DETAILED OFFICE 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 .
Claims Status
Applicant’s amendment to the claims, filed on November 18, 2025, is
acknowledged. This listing of the claims replaces all prior versions and listings of the
claims.
Claims 11-12 and 16-24 are pending and are being examined on the merits.
Claims 1-10 are cancelled.
Applicant’s remarks and declaration under 37 CFR 1.132 filed November 18, 2025 in response to the non-final rejection mailed May 20, 2025, are acknowledged and have been fully considered.
Previous rejections of claims 13-15 are withdrawn in view of the instant amendment to cancel these claims.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Election/Restrictions
Applicants’ election, filed 4/22/2025, electing Group II without traverse and election of the species (see below) without traverse is acknowledged.
The elected species are:
Species Group 1: Escherichia
Species Group 2: folP-glM
Species Group 3: a σ factor regulated promoter, such as σ32, σB, or σS factor regulated promoter.
Species Group 4: Human Growth hormone.
Claims 11-12 and 16-24 and elected species are under examination.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 3/11/2026 is in compliance, except where references are lined through, with the provisions of 37 CFR 1.97. Accordingly, the IDS has been considered by the examiner and those references therein have been indicated as such.
Claim Objections
Claims 11, 16, and 21 are newly objected to because of the following informalities:
Claim 11 is objected to because of the recitation of “introducing the cell comprising a substrate for the production of said product” in step ii). In the interest of improving claim form, the Office suggests amending the recited phrase to “introducing a substate substrate to the cell for the production of said product.”
Claim 16 is objected to because of the recitation of “cell comprises” in line 1. In the interest of improving claim form, the Office suggests amending the recited phrase to “cell further comprises.”
Claim 21 is objected to because of the recitation of “RBS” without first writing out the full phrase for which the abbreviation “RBS” is used. In the interest of improving claim form, the Office suggests amending “RBS” to “ribosomal binding site (RBS)”.
Claim Rejections - 35 USC § 102
The rejection of claim 11 under 35 U.S.C. 102(a)(1) as being anticipated by Rugbjerg et al. (Synthetic addiction extends the productive life time of engineered Escherichia coli populations, PNAS, published March 6, 2018, Vol. 115, No. 10, p. 2347-2352; cited on the IDS from 9/20/2022; hereafter “Rugbjerg”) is withdrawn because the Rugbjerg reference does not teach the newly added limitation of “wherein the burden-sensing promoter is not directly induced by the product itself.”
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.
Claim 22 is rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 22 is indefinite because of the use of exemplary claim language in the phrase “such as” in line 2. The phrase “such as” renders the claims indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. Description of examples or preferences is properly set forth in the specification rather than the claims. If stated in the claims, examples and preferences may lead to confusion over the intended scope of a claim. See MPEP § 2173.05(d). For the purpose of compact prosecution, the limitations following “such as” are interpreted as optional limitations.
Claim Rejections - 35 USC § 103
The previous rejection of claim 12 under 35 U.S.C. 103 as being unpatentable over Rugbjerg, in view of Champion et al. (Proteomics, published 2003, Vol. 13, p. 1365-1373; cited on the Form PTO-892 mailed May 20, 2025; hereafter “Champion”), Rodrigues et al. (Biochemical Engineering Journal, published 2017, Vol. 125, p. 41-49; cited on the Form PTO-892 mailed May 20, 2025; hereafter “Rodrigues”), and Kim et al. (PLOS ONE, published 2013, Vol. 8, No. 1; cited on the Form PTO-892 mailed May 20, 2025; hereafter “Kim”) is withdrawn because the recited references do not teach the newly added limitation to claim 11 “wherein the burden-sensing promoter is not directly induced by the product itself.”.
Claims 11-12 and 16-24 are newly rejected under 35 U.S.C. 103 as being unpatentable over Rugbjerg in view of Champion, Rodrigues, Kim, and Tao et al. (Front. Microbiol., published 2015, Vol. 6, No. 2; cited on the attached Form PTO-892; hereafter “Tao”).
Regarding claim 11 step i) and claims 12, 16, 18, 20, and 22, Rugbjerg teaches that microbial cells engineered for chemical synthesis and natural extraction frequently exhibit reduced cell fitness due to the loads of production (p. 2347, col 1, para 1; meeting claim limitations of independent claim 11). Production loads typically arise from metabolic depletions in addition to toxicities from pathway intermediates and end products (p. 2347, col 1, para 1).
Rugbjerg teaches the synthetically addicted Escherichia coli (abbreviated E. coli) production cells limit the metabolic burden associated with high-yield bioproduction of metabolites (Abstract; p. 2347, col 2, box labeled “Significance”; p. 2358, col 2, para 2). The E. coli engineered to produce mevalonic acid utilize a product-responsive pBAD promoter that controls the expression of two essential genes (folp and glmM) (Abstract; p. 2348, col 1, para 2 – p. 2350, col 1, para 1; p. 2352, col 1, para 2; Figure 2 – see image below).
