Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
This application is a US national phase of PCT/EP2022/073110 filed August 18,
2022, with foreign priority application EP21191995.6, filed August 18, 2021. Applicant's
Preliminary amendment filed July 26, 2024 is acknowledged. Claims 3-11, 13, and 16
are amended. Currently claims 1-16 are pending.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 1-10, and species: 1) Bacillus, 2) promoter upregulated by oxyR, 3) Nutritional, 4) protease; in the reply filed on May 1, 2026 is acknowledged.
Claims 11-16 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Group, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on May 1, 2026.
Claims 1-10 are under examination.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Drawings
The drawings are objected to because the drawings are labeled “Figure” instead of “FIG.”. See MPEP 608.02 V. DRAWING STANDARDS (u):
(u) Numbering of views.
(1) The different views must be numbered in consecutive Arabic numerals, starting with 1, independent of the numbering of the sheets and, if possible, in the order in which they appear on the drawing sheet(s). Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter. View numbers must be preceded by the abbreviation "FIG." Where only a single view is used in an application to illustrate the claimed invention, it must not be numbered and the abbreviation "FIG." must not appear.
(2) Numbers and letters identifying the views must be simple and clear and must not be used in association with brackets, circles, or inverted commas. The view numbers must be larger than the numbers used for reference characters.
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
Claims 7-8 are objected to because of the following informalities:
Claim 7, line 2, the acronym, RBS, needs to be recited in full name “ribosomal binding site (RBS)” at the first occurrence.
Claim 8, line 5, the acronym, UPR, needs to be recited in full name “unfolded protein response (UPR)” at the first occurrence.
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.
Claims 1-10 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 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.
Claims 1-10 recite a microbial production cell genetically engineered to synthesize a product comprising a first essential gene operably linked to a first burden-sensing promoter and a second essential gene operatively linked to a second burden-sensing promoter, wherein both promoters are heterologous to their respective gene, and wherein synthesis of the product confers a burden to the cell, and wherein expression of each promoter is upregulated when induced by said burden relative to a basal level expression of essential gene when the promoter is not induced. Claim 3 recites the microbial cell can be a bacterium, yeast, or filamentous fungus, and recites specific species of each category. Claim 7 recites the essential gene is operably linked to a synthetic ribosomal binding sequence (RBS) whose sequence is selected to modify the translational strength of the essential gene independent of induction of the promoter. Claim 8 recites the burden-sensing promoter can be a ribosomal RNA promoter, a promoter upregulated by oxyR, a promoter comprising a UPR element upregulated by HAC1, or a DNA damage-sensing promoter. Claims 2 and 9-10 recite the microbial cell’s fitness cost and product yield. The specification defines ‘essential gene’ if down-regulated, leads to reduction in the growth rate of the microbial production cell under production conditions, and is essential for growth irrespective of the nutrient composition of the production conditions, and identifies non-limiting examples in E. coli, Bacillus, Saccharomyces, Aspergillus, and Corynebacterium (pg. 10, lines 1-20; pgs. 17-19, Table 1). The specification defines ‘burden-sensing promoter’ as a promoter induced by said burden, and identifies examples of promoters of E. coli, S. cerevisiae, Bacillus, Aspergillus, and Corynebacterium (pg. 8, lines 14-35; pgs. 23-26, Table 2). To satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention. See, e.g., Moba, B.V. v. Diamond Automation, Inc., 325 F.3d 1306, 1319, 66 USPQ2d 1429, 1438 (Fed. Cir. 2003); Vas-Cath, Inc. v. Mahurkar, 935 F.2d at 1563, 19 USPQ2d at 1116.