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 Status
Responsive to the claim set filed 06/10/2024, claims 1-20 are pending in the application and are presently considered.
Information Disclosure Statement
The information disclosure statements (IDS) filed on 08/05/2024 and 09/11/2024 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Drawings
The drawings are objected to because: in FIG. 3, the shading of the dots and the elements in the corresponding legend (representing “up-regulated”, “down-regulated” and “no change”) cannot be distinguished from one another.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 1 is objected to because of the following informalities: “acid-tolerant genes” is recited in line 2 which clearly provides antecedent basis for “the acid tolerant gene” recited in lines 2-3. For consistency, please amend the claim to make both of these recitations either singular or plural (e.g., “wherein the acid tolerant genes comprise[s]”). Appropriate correction is required.
Claim 14 is objected to because of the following informalities: “the step of acclimation” recited in line 2 clearly refers to the step of “acclimating a microorganism” recited in claim 11. For consistency, please amend “the step of acclimation” in line 2 to “the step of acclimating the microorganism”. Appropriate correction is required.
Claim Interpretation
Claim 16 recites the limitation “wherein the plurality of acidic culture solutions with different pH values are used to culture the microorganism for 10 days to 20 days respectively” (Emphasis added). Claim 14, from which claim 16 depends, recites “preparing a plurality of acidic culture solutions with different pH values ranging from pH 6.5 to pH 4” and “culturing the microorganism sequentially with the culture solution with a high pH value to a low pH value from the plurality of acidic culture solutions”. Here, the use of the term “respectively” is interpreted to mean that each of the acidic culture solutions with different pH values are used to culture the microorganism for 10 to 20 days. For example, the microorganism is cultured in the first acidic culture solution for 10 to 20 days, the second acidic culture solution for 10 to 20 days, etc.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 7 is 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 7 recites “polyketide synthase (octaketide synthase complex antDEFBG)” which renders the claim indefinite, because it is not clear whether the phrase recited in parentheses is a further limitation or merely an example. A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 7 recites the broad recitation “polyketide synthase”, and the claim also recites “octaketide synthase complex antDEFBG” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Applicant is reminded that the description of examples and preferences is properly set forth in the specification rather than in a single claim. A narrower range or preferred embodiment may also be set forth in another independent claim or in a dependent claim.
Claim Rejections - 35 USC § 112(a) – 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 1-8 and 19-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a genetically engineered Escherichia coli comprising a higher expression level of acid-tolerant genes compared to its wild-type, does not reasonably provide enablement for a genetically engineered microorganism derived from ANY source microorganism. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement requires that the specification teach those in the art to make and use the
invention without undue experimentation. Factors to be considered in determining whether a
disclosure would require undue experimentation include (1) the nature of the invention, (2) the state of the prior art, (3) the predictability or lack thereof in the art, (4) the amount of direction or guidance present, (5) the presence or absence of working examples, (6) the quantity of experimentation necessary, (7) the relative skill of those in the art, and (8) the breadth of the claims.
Summary of the Problem
The claims are drawn to a broad genus of genetically engineered microorganisms, wherein ANY source microorganism has been made to express at least one of three genes found to be up-regulated in acid-tolerant E. coli. The claims require that the genetically engineered microorganism expresses at least one of the three genes, while not all microorganisms possess these genes. The claims also require that the engineered strain is tolerant to acidic environments in the range of pH 3 to pH 5. Here, the claims are not enabled for their full scope, because: (1) the recited genes are not known in the art to have the effect of increasing acid-tolerance, (2) the effects of introducing these genes to heterologous microorganisms are not reported in the art as conferring acid-tolerance, (3) the Examples of the specification use a single strain of E. coli as the source microorganism, which natively possesses these genes, while other microorganisms do not, and (4) the specification provides no guidance or direction regarding the overexpression of these genes in heterologous species.
As set forth below, numerous pertinent factors, to which the specification provides little
to no guidance regarding, are required in order for even a skilled artisan to be able to make and
use the invention as claimed, such that any attempt to do so warrants undue experimentation
without a reasonable expectation of success. Given the breadth of the claimed genus and the unpredictability in the art, the specification as filed does not reasonably provide sufficient guidance, direction, and/or exemplification for a person of ordinary skill to make and/or use the claimed invention using ANY microorganism.