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Rugbjerg teaches that AraC can act as both a repressor and an activator as well as that L-arabinose induces AraC (p. 2350, col 1, para 5; p. 2348, col 2, para 2). The precursor cells employing L-arabinose sensitive AraC developed by Rugbjerg were cultured in the presence of the AraC inducer L-arabinose (p. 2347, col, 2 para 2; p. 2353, col 1, para 2). Rugbjerg then modified the AraC to be mevalonic acid-responsive (AraCmev) (p. 2348, col 2, para 3). An ordinary artisan before the effective filing date would have immediately interpreted the production systems depicted in Figure 2, using the arabinose-responsive AraC or the mevalonic acid-responsive biosensor AraCmev, used by Rugbjerg activate the essential genes in the system.
Regarding claim 11 step ii), the single colonies of cells are cultured in M9 medium (p. 2348, col 2, para 3; p. 2351, col 2, para 5; p. 2352, col 1, para 3). An ordinary artisan before the effective filing date would have immediately recognized the single colonies used to culture the strains taught by Rugbjerg comprises production cells arising from a single cell.
Regarding claim 11 step iii), the mevalonic acid produced by the production cell produced was quantified via HPLC (p. 2352, col 2, para 2).
Regarding claim 19, Rugbjerg teaches the essential genes, folp and glmM, are non-conditionally essential genes (Abstract; p. 2348, col 1, para 2).
Regarding claim 21, Rugbjerg teaches introducing four ribosome-binding sequences (RBSs) with different RBS strengths into the operon that regulate the expression of the essential genes (p. 2348, col 2, para 2-3; Figure 2).
Regarding claims 23-24, Rugbjerg teaches the mevalonic product-addicted production organism retains high-yield (>95%) mevalonic acid production through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, at which time the control nonaddicted strain exhibits severely diminished production (<5%) (Abstract; p. 2349, col 1, para 2 - p. 2349, col 2, para 2; Figure 3; Table 3; Table S4).
Rugbjerg does not explicitly teach or suggest wherein the AraCmev controlled pbad-folP-glmM system comprises a burden-sensing promoter that is not directly induced by the product itself. Regarding claim 12, Rugbjerg does not teach or suggest production of human growth hormone as a product.
Champion teaches E. coli cells producing human growth hormone (hGH) increase expression of inclusion body binding protein A (IbpA) six-fold (Abstract; p. 1366, col 1, para 2; p. 1370, col 1, para 2; Figures 1-2). IbpA is a heat shock protein shown to bind insoluble heterologous proteins that accumulate in the cytoplasm (p. 1370, col 1, para 2). Since 10% of recombinant hGH typically accumulates as a cytoplasmic precursor, this accumulation is likely to trigger overexpression of IbpA (p. 1370, col 1, para 2). Champion teaches recombinant hGH is an FDA approved replacement for endogenous growth hormone for patients with growth hormone deficiency (p. 1365, col 2, para 2 – p. 1366, col 1, para 1).
Kim teaches high-level expression of recombinant human growth hormone (hGH) in E. coli leads to the formation of insoluble aggregates as inclusion bodies (Abstract). Kim teaches cloning the human growth hormone (hGH) gene into an IPTG inducible pET-28a plasmid and recombinantly expressing hGH in E. coli transformed with the plasmid (p. 1, col 2, para 3 – p. 2, col 1, para 1). Kim teaches a method to recover and purify active recombinant human growth hormone (hGH) expressed in E. coli (Abstract; p. 1, col 1, para 1 - p. 1, col 2, para 2; p. 2, col 1, para 3 – p. 2, col 2, para 1).
Rodrigues teaches the use of an ibpA promoter (hereafter “pibpA”) to induce gene expression in E. coli (Abstract; p. 46, col 1, para 1). Rodrigues also teaches production of heterologous proteins causes metabolic stress (p. 47, col 1, para 1).
Tao teaches wherein the expression of ibpA is regulated by the σ32 protein (p. 8, col 1, para 2).
In view of the combined teachings of Rugbjerg, Champion, Rodrigues, Kim, and Tao, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the pBAD promoter that regulates expression of the essential genes folP and glmM taught by Rugbjerg with the ibpA promoter (pibpA) taught by Rodrigues, use the modified pibpA-folP-glmM system in the E. coli that Kim taught to overexpress recombinant hGH, to recover the hormone; thereby arriving at the invention of claims 11-12, 16, and 18-24.
Rugbjerg taught the use of a pBAD promoter to regulate the expression of folP and glmM in E. coli cells reduces burden and improves long-term product production in cells. Champion taught the heat shock protein ibpA binds insoluble heterologous proteins in the cytoplasm, hGH generating E. coli cells exhibit increased ibpA levels, and that recombinant expression of hGH accumulation in the cytoplasm is likely to trigger the overexpression ibpA. A simple substation of pBAD in the pBAD-folP-glmM system taught by Rugbjerg for the ibpA promoter (pibpA) and inclusion of the operon in the hGH producing cell of Kim would permit regulation the expression of the folP and glmM based on the burden associated with the accumulation of recombinant hGH and its propensity to aggregate, which would resultingly increase the productive lifespan of the cell and allow high-capacity production of hGH over many generations.
An ordinary artisan would have had a reasonable expectation of success of recovering recombinant hGH from E. coli because Kim taught recovering and purifying recombinant hGH from E. coli. An ordinary artisan would have had