DETAILED ACTION
Election/Restrictions
Applicant's election with traverse of Group I, claims 1-7, 10-13, 16, 17, 19, 21, 27, 29 and 31, in the reply filed on 10/15/25 is acknowledged. The traversal is on the ground(s) that:
Applicant respectfully traverse the restriction requirement of non-elected claims 33 and 36 (which resemble the process of claim 12). The arguments presented on 10/15/25 are found persuasive and claims 33 and 36 have been rejoined.
Claims 1-7, 10-13, 16, 17, 19, 21, 27, 29, 31, 33 and 36 are currently pending.
Claim Objections
Claims 19 and 33 are objected to because of the following informalities:
Claim 19 comprises duplicate features “wherein, under conditions of depleting of a limiting nutrient from a growth medium in which the genetically modified microorganism is growing, a stationary phase or non-dividing cellular state is induced” is repeated.
Claim 33 depends from a canceled claim.
Appropriate correction is required.
Claim Rejections - 35 USC § 112-2nd paragraph
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 1-7, 10-13, 16, 17, 19, 21, 27, 29, 31, 33 and 36 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites a genetically modified E. coli microorganism comprises “a production pathway comprising citramalate synthase for the production of citramalate,
1) a conditional induction of SoxS;
2) a conditionally triggered synthetic metabolic valve that silences gene expression of the citrate synthase (gItA) or glucose-6-phosphate-dehydrogenase (zwf) gene(s)” or 3) “a conditionally triggered synthetic metabolic valve that enables selective proteolysis of the citrate synthase (gItA) or glucose-6-phosphate-dehydrogenase (zwf) enzyme(s)”. First, it is unclear how the E. coli comprises “a conditional induction of SoxS.” What is the structure of this conditional induction? How/when would SoxS would be Induced? Claim 1 does not actually specify how/when SoxS would be Induced (e.g., the synthetic metabolic valves are conditionally triggered under aerobic or partially aerobic conditions during the stationary phase or non-dividing cellular state, but no mention of the structure or the conditions for induction of SoxS).
Further, the use and placement of the “or”s in the claim do not provide a modified E.coli capable of performing the desired functions because the claim could have just 1), just 2), or just 3) above. The conditionally triggered induction of SoxS is not required in this manner, as the claim could just include part b) or part c). This leaves the claim incredibly confusing and the metes and bounds of the claim are not readily understood.
At the same time, it is unclear what are said “conditionally triggered synthetic metabolic valves” for silencing gene expression and for enabling selective proteolysis. Moreover, it is unclear how said synthetic metabolic valves are conditionally triggered. The use of the term “production pathway” is also unclear because the metes and bounds of this term is not readily understood. It is known, for example, that metabolic pathway for citramalate synthesis in Escherichia coli, expressing cimA, coding citramalate synthase, comprises key enzymes (and corresponding genes}: citrate synthase (gltA), 3-isopropyimalate dehydratase (leuC), acetate kinase (ackA), phosphotransacetylase (pia), acetyl-CoA synthetase (acs), pyruvate oxidase (poxB), phosphoenolpyruvate synthase (ppsA). However, it is not provided in the claim or the examples of the description that “the production pathway comprising citramalate synthase for the production of citramalate” comprises, for example, acetyl-CoA synthetase (acs) gene.
Claims 1 and 19 are also vague and indefinite because the presence of both “a conditionally triggered synthetic metabolic valve that silences gene expression of the citrate synthase (gilA), or glucose-6-phosphate-dehydrogenase (zwf gene(s)” and “a conditionally triggered synthetic metabolic valve that enables selective proteolysis of the citrate synthase (gltA), or glucose-6-phosphate-dehydrogenase (zwf) enzyme(s)”. In this regard, it is unclear how silencing expression and enabling selective proteolysis are performed for the same gene simultaneously so the claims are unclear.
The Examiner also notes that the features of claims 1 and 19 “pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme activity is increased within the genetically modified microorganism...” are unclear because it is unclear compared to what the activity is “increased”, as well as what modifications the microorganism comprises for “increasing activity’.
Claim 3 is vague and indefinite because it does not recite a gene which encodes pyruvate ferredoxin oxidoreductase. The mere recitation of a name to describe the invention is not sufficient to satisfy the Statute's requirement of adequately describing and setting forth the inventive concept. The claim should provide any structural properties, such as the nucleic acid sequence or if it is very well known in the art the actual designation/name of the gene, which would allow for one to identify the gene without ambiguity. The mere recitation of a name does not adequately define the claimed product. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow for one to identify what is being claimed. Appropriate clarification and/or correction is required.