The nature of the invention / breadth of the claims
Claim 1 recites a genetically engineered microorganism, comprising a higher expression level of acid-tolerant genes compared to a source microorganism, wherein the acid-tolerant genes comprise at least one of dsdA, dcuC and glaA. Here, the “source microorganism” is interpreted to include ANY microorganism. Dependent claim 4 requires that the genetically engineered microorganism has tolerance to an acidic environment, wherein the pH value of the acidic environment ranges from pH 3 to pH 5. Because dependent claim 4 does not provide any further structural limitations, it is understood that the engineered microorganism of the parent claim must also possess this tolerance.
Dependent claim 3 limits the genetically engineered microorganism of claim 1 to being derived from the source microorganism comprising Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Pseudomonas putida, Yarrowia lipolytica, Saccharomyces cerevisiae, or Pichia pastoris. Examiner notes that Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis and Pseudomonas putida are species of bacteria, while Yarrowia lipolytica, Saccharomyces cerevisiae and Pichia pastoris are species of yeasts.
Dependent claims 19-20 are directed to a method of using the genetically engineered microorganism of claim 1 to produce a target chemical, wherein the microorganism is cultured in an acidic culture solution in the range of pH 3 to pH 5.
In view of the prior art of record, the dsdA, dcuC and glaA are not naturally present in all microorganisms (discussed further below). Hence, the scope of the claims necessarily include microorganisms that have been genetically engineered to possess these genes in order for them to be expressed at any level.
The state of the prior art / the predictability or lack thereof in the art
Vorachek-Warren et al. (cited on Form 892) teach that the dsdA gene is derived from Escherichia coli and encodes a D-serine deaminase which conferred resistance to D-serine and the ability to use D-serine as a nitrogen source in genetically-manipulated Saccharomyces cerevisiae (see Abstract). However, the prior art is silent regarding this gene having any role in acid-tolerance. Xu et al. (cited on Form 892) teach that several regulatory systems are known to control acid resistance in E. coli, enabling the bacteria to survive under acidic conditions without growth, such as the acid-tolerance response (ATR) system and its regulatory circuit (see Abstract). However, none of the genes recited in claim 1 are reported to be involved in the acid-tolerance response system of E. coli.
Abo-Amer et al. (cited on Form 892) teach that there are four known C4-dicarboxylate transporters in E. coli, one of which is DcuC, which enable E. coli to use C4-dicarboxylates (succinate, fumarate, and malate) and aspartate as sole carbon and energy sources (see pg. 1879, col. 1, para. 1). According to Karapetyan et al. (cited on Form 892), active DcuD is important for H± efflux at pH 7.5 (see Abstract). However, the prior art is otherwise silent regarding dcuC having any role in acid-tolerance, let alone at acidic levels (e.g., pH 3 to 5).
Alkis et al. (cited on Form 892) disclose that the glaA gene from Aspergillus niger (a filamentous fungus), which is responsible for encoding the glucoamylase enzyme, was cloned into a plasmid and transferred into P. pastoris (see Abstract), and the isolated/purified enzyme was active across a wide pH range of pH 5 to pH 10 (see pg. 124, col. 1, para. 1). Kumar et al. (cited on Form 892) teach that fungal glucoamylase from Aspergillus spp., which is widely used in commercial starch processing, has optimal activity under acidic conditions of pH 4.0-4.5 (see pg. 238, col. 2, para. 2). However, neither reference suggests the gene encoding this enzyme to confer acid-tolerance to the microorganism itself. Conversely, Geoghegan et al. (cited on Form 892) disclose that the transcription factor WarA was identified in Aspergillus fumigatus as being required for resistance to a range of weak acids, and the deletion of the orthologous gene in Aspergillus niger showed that several genes were significantly upregulated in the wild type compared with the mutant (see Abstract). Among these genes was a putative ABC-type transporter found to be required for weak-acid resistance in A. niger and to be a functional homologue of the Saccharomyces cerevisiae protein Pdr12p (see Abstract). However, none of the genes recited in claim 1 are reported to be involved in the acid-tolerance of Aspergillus or Saccharomyces.
Hence, while there may be some predictability in the art when transforming microorganisms with heterologous genes, such as dsdA, dcuC and glaA, these genes are not reported in the art as conferring the acid-resistance required by the claimed invention to either the microorganisms they are derived from or to any microorganism they have been introduced to. There remains an open question as to what effect the overexpression of these genes in ANY microorganism would have. Given the vast range of optimum growth conditions across all microorganisms known in the art, one of ordinary skill would not have a reasonable expectation that any source microorganism engineered to overexpress the claimed genes would possess the functions required by the claimed invention, particularly in the absence of sufficient guidance, direction and/or exemplification.
