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 .
Amendments Received
Amendments to the claims were received and entered on 11/19/2025.
Status of Claims
Claims 1-20 are currently pending.
Claims 1-2 and 6-20 are under consideration, as claims 3-5 are withdrawn.
Priority
Acknowledgment is made of applicant’s claim for benefit under 35 U.S.C. 119(e) of Provisional application No. 63/608,786, filed on December 11, 2023. The present application and all claims are being examined with an effective filing date of December 11, 2023. In future actions, the effective filing date may change due to amendments or further review of priority documents.
Withdrawn Objections
In view of Applicant’s amendments, objections to claims 19 and 20 are hereby withdrawn.
Claim Objections
Claim 18 is objected to because of the following informalities:
The newly amended claim currently recites “The strain of C. necator of claim 1, wherein the strain of C. necator is modified genetic modification”. Please amend the claim to recite “modified by genetic modification”.
Appropriate correction is required.
Maintained/Modified Rejections Necessitated by Amendment
Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 18 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
For purposes of examination, the claim is interpreted as requiring a strain that is modified by genetic modification. Claim 1, however, already recites a strain of C. necator comprising a heterologous transgene, which constitutes a genetic modification. Accordingly, claim 18 fails to further limit the scope of claim 1.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Response to Arguments for Rejections under 35 USC § 112(d)
In the response filed on 11/19/2025, Applicant argues that amendment of claim 18 to delete the option of adaptive laboratory evolution cures the rejection under 35 USC 112(d). This argument is not persuasive. As amended, claim 18 recites that the strain of claim1 is modified by genetic modification. However, claim1 already recites a strain comprising a heterologous transgene, necessarily constituting genetic modification. Therefore, the limitation of claim 18 merely restates subject matter already required by claim 1 and does not further limit the scope of the claim. Accordingly, the rejection is maintained.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 6-12 and 17-19 are rejected under 35 U.S.C. 103 as being unpatentable over Averesch et al. (WO2023283609, cited in a previous office action) and Neves et al. (Use of enzymes in extraction of polyhydroxyalkanoates produced by Cupriavidus necator, Biotechnol Progress, 2012, 28: 1575-1580, cited in a previous office action).
The examiner further relies on Poladyan et al. (Growth of the facultative chemolithoautotroph Ralstonia eutropha on organic waste materials: growth characteristics, redox regulation and hydrogenase activity, Microb Cell Fact (2019) 18:201, cited in PTO-892) as evidentiary support for the general knowledge that C. necator grows well at mesophilic temperatures, as discussed below with respect to claim 19.
Claim 12 is included because while the claim recites “wherein the carbohydrase is a cellulase, a β-glucanase, a xylanase, an amylase, or a galactosidase”, it does not require that the digestive enzyme be a carbohydrase. Claim 12 is still drawn to a strain of C. necator, comprising a heterologous digestive enzyme selected from a protease…”, as recited in claim 1 (upon which claim 12 depends).
Claims 1-2, 6-10 are rejected together because they share the same core inventive concept – a strain of C. necator engineered to express one or more heterologous proteases – and differ only in the number of protease-encoding transgenes or the expression of heterologous proteases.
Regarding claims 1-2, 6-10 and 12, Averesch et al. teaches microorganisms for producing polyhydroxyalkanoate (PHA) compounds, wherein in some cases the species of the microorganism is Cupriavidus necator that has been genetically modified to include PHA synthase genes (Abstract). Averesch et al. further teaches that “in certain embodiments, the phaC gene is heterologous to the microorganism…is a mutant PHA synthase from Pseudomonas sp. MBEL 6‑19” (Specification, pg. 7, para 0041). Averesch et al. discloses a ΔphaC1 mutant of C. necator complemented with a heterologous PHA synthase gene (phaC1437) from Pseudomonas sp. MBEL 6‑19, in combination with heterologous transferase genes, hadA from Clostridium difficile or pct540 from Clostridium propionicum, resulting in copolymer production with altered monomer composition (pg. 19, para 0076). Averesch et al. also teaches that cell lysis and protein extraction from C. necator biomass was carried out using proprietary lysis reagents in combination with protease inhibitor cocktails (pg. 23–24, para 0088-0089), demonstrating that proteases are recognized as potent agents for cell disruption in processing C. necator. However, Averesch et al. does not teach the heterologous expression of protease, or a gene (and a second and third gene) encoding a protease, in C. necator.