Claim 7 is vague and indefinite due to the description “sugar transporter”. The mere recitation of a name to describe the invention is not sufficient to satisfy the Statute's requirement of adequately describing and setting forth the inventive concept. The claim should provide any structural properties, such as the nucleic or amino acid sequence or if it is very well known in the prior art the actual designation/name of the transporter, which would allow for one to identify the product without ambiguity. The mere recitation of a name does not adequately define the claimed product. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow for one to identify what is being claimed. Appropriate clarification and/or correction is required.
Claim 11 is vague and indefinite due to the recitation of “a CASCADE guide array’ is. Furthermore, said term was not revealed by the Examiner in the prior art. Said circumstances claim 11 employs the term "CASCADE guide array”. This is internal nomenclature and as such claim 11 is unclear on its own. While the specification can be used to provide definitive support, the claims are not read in a vacuum. Rather, the claim must be definite and complete in and of itself. Limitations from the specification will not be read into the claims. The claims as they stand are incomplete and fail to provide adequate structural properties to allow for one to identify what is being claimed. Appropriate clarification and/or correction is required.
Claim 12 and 33 recite “a rate of 30 g/L or greater”; however, this is vague and indefinite because the rate of production is determined by how much organic matter is synthesized, over what period of time and in what volume, or over what area, in this connection, the parameter, which is taken into account in a production rate, is time, however, it is not provided in the claim.
Claim 36 recites “a rate at or greater than 100 g/L”; however, this is vague and indefinite because the rate of production is determined by how much organic matter is synthesized, over what period of time and in what volume, or over what area, in this connection, the parameter, which is taken into account in a production rate, is time, however, it is not provided in the claim.
Claim Rejections - 35 USC § 112-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 1-7, 10-13, 16, 17, 19, 21, 27, 29, 31, 33 and 36 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 claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The claims broadly recite for example:
A genetically modified microorganism (any microorganism in claim 19; E. coli in claim 1) comprising: a production pathway comprising citramalate synthase for the production of citramalate,
a conditional induction of SoxS: a conditionally triggered synthetic metabolic valve that silences gene expression of the citrate synthase (gltA), or glucose-6-phosphate-dehydrogenase (zwf) gene(s);
or a conditionally triggered synthetic metabolic valve that enables selective proteolysis of the citrate synthase (gltA) or glucose-6-phosphate-dehydrogenase (zwf) enzyme(s);
wherein the synthetic metabolic valve(s) of the microorganism are conditionally triggered during a stationary phase or non-dividing cellular state;
wherein, under conditions of depleting of a limiting nutrient from a growth medium in which the genetically modified microorganism is growing, a stationary phase or non-dividing cellular state is induced;
wherein pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme activity is increased within the genetically modified microorganism under aerobic or partially aerobic conditions during the stationary phase or non-dividing cellular state to produce an acetyl CoA pool; and
wherein sugar uptake is enhanced within the genetically modified microorganism, when compared to a non-genetically modified microorganism.
The specification recites that the inventors created synthetic metabolic valves, to dynamically reduce levels of the first committed step in each pathway, namely citrate synthase (GItA, “G”, encoded by the gltA gene) and glucose 6-phosphate dehydrogenase (Zwf, “Z”, encoded by the zwf gene). They show that dynamic control over these two enzymes improves stationary phase production of pyruvate, and citramalate, and have applicability in the production of numerous products requiring pyruvate and/or acetyl-CoA. The native E. coli Type I-E Cascade/CRISPR system is
used for gene silencing (See FIG3Ci-iii). Targeted proteolysis is implemented by linking the expression of the chaperone SspB to phosphate deprivation. SspB, when induced, binds to C- terminal DAS+4 peptide tags on any target protein and causes degradation by the ClpXP protease of FE. coli (FIG 3D). Using engineered strains, as Figure 1E demonstrates, protein levels can be controlled in a two-stage process, as exemplified by turning “ON” GFP and “OFF” constitutively expressed mCherry. While, in this case, the combination of gene silencing with proteolysis results in the largest rates of protein degradation (FIG 3F-G), the impact of each approach and specific decay rates, will vary depending on the target gene/enzyme and its specific natural turnover rates and expression levels.
Instant claims 1 and 19 require that pyruvate-flavodoxin/ferredoxin oxidoreductase (PFO) activity is increased during the stationary phase, and sugar uptake is increased. The instant specification shows that these properties are conferred by cell-cycle specific downregulation of gltA and zwf respectively. However, the instant claims only require that any one of gltA or zwf and SoxS are selectively downregulated. To the extent that gltA and zwf do not both have to be downregulated, the claims are therefore inconsistent with the teachings of the instant specification. Furthermore, the description indicates that SoxS needs to be upregulated (example 6) in order to enhance ACoA flux. Therefore, the feature that SoxS is downregulated appears to be inconsistent with the description and not enabled as written.
