DETAILED ACTION
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 .
Claim Objections
Claim 22 is objected to because of the following informalities:
Claim 22, line 4, recites “one or more biosynthetic pathway genes” and in line 12 recites “the biosynthetic pathway genes.” Consistent claim terminology should be used throughout the claim such chat “one or more biosynthetic pathway genes” that includes a singular gene and “the biosynthetic pathway genes” that is limited to plural genes should be recited consistently in the claim.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claim 22 is 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.
“New or amended claims which introduce elements or limitations that are not supported by the as-filed disclosure violate the written description requirement. See, e.g., In re Lukach, 442 F.2d 967, 169 USPQ 795 (CCPA 1971) (subgenus range was not supported by generic disclosure and specific example within the subgenus range); In re Smith, 458 F.2d 1389, 1395, 173 USPQ 679, 683 (CCPA 1972) (an adequate description of a genus may not support claims to a subgenus or species within the genus)”; MPEP 2163(I)(B). “While there is no in haec verba requirement, newly added claims or claim limitations must be supported in the specification through express, implicit, or inherent disclosure.” MPEP 2163(I)(B).
“With respect to newly added or amended claims, applicant should show support in the original disclosure for the new or amended claims. See, e.g., Hyatt v. Dudas, 492 F.3d 1365, 1370, n.4 (Fed. Cir. 2007) (citing MPEP § 2163.04 which provides that a "simple statement such as ‘applicant has not pointed out where the new (or amended) claim is supported, nor does there appear to be a written description of the claim limitation ‘___’ in the application as filed’ may be sufficient where the claim is a new or amended claim, the support for the limitation is not apparent, and applicant has not pointed out where the limitation is supported."); see also MPEP §§ 714.02 and 2163.06 ("Applicant should ... specifically point out the support for any amendments made to the disclosure."); and MPEP § 2163.04.” MPEP 2163(II)(A).
New claim 22 was presented in an amendment dated 07/30/2026. Applicant has not pointed where the limitations of new claim 22 is supported in the as-filed specification other than a generic statement that “New claims 21-22 are presented, and fully supported by the Specification as originally filed including the original claims,” which is not sufficient to point out where the limitations of claim 22 are supported. Further, where all of the limitations of claim 22 is supported in not apparent.
The specification discloses:
[13] In any of the embodiments disclosed herein, the method can further comprise forming the unpurified mixture of biosynthetic pathway enzymes by a process that involves forming a mixture comprising a cell lysate, one or more biosynthetic pathway genes, one or more cofactors, and one or more energy molecules, and agitating the mixture to allow cell-free expression of the biosynthetic pathway genes to produce the unpurified mixture of biosynthetic pathway enzymes.
[17] In any of the embodiments disclosed herein, the reaction mixture can further comprise NH3 and bicarbonate, and the method can further comprise reacting at least the biocatalyst with the NH3, the bicarbonate, and the first reaction product to produce a second reaction product.
The mixture of paragraph [13] of the specification does not describe presence of bicarbonate and NH3 as required by claim 22. Paragraph [17] of the specification describes a reaction mixture for forming a product that requires bicarbonate and NH3, but does not require a cell lysate nor biosynthetic pathway genes. While the unpurified mixture of biosynthetic pathway enzymes as in paragraph [13] may be added without purification to produce a reaction mixture as in paragraph [17], or vice versa, the resulting reaction mixture is not agitated to allow for cell-free expression of the biosynthetic pathway genes. Rather, any cell-free expression including agitation is done prior to addition of bicarbonate and NH3 to form a reaction mixture as set forth in para. [17].
For example, the specification emphasizes dilution of the biocatalyst after cell-free expression occurs prior to forming any reaction mixture. Example 3, para. [84], of the specification provides: “In Step 2, the CFE-based biocatalyst is diluted with water or inexpensive buffer to volumetrically expand the reaction. To initiate the chemical synthesis (Step 3), the substrates [e.g. bicarbonate and HN3] and cofactors are added to the reaction.” While such a “Step 2” forms a mixture containing cell free expression components (i.e. cell lysate, one or more biosynthetic pathway genes) and substrates and cofactors for product formation (i.e. NH3, bicarbonate), such a mixture in “Step 2” is not agitated to allow cell-free expression of the biosynthetic pathway where any such cell-free expression occurs prior to “Step 2” in “Step 1” wherein “the transcription-translation conditions optimal in the CFE . . . are maintained” (i.e. prior to any dilution or addition of bicarbonate and NH3).