The amount of direction or guidance present
The specification discloses that by screening and establishing strains that are tolerant to acidic environments and can utilize acetic acid as a carbon source, the prepared genetically engineered strains can be better utilized to produce a target chemical (see pg. 6, para. [0023]). The specification discloses that the genetically engineered microorganism has a higher expression level of at least one of the dsdA, dcuC and glaA genes (pg. 6, para. [0024]), and the source microorganism of the genetically engineered microorganism may include the species as recited in claim 3 (see pg. 7, para. [0027]). The specification discloses a novel genetically engineered E. coli strain is provided with the deposit number BCRC 940699, which has a higher expression level of acid-tolerant genes than the source microorganism, wherein the genes “may include at least one of dsdA, dcuC and glaA” (see pg. 11, para. [0036]).
However, there is zero discussion in Applicant’s disclosure regarding the effects of dsdA, dcuC and glaA in any microorganism outside of E. coli. Furthermore, there is zero description regarding what structure/function relationship relates the presence of these genes to the acid-tolerance of the genetically engineered microorganism. There is also no discussion regarding the introduction of these genes to a source microorganism wherein the gene(s) are naturally absent, which is relevant to the broad genus Applicant has claimed, because not all microorganisms are known to possess these specific genes. In view of the prior art of record, the effects of these genes in heterologous organisms is largely unknown, particularly as they relate to the acid-tolerance of these organisms.
The presence or absence of working examples
In Example 1, the inventors disclose the acclimating of Escherichia coli K-12 to obtain acid-tolerant strains. Here, the E. coli strain was first inoculated into a culture solution at pH 6.5 for 24 hours. After confirming that the strains have continued to grow, 10% of the strains were transferred to new culture solutions (transferred into culture solutions of pH 6, pH 5.5, pH 5, pH 4.5 and pH 4 in sequence). The strains were cultured under the environments of each pH value for 14 days until it was observed that the strains could survive in that pH environment. See pg. 18, para. [0065].
In Example 2, the inventors disclose that Next Generation Sequencing (NGS) was used to analyze the transcriptome of the acclimated strains. The inventors proposed that the expression changes of all genes in the genome may be measured to obtain genes that may be related to the strain’s ability to tolerate acidic environments. The inventors compared the expressed genes of native Escherichia coli with the acid-tolerant strains, and found that the expression of 221 genes was up-regulated in the acid-tolerant strains compared to native E. coli. Genes with “high differential expression levels” are listed in Table 1. See pgs. 19-20, paras. [0068]-[0071].
Hence, Applicant’s Examples are directed to a single species of the invention (Escherichia coli), and the claimed “acid-tolerant genes” are based on comparing the expression levels of genes in the acid-tolerant strains of E. coli with those of native E. coli. Applicant does not disclose any reduction to practice involving any other species of the claimed invention, nor is there any disclosure in the specification that relates the expression levels of these genes to any other organism. Furthermore, the three claimed genes that were overexpressed were among 221 genes that were upregulated in the acid-tolerant strains of E. coli. No experiments were performed to elucidate whether the overexpression of these three genes were responsible for acid-tolerance in E. coli, let alone whether the overexpression these genes in other species would have this effect.
The quantity of experimentation necessary / The relative skill in the art
What Applicant has shown is a starting point from which, when given the bid to do so by the instant claims, someone else skilled in the art may pick up a path of studies that may lead to the completion of the claimed invention. However, it is not sufficient for the specification to provide merely “a starting point, a direction for further research”; it must provide “reasonable detail” sufficient to enable a person of ordinary skill in the art to make or use the invention. Automotive Technologies Intern., Inc. v. BMW of North America, Inc., 501 F.3d 1274, 1284 (Fed. Cir. 2007).
In the instant case, it is unknown whether any given microorganism overexpressing dsdA, dcuC and glaA would have the necessary functions of the claimed invention (i.e., tolerance to an acidic environment ranging from pH 3 to pH 5), and a person of ordinary skill in the art would not have a reasonable expectation of success in making or using the claimed invention. While Applicant’s methods may be enabling for E. coli, as demonstrated in Applicant’s Examples, the instant specification does not provide sufficient guidance in order for one to make and use the claimed invention using ANY source microorganism.