Additionally, claim 12 relates to production of any protein product. At the same time, it is the microorganism of claim 1 that produces the product at a rate of 30 g/L or greater. However, the examples in the instant specification disclose production of pyruvate with a yield of more than 30 g/L and production of citramalate with a yield of more than 100 g/L (see Example 3). This demonstrates achievement of the engineered microorganism and not the production of any other heterologous proteins.
On the basis of Example 3, wherein it is indicated that they implement CRISPRi/Cascade based gene silencing or implement a targeted proteolysis by linking the expression of the chaperone SspB to phosphate deprivation; however, the claimed genetically modified E. coli/microorganism comprises “a conditional induction of SoxS”, at the same time it is unclear how the genetically modified microorganism which is modified for a ‘conditional induction’ of SoxS. Example 6, wherein engineering an extra copy of SoxS on the chromosome, induced by the low phosphate inducible yibD gene promoter, is disclosed, but nothing beyond that. The similar remarks also concern claims 1 and 19 where the E. coli/microorganism comprises “a conditionally triggered synthetic metabolic valve that silences gene expression of the citrate synthase (gltA), or glucose-6- phosphate-dehydrogenase (zwf) gene(s)” or “a conditionally triggered synthetic metabolic valve that enables selective proteolysis of the citrate synthase (gItA), or glucose-6-phosphate- dehydrogenase (zwf) enzyme(s). At the same time, it is unclear what are said “conditionally triggered synthetic metabolic valves” for silencing gene expression and for enabling selective proteolysis. The instant specification only teaches, for example, in Example 3, wherein it is indicated that they implement CRISPRi/Cascade based gene silencing or implement a targeted proteolysis by linking the expression of the chaperone SspB to phosphate deprivation.
Additionally, the instant claims may be interpreted as relating to an E. coli or any modified microorganism comprising:’
A, B and D; or
A, C, and D,
Wherein A is a conditional induction of SoxS, B is a production pathway comprising citramalate synthase for the production of citramalate, C is a conditionally triggered synthetic metabolic valve that silences gene expression of gltA and/or zwf genes, and D is a conditionally triggered synthetic metabolic valve that enables selective proteolysis of gltA and/or zwf enzymes.
Based on Fig. 5A, the applicant’s intention appears to be the former, and thus, a suitable rectification to overcome the clarity objection would he to add in the conjunction “and”. The instant specification presents findings that:
synthetic metabolic “G” valve that dynamically reduces level of citrate synthase encoded by the gltA gene) would enhance sugar uptake (Example 3);
a synthetic metabolic “Z" valve that dynamically reduces level of glucose-6- phosphate dehydrogenase (encoded by the zwf gene would increase pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme (PFO activity (Example 3); and
induction/activation of soxS Improves acetyl-CoA flux; via PFO activity (Example 8).
It is noted claim 1 does not actually specify how/when SoxS would be Induced (cf. the synthetic metabolic valves are conditionally triggered under aerobic or partially aerobic conditions during the stationary phase or non-dividing cellular state).
Thus, pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme activity and sugar uptake to be increased within the genetically modified microorganism, as defined in/required by the characterizing portion of claim 1, both the “G" and “Z" valves must have been essential. However, since claim 1 currently encompasses conditions where only the “G" or the “Z” valve is present, the claim lacks enablement. Further, the instant specification only enables the use of E.coli and not any microorganism. However, claim 19 allows for the use of any genetically modified microorganism, which includes microorganisms belonging to any genus or species. in this claim, the "product" includes any chemical substance. In contrast, the examples in the detailed description of the invention merely state that a genetically modified microorganism for producing citramalic acid was produced using Escherichia coli as a host microorganism. Qn the other hand, in general, the endogenous metabolic enzymes of a microorganism vary depending on the genus and species to which the microorganism belongs, and the raw materials consumed and their metabolic products also differ greatly. Considering this, it is not the case that a microorganism belonging to any genus or species has a “production pathway including at least one enzyme for producing a product from an acety!-CoA precursor" for a desired “product” as defined in claim 19. Even if a microorganism has “one enzyme” involved in the “production pathway,” the “production pathway” of the desired “product is usually composed of multiple metabolic enzymes. in this case, in order to work the invention of claim 19, it is necessary to examine whether or not a group. of microorganisms belonging to any genus or species has a "production. pathway including at least one enzyme for producing a product from an acetyl-CoA precursor" for a desired "product," and if not to identify the missing metabolic enzymes and then introduce them into the microorganism, which requires trial and error beyond the extent that a person skilled in the art can expect. In addition, although claim 21 and 28 specify that “the genetically modified microorganism is an Enterobacter microorganism,” the instant specification does not disclose any working examples using “Enterobacter microorganisms" or anything equivalent thereto. Furthermore, even if not even one working example is disclosed in the detailed description of the invention, it cannot be said that it is clear in light of the common general technical knowledge that an Enterobacter microorganism belonging to a different genus from Escherichia coli can be used as a host microorganism for producing citramalic acid in an equivalent manner to Escherichia coli.