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described by their complete structure. In the instant case, the specification merely gives working examples of E. coli transformed with a burden-sensing promoter (PmutM, Pvccv or Pvcix), which substituted for the native promoter of the essential gene operon folP-glmM, that included an RBS between the promoter and essential gene (Example 1); in Example 2, the promoters used were Pvccv, PvciX, PibpA, PgrpE, PldhA and PybbN; in Example 4 the promoter used was PcspD or PmutM the controlled folP-glmM. Example 5 describes engineering S. cerevisiae with promoters of ribosomal RNA genes (RPL3 (SEQ ID NO.: 95), RPL6A (SEQ ID NO.: 96) and RPL28 (SEQ ID NO.: 97)), DNA damage sensing promoters (OGG1 (SEQ ID NO.: 98), RAD51 (SEQ ID NO.: 99) and RAD54 (SEQ ID NO.: 100)), and unfolded protein response (UPR) promoters upregulated by the HAC1 transcription factor (e.g. KAR2 (SEQ ID NO.: 91), PDI1 (SEQ ID NO.: 92), SSA1 (SEQ ID NO.: 93) or FPR2 (SEQ ID NO.: 94)); that are introduced in front of a native growth regulating gene URA3. Example 6 describes engineering Pichia pastoris with the promoters pPDI1, pFPR2, pRPL3 and pRPL6A; that are introduced in front of an integrated construct comprising a kanMX conditionally selectable G418 resistance gene operably linked to one of the promoters; and in Example 12, P. pastoris is engineered with variant promoter Pgsh2 operably linked with different translation initiation site (TIS) sequences and the essential gene cia1 . Example 8 describes engineering Bacillus subtilis with the promoters PhrcA or PperR upstream of the essential gene iscU or accC; and in Example 9 the accC gene was operably linked to promoters PctsR, PdnaK and PhrcA. In Example 13, Aspergillus oryzae is engineered to include promoter sequences from gene homologs of bipA, clxA, and agsA upstream of essential gene ERG10, PFS2, or TUB1, integrated with 4 different TIS variants. The described essential genes and promoters in the working examples and in Tables 1-2 (with corresponding SEQ ID NO’s) are the only species whose complete structure is disclosed. While the genus encompasses a large number of variants that have the same activity as ‘essential gene’ or ‘burden-sensing promoter’ in kind and the genus encompasses a large number of variants that have a different structure, the specification does not describe the complete structure of a representative number of species of the large genus of ‘essential gene’ variants or ‘burden-sensing promoter’ variants or functional equivalents thereof.
Next, then, it is determined whether a representative number of species have been sufficiently described by other relevant identifying characteristics (i.e., other than nucleotide sequence or amino acid sequence), specific features and functional attributes that would distinguish different members of the claimed genus. In the instant case, the only other identifying characteristic of an ‘essential gene’ as suitable genes encode enzymes responsible for synthesizing cell wall constituents, and preferably neither encodes a desired product to be expressed by the production strain; nor encodes a protein that facilitates synthesis of a desired product or intermediate thereof in a heterologous pathway to be expressed by the production strain; and the only other identifying characteristic of a ‘burden-sensing promoter’ is operably linked in a microbial production cell, and when cultured under production conditions as compared to a mutant derivative of said microbial production cell that synthesizes essentially none, or at least 50 % less, of the intended product of the production cell, such mutant derivative includes a non-productive escape mutant isolated following long-term cultivation of a population of cells derived from said production cell, and burden-sensing promoters may also be hybrids, scrambled or truncated versions of such natural promoters as long as such promoters still maintain the response to burden. Such broad limitations cannot be an identifying characteristic for the claimed diverse genus of ‘essential gene’ and ‘burden-sensing promoter’ since by Applicant’s definition of variant or functional equivalent thereof all members of the claimed genus will have that characteristic.
The inventions of claims 3 and 8 require the use of the inventions of claims 1-2, 4-7 and 9-10, and therefore are likewise rejected under 35 U.S.C. 112, first paragraph, as failing to comply with the written description requirement.
In conclusion, Applicant’s disclosure of the species of ‘essential gene’ and ‘burden-sensing promoter’ of the claimed broad genus is not deemed sufficient to reasonably convey to one skilled in the art that Applicant was in possession of the claimed broad genus at the time the application was filed. Thus, it is concluded that the written description requirement is not satisfied for the claimed genus.
Claim Interpretation
Claim 1 recites a first and second essential gene operably linked to a first and second burden-sensing promoter, wherein the promoter is heterologous with respect to the essential gene. Claim 4 then recites the first and second essential genes are identical; and claim 5 recites the first and second promoters are different. Thus the examiner interprets claim 1 as reciting an embodiment wherein the first and second essential genes are different and both are operably linked to a single/same burden sensing promoter, for purposes of applying prior art.
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.
Claims 1-4 and 6-10 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Danmarks Tekniske Universitet (US 20230323284 A1, filed 2/12/2021, hereinafter “Danmarks”).