Claim 22 specifically requires that a mixture comprising all of a cell lysate, NH3, bicarbonate, one or more biosynthetic pathway enzymes and a cofactor or energy molecule that is suitable to be agitated to produce an unpurified mixture of biosynthetic pathway enzymes. For the reasons set forth above it is not apparent wherein such a mixture is supported by the as filed specification.
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.
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.
Claim(s) 1, 3, 4, 6, 7, 9-11, 13-16, 19 and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (Enzymatic Electrosynthesis of Glycine from CO2 and NH3, Angewandte Chemie Int. Ed. 62, Feb. 2023, e202218387) (published 02/23/2023) further in view of Lim et al. (Cell-free synthesis of industrial chemicals and biofuels from carbon feedstocks, Current Opin. Biotechnol. 73, 2022, 158-63), Karim et al. (In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design, Nature Chem. Biol. 16, 2020, 912-19), Uniprot, Accession No. P27248, 2022, www.uniprot.org, and Uniprot, Accession No. C7C8C7, 2022, www.uniprot.org.
Wu, abstract, states:
Enzymatic electrosynthesis has gained more and more interest as an emerging green synthesis platform, particularly for the fixation of CO2. However, the simultaneous utilization of CO2 and a nitrogenous molecule for the enzymatic electrosynthesis of value-added products has never been reported. In this study, we constructed an in vitro multienzymatic cascade based on the reductive glycine pathway and demonstrated an enzymatic electrocatalytic system that allowed the simultaneous conversion of CO2 and NH3 as the sole carbon and nitrogen sources to synthesize glycine. Through effective coupling and the optimization of electrochemical cofactor regeneration and the multienzymatic cascade reaction, 0.81 mM glycine was yielded with a highest reaction rate of 8.69 mg L−1 h−1 and faradaic efficiency of 96.8 %. These results imply a promising alternative for enzymatic CO2 electroreduction and expand its products to nitrogenous chemicals.
“To achieve the simultaneous utilization of CO2 and NH3 as the sole carbon and nitrogen sources, an in vitro multienzymatic-cascade-based enzymatic electrocatalytic system was designed, consisting of four modules (Figure 1A): i) CO2 is reduced to formic acid under catalysis by a formate dehydrogenase (FDH); ii) formic acid is converted into 5,10-methylene-THF sequentially though catalysis by a formate-tetrahydrofolate ligase (FtfL), a methenyltetrahydrofolate cyclohydrolase (FchA) and a methylenetetrahydrofolate dehydrogenase (MtdA); iii) the endogenous enzymes of the GCS (GcvT, GcvH GcvP and GcvL) condense 5,10-methylene-THF with another CO2 and NH3 molecule to produce glycine; and iv) electrochemical cofactor regeneration allows the efficient reduction of NADH from NAD+. Additionally, a polyphosphate kinase (PPK) is added to enable the regeneration of ATP from ADP at the expense of polyphosphate. As a result, all cofactors, including the reducing power, ATP, and THF, are well-balanced. The overall reaction can be written as:” Wu, page 2, right col.