Genentech Inc. v. Novo Nordisk A/S (CAFC) 42 USPQ2d 1001 clearly states: “Patent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable. See Brenner v. Manson, 383 U.S. 519, 536, 148 USPQ 689, 696 (1966) (stating, in context of the utility requirement, that "a patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.") Tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.”
Claim Rejections - 35 USC § 112- Written Description
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 1-7, 10-13, 16, 17, 19, 21, 27, 29 and 31, 33 and 36 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.
The claims broadly recite for example:
A genetically modified microorganism (any microorganism in claim 19; E. coli in claim 1) comprising: a production pathway comprising citramalate synthase for the production of citramalate,
a conditional induction of SoxS: a conditionally triggered synthetic metabolic valve that silences gene expression of the citrate synthase (gltA), or glucose-6-phosphate-dehydrogenase (zwf) gene(s);
or a conditionally triggered synthetic metabolic valve that enables selective proteolysis of the citrate synthase (gltA) or glucose-6-phosphate-dehydrogenase (zwf) enzyme(s);
wherein the synthetic metabolic valve(s) of the microorganism are conditionally triggered during a stationary phase or non-dividing cellular state;
wherein, under conditions of depleting of a limiting nutrient from a growth medium in which the genetically modified microorganism is growing, a stationary phase or non-dividing cellular state is induced;
wherein pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme activity is increased within the genetically modified microorganism under aerobic or partially aerobic conditions during the stationary phase or non-dividing cellular state to produce an acetyl CoA pool; and
wherein sugar uptake is enhanced within the genetically modified microorganism, when compared to a non-genetically modified microorganism.
The specification recites that the inventors created synthetic metabolic valves, to dynamically reduce levels of the first committed step in each pathway, namely citrate synthase (GItA, “G”, encoded by the gltA gene) and glucose 6-phosphate dehydrogenase (Zwf, “Z”, encoded by the zwf gene). They show that dynamic control over these two enzymes improves stationary phase production of pyruvate, and citramalate, and have applicability in the production of numerous products requiring pyruvate and/or acetyl-CoA. The native E. coli Type I-E Cascade/CRISPR system is
used for gene silencing (See FIG3Ci-iii). Targeted proteolysis is implemented by linking the expression of the chaperone SspB to phosphate deprivation. SspB, when induced, binds to C- terminal DAS+4 peptide tags on any target protein and causes degradation by the ClpXP protease of FE. coli (FIG 3D). Using engineered strains, as Figure 1E demonstrates, protein levels can be controlled in a two-stage process, as exemplified by turning “ON” GFP and “OFF” constitutively expressed mCherry. While, in this case, the combination of gene silencing with proteolysis results in the largest rates of protein degradation (FIG 3F-G), the impact of each approach and specific decay rates, will vary depending on the target gene/enzyme and its specific natural turnover rates and expression levels.
Claims 1 and 19 require that PFO activity is increased during the stationary phase, and sugar uptake is increased. The specification shows that these properties are conferred by cell-cycle specific downregulation of gltA and zwf respectively. However, claim 7 only requires that any one of gltA, zwf and SoxS are selectively downregulated. To the extent that gltA and zwf do not both have to be downregulated, the claims are therefore inconsistent with the description. Furthermore, the description indicates that SoxS needs to be upregulated (example 6) in order to enhance ACoA flux. Therefore, the feature that SoxS is downregulated appears to be inconsistent with the description. It would require one skilled in the art undue experimentation to carry out across their full scope.
Claims 12 and 33 relate to production of any protein product. At the same time, it is the microorganism of claim 1 that produces the product at a rate of 30 g/L or greater. However, the examples of implementation of the invention disclose production of pyruvate with a yield of more than 30 g/L and production of citramalate with a yield of more than 100 g/L (see Example 3). This demonstrates achievement of the technical result of the invention and does not disclose production of any protein products. The claims should be limited to the production of pyruvate and citramalate, respectively.
