaert in a separate plasmid. One would be motivated to do so because Aesaert discloses that the enzymes are both part of the sialylation pathway (page 27 lines 16-21). There would be a reasonable expectation of success because Aesaert demonstrates constructing a plasmid comprising the enzymes together.
Regarding claim 6, Aesaert teaches a cell that is a bacterium or yeast, preferably said bacterium is an E. coli strain. See claim 27 of Aesaert.
Aesaert does not teach Escherichia coli BL21 (DE3).
Jennewein teaches E. coli BL21(DE3). See, for example, table 1 on page 27.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the S. cerevisiae (yeast) in example 18 of Aesaert with the E. coli BL21 (DE3) of Jennewein. One would be motivated to do so because Jennewein suggests that the E. coli Bl21 (DE3) strain can be used for the overproduction of sialyltransferase enzymes (page 41 lines 2-4). There would be a reasonable expectation of success because Jennewein demonstrates integrating glmS, gna1, slr1975, neuB and ppsA into the genome of the E. coli Bl21 (DE3) strain.
Regarding claim 8, Aesaert teaches a method comprising cultivating a cell to produce sialylated di- and/or oligosaccharide, and preferably separating said sialylated di-and/or oligosaccharide from said cultivation. See claim 32 of Aesaert. The method further comprising adding to the culture medium a lactose feed. See claims 34, 38 and 40 of Aesaert. Aesaert teaches a sialylated di- and/or oligosaccharide selected from a group that includes mammalian milk oligosaccharide. See claim 14 of Aesaert. Mammalian milk oligosaccharide refers to oligosaccharides including 6’-sialyllactose, and 3’-sialyllactose. See page 22 lines 1-3. In example 18, Aesaert teaches producing 3’-siallyactose with a modified S. cerevisiae strain comprising an expression plasmid, where the strain is evaluated in a growth experiment on a medium comprising lactose. After incubation, the culture broth is harvested and the sugars are analyzed. See page 104 lines 15-31.
Jennewein teaches a method for producing a sialylated oligosaccharide that is selected from a group that includes 3’-sialyllactose and 6’-sialyllactose. See claims 1 and 2 of Jennewein. The cell is cultivated in the presence of lactose. See claim 6 of Jennewein. The sialylated oligosaccharide can be recovered from the fermentation broth. See page 17 lines 25-26. In example 4, Jennewein teaches growing E. coli BL21 (DE3) #1363. Lactose is added after one hour of incubation. See the paragraph spanning pages 36-37.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to modify the plasmid containing S. cerevisiae (yeast) cell in example 18 of Aesaert to a plasmid containing bacterium as suggested by Aesaert, and to further apply the method of Aesaert and/or Jennewein to that modified host cell; because both Aesaert and Jennewein teaches culturing cells in the presence of lactose and separating or recovering 3’-sialyllactose and 6’-sialyllactose.
Regarding claim 10, Aesaert teaches IPTG as an expression inducer. See page 8 lines 22-23.
Aesaert does not teach inducing culture with IPTG when cultured to OD value of 0.6 to 0.8; and/or supplementing 2 g/L MgSO4·7H-2O, 20 g/L glycerol, 1mL/L trace element stock solution and 5 g/L lactose after the induction culture.
Jennewein teaches inducing gene expression by the addition of IPTG when the E. coli BL21(DE3) #1363 culture reaches an OD600 of 0.1 to 0.3. See example 4 page 36 lines 16-17 and example 5 page 39 lines 5-6. In example 8, Jennewein teaches adding lactose to cultures that reached an OD600 of 0.5 to 1. After 24 hours of incubation, IPTG is added to induce gene expression. See page 43 lines 21-22.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to apply the IPTG induction step of Jennewein to the modified plasmid containing bacterium suggested by Aesaert. One would be motivated to do so because both Aesaert and Jennewein suggest that IPTG can be used to induce gene expression. There would be a reasonable expectation of success because Jennewein demonstrates adding IPTG to an E. coli BL21(DE3) #1363 culture at an OD600 of 0.1 to 0.3.
Regarding claim 11, Aesaert teaches growing host strains at 30˚C and shaking at 200 rpm. See page 94 lines 31-32
Jennewein teaches growing E. coli BL21 (DE3) #1363 at 30˚C in shake flasks. See page 36 lines 12-13.
Aesaert and Jennewein do not teach 250 rpm.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to optimize 200 rpm shaking of Aesaert. A person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because Aesaert teaches a 200 rpm from which one could optimize. MPEP 2144.05(II)(A) states that “[w]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Regarding claim 12, Aesaert teaches modifying an E. coli strain by knocking out nanA (N-acetylneuraminate lyase), nanE and nanK (N-acetylneuraminate kinase). See, for example, page 95 lines 6-7, page 97 line 4, page 98 lines 11-12.
Jennewein teaches deleting nanA, nanK and nanE from E. coli. See example 1 spanning pages 10-11.
Regarding claim 15, Jennewein teaches a sialyltransferase gene with the accession number BAF91416. See table on page 35.
Claims 9 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Aesaert (WO 2022/034080 published 02/17/2022, and effectively filed 08/10/2021), and Jennewein (WO 2019/020707), as applied to claims 1-2, 4-6, 8, 10-12, and 15 above, and further in view of Cramer (CN 107099559A, translation provided herein).