While the skill in the art is high, the predictability in the art is low due to the diversity of microorganisms, their genetics, and the complexity of biological systems. In order to practice the full scope of the claimed invention, a person of ordinary skill would be required to introduce the genes of dsdA, dcuC and/or glaA to a reasonably high number of microorganisms and discover for themselves whether this modification is sufficient, whether only certain genes confer the claimed functions, or whether further steps are required to arrive at the claimed invention. Here, the scope of the claims comprise are broad genus, while there is not enough predictability in the art for one to determine which embodiments would be operative without undue experimentation.
Conclusion
The amount of guidance, direction, and exemplification disclosed in the specification as filed would not have been sufficient to have enabled the skilled artisan to make and/or use the claimed invention at the time the application was filed without undue and/or unreasonable experimentation.
“Enabling the full scope of each claim is part of the quid pro quo of the patent bargain. A patentee who chooses broad claim language must make sure the broad claims are fully enabled. The scope of the claims must be less than or equal to the scope of the enablement to ensure that the public knowledge is enriched by the patent specification to a degree at least commensurate with the scope of the claims.” Sitrick v. Dreamworks, LLC, 516 F.3d 993, 999 (Fed. Cir. 2008).
In deciding In re Fisher, 166 USPQ 18, 24 (CCPA 1970), the Court indicated the more unpredictable an area is, the more specific enablement is necessary in order to satisfy the statute. “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.” Genentech Inc. v. Novo Nordisk A/S, 42 USPQ2d 1001, 1005 (CA FC 1997).
In conclusion, upon careful consideration of the factors used to determine whether undue experimentation is required, in accordance with the Federal Circuit decision of In re Wands, 858 F.2d at 737, 8 USPQ2d at 1404 (Fed. Cir. 1988), the amount of guidance, direction, and exemplification disclosed in the specification, as filed, is not deemed sufficient to have enabled the skilled artisan to make and/or use the full scope of the claimed invention at the time the application was filed without undue and/or unreasonable experimentation.
Claims 9-10 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.
It is apparent that the microorganism having the deposit number BCRC 940699 is required to practice the claimed invention because it is a necessary limitation for the success of the invention as stated in the claims. As a required element it must be known and readily available to the public or obtainable by a repeatable method set forth in the specification, or otherwise readily available to the public. If it is not so obtainable or available, the enablement requirements of 35 U.S.C. § 112, first paragraph, may be satisfied by a deposit of BCRC 940699. See 37 CFR 1.802.
If a deposit is made under the terms of the Budapest Treaty, then an affidavit or declaration by applicants or someone associated with the patent owner who is in a position to make such assurances, or a statement by an attorney of record over his or her signature, stating that the deposit has been made under the terms of the Budapest Treaty and that all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of a patent, would satisfy the deposit requirements. See 37 CFR 1.808.
If a deposit is not made under the terms of the Budapest Treaty, then an affidavit or declaration by applicants or someone associated with the patent owner who is in a position to make such assurances, or a statement by an attorney of record over his or her signature, stating that the deposit has been made at an acceptable depository and that the following criteria have been met:
(a) during the pendency of this application, access to the invention will be afforded to one determined by the Commissioner to be entitled thereto;
(b) all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon granting of the patent;
(c) the deposit will be maintained for a term of at least thirty (30) years and at least five (5) years after the most recent request for the furnishing of a sample of the deposited material;
(d) a viability statement in accordance with the provisions of 37 CFR 1.807; and
(e) the deposit will be replaced should it become necessary due to inviability, contamination or loss of capability to function in the manner described in the specification.
In addition, the identifying information set forth in 37 CFR 1.809(d) should be added to the specification. See 37 CFR 1.803 - 37 CFR 1.809 for additional explanation of these requirements.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1 and 3 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Meng et al. (cited on Form 892), hereafter, “Meng”.
Regarding claim 1, Meng teaches a genetically engineered E. coli, WSA157, which was modified by replacing the upstream regulated region of the dcuC operon with a strong constitutive promoter resulting in strain WSA161, which increased its specific growth rate by 59% compared to WSA157 (see pg. 8, col. 1, para. 1). Meng teaches that synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield (see Abstract). Hence, Meng teaches a genetically engineered microorganism, comprising a higher expression level of the dcuC gene, which meets the limitations of claim 1.
Regarding claim 3, Meng teaches the genetically engineered microorganism is E. coli, as discussed above.
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.
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.