The applied reference has a common joint inventor and joint assignee with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2). This rejection under 35 U.S.C. 102(a)(2) might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C. 102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B) if the same invention is not being claimed; or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed in the reference and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement.
Regarding claim 1, Danmarks teaches a microbial production cell genetically engineered to synthesize a product, said microbial cell further comprising: a. an essential gene operably linked to a burden-sensing promoter, wherein said promoter is heterologous with respect to said essential gene; wherein synthesis of the product confers a burden on said cell, and wherein expression of said essential gene is up-regulated when said burden-sensing promoter is induced by said burden relative to a basal level expression of said essential gene when said burden-sensing promoter is not induced, wherein the essential operon is folP-glmM (claims 1 and 6). The Examiner interprets instant claim 1 as reciting an embodiment wherein the first and second essential genes are different and both are operably linked to the same first and second burden sensing promoters, thus Danmarks anticipates the claim.
Regarding claim 2, Danmarks teaches said burden conferred by synthesis of said product, when said essential gene in the cell is operably linked to its native promoter, has a fitness cost measured as a percent reduction in the exponential phase growth rate of the microbial production cell selected from among ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥35% and ≥45% relative to a corresponding non-producing microbial cell, which anticipates the claim (claim 3).
Regarding claim 3, Danmarks teaches the microbial production cell is Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Propionibacterium, Bacteroides, and Bifidobacterium; or ii. a yeast belonging to a genus selected from among Saccharomyces, Kluyveromyces, Candida, Pichia, Komagataella, Cryptococcus, Debaromyces, Hansenula, Yarrowia, ZygoSaccharomyces and SchizoSaccharomyces; or iii. a filamentous fungus selected from among Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma Thermomyces, Streptomyces and Aspergillus, thus anticipates the claim (claim 4).
Regarding claim 4, Danmarks teaches the microbial production cell according to claim 1, wherein the cell is a bacterium and the essential gene is an E. coli gene or operon selected from among folP-glmM, glmM, murI, asd, thyA, usA, rpoD, nusG, rpsU, accD, degs, fldA, ftsN, hflB, lolA, mraY, mreD, murA, murB, murF, nadD, rplV and rpsG or a homologue thereof. The operon ‘folP-glmM’ as indicated in Danmarks is one ‘essential gene’ as recited in claim 6, thus the Examiner interprets instant claim 1 as reciting an embodiment wherein the first and second essential genes are identical (i.e. the same) and both are operably linked to a single/same burden sensing promoter.
Regarding claim 6, Danmarks teaches the essential gene is a non-conditional essential gene (claim 5).
Regarding claim 7, Danmarks teaches the essential gene is operably linked to a synthetic RBS whose sequence is selected to modify the translational strength of the essential gene independent of induction of said burden-sensing promoter (claim 7).
Regarding claim 8, Danmarks teaches the burden-sensing promoter is selected from among: i. a σ factor regulated promoter, such as a σ32, σB or σS factor regulated promoter, ii. a ribosomal RNA promoter, iii. an HAC1-upregulated promoter comprising a UPR element, and iv. a DNA damage-sensing promoter (claim 8).
Regarding claim 9, Danmarks teaches the cell is characterized by an increased product yield after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 generations of cell division from a single cell, as compared to a parent microbial production cell lacking said essential gene operably linked to a burden-sensing promoter (claim 9).
Regarding claim 10, Danmarks teaches the cell is characterized by an increased product yield of at least 10, 25, 50, or 80% following at least 50 generations of cell division from a single cell as compared to a parent microbial production cell lacking said essential gene operably linked to a burden-sensing promoter (claim 10).
Thus, Danmarks anticipates the claims 1-4 and 6-10.
Claims 1-3, 6-7, and 9-10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Rugbjerg et al. (PNAS, 2018, vol. 115, no. 10, pgs. 2347-2352, cited in IDS filed 1/17/2025, hereinafter “Rugbjerg”).