The claims fail to indicate the inventive concept, for example, on the basis of Example 3, wherein it is indicated that they implement CRISPRi/Cascade based gene silencing or implement a targeted proteolysis by linking the expression of the chaperone SspB to phosphate deprivation..
Additionally, the instant claims may be interpreted as relating to an E. coli or any modified microorganism comprising:’
A, B and D; or
A, C, and D,
Wherein A is a conditional induction of SoxS, B is a production pathway comprising citramalate synthase for the production of citramalate, C is a conditionally triggered synthetic metabolic valve that silences gene expression of gltA and/or zwf genes, and D is a conditionally triggered synthetic metabolic valve that enables selective proteolysis of gltA and/or zwf enzymes.
Based on Fig. 5A, the applicant’s intention appears to be the former, and thus, a suitable rectification to overcome the clarity objection would he to add in the conjunction “and”. The instant specification presents findings that:
synthetic metabolic “G” valve that dynamically reduces level of citrate synthase encoded by the gltA gene) would enhance sugar uptake (Example 3);
a synthetic metabolic “Z" valve that dynamically reduces level of glucose-6- phosphate dehydrogenase (encoded by the zwf gene would increase pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme (Pfo activity (Example 3); and
induction/activation of soxS Improves acetyl-CoA flux;, via Pfo activity (Example 8).
It is noted claim 1 does not actually specify how/when SoxS would be Induced (cf. the synthetic metabolic valves are conditionally triggered under aerobic or partially aerobic conditions during the stationary phase or non-dividing cellular state).
Thus, pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme activity and sugar uptake to be increased within the genetically modified microorganism, as defined in/required by the characterizing portion of claim 1, both the “G" and “Z" valves must have been essential. However, since claim 1 currently encompasses conditions where only the “G" or the “Z” valve is present, the claim lacks enablement. Accordingly, the instant claims are not enabled by the instant specification.
Further, claim 19 allows for the use of any genetically modified microorganism, which includes microorganisms belonging to any genus or species. in this claim, the "product" includes any chemical substance. In contrast, the examples in the detailed description of the invention merely state that a genetically modified microorganism for producing citramalic acid was produced using Escherichia coli as a host microorganism. Qn the other hand, in general, the endogenous metabolic enzymes of a microorganism vary depending on the genus and species to which the microorganism belongs, and the raw materials consumed and their metabolic products also differ greatly. Considering this, it is not the case that a microorganism belonging to any genus or species has a “production pathway including at least one enzyme for producing a product from an acety!-CoA precursor" for a desired “product” as defined in claim 19. Even if a microorganism has “one enzyme” involved in the “production pathway,” the “production pathway” of the desired “product is usually composed of multiple metabolic enzymes. in this case, in order to work the invention of claim 19, itis necessary to examine whether or not a group. of microorganisms belonging to any genus or species has a "production. pathway including at least one enzyme for producing a product from an acetyl-CoA precursor" for a desired "product," and if not to identify the missing metabolic enzymes and then introduce them into the microorganism, which requires trial and error beyond the extent that a person skilled in the art can expect. In addition, although claim 21 and 28 specify that “the genetically modified microorganism is an Enterobacter microorganism,” the instant specification does not disclose any working examples using “Enterobacter microorganisms" or anything equivalent thereto. Furthermore, even if not even one working example is disclosed in the detailed description of the invention, it cannot be said that it is clear in light of the common general technical knowledge that an Enterobacter microorganism belonging to a different genus from Escherichia coli can be used as a host microorganism for producing citramalic acid in an equivalent manner to Escherichia coli.
To fulfill the written description requirements set forth under 35 USC § 112, first paragraph, the specification must describe at least a substantial number of the members of the claimed genus, or alternatively describe a representative member of the claimed genus, which shares a particularly defining feature common to at least a substantial number of the members of the claimed genus, which would enable the skilled artisan to immediately recognize and distinguish its members from others, so as to reasonably convey to the skilled artisan that Applicant has possession the claimed invention. Applicants have not described the genus of claimed nucleotides such that the specification might reasonably convey to the skilled artisan that Applicants had possession of the claimed invention at the time the application was filed.