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Experimental procedures on pages 3-4 of supplemental material of Wu provides:
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The above is a description of a method of converting formate to a desired compound being glycine (second product) by first producing a first product being 5,10-methylenetetrahydrofolate (5,10-methylene-THF) by providing a reaction mixture being a biocatalyst comprising the various purified enzymes described by Wu and formate and reacting the biocatalyst with formate to produce 5,10-methylenetetrahydrofolate (first product) and glycine (second product). The reaction mixture further contains 5 mM ATP as an energy molecule, 50 mM NH4Cl that is the source of NH3 in the equation cited from Wu, page 2, right col., and 50 mM NaHCO3 bicarbonate. “Reaction conditions were subsequently optimized to further improve glycine synthesis. To simplify the operation, 10 mM formate was used for the optimization of modules ii and iii. First, different concentrations of HCO3- were evaluated in the presence of 50 mM NH4Cl. At 10 mM NaHCO3, the production of glycine was only 0.30 mM (Figure 3A). With NaHCO3 increased to 50 mM, the amount of glycine rose to 0.61 mM.” Wu, page 3, right col. That is, the biocatalyst is reacted with NH3, bicarbonate and 5,10-methylenetetrahydrofolate (first product) to produce the second product glycine, as shown in Fig. 1 of Wu.
The preceding meets the features of at least claims 1, 6, 7, 10, and 11 except for the biocatalyst being an unpurified mixture of biosynthetic pathway enzymes.
As discussed above, Wu teaches purification of individual enzymes and their combination for forming a biocatalyst for production of glycine.
Lim, abstract, teaches:
“The power of biological systems can be harnessed with higher efficiency when biosynthetic reactions are decoupled from cellular physiology. This can be achieved by cell-free synthesis, which relies on the in vitro use of cellular machinery under optimized reaction conditions. As exemplified by the recent development of mRNA vaccines and therapeutics, the cell-free synthesis of biomolecules is fast, efficient and flexible. Cell-free synthesis of industrial chemicals and biofuels is drawing considerable attention as a promising alternative to microbial fermentation processes, which currently show low conversion yields and toxicity to host cells. Here, we provide a brief overview of the history of cell-free synthesis systems and the state-of-the-art cell-free technologies used to produce diverse chemicals and biofuels. We also discuss the future directions of cell-free synthesis that can fully harness the synthetic power of biological systems.”
“Instead of relying on the use of purified enzymes, cell free metabolic pathways can also be operated using the crude cell lysate. Given the endogenous cofactor regeneration in cells, the cell lysate-based systems can be used to regulate cofactor balance in artificial pathways without implementing separate regulation modules.” Lim, page 160, left col. “The speed of pathway development can be accelerated by modular assembly of cell lysates that contain pathway-specific enzymes produced via cell-free protein synthesis. Recently, Karim et al. used this strategy successfully to optimize 3-hydroxybutyrate and n-butanol biosynthetic pathways.” Lim, pages 161-62.
“Cell-free biosynthesis is a promising approach for production of industrial chemicals and biofuels. Because the parameters of pathway design are not constrained by the need to maintain cell viability, cell-free synthesis systems can accommodate artificial pathways designed entirely for simplified and resource-effective production of the desired chemical products. While construction of cellfree biosynthesis systems involves additional enzyme-and cofactor-related costs, this limitation can be
addressed by using crude lysates instead of purified enzymes.” Lim, page 162, left col.
Karim, as cited by Lim, in Fig. 1 shows cell-free enzyme/protein synthesis of various enzymes for production of 3-hydroxybutyrate and n-butanol biosynthetic pathways. Karim, Methods provides:
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CFPS is cell-free protein synthesis, wherein as illustrated in Fig. 1 of Karim, CFPS is performed in a cell lysate: “Lysates are enriched with pathway enzymes via overexpression before lysis or by CFPS
post-lysis and mixed to assemble enzymatic pathway combinations.” The description that “CFPS reactions were mixed” is within the broadest meaning of “agitating” as recited. Regardless, upon combining materials to form a CFPS reaction, an ordinarily skilled artisan would readily understand that it is appropriate to mix the components wherein any degree of mixing is within the broadest reasonable meaning of agitating.
As discussed, Wu teaches purification of individual enzymes and their combination for forming a biocatalyst for production of glycine. However, Lim teaches that “Instead of relying on the use of purified enzymes, cell free metabolic pathways can also be operated using the crude cell lysate. “Given the endogenous cofactor regeneration in cells, the cell lysate-based systems can be used to regulate cofactor balance in artificial pathways without implementing separate regulation modules” and “While construction of cellfree biosynthesis systems involves additional enzyme-and cofactor-related costs, this limitation can be addressed by using crude lysates instead of purified enzymes.” Lim, page 160, left col, and page 162, left col.