Regarding claims 1, 3, 6-7, Rugbjerg teaches synthetic addiction extends productive life time of engineered E. coli populations by coupling high-yield production of a desired metabolite to expression of nonconditionally essential genes, such as controlling the expression of two nonconditionally essential genes with a mevalonic acid biosensor (abstract). Rugbjerg teaches a strain obtained by recombining "pBAD-RBS-folP-glmM", which is a construct containing a promoter pBAD that depends on a mevalonate biosensor AraCmev, a ribosome binding site RBS, and essential genes folP-glmM (instant specification defined as non-conditional), with chromosomes of E. coli overexpressing the mevalonate pathway (pg. 2348, col. 2, para 3). Although Rugbjerg does not explicitly state the strain produces a ‘product’, Rugbjerg teaches that the fitness of a cell is often reduced by "product load" in a recombinant strain (pg. 2347, col. 1, para 1), thus the "product" is described as a "load" in Rugbjerg, and the promoter depending on the product biosensor is a load-sensitive promoter; therefore, "pBAD" and "folP-glmM", which corresponds to the "first burden-sensing promoter" and the "second burden-sensing promoter", and the "first essential gene" and the "second essential gene" of the invention.
Regarding claim 2, Rugbjerg teaches three clones with high fitness, e3.5, e3.5m, and e3.6, displayed addiction to L-arabinose in liquid cultures (Fig. 2B) with various degrees of system fitness cost, compared with the control strain AraC with wild-type folP-glmM promoter, and in the absence of L-arabinose, e3.5m displayed >40% reduction in average growth rate (Fig. 2B), indicating significant addiction to L-arabinose (pg. 2348, col. 2, para 2), which falls within the range recited, thus anticipating the claim.
Regarding claims 9-10, Rugbjerg teaches comparison of the addicted e3.9 strain to the nonaddicted control strain pe1 with wild-type folP-glmM promoter, and found initial production in product-addicted and nonaddicted strains was equal at, respectively, 2.9 and 3.1 g/L mevalonic acid, however the pe1 lineages gradually lost mevalonic acid production (Fig. 3B), likely due to selective enrichment of nonproducing cells in the populations, production declines corresponded to a half-life of the population productivity of 50 generations, wherein all four nonaddicted lineages, mevalonic acid production ultimately fell to below 5% within the accumulated 95 generations studied (pg. 2349, col. 2, para 2). In contrast, the four product-addicted lineages of e3.9 retained their initial low growth rates and effectively endured 95 generations without statistically significant improvements of maximum growth rates (Fig. 3C). Coherent with not gaining fitness, the four product-addicted lineages remained >95% productive at the end of the large-scale simulated fermentation at generation 95 (Fig. 3D) (pg. 2349, col. 2, para 2), which anticipates the increased product yield after 50 generations compared to the parent strain lacking the load-sensitive promoters of claim 9, and falls within the range recited in claim 10, thus anticipating the claims.
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.
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 4-5 and 8 are rejected under 35 U.S.C. 103 as being unpatentable over Rugbjerg as applied to claims 1-3, 6-7, and 9-10 above, and further in view of Shirakawa et al. (Gene, 28 (1984) 127-132, hereinafter “Shirakawa”), as evidenced by Yuan et al. (Metabolic Engineering 8 (2006) 79–90, hereinafter “Yuan”).
Regarding claims 4-5, As discussed above, Rugbjerg teaches genetically engineered cells wherein an essential gene is operably linked to a burden-sensing promoter. Rugbjerg teaches a weakness of the system is the concurrent regulation of two essential genes in an operon, which means that a single mutation in the sensor promoter could cause escape despite two actuating genes (pg. 2351 col.1, para 3). Rugbjerg does not teach the first and second essential genes are identical, nor that the burden-sensing promoters are different.
However, Shirakawa teaches a novel expression vector using the 236-bp promoter-operator fragment of the recA gene (PrecA) of E. coli and assessed for its strength by placing upstream of the cro-lacZ gene, wherein under noninducing conditions, the promoter was regulated by the LexA protein, and upon induction by nalidixic acid, high expression of B-galactosidase activity was observed for an extended period (abstract). Shirakawa teaches the recA gene product is a multi-functional protein that is essential for genetic recombination and SOS-function, and when the cell is treated with substances that damage DNA (i.e. nalidixic acid), high level of production of RecA protein takes place (pg. 128, col.1, para 1). Therefore, Shirakawa teaches the portable recA promoter allows strong expression of any gene preceded by a SD sequence (Shine-Dalgarno sequence), and induction by nalidixic acid is easy, and causes no impairment of the protein synthesis (pg. 131, col.2, para 2).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify Rugbjerg’ genetically engineer E. coli by introducing a second different heterologous promoter (PrecA) upstream of and a SD sequence preceding the folp-glmM gene, as taught by Shirakawa. One of ordinary skill in the art would have been motivated to include an additional burden-response promoter (PrecA) that is activated/induced under DNA damaging conditions, thus resulting in controlled expression of the essential gene under various altered environmental conditions, i.e. accumulated arabinose and mevalonic acid (pBAD-AraCmev) or DNA damage from nalidixic acid (PrecA). Therefore, the engineered E. coli would effectively comprise different first and second promoters that activate the same (identical) essential gene, which meets the limitations of claims 4-5.