The purpose of the "written description" requirement is broader than tomerely explain how to "make and use"; the applicant must convey with reasonableclarity to those skilled in the art that, as of the filing date sought, he or she was inpossession of the invention. The invention is, for purposes of the "writtendescription" inquiry, whatever is now claimed. See Vas-Cath, Inc. v. Mahurkar,935 F.2d 1555, 1563-64, 19 USPQ2d 1111, 1117 (Federal Circuit, 1991).Furthermore, the written description provision of 35 USC § 112 is severable fromits enablement provision; and adequate written description requires more than amere statement that it is part of the invention and reference to a potential methodfor isolating it. The nucleic acid itself is required. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. V. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016. The Guidelines for Examination of Patent Applications Under the 35 U.S.C. 112, paragraph 1, "'Written Description" Requirement (66 FR 1099-1111, January 5,2001) state, "[p]ossession may be shown in a variety of ways including description of an actualreduction to practice, or by showing the invention was 'ready for patenting' such asby disclosure of drawings or structural chemical formulas that show that the invention was complete, or by describing distinguishing identifying characteristics sufficient to show that the applicant was in possession of the claimed invention" (Id. at 1104). Moreover, because the claims encompass a genus of variant species, an adequate written description of the claimed invention must include sufficient description of at least a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics sufficient to show that Applicant was in possession of the claimed genus. However, factual evidence of an actual reduction to practice has not been disclosed by Applicant in the specification; nor has Applicant shown the invention was "ready for patenting" by disclosure of drawings or structural chemical formulas that show that the invention was complete; nor has Applicant described distinguishing identifying characteristics sufficient to show that Applicant were in possession of the claimed invention at the time the application was filed. The Guidelines further state, "[f]or inventions in an unpredictable art, adequatewritten description of a genus which embraces widely variant species cannot beachieved by disclosing only one species within the genus'" (Id. at 1106);accordingly, it follows that an adequate written description of a genus cannot beachieved in the absence of a disclosure of at least one species within the genus.
The scope of the claim includes numerous structural variants, and the genus is highly variant because a significant number of structural differences between genus members is permitted. The specification does not describe any members of the claimed genus by complete structure. One of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the genus, and thus, that the applicant was not in possession of the claimed genus. The claimed subject matter is not supported by an adequate written description because a representative number of species has not been described.
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.
Claim(s) 1-7, 10-13, 16, 17, 19, 21, 27, 29, 31, 33 and 36 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al (US2019/093135, published on March 28, 2019) and Pharkya et al (CN107208118A, published on September 26, 2017; published as WO2016044713A1; provided by Applicants) and Lynch et al (CN116536960A, published on December 30, 2019, also published as WO2018156646A1; provided by Applicants).
Wu discloses a genetically modified E. coli microorganism producing citramalate and comprising citramalate synthase (including cimA3.7), a conditionally expressed citrate synthase and/or glucose-6-phosphate-dehydrogenase with a reduced activity, poxB with a reduced activity, at the same time, it discloses a possibility to produce citramalate no less than 35 g/L in 132 hours based on batch culture conditions; see [0003], [0005]-[0006], [0008], [0020], [0056], [0107], claims 1, 7, 12, 22, 23, 33, example 2). Instant Claims 1 and 19 are drawn to a genetically modified E. coli microorganism (and any microorganism) which discloses a metabolically engineered Escherichia coli expressing the cimA gene coding citramalate synthase. Knockouts in the gltA, leuC and ackA genes coding citrate synthase, 3-isopropylmalate dehydratase, and acetate kinase, respectively, were helpful in achieving high citramalate yield. In addition, the two precursors for citramalate, pyruvate and acetyl-CoA, are generated through the glycolytic pathway, and increasing the flux through glycolysis might improve citramalate productivity and yield. Glycolytic flux may also be improved by a deletion in glucose 6-phosphate dehydrogenase (zwf) (see paragraphs 187 and 190 of the description of Wu). It can be seen that Wu has disclosed that knockouts in the gltA and zwf can improve the yield of citramalate.
However, instant claims 1 and 19 differ from Wu in that they further recite that the genetically modified microorganism comprises a conditional induction of SoxS, increased pyruvate-flavodoxin/ferredoxin oxidoreductase enzyme activity, enhanced sugar uptake, as well as the growth conditions of the microorganism and conditionally triggered synthetic metabolic valve.
Accordingly, the instant claims provide another genetically modified Escherichia coli that promotes the production of citramalate. Regarding the aforementioned distinguishing technical features, Wu also discloses that the glycolysis pathway can increase the production of pyruvate and acetyl-CoA, improving the production of citramalate. Therefore, in order to provide genetically modified microorganisms that promote the production of citramalate, those of ordinary skill in the art would have the motivation to increase the sugar uptake of microorganisms to promote the glycolysis pathway, thus promoting the production of citramalate.