Claim(s) 2 and 4-5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Meng as applied to claims 1 and 3 above, and further in view of Lin et al. (cited on Form 892), hereafter, “Lin”.
Regarding claim 2, Meng discloses a rational approach to increase succinate yield in Escherichia coli by introducing genes from Corynebacterium glutamicum for succinate production and making sequential modifications to overexpress several other genes, including dcuC, to generate mutant strains (see Abstract). In particular, Meng discloses that dcuC overexpression increased succinate yield and the specific growth rate of the E. coli, demonstrating that activation of Dcu transportations effectively accelerate the anaerobic succinate transport out of E. coli (see pg. 8, col. 1, para. 1). Meng teaches that succinate and its derivatives are widely used as specialty chemicals in the food, pharmaceutical, and cosmetic industries (see pg. 2, col. 1, para. 1).
Meng also discloses the inactivation of acetate formation genes to remove byproduct formation and improve succinate yield (see Abstract). Meng teaches that the presence of acetate at high concentrations is undesirable in the fermentation broth since it interferes with the target succinate biosynthesis and increases the cost of succinate purification (see pg. 9, col. 2, para. 1).
Meng does not teach the genetically engineered microorganism comprising at least one exogenous nucleotide sequence encoding acetyl-CoA synthetase (ACS).
Lin teaches the overexpression of acetyl-CoA synthetase (ACS) in Escherichia coli showed significant reduction in acetate during glucose fermentation, and also greatly enhanced acetate assimilation when acetate was used as a carbon source. Lin teaches the native acs gene was cloned and overexpressed in E. coli, which resulted in the E. coli showing a significant reduction in acetate during glucose fermentation. Lin teaches these features of the genetically engineered E. coli are ideal for applications in metabolic engineering. See Abstract.
Lin teaches E. coli is known to produce significant amounts of acetate during excess glucose fermentation in high cell density cultures (see pg. 870, col. 1, Introduction). Lin teaches that high acetate accumulation is harmful to cell growth as it decouples transmembrane pH gradients, and this negatively affects internal osmotic pressure, intracellular pH and amino acid synthesis (see pg. 870, col. 1, Introduction to col. 2, para. 1). Lin teaches that enhancing the capability of E. coli to assimilate acetate would reduce harmful effects of acetate, recycle wasted carbon, and increase carbon flux toward desired pathways (see pg. 870, col. 2, para. 2).
Lin teaches that many favorable product formation pathways branch from the acetyl-CoA node in the central metabolic network of E. coli, and the application of ACS overexpression in increasing the flux toward acetyl-CoA from acetate assimilation can be potentially useful for enhancing the production of these products. The production of native metabolites such as the specialty chemical succinate can also be increased by empowering the strain with efficient acetate assimilation via ACS overexpression. One example of such an application is in genetically engineered E. coli strains that can solely produce succinate under aerobic conditions. Overall, ACS overexpression in E. coli offers the advantages of reduced acetate accumulation and enhanced acetate assimilation, both of which can improve cell growth and product formation. See pg. 873, col. 2, para. 2.
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Meng and Lin, because both references teach genetically engineered E. coli with reduced acetate formation and increased product yield of succinate. One would have recognized from both disclosures the advantages of reducing acetate accumulation and would have been motivated to apply the solution taught by Lin to further enhance the growth and product formation of the mutant strains taught by Meng. As both references disclose the results of these solutions (i.e., overexpression of DcuC, cloning of a gene encoding ACS) effectively enhance the growth of the microorganism and/or the production of desirable products (e.g., succinate), one would have recognized there to be a reasonable expectation of success when applying these teachings in combination. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103). Furthermore, it is well within the ordinary skill in the art to clone a native gene and to exogenously introduce additional copies into a bacterium, such as the well-studied E. coli, to overexpress the encoded enzyme.
Regarding claim 4, Meng and Lin do not explicitly teach the genetically engineered microorganism having a tolerance to an acidic environment, wherein the pH value of the acidic environment ranges from pH 3 to pH 5. However, where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). "Products of identical chemical composition can not have mutually exclusive properties." In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). In the instant case, no further structure of the microorganism is recited in the present claim, and the acid-tolerance of the microorganism is an inherent property that would be expected to be present when overexpressing the same acid-tolerant genes as in the instantly claimed microorganism. Because the limitations of the claimed product are necessarily present in the prior art combination, the functional limitations of the asserted claim are inherently met by the combination of references. See MPEP 2112.