Regarding claim 8, Rugbjerg teaches genetically engineered cells wherein essential genes are operably linked to a burden-sensing promoter (pBAD-RBS-folP-glmM). Rugbjerg does not teach the burden-sensing promoter is a DNA damage-sensing promoter.
However, Shirakawa teaches a novel expression vector using the 236-bp promoter-operator fragment of the recA gene (PrecA) of E. coli and assessed for its strength by placing upstream of the cro-lacZ gene, wherein under noninducing conditions, the promoter was regulated by the LexA protein, and upon induction by nalidixic acid, high expression of B-galactosidase activity was observed for an extended period (abstract). Shirakawa teaches the recA gene product is a multi-functional protein that is essential for genetic recombination and SOS-function, and when the cell is treated with substances that damage DNA (i.e. nalidixic acid), high level of production of RecA protein takes place (pg. 128, col.1, para 1). Therefore, Shirakawa teaches the portable recA promoter allows strong expression of any gene preceded by an SD sequence (Shine-Dalgarno sequence), and induction by nalidixic acid is easy, and causes no impairment of the protein synthesis (pg. 131, col.2, para 2). As evidenced by Yuan, endogenous promoter replacement with heterologous promoters of endogenous genes is a conventional genome engineering technique (title, pg. 83, col. 2, para 2).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date to modify the genetically engineered E. coli by replacing the endogenous promoter of “folp-glmM” with a heterologous burden-sensing promoter as taught by Rugbjerg, with the DNA damage-sensing PrecA promoter taught by Shirakawa with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to replace known promoters that are sensitive to different environmental conditions to effectively activate and express essential genes to improve fitness costs when producing a desired product as taught by Rugbjerg, since heterologous promoter replacement is a well-understood and routine practice in the art, as disclosed by Yuan.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1-4 and 6-10 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of copending Application No. 17/796285 (reference application). Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims anticipate the instant claims.
Claim 1 of ‘285 recites a microbial production cell genetically engineered to synthesize a product, said microbial cell further comprising: a. an essential gene operably linked to a burden-sensing promoter, wherein said promoter is heterologous with respect to said essential gene; wherein synthesis of the product confers a burden on said cell, and wherein expression of said essential gene is up-regulated when said burden-sensing promoter is induced by said burden relative to a basal level expression of said essential gene when said burden-sensing promoter is not induced, and claim 6 of ‘285 recites wherein the essential operon is folP-glmM. The Examiner interprets instant claim 1 as reciting an embodiment wherein the first and second essential genes are different and both are operably linked to the same first and second burden sensing promoter.
Regarding instant claim 2, claim 3 of ‘285 recites said burden conferred by synthesis of said product, when said essential gene in the cell is operably linked to its native promoter, has a fitness cost measured as a percent reduction in the exponential phase growth rate of the microbial production cell selected from among ≥5%, ≥10%, ≥15%, ≥20%, ≥25%, ≥35% and ≥45% relative to a corresponding non-producing microbial cell, which anticipates the claim.
Regarding instant claim 3, claim 4 of ‘285 recites the microbial production cell is Escherichia, Lactobacillus, Lactococcus, Corynebacterium, Bacillus, Acetobacter, Acinetobacter, Pseudomonas; Propionibacterium, Bacteroides, and Bifidobacterium; or ii. a yeast belonging to a genus selected from among Saccharomyces, Kluyveromyces, Candida, Pichia, Komagataella, Cryptococcus, Debaromyces, Hansenula, Yarrowia, ZygoSaccharomyces and SchizoSaccharomyces; or iii. a filamentous fungus selected from among Penicillium, Rhizopus, Fusarium, Fusidium, Gibberella, Mucor, Mortierella, Trichoderma Thermomyces, Streptomyces and Aspergillus, thus anticipates the claim.