Moreover, Pharkya et al discloses that pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the reversible oxidation. Of pyruvate to form acetyl-CoA, while the reduced ferredoxin produced by PFOR generates NADH or NADPH. Ydbk is a ferredoxin oxidoreductase in Escherichia coli (see paragraph 424 of Wu and paragraph bridging page 136 and page 137), that is, Wu provides technical inspiration that ydbk can promote the production of acetyl-CoA. By promoting the production of citramalate precursor, those of ordinary skill in the art would reasonably expect that more citramalate can be produced under the action of citramalate synthase.
Pharkya also discloses a recombinant microorganism using one or more synthetic metabolic valves (using the CASCADE System and controlled proteolysis), thereby enabling the dynamic control over metabolic pathways. Other embodiments of the invention are directed to multi-stage bioprocesses that utilize genetically modified microorganism that in turn utilize one or more synthetic metabolic valves that enable dynamic flux control. Still in other embodiments of the invention, the transitions between stages in multistage bioprocesses using genetically modified microorganisms are controlled by the addition of chemical inducers or by the control of key nutrient levels (see [0006]-[0007], [0012]). It is also disclosed that to function more efficiently, a microorganism may comprise one or more gene deletions. For example, in E. coli, the genes encoding the lactate dehydrogenase (IdhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate- formate lyase (pflB), methyiglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), the clpXP protease specificity enhancing factor (sspB), the ATP- dependent Lon protease (lon), the outer membrane protease (ompT), the arcA transcriptional dual regulator (arcA), and the iclR transcriptional regulator (icIR) may be disrupted, including deleted (see [0085]). It is also disclosed that a microorganism may comprise one or more synthetic metabolic valves, composed of enzymes targeted for controlled proteolysis, expression silencing or a combination of both controlled proteolysis and expression silencing. For example, one enzyme encoded by one gene or a combination of numerous enzymes encoded by numerous genes in E. co/f may be designed as synthetic metabolic valves to alter metabolism and improve product formation. Representative genes in E. coli may include but are not limited to the following: fabl, zwf, gltA, ppc, udhA, Ipd, sucD, aceA, pBiA, lon, rpoS, tktA or tktB (See [0086], examples). Therefore, use of the synthetic metabolic valves in microorganisms under certain conditions is known to produce various products, and in this regard, production of such a microorganism is evident. Example 4, discloses construction of E. coli strains with low phosphate induction of the ssB chaperone. These strains all lack pflB and poxB (paragraph 114). GltA and zwf were also marked for controlled proteolysis using DAS4 tags (paragraph 116) Specifically, strains DLF_0167, DLF_0048, DLF_0044, DLF_0288 and DLF_0290 ail fall within the scope of claim 19. Claim 4 differs from these strains in that it requires a citramalate synthase pathway. See [0006]-[0007], [0012], [0085)- [0086], examples).
However, Wu discloses using these metabolically controlled strains in a variety of synthesis pathways; citramalate synthesis was known and therefore appears to be a mere design choice. The features of the various dependent claims appear to be either found in the disclosure of Wu (e.g. synthetic metabolic valves using CASCADE) or inherent functions of the strains disclosed in Wu (e.g. induction of PFO downstream from zwf inhibition) or routine alternatives thereof (e.g. choice of microorganism).
In addition, Lynch et al discloses microorganism genetically modified to comprise: a production pathway comprising at least one enzyme for the biosynthesis of a product, and a combination of multiple synthetic metabolic valves. The synthetic metabolic valves comprise one or more or a combination of the following: controlled gene silencing and controlled proteolysis. The development of platform microbial strains that utilize synthetic metabolic valves can decouple growth from product formation. These strains enable the dynamic control of metabolic pathways, including those that when altered have negative effects on microorganism growth. Dynamic control over metabolism is accomplished via a combination of methodologies including but not limited to transcriptional silencing and controlled enzyme proteolysis. These microbial strains are utilized in a multi-stage bioprocess encompassing at least two stages. In the first stage in which microorganisms are grown and metabolism can be optimized for microbial growth and at least one other stage in which growth can be slowed or stopped, and dynamic changes can be made to metabolism to improve production of desired product, such as a chemical or fuel. The transition of growing cultures between stages and the manipulation of metabolic fluxes can be controlled by artificial chemical inducers or preferably by controlling the level of key limiting nutrients. One or more of the following enables these synthetic valves: 1) transcriptional gene silencing or repression technologies in combination with 2) inducible enzyme degradation and 3) nutrient limitation to induce a stationary or non-dividing cellular state. SMVs are generalizable to any pathway and microbial host. These synthetic metabolic valves allow for novel rapid metabolic engineering strategies, wherein a rate of the biosynthesis of the product is increased in a productive stationary phase upon a depletion of a nutrient, wherein the depletion of the nutrient induces the multiple synthetic metabolic valves (see paragraphs 80-82 and 91, and paragraphs 0080-0082 and 0091 of Lynch WO2018156646A1). From the above, it can be seen that Lynch discloses synthetic metabolic valves for silencing or proteolysis may be used, and the synthetic metabolic valve is induced by depletion of a nutrient to keep cells in a stationary or dividing state.