Regarding claim 5, Meng teaches that inactivating the acetate formation gene ackA-pta promoted succinate yield but decreased the cells’ overall fitness (see Abstract; pg. 9, col. 2, para. 2). Meng discloses that heterogeneously expressing pyc, encoding pyruvate carboxylase (PYC), from C. glutamicum led to higher succinate yield and restored the ability of the engineered strain to ferment glucose (see Abstract; pg. 9, col. 2, para. 2). Hence, Meng teaches the genetically engineered microorganism comprising an exogenous nucleotide sequence (i.e., pyc) encoding an enzyme (i.e., PYC) related to the synthesis of a target chemical (i.e., succinate). Thus, it would have been obvious for the exogenous nucleotide sequence encoding ACS to further comprise a sequence encoding an enzyme related to the synthesis of a target chemical.
Claim(s) 6 and 7 is/are rejected under 35 U.S.C. 103 as being unpatentable over Meng and Lin as applied to claims 1-5 above, and further in view of Philippe at al. (US 20210261992 A1; cited in the IDS filed 08/05/2024), hereafter, “Philippe”.
Regarding claim 6, Meng teaches a genetically engineered E. coli comprising a higher expression level of the dcuC gene (as discussed regarding claim 1), and Lin teaches a genetically engineered E. coli comprising a cloned acs gene encoding acetyl-CoA synthetase (ACS) (as discussed regarding claim 2). Meng also teaches the genetically engineered E. coli further comprising an exogenous nucleotide sequence encoding an enzyme related to the synthesis of a target chemical, as discussed regarding claim 5. Meng also teaches that dcuC overexpression increased the specific growth rate of the E. coli by 59% and specific glucose uptake by 7% over the corresponding measurements for the source E. coli (see Meng at pg. 8, col. 1, para. 1).
Meng and Lin do not teach the genetically engineered microorganism, wherein the target chemical comprises carmine, para-aminobenzoic acid (PABA), indigo blue, melanin, or a combination thereof.
Philippe teaches that the natural pigment carmine, the aluminum salt of carminic acid, is one of the most frequently used colorants of food, beverages, medicine, cosmetics, and textiles (see pg. 1, para. [0003]). Philippe teaches that current industrial production of carmine involves the harvesting of carminic acid (CA) from cochineal insects grown on Opuntia ficus-indica cactus plants in commercial plantations (see pg. 1, para. [0004]). This source is relatively expensive and subject to undesirable quality variation and price fluctuation, and the extraction process results in some amount of insect protein contaminating the colorant product, creating a risk for allergy-related problems (see pg. 1, paras. [0004]-[0005]). This has prompted the exploration of synthetic chemistry approaches to the production of carmine; however, the expense of these processes prohibits their broad application (see pg. 1, para. [0005]).
Philippe teaches a host cell for producing carminic acid, the host cell expressing an enzymatic pathway for biosynthesis of carminic acid from polyketide building blocks (see claim 1), wherein the host cell is Escherichia coli (see claim 5). Philippe teaches the host cell expresses one or more enzymes of a bacteria, fungus, plant or insect species (see claim 8), wherein the enzyme possesses octaketide synthase and cyclase activities (see claim 13). Philippe also teaches that one of the enzymes expressed by the host cell is a recombinant polyketide synthase that utilizes Acetyl-CoA as an initial building block for carminic acid synthesis (see claim 7; FIG. 2; pg. 2, para. [0026]). As the host cell is engineered to express one or more enzymes derived from other species, it is understood that the host cell comprises an exogenous nucleotide sequence encoding enzyme(s) (e.g., polyketide synthase) related to the synthesis of carmine.
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Meng, Lin and Philippe for at least the following reasons: (1) Philippe teaches that carmine is an industrially useful product and previous methods are expensive, subject to quality and price fluctuations, and create a risk for allergy-related problems; (2) Philippe teaches a genetically engineered E. coli comprising exogenous genes encoding enzymes related to carmine synthesis; (3) Meng teaches that overexpression of dcuC increases specific growth rate and glucose uptake in E. coli; (4) Lin teaches that E. coli is known to produce acetate during glucose fermentation, and the overexpression of acetyl-CoA synthetase in E. coli significantly reduces acetate during glucose fermentation, reducing harm to cell growth; (5) Lin teaches that many favorable product formation pathways branch from Acetyl-CoA, while Philippe teaches an exogenous enzyme that utilizes Acetyl-CoA as a building block for carminic acid synthesis; and (6) all references relate to optimizing genetically engineered E. coli in bioprocesses to produce useful products.