Neves et al. teaches that poly(3‑hydroxybutyrate) (P(3HB)) and its copolymer poly(3‑hydroxybutyrate‑co‑3‑hydroxyvalerate) (P(3HB‑co‑3HV)) are biodegradable thermoplastic polymers synthesized by C. necator, and evaluates the use of several proteases (including Corolase® L10, Alcalase® 2.4L, Corolase® 7089, and Protemax® FC) and glycosidases for their recovery. Neves et al. further teaches that “the results showed that the use of enzymes for the polymer extraction is an efficient process that assists in the cell disruption of Cupriavidus necator” (Abstract). It would be obvious to a person of ordinary skill in the art that internal expression of such proteases in C. necator could obviate the need for adding proteases externally, streamlining downstream processing.
An invention would have been obvious to a person of ordinary skill in the art if some teaching in the prior art would have led that person to combine prior‑art reference teachings to arrive at the claimed invention. Before the effective filing date of the claimed invention, Averesch et al. demonstrates that C. necator can be genetically engineered to express diverse heterologous genes using established recombinant DNA techniques. Averesch et al. also evidences an awareness of the role and potency of proteases in cell lysis by describing the use of protease inhibitors during extraction, and describes conventional chemical lysis for PHA extraction, which would prompt a person of ordinary skill in the art to consider the direct use of proteases, including engineering the organism to produce them. Neves et al. teaches that proteases are useful for enzymatic digestion and cell lysis to aid in product recovery from C. necator biomass. Given the well‑known benefits of using multiple proteases to increase degradation rate, broaden substrate specificity, or improve efficiency in biomass processing, it would have been obvious to a person of ordinary skill in the art to incorporate multiple copies (three or more) of protease‑encoding transgenes into the engineered C. necator strain of Averesch et al., to internalize enzymatic cell lysis capability and, to add the additional heterologous protease‑encoding transgenes to further improve enzymatic cell disruption and downstream product extraction efficiency. A person of ordinary skill in the art would have had a reasonable expectation of success because both the genetic modification methods for C. necator and the use of multiple proteases in biomass processing were well established. Therefore, the invention as a whole would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention.
Regarding claim 11, savinase and keratinase are each well‑known commercial proteases derived from Bacillus species, widely used in industrial applications for protein degradation due to their high stability and activity. It would have been obvious to a person of ordinary skill in the art to select savinase or keratinase as the protease expressed in the recombinant C. necator of Averesch et al., in view of Neves et al.’s teaching of protease utility in C. necator processing, because these enzymes were known in the art to be suitable, effective, and commercially available for cell disruption and protein hydrolysis. Selection of one known protease for another with the same intended function would have been a routine substitution with predictable results. Therefore, it would be obvious for a person of ordinary skill in the art, to make these substitutions and expect predictable results (see MPEP 2144.06, “Substituting equivalents known for the same purpose”).
Regarding claim 17, codon optimization of heterologous genes for a specific host organism is a routine and well‑known practice in the art to improve transcriptional and translational efficiency. It would have been obvious to a person of ordinary skill in the art to codon‑optimize the heterologous protease gene for C. necator in the recombinant strains of Averesch et al., in view of Neves et al.’s teaching of protease utility in biomass processing, in order to enhance expression levels and enzymatic activity. Such optimization is a conventional design consideration that predictably improves heterologous protein production.
Regarding claim 18, given that claim 1 requires the strain to comprise a heterologous transgene encoding a digestive enzyme, the strain is inherently genetically modified. As described above, Averesch et al. discloses a Cupriavidus necator that has been genetically modified with a heterologous PHA synthase gene.