Regarding claim 9, Aesaert teaches a Luria Broth medium for E. coli containing tryptone peptone and yeast extract. Aesaert teaches a minimal medium for E. coli cultivation comprising 30 g/L glycerol, 2.993 g/L KH2PO4- and 7.315 g/L K2HPO4. See page 82 lines 18-24.
Jennewein teaches E. coli BL21 (DE3). See example 4 on page 36 in lines 12-13.
Aesaert and Jennewein do not teach a medium comprising 12 g/L trypsin, 24 g/L yeast extract, 4 mL/L glycerol, 2.31 g/L KH2PO4 and 12.54 g/L K2HPO4.
Cramer teaches transforming E. coli BL21 cells with a pET28a and culturing in a TB medium (23.6 g/L yeast extract, 11.8 g/L trypsin [potential mistranslation], 9.4 g/L K2HPO4, 2 g/L KH2PO4 and 4 mL/L glycerol). See section 7.2 on page 26 of the translation. Cramer teaches vector systems and recombinant microorganisms capable of encoding/ expressing enzymes. See the second to last paragraph on page 2 of the translation.
Aesaert, Jennewein and Cramer do not teach a medium comprising 12 g/L trypsin, 24 g/L yeast extract, 2.31 g/L KH2PO4 and 12.54 g/L K2HPO4.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the S. cerevisiae (yeast) in example 18 of Aesaert with the E. coli BL21 (DE3) cell of Jennewein; to further replace medium taught by Aesaert with the TB medium for E. coli BL21 (DE3) taught by Cramer; and to further optimize the concentrations of the TB medium components. One would be motivated to do so because Cramer suggests that the TB medium is suitable for E. coli BL21(DE3) cells, which is the same E. coli strain taught by Jennewein. There would be a reasonable expectation of success because the TB medium of Cramer and the E. coli medium of Aesaert include similar components including glycerol, KH2PO4- and K2HPO4. One would be further motivated to optimize the concentrations of the components because a person of ordinary skill in the art has good reason to pursue the known options within their technical grasp. There would be a reasonable expectation of success because the concentrations in the TB medium of Cramer are reasonably close to the instantly claimed concentrations. In other words, the 11.8 g/L trypsin of Cramer is reasonably close to the instantly claimed 12 g/L; the 23.6 g/L yeast extract of Cramer is reasonably close to the instantly claimed 24 g/L; the 2 g/L KH2PO4 of Cramer is reasonably close to the instantly claimed 2.31; and the 9.4 g/L of K2HPO4 of Cramer is reasonably close to the instantly claimed 12.54 g/L.
Regarding claim 16, Cramer teaches transforming E. coli BL21 (DE3) with a pET28a vector. See page 26 of the translation. Cramer teaches amplifying with a T7 promoter. See lines 4-5 on page 23.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the pRS420 plasmid of Aesaert with the pET28a plasmid of Cramer. One would be motivated to use the pET28a plasmid of Cramer because Cramer suggests that the plasmid uses a T7 promoter, which is the same promoter taught by Jennewein. There would be a reasonable expectation of success because Cramer demonstrates transforming E. coli BL21 (DE3) with pET28a.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Aesaert (WO 2022/034080 published 02/17/2022, and effectively filed 08/10/2021), and Jennewein (WO 2019/020707), as applied to claims 1-2, 4-6, 8, 10-12, and 15 above, and further in view of Li (CN 110396532A, translation provided herein).
Regarding claim 13, Aesaert, in example 18, teaches producing 3’sialyllactose with a modified S. cerevisiae (yeast) strain that includes a pRS420-derived yeast expression plasmid comprising constitutive transcriptional units for glmS, a phosphatase selected from a group that includes YqaB, N-acetylglucosamine 2-epimerase from B. ovatus, NeuB, N-acylneuraminate cytidylyltransferase and a beta-galactoside alpha-2,3-sialytransferase. See page 104 lines 15-27.
Jennewein teaches E. coli BL21 (DE3) harboring plasmids encoding sialyltransferases. See example 4 on page 36 in lines 12-13.
Aesaert and Jennewein do not teach a plasmid vector 1 that is pACYCDuet.
Li teaches a method for preparing sialic acid lactose comprising constructing a multi-enzyme co-expression system into pACYCDuet-1 vector. See example 1 [0070-0072]. The α-2,3-sialytransferase gene is constructed into the plasmid. The recombinant plasmid is transferred into [E. coli] BL21 (DE3). See [0073]-[0074], [0086] and see figure 4 for the 3’sialyllactose produced.
It would have been obvious to a person of ordinary skill in the art prior to the effective filing date of the instantly claimed invention to replace the S. cerevisiae (yeast) in example 18 of Aesaert with the E. coli BL21 (DE3) cell of Jennewein, and further replace the pRS420 plasmid of Aesaert with the pACYCDuet of Li. One would be motivated to do so because Li teaches using the pACYCDuet plasmid for expressing α-2,3-sialytransferase and producing 3’sialyllactose. There would be a reasonable expectation of success because Li demonstrates transferring the pACYCDuet plasmid into E. coli BL21 (DE3), which is the same bacterial strain taught by Jennewein.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/K.C.B./Examiner, Art Unit 1657