Regarding instant claim 4, claim 6 of ‘285 recites the microbial production cell according to claim 1, wherein the cell is a bacterium and the essential gene is an E. coli gene or operon selected from among folP-glmM, glmM, murI, asd, thyA, usA, rpoD, nusG, rpsU, accD, degs, fldA, ftsN, hflB, lolA, mraY, mreD, murA, murB, murF, nadD, rplV and rpsG or a homologue thereof. The operon ‘folP-glmM’ as indicated in ‘285 is one ‘essential gene’ as recited in claim 6, thus the Examiner interprets instant claim 1 as reciting an embodiment wherein the first and second essential genes are identical (i.e. the same) and both are operably linked to a single/same burden sensing promoter, thus anticipates the claim.
Regarding instant claim 6, claim 5 of ‘285 recites the essential gene is a non-conditional essential gene.
Regarding instant claim 7, claim 7 of ‘285 recites the essential gene is operably linked to a synthetic RBS whose sequence is selected to modify the translational strength of the essential gene independent of induction of said burden-sensing promoter.
Regarding claim 8, claim 8 of ‘285 recites the burden-sensing promoter is selected from among: i. a σ factor regulated promoter, such as a σ32, σB or σS factor regulated promoter, ii. a ribosomal RNA promoter, iii. an HAC1-upregulated promoter comprising a UPR element, and iv. a DNA damage-sensing promoter.
Regarding instant claim 9, claim 9 of ‘285 recites the cell is characterized by an increased product yield after at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 generations of cell division from a single cell, as compared to a parent microbial production cell lacking said essential gene operably linked to a burden-sensing promoter.
Regarding instant claim 10, claim 10 of ‘285 recites the cell is characterized by an increased product yield of at least 10, 25, 50, or 80% following at least 50 generations of cell division from a single cell as compared to a parent microbial production cell lacking said essential gene operably linked to a burden-sensing promoter.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Claims 1-10 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-10 of copending Application No. 17/796285 in view of Rugbjerg and Shirakawa, as evidenced by Yuan.
Claims 1-10 of ‘285 recite an obvious variation of instant claims 1-4 and 6-10.
Regarding instant claim 5, ‘285 does not recite a first and second burden-sensing promoter that are different from one another.
However, Rugbjerg teaches genetically engineered cells wherein an essential gene is operably linked to a burden-sensing promoter. Rugbjerg teaches a weakness of the system is the concurrent regulation of two essential genes in an operon, which means that a single mutation in the sensor promoter could cause escape despite two actuating genes (pg. 2351 col.1, para 3).
Shirakawa teaches a novel expression vector using the 236-bp promoter-operator fragment of the recA gene (PrecA) of E. coli and assessed for its strength by placing upstream of the cro-lacZ gene, wherein under noninducing conditions, the promoter was regulated by the LexA protein, and upon induction by nalidixic acid, high expression of B-galactosidase activity was observed for an extended period (abstract). Shirakawa teaches the recA gene product is a multi-functional protein that is essential for genetic recombination and SOS-function, and when the cell is treated with substances that damage DNA (i.e. nalidixic acid), high level of production of RecA protein takes place (pg. 128, col.1, para 1). Therefore, Shirakawa teaches the portable recA promoter allows strong expression of any gene preceded by an SD sequence (Shine-Dalgarno sequence), and induction by nalidixic acid is easy, and causes no impairment of the protein synthesis (pg. 131, col.2, para 2).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to genetically engineer an E. coli with a heterologous promoter operably linked to an essential gene (folp-glmM) as recited in ‘285, and modify the chromosome by introducing a second different heterologous promoter (PrecA) upstream of the folp-glmM gene modified with a SD sequence as taught by Shirakawa with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to include an additional burden-response promoter (PrecA) that is activated/induced under DNA damaging conditions, thus resulting in controlling expression of the essential gene under various altered environmental conditions, as well as reduce weakness of the system with the concurrent regulation of two essential genes in an operon, which reduces risk of a single mutation in the sensor promoter that could cause escape despite two actuating genes as taught by Rugbjerg. Therefore, the engineered E. coli would effectively comprise different first and second promoters that activate the essential gene, which meets the limitation of claim 5.
This is a provisional nonstatutory double patenting rejection.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657
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