Regarding claim 19, Averesch et al. teaches precultures of the engineered Cupriavidus necator in two steps: wherein the first step comprises “starting with fructose as a carbon source”, the medium is inoculated with C. necator from solid RB + kan (heterotrophic growth) and “incubated at 30°C with shaking at 200 rpm overnight”. In the second step, those cultures were transferred to autotrophic conditions…”. C. necator was cultivated under autotrophic conditions in a bioelectrochemical system operated without a membrane, with a three‑electrode setup, stirred at 300 rpm, and continuously supplied with CO₂ gas at 1 mL/min to the reactor medium (pg. 22-23, paragraph 0086-0087). Although Averesch et al. does not expressly disclose the temperature employed during the autotrophic phase, it teaches that the heterotrophic pre-culture was performed at 30°C. It would have been obvious to a person of ordinary skill in the art to conduct the subsequent autotrophic growth step at a workable growth temperature within the known growth range (e.g., 37°C-40°C) because C. necator is known to grow well at this temperature, as evidenced by Poladyan et al. Specifically, Poladyan et al. teaches that R. eutropha (i.e., C. necator) cultures are routinely grown at 37 °C for precultures (pg. 3, Materials and Methods), demonstrating that 37 °C is a known and workable cultivation temperature for C. necator strains. Thus, selection of a temperature from 37–40 °C for the autotrophic growth step would have been a routine optimization of a result‑effective variable, and would have been obvious to a person of ordinary skill in the art. Using the same or similar temperature would have been a predictable choice to maintain cell viability and growth. Additionally, 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. Optimization of parameters is a routine practice that would be obvious for a person of ordinary skill in the art to employ. "[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). See MPEP 2144.05 II. It would have been customary for an artisan of ordinary skill to determine the optimal temperature of culture conditions to achieve the desired results. Thus, absent some demonstration of unexpected results from the claimed parameters, the optimization of temperature would have been obvious at the time of applicant's invention. Therefore, the claimed invention, as a whole, would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the invention., because the combined teachings of the prior art are fairly suggestive of the claimed invention.
Claims 13-16 are rejected under 35 U.S.C. 103 as being unpatentable over Averesch et al. and Neves et al., as applied to claim 1 above, and further in view of Contesini et al. (An overview of Bacillus proteases: from production to application (2018) Critical Reviews in Biotechnology, 38(3), p. 321–334, cited in a previous office action).
The teachings of Averesch et al. and Neves et al. as they apply to claim 1 have already been discussed above. Briefly, Averesch et al. teaches genetic engineering of C. necator to express heterologous genes, and Neves et al. teaches that proteases are useful for cell disruption and product recovery from C. necator biomass, thereby motivating a person of ordinary skill in the art to modify the Cupriavidus necator strain of Averesch et al. to express one or more heterologous protease(s).
Claim 16 is included because while the claim recites “wherein the eukaryote is a yeast”, it does not require that the source of the digestive enzyme be a eukaryote. Claim 16 is still drawn to the strain of C. necator of claim 1, wherein the heterologous digestive enzyme is from a bacteria, as recited in claim 13 (upon which claim 16 depends).
Regarding claims 13-16, Contesini et al. teaches that the genus Bacillus is the most important bacterial source of industrial proteases, capable of producing high yields of neutral and alkaline proteolytic enzymes with desirable properties such as stability to extreme temperatures, pH, organic solvents, detergents, and oxidizing agents (Abstract). Contesini et al. further teaches that several Bacillus species, including Bacillus subtilis and Bacillus licheniformis, are well‑studied commercial producers of proteases across diverse industries, and additionally, have been granted “GRAS” (Generally Recognized as Safe) status by the FDA (pg. 322, left column last para-right column, first and third para). Given these known advantages, a person of ordinary skill in the art, in view of Neves et al.’s teaching of protease function in biomass processing, would have been motivated to select a Bacillus‑derived protease gene from any of the species recited in claim 15 (Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, or Bacillus lentus) as the heterologous protease in the engineered C. necator of Averesch et al. The selection of such a protease from among well-known industrial Bacillus species would have been a routine choice among recognized equivalents, with predictable results.
Claim 20 is rejected under 35 U.S.C. 103 as being unpatentable over Averesch et al. and Neves et al., as applied to claim 1 above, and further in view of Chen et al. (Construction of Cupriavidus necator displayed with superoxide dismutases for enhanced growth in bioelectrochemical systems. Bioresour. (2023) Bioprocess. 10:36, pg. 1-12, cited in a previous office action)
The teachings of Averesch et al. and Neves et al. as they apply to claim 1 have already been discussed above. Briefly, Averesch et al. teaches genetic engineering of C. necator to express heterologous genes, and Neves et al. teaches that proteases are useful for cell disruption and product recovery from C. necator biomass, thereby motivating a person of ordinary skill in the art to modify the Cupriavidus necator strain of Averesch et al. to express one or more heterologous protease(s).
Claim 20 recites a C. necator strain having a hydrogen-to-carbon dioxide uptake ratio lower than that of a wild-type C. necator strain cultured under corresponding temperatures and conditions. The term “hydrogen-to-carbon dioxide uptake ratio” is understood as the relative hydrogen consumption rate to carbon dioxide consumption rate during autotrophic growth.