Wu, as discussed, teaches a cell-free system of purified enzymes for production of glycine, as discussed, that also requires addition of cofactors such as ATP, NADH, NADPH, PLP, etc., as discussed. Lim directly teaches that the cost of additional cofactors can be mitigated by use of a crude lysate rather than purified enzymes. It is noted that the enzymes/biocatalyst of Wu is produced recombinantly in E. coli, followed by lysis of E. coli and purification of enzymes by Ni-NTA resin. However, in view of the teachings of Lim, an ordinarily skilled artisan would have been motivated to employ the enzymes/biocatalysts of Wu as a crude lysate, rather than purified, to achieve at least the advantages of cost savings as taught by Lim. Implementation of the same meets the features of claim 1 (previous claim 2) reciting a biocatalyst being an unpurified mixture of biosynthetic pathway enzymes.
Regarding claims 9 and 21, the Experimental Methods of Wu describe placing the various enzymes described in a reaction mixture of 1 mL (or 10 mL) that is within the broadest reasonable meaning of the biocatalyst (i.e. enzymes) being in “diluted form” as far as the purified enzymes are more concentrated prior to addition to the reaction mixture and become diluted in the reaction mixture. That is:
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As discussed, Wu teaches that purified enzymes, such as MeFtfL, are prepared with “The protein concentration was determined by Bradford method” (i.e. undiluted biocatalyst). Wu, Experimental Procedures, page 3. Due to the number of components combined to form the reaction for glycine synthesis from CO2, it is clear that each component including enzymes (e.g. MeFtfL) undergoes substantial dilution upon addition to the reaction. “Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. "[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." MPEP 2144.05(II)(A). In view of Wu teaching the general condition that a more concentrated enzyme/biocatalyst is diluted into a reaction mixture, it is not inventive to discover that a biocatalyst enzyme from a first undiluted solution can workably be diluted at least 10-fold prior to reaction with formate. Fig. 1 of Wu shows that the FtfL enzyme utilizes formate and THF substrates to produce 10-formyl-THF. Similarly, regarding claim 21, “volumetric expansion of up to 200-fold” is an upper limit such for example, a volumetric expansion of 10-fold, 20-fold, etc., meets the features of claim 21. As just discussed, it is not inventive to discover that a biocatalyst enzyme from a first undiluted solution can workably be diluted (volumetric expansion) of 200-fold or less as a workable range, wherein addition of enzyme/biocatalyst to the reaction mixture containing 100 mM phosphate buffer is dilution with a buffer.
Regarding claim 19, Wu describes formate being produced by reduction of CO2: “To confirm the
proposed enzymatic electrocatalytic system, we first verified module i for the transformation (i.e. reduction) of CO2 into formate.” Wu, page 2, right col; see also, Wu, abstract.
Regarding claim 3, Lim further teaches “The speed of pathway development can be accelerated by modular assembly of cell lysates that contain pathway-specific enzymes produced via cell-free protein synthesis.” As such, when cell lysates are employed to form a biocatalyst (i.e. synthesis pathway), Lim teaches that it is a known technical solution to express needed enzymes forming such biocatalyst or synthesis pathway by cell-free protein synthesis. Karim, which is cited for additional experimental detail, teaches that at least one DNA/gene encoding an appropriate enzyme or biosynthetic pathway gene is combined with cell lysate to produce a cell-free protein synthesis reaction that is combined with other needed material inputs required. In Karim, this includes glucose, NAD, and CoA. Wu, as discussed, explains that cell-free synthesis of serine requires addition of at least energy molecule ATP and cofactors NADH, NADPH, bicarbonate and PLP. Whether the cell-free reaction system is purified enzymes or a cell-lysate containing enzymes produced by CFPS, the same energy molecules and cofactors as taught by Wu are required for any synthesis. As discussed, Karim teaches that it is appropriate to mix CFPS reactions producing different enzymes to form an overall biocatalyst or synthesis pathway wherein any mixing is within the broadest reasonable meaning of “agitating.” As such, wherein the glycine-production pathway of Wu is implemented by CFPS of appropriate enzyme/biocatalyst by CFPS as guided by Lim and Karim, a mixture comprising: a cell lysate, one or more biosynthetic pathway genes, one or more cofactors, and one or more energy molecules; and agitating the mixture to allow cell-free expression of the biosynthetic pathway genes to produce the unpurified mixture of biosynthetic pathway enzymes, as recited in claim 3. An ordinarily skilled artisan at the time of filing would have been motived to do this to realize cost savings over use of purified enzymes and to accelerate pathway development by employing cell-free protein synthesis as taught by Lim and Karim as discussed above.