Thus, a person of ordinary skill would have recognized that the teachings of Meng and Lin would have been advantageous when combined with those of Philippe to provide a microorganism that may be more effective in producing carmine. There would have been a reasonable expectation for success, as all references relate to the production of useful products using genetically engineered E. coli where modulation of many of the same factors are disclosed (e.g., increased glucose uptake, decreased acetate accumulation, increased availability of Acetyl-CoA), which would be expected to enhance cell growth and improve product yield in view of the above references. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 7, Philippe teaches the host cell expresses a recombinant polyketide synthase which is related to the synthesis of carmine, as discussed above.
Claim(s) 6 and 8 is/are rejected under 35 U.S.C. 103 as being unpatentable over Meng and Lin as applied to claims 1-7 above, and further in view of Anderson et al. (US 20170211104 A1; cited on Form 892), hereafter, “Anderson”.
Regarding claim 6, Meng teaches a genetically engineered E. coli comprising a higher expression level of the dcuC gene (as discussed regarding claim 1), and Lin teaches a genetically engineered E. coli comprising a cloned acs gene encoding acetyl-CoA synthetase (ACS) (as discussed regarding claim 2). Meng also teaches the genetically engineered E. coli further comprising an exogenous nucleotide sequence encoding an enzyme related to the synthesis of a target chemical, as discussed regarding claim 5. Meng also teaches that dcuC overexpression increased the specific growth rate of the E. coli by 59% and specific glucose uptake by 7% over the corresponding measurements for the source E. coli (see Meng at pg. 8, col. 1, para. 1). As previously discussed, Lin teaches that many favorable product formation pathways branch from Acetyl-CoA, and the overexpression of acetyl-CoA synthetase in E. coli significantly reduces acetate during glucose fermentation, reducing harm to cell growth.
Meng and Lin do not explicitly teach a genetically engineered microorganism, wherein the target chemical comprises carmine, para-aminobenzoic acid (PABA), indigo blue, melanin, or a combination thereof.
Anderson teaches compositions and methods for the biosynthetic production of acetaminophen, p-aminophenol and p-aminobenzoic acid (PABA) (see Abstract). Anderson teaches that acetaminophen is a popular analgesic, considered by WHO as an “essential medicine” that should “be available at all times in adequate amounts and in appropriate dosage forms (see pg. 1, para. [0004]). Anderson teaches that there is no synthetic chemistry route to produce acetaminophen that does not involve one or more hazardous agents, causing risk to the health of production workers, and these processes also require the use of organic solvents imposing additional environmental burden (see pg. 1, para. [0005]).
Anderson teaches a non-naturally occurring microbial organism comprising at least three exogenous genes encoding acetaminophen pathway enzymes expressed in a sufficient amount to produce acetaminophen, wherein said acetaminophen pathway comprises (i) an enzyme that converts chorismic acid to p-aminobenzoic acid (PABA), (ii) an enzyme that converts p-aminobenzoic acid to p-aminophenol, and (iii) an enzyme that coverts p-aminophenol to acetaminophen (see claim 1). Anderson teaches the modified microorganism may be yeast, bacteria, or fungi (see pg. 9, para. [0115]). Anderson discloses methods of producing p-aminobenzoic acid (PABA) via fermentation which comprise providing a fermentation media comprising a carbon substrate (see pg. 2, para. [0026]). Anderson discloses FIG. 1, which shows the biosynthetic pathway encoded by strains of the disclosure (see pg. 3, para. [0040]):
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Anderson teaches the engineered organisms have two to five heterologous genes, which may include pabA, pabB, and pabC (see pg. 3, para. [0032]). Anderson teaches that enzymes that modify chorismic acid to form p-aminobenzoic acid (PABA) include glutamine amidotransferase (PabA), 4-amino-4-deoxychorismate synthase (PabB), and PabC (see pg. 3, para. [0033]).
Thus, Anderson teaches a genetically engineered microorganism comprising exogenous nucleotide sequence(s) encoding enzyme(s) related to the synthesis of para-aminobenzoic acid (PABA).