Therefore, in order to provide another genetically modified Escherichia coli that promotes the production of citramalate, and on the basis that Wu discloses knockouts in the gltA and zwf can improve the yield of citramalate, those of ordinary skill in the art would have the motivation to use the synthetic metabolic valve in Lynch to silence or hydrolyze gltA and zwf proteins, and conditionally trigger the synthetic metabolic valve. Moreover, SoxS induction can induce the expression of genes such as ferredoxin oxidoreductase. Based on Wu and Pharkya, those skilled in the art would have the motivation to induce SoxS to promote the expression of ferredoxin oxidoreductase. It would have been prima facie obvious to those of ordinary skill in the art to produce the claimed modified E. coli on the basis of the teachings of in combination with Pharkya and Lynch and commonly used technical means in the art.
It is also disclosed that the pta, ackA, and poxB coding regions, which code respectively for phosphotransacetylase, acetate kinase, and pyruvate oxidase, respectively, mediate the conversion of acetyl CoA or pyruvate to acetate. Mutations of pta, ackA, and poxB coding regions resulted in increased amounts of citramalate (see paragraph 64 of the description of Wu). Therefore, in order to promote the level of citramalate, those skilled in the art would have the motivation to delete the poxB gene in the microorganisms as recited in instant claim 2. Moreover, those skilled in the art know that the pyruvate formate lyase plb can promote the production of acetyl CoA from pyruvate, and the deletion of pflb can also promote the production of citramalate precursor.
Claims 3 and 4 disclose overexpression of a gene encoding pyruvate-flavodoxin/ferredoxin oxidoreductase, and more specifically the ybdk gene. Both Wu and Pharkya teach that ydbk can promote the generation of acetyl CoA. By promoting the production of citramalate precursor, it is foreseeable for those skilled in the art to produce more citramalate under the action of citramalate synthase. Moreover, Lynch has already disclosed a method for decoupling strain growth from product formation to improve product yield. Therefore, those of ordinary skill in the art would have the motivation to induce gene expression and promote product formation during the stationary phase of cells.
Claims 5, 6, 10 and 11 are made obvious as Pharkya provides technical inspiration that ydbk can promote the production of acetyl CoA. By promoting the production of citramalate precursor, more citramalate can be produced under the action of citramalate synthase, which can be expected by those of ordinary in the art. However, how to increase the activity of ydbk belongs to a mechanism definition and will not affect the structure/composition of the genetically modified E. coli microorganism. Therefore, how to increase the activity of ydbk does not have a substantial defining effect. Wu also discloses that the glycolysis pathway can increase the production of pyruvate and acetyl CoA, improving the production of citramalate. Therefore, in order to provide a genetically modified microorganism that promotes the production of citramalate, those of ordinary skill in the art would have the motivation to increase the sugar uptake of the microorganism to promote the glycolysis pathway, so as to promote the production of citramalate. Moreover, increasing the activity of sugar transporters to increase sugar uptake Is a common technical means in the art, and PTS is also a common sugar transporter. The use of CRISPR interference to achieve gene silencing, including silencing multiple target genes by encoding small guide RNAs in the microorganism, as well as different numbers of promoters, is also a routine operation in the art. Claim 12 requests protection for a bioprocess for production of a protein product from the genetically modified microorganism of clam 1. On the basis that the genetically modified microorganism is obvious, Lynch discloses synthetic metabolic valves for silencing or proteolysis may be used, and the synthetic metabolic valve is induced by depletion of a nutrient to keep cells in a stationary or dividing state. On the basis of Wu, those of ordinary skill in the art would have the motivation to use the synthetic metabolic valve in Lynch to silence or hydrolyze gltA and zwf proteins, and conditionally trigger the synthetic metabolic. valve. As for the production rate of the product, those of ordinary skill in the art can make routine optimization and adjustments as needed.
Regarding claims 13, 16 and 17, Wu has already disclosed a metabolically engineered Escherichia coli expressing the cimA gene coding citramalate synthase for producing citramalate and knockouts in the gltA and zwf can improve the yield of citramalate. Moreover, Pharkya teaches that ydbk can promote the production of acetyl CoA. By promoting the production of citramalate precursor and under the action of citramalate synthase, of ordinary skill in the art could optimize its