Regarding claim 4, Wu, supplemental page 20,, Table S2, provides that one gene expressed for forming the enzymes/biocatalyst described therein is:
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, which translates to the following sequence that is nearly identical to SEQ ID NO: 2 other than the underlined portion:
MHHHHHHGAMGAGNETIETFLDGLASSAPTPGGGGAAAISGAMGAALVSMVCNLTIGKKKYVEVEADLKQVLEKSEGLRRTLTGMIADDVEAFDAVMGAYGLPKNTDEEKAARAAKIQEALKTATDVPLACCRVCREVIDLAEIVAEKGNLNVISDAGVAVLSAYAGLRSAALNVYVNAKGLDDRAFAEERLKELEGLLAEAGALNERIYETVKSKVN
Wu, supplemental page 3, explains that “the His-tagged enzymes were purified by Ni-NTA resin columns. An ordinarily skilled artisan would have recognized the six consecutive H residues as being the reference His-tag. At the time of filing an ordinarily skilled artisan in modifying Wu to not utilize purified enzymes would recognize that a His tag that functions to allow for purification with a Ni-NTA resin is not required and can be omitted wherein purification is omitted. Uniport C7C8C7 evidences that the corresponding wild-type methenyltetrahydrofolate cyclcohydrolase from M. extorquens has a sequence identical to SEQ ID NO: 2. As such, wherein Wu is modified to not employ purified enzymes, an ordinarily skilled artisan at the time of filing would have been motivated to express this gene in a form omitting a His-tag or with the sequence of SEQ ID NO: 2 as shown in Uniprot C7C8C7.
Claim 4 is understood as reciting a Markush group requiring only one of the recited enzymes. However, if it is argued that claim 5 requires two or more of the recited enzymes, Table S2 (page 21) of Wu provides a gene for E. coli T protein (Ectprotein) that translates to the following amino acid sequence:
MGSSHHHHHHMAQQTPLYEQHTLCGARMVDFHGWMMPLHYGSQIDEHHAVRTDAGMFDVS
HMTIVDLRGSRTREFLRYLLANDVAKLTKSGKALYSGMLNASGGVIDDLIVYYFTEDFFR
LVVNSATREKDLSWITQHAEPFGIEITVRDDLSMIAVQGPNAQAKAATLFNDAQRQAVEG
MKPFFGVQAGDLFIATTGYTGEAGYEIALPNEKAADFWRALVEAGVKPCGLGARDTLRLE
AGMNLYGQEMDETISPLAANMGWTIAWEPADRDFIGREALEVQREHGTEKLVGLVMTEKG
VLRNELPVRFTDAQGNQHEGIITSGTFSPTLGYSIALARVPEGIGETAIVQIRNREMPVK
VTKPVFVRNGKAV
Such sequence it it’s wild-type form is identical to recited SEQ ID NO: 7 except for the underlined portion. Uniprot P27248 evidences that such underlined portion is as addition to the wild-type sequence of the enzyme. As such, wherein Wu is modified to not employ purified enzymes, an ordinarily skilled artisan at the time of filing would have been motivated to express this T protein gene in a form omitting a His-tag or with the sequence of SEQ ID NO: 7 as shown in Uniprot P27248.
Regarding claims 13 and 14, as discussed, Wu teaches that ATP, NADH, NADPH and PLP is to be added to any cell-free reaction mixture for producing serine from formate which is required whether the individual enzymes are purified or not.