It would have been obvious at the time of filing for a person of ordinary skill in the art to have arrived at the claimed invention by combining the teachings of Meng, Lin and Anderson for at least the following reasons: (1) Anderson teaches that para-aminobenzoic acid (PABA) is an intermediate in processes to produce acetaminophen, which is an essential drug in high demand, wherein there is a need in the art for safer and more economical production methods; (2) Anderson teaches genetically engineered microorganisms, including bacteria, comprising exogenous genes encoding enzymes related to PABA synthesis and methods comprising a fermentation media comprising a carbon substrate; (3) Meng teaches that overexpression of dcuC increases specific growth rate and glucose uptake in E. coli; (4) Lin teaches that E. coli is known to produce acetate during glucose fermentation, and the overexpression of acetyl-CoA synthetase in E. coli significantly reduces acetate during glucose fermentation, reducing harm to cell growth; (5) Lin teaches that many favorable product formation pathways branch from Acetyl-CoA, while Anderson teaches Acetyl-CoA as a substrate in the conversion of PABA to acetaminophen; and (6) all references relate to optimizing genetically engineered microorganisms in bioprocesses to produce useful products.
Thus, a person of ordinary skill would have recognized that the teachings of Meng and Lin would have been advantageous when combined with those of Anderson to provide a microorganism that may be more effective in producing para-aminobenzoic acid (PABA) and acetaminophen. There would have been a reasonable expectation for success, as all references relate to the production of useful products using genetically engineered E. coli where modulation of many of the same factors are disclosed (e.g., increased glucose uptake, decreased acetate accumulation, increased availability of Acetyl-CoA), which would be expected to enhance cell growth and improve product yield in view of the above references. Hence, the combination would have been readily apparent and deemed to be a mere (A) combining of prior art elements according to known methods to yield predictable results (see MPEP 2143(I): Rationales to support rejections under 35 U.S.C. 103).
Regarding claim 8, Anderson teaches the genetically engineered microorganism, wherein the enzyme related to the synthesis of PABA include glutamine amidotransferase (PabA) and 4-amino-4-deoxychorismate synthase (PabB), as discussed above.
Claim(s) 11-13 and 17-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lin and Philippe, and in further view of Guan et al. (cited on Form 892), hereafter, “Guan”, and as further evidenced by Kang et al (cited on Form 892), hereafter, “Kang”.
Regarding claim 11, (a) Guan teaches that adaptive evolution is a powerful tool for strain improvement, during which the tolerant strains are repeatedly transferred into fresh broth and the pH is lowered gradually (see pg. 58, col. 1, para. 1). Guan teaches that higher acid tolerance adopted by microbial producers enhances their stability during the synthetic process in which acids accumulate (see pg. 51, col. 1 to col. 2, para. 1). Guan teaches that organic acids are formed during most microbial fermentation processes as either products or by-products, which acidify the microbial environment, and that great quantities of acetic acid may be released during biomass utilization in industrial production, which leads to increased acid stress (see pg. 51, col. 2, para. 2).
(b) As previously discussed, Lin teaches that introducing a cloned acs gene in E. coli resulted in a significant reduction in acetate during glucose fermentation, enhanced the capability of E. coli to use acetate as a substrate, and reduced harmful effects. Lin also teaches that many favorable product formation pathways branch from the acetyl-CoA node in the central metabolic network of E. coli.
(c) As previously discussed, Philippe teaches genetically engineered E. coli comprising exogenous genes encoding enzymes related to carmine synthesis, in methods to produce carmine, an industrially useful product. Philippe also teaches that the exogenous enzyme utilizes Acetyl-CoA as a building block for carminic acid synthesis.
As relevant to the above disclosures, it is understood that carmine (i.e., carminic acid) and acetate (i.e., acetic acid) are organic acids that are produced during microbial fermentation (see Kang at pg. 49, col. 2, para. 2; pg. 56, Table 2).
It would have been obvious for a person of ordinary skill to have arrived at the claimed invention by combining the above references for at least the following reasons: (1) Philippe teaches that carmine is an industrially useful product and previous methods are expensive, subject to quality and price fluctuations, and create a risk for allergy-related problems; (2) Philippe teaches a genetically engineered E. coli comprising exogenous genes encoding enzymes related to carmine synthesis; (3) Lin teaches that E. coli is known to produce acetate during glucose fermentation, and the overexpression of acetyl-CoA synthetase in E. coli significantly reduces acetate during glucose fermentation, reducing harm to cell growth; (4) Lin teaches that many favorable product formation pathways branch from Acetyl-CoA, while Philippe teaches an exogenous enzyme that utilizes Acetyl-CoA as a building block for