Regarding claim 20, Chen et al. discloses that C. necator grows autotrophically on H₂, CO₂, and O₂ and produces four distinct NiFe hydrogenases, each serving unique physiological roles in hydrogen oxidation and autotrophic metabolism. For example, the membrane-bound hydrogenase (hoxGKZ) and soluble hydrogenase (hoxHYUF) function to oxidize hydrogen and generate reducing power for CO₂ fixation via the Calvin–Benson–Bassham cycle (all of pg. 2 and pg. 6, bottom left column-right column). Chen et al. further teaches that promoter strengthening in a modified strain (CMS) increases hydrogenase expression relative to wild-type H16, demonstrating that hydrogenase activity can be altered by genetic modification (pg. 6, right column). Chen et al. also teaches the significance of utilizing CO₂ as a feedstock to synthesize bio-based products, particularly single cell protein (SCP) as an alternative food or feed, and identifies bioelectrical systems (BES) as a promising platform for C. necator to directly produce SCP from CO₂ (Abstract and pg. 2)
An invention would have been obvious to a person of ordinary skill in the art if some teaching in the prior art would have led that person to combine prior art reference teachings to arrive at the claimed invention. Before the effective filing date of the claimed invention, given Chen et al.’s disclosure that hydrogenases govern hydrogen oxidation and thereby influence the balance of H₂ and CO₂ utilization in C. necator, and its explicit teaching that improving CO₂ conversion efficiency is significant for SCP production in BES, it would have been obvious to a person of ordinary skill in the art to modulate hydrogenase activity to reduce hydrogen oxidation that does not contribute to CO₂ fixation. Chen et al. demonstrates that hydrogenase activity can be altered by genetic modification, and said practitioner would have readily predicted that applying such modification to reduce unnecessary hydrogen oxidation would result in a C. necator strain having a hydrogen to carbon dioxide ratio lower than that of a wild type strain under the same conditions, with a reasonable expectation of success. The prior art provides both the identification of the relevant metabolic components and a clear motivation to optimize gas uptake ratios for enhanced biosynthetic efficiency, rendering the claimed strain obvious.
Response to Arguments for Rejections under 35 USC § 103
In the response filed 11/19/2025, applicant argues that Averesch et al. and Neves et al. teach away from the claimed invention because the cited references describe the use of enzymes, including proteases, for cell disruption during product recovery, whereas applicant’s claimed strain contains a heterologous digestive enzyme within the strain to enhance nutritional properties. Applicant asserts that the references therefore teach the “direct opposite” of the claimed invention. Applicant’s arguments have been fully considered and have been found to be not persuasive.
A reference teaches away only when it criticizes, discredits, or otherwise discourages the claimed solution. The mere fact that a reference teaches a different use or purpose for a known element does not constitute teaching away. Applicant has not identified, and the Examiner does not find, any disclosure in Averesch et al. or Neves et al. that explicitly criticizes, discredits, or discourages the intracellular expression of heterologous digestive enzymes in C. necator.
Claim 1 is directed to a strain of C. necator comprising a transgene encoding a heterologous digestive enzyme. The claim does not recite any limitation regarding the intended use of the strain, enhancement of nutritional value, prevention of cell lysis, or retention of intact cells. Arguments directed to intended purpose described in the specification do not limit the claim where such limitations are not positively recited.
As set forth in the Office Action, Averesch et al. teaches that C. necator can be genetically engineered to express heterologous genes using established recombinant techniques, and Neves et al. teaches the use of proteases in processing C. necator biomass. In view of the recognized utility of proteases in biomass degradation and processing, it would have been obvious to a person of ordinary skill in the art to incorporate a protease-encoding transgene into the engineered C. necator strain of Averesch et al., thereby internalizing enzymatic capability. Applicant’s arguments do not rebut this articulated rationale for combination.
Therefore, the rejections of claims 1-2, 6-20 under 35 U.S.C. §103 are maintained.
Conclusion
No claim is in condition for allowance.
THIS ACTION IS MADE FINAL. 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 NAGHMEH NINA MOAZZAMI whose telephone number is (703)756-4770. The examiner can normally be reached Monday-Friday, 9:00-5:00.
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/NAGHMEH NINA MOAZZAMI/Examiner, Art Unit 1652
/ROBERT B MONDESI/Supervisory Patent Examiner, Art Unit 1652