Regarding claim 15 and 16, Wu teaches enzyme expression in E. coli. Karim (methods, first page, left col.) also teaches that “E. coli BL21(DE3) (NEB) was used for preparation of cell extracts, which were used to express all exogenous proteins in vitro.” “For the cell-free work, the pJL1 plasmid (Addgene, 69496) was used.” Wu, methods, first page, left col. Fig. 1 of Karim also illustrates circular plasmids for in vitro enzyme synthesis. As such, the cited prior art directly suggests that any CFPS or in vitro enzyme synthesis is performed with an E. coli lysate with one or more plasmids having the biosynthetic gene(s). It is noted that claim 3 recites “one or more biosynthetic pathway genes” such that all of claims 3, 15 and 16 are understood as requiring only one such gene. Regardless, Karim teaches that it is appropriate to mix individual CFPS reactions together such that a lysate with plural such genes are produced.
Claim(s) 1, 3, 4, 6, 7, 9-11, 13-19 and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (Enzymatic Electrosynthesis of Glycine from CO2 and NH3, Angewandte Chemie Int. Ed. 62, Feb. 2023, e202218387) (published 02/23/2023), Lim et al. (Cell-free synthesis of industrial chemicals and biofuels from carbon feedstocks, Current Opin. Biotechnol. 73, 2022, 158-63), Karim et al. (In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design, Nature Chem. Biol. 16, 2020, 912-19), Uniprot, Accession No. P27248, 2022, www.uniprot.org, and Uniprot, Accession No. C7C8C7, 2022, www.uniprot.org, as applied to 1, 3, 4, 6, 7, 9-11, 13-16, 19 and 21 above, and further in view of Koglin et al. (U.S. 2019/0309311 A1).
Regarding claims 17 and 18, as discussed, Lim teaches cell free protein synthesis from an apparently circular plasmid DNA. Koglin, abstract, relates to “an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system.” Regardless of the specific improvements taught by Koglin, Koglin, para. [0004], teaches:
More specifically, cell-free expression systems generate target molecules and complexes such as RNA species and proteins without using living cells. A typical cell-free expression system may utilize the biological components/machinery found in cellular lysates to generate target molecules from DNA containing one or more target genes. Common components of a typical cell-free expression system reaction may include a cell extract generally derived from a call culture lysate, an energy source such as ATP, a supply of amino acids, cofactors such as magnesium, and the nucleic acid synthesis template with the desired genes, typically in the form of a plasmid synthesis template, or linear expression (or synthesis) template (LET or LST). A cell extract may be obtained by lysing the cell of interest and removing the cell walls, genomic DNA, and other debris through centrifugation or other precipitation methods. The remaining portions of the lysate, or cell extract may contain the necessary cell machinery needed to expresses the target molecule.
As such, Koglin teach that it is established in the prior art that either a circular plasmid or a linear DNA template can serve as the gene for expression in cell-free expression systems. As such, at the time of filing in addition to using a plasmid as taught by Lim, an ordinarily skilled artisan would have been motivated to utilize any appropriate gene structure including such gene for cell-free expression provided as a linear DNA as recited in claim 17 or both a plasmid and linear DNA as recite in claim 18. An ordinarily skilled artisan at time of filing would have been motivated to do the same since Koglin teach that either plasmids or linear DNA can be used in cell free expression systems based upon cell lysates. As far as it is understood that plural copies of the gene are provided in any cell free expression system, it is a matter of design choice to provide such gene as both plasmid and linear DNA.
Claim(s) 1, 3, 4, 6-16, and 19-21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (Enzymatic Electrosynthesis of Glycine from CO2 and NH3, Angewandte Chemie Int. Ed. 62, Feb. 2023, e202218387) (published 02/23/2023), Lim et al. (Cell-free synthesis of industrial chemicals and biofuels from carbon feedstocks, Current Opin. Biotechnol. 73, 2022, 158-63), Karim et al. (In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design, Nature Chem. Biol. 16, 2020, 912-19), Uniprot, Accession No. P27248, 2022, www.uniprot.org, and Uniprot, Accession No. C7C8C7, 2022, www.uniprot.org, as applied to 1, 3, 4, 6, 7, 9-11, 13-16, 19 and 21 above, and further in view of Kim et al. (Growth of E. coli on formate and methanol via the reductive glycine pathway, Nature Chem. Biol. 16, 2020, 538-45).
Regarding claims 8, 12 and 20, Kim teaches an E. coli host engineered to convert formate into glycine, serine and/or pyruvate via a 5,10-methjylene-THF intermediate as follows:
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With reference to Fig. 1 of Kim, Fig. 1 shows show incorporation of carbon from formate into 5,10-methylene-THF and then production of glycine by enzymes of a glycine cleavage system, which is a pathway apparently identical to how formate is converted into glycine as in Fig. 1A of Wu.
Kim, Fig. 1 (and text cited above), shows that an additional enzymatic step can be employed to glycine to serine and serine to pyruvate as follows:
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Kim does not relate directly to cell-free systems as taught in Wu. Regardless, Wu teaches cells free systems that successfully produces 5,10-methylene-THF and glycine from formate with the same reactions as shown in Fig. 1 of Kim. As far as Kim teaches that it is advantageous to further convert glycine so produced to further C3 products of serine (third product) and pyruvate (fourth product), an ordinarily skilled artisan at time of filling would have been motivated to modify the cell-free biocatalyst as taught by Wu to include the further SHMT (glyA) and serine deaminase enzymes to produce further products of serine and pyruvate that Kim teach are beneficial to produce from an initial formate carbon source through 5,10-methylene-THF and glycine intermediates. It is noted that the cell free system of Wu produces 5,10-methylene-THF that can be used by SHMT for conversion of glycine to serine. That is, providing SHMT to the biocatalyst of Wu would predictably allow for reacting the same with 5,10-methylene-THF (first product) and glycine (second product) to produce serine (third product) as recited in claims 8 and 12. Further reacting of a biocatalyst including serine deaminase with serine (third product) produce pyruvate (fourth product) meets the features of claim 20.
Response to arguments
Applicant argues:
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e. specific ten-enzyme formate to glycine/serine pathway) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Wu and not Lim is cited for teaching an enzymatic formate to glycine pathway.
Applicant argues:
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e. co-expression of any enzymes nor other details of paras. [0093]-[0097] of the specification) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Further, the abstract of Wu states that 0.81 mM glycine was produced wherein the production of any amount of glycine meets the limitation of the claims. There is no technical reason why substitution of the purified enzymes demonstrated by Wu with enzymes that are either unpurified or produced by cell-free expression would yield significantly different results.
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e. gcvH expression from linear DNA, any specific promoter, any requirement for DNA molar ratios of specific genes) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues:
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The prior art provides motivation for substitution of unpurified enzymes produced by cell-free expression. “Instead of relying on the use of purified enzymes, cell free metabolic pathways can also be operated using the crude cell lysate. Given the endogenous cofactor regeneration in cells, the cell lysate-based systems can be used to regulate cofactor balance in artificial pathways without implementing separate regulation modules.” Lim, page 160, left col. “The speed of pathway development can be accelerated by modular assembly of cell lysates that contain pathway-specific enzymes produced via cell-free protein synthesis.” Lim, pages 161-62.
Applicant argues:
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In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e. the specific structure that allows for 39% conversion) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). It is noted that an embodiment with 0.1% conversion of formate would meet the limitations of all of the rejected claims.
Applicant argues:
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There is no recitation in the claims of an “expression host.” The specific enzyme identities, energy molecules and cofactors recited in the claims are taught by Wu as stated in the body of the rejections.
Regarding arguments that Kim does not disclose a cell-free system or unpurified enzymes, Lim and Karim are relied upon for teaching such features. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Applicant argues:
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As far as Wu teaches an in vitro and cell-free system to convert formate or CO2 to glycine, an ordinarily skilled artisan would have expected success in further converting glycine to serine using enzymes known in the art to carryout such a transformation further applied in vitro.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TODD M EPSTEIN whose telephone number is (571)272-5141. The examiner can normally be reached Mon-Fri 9:00a-5:30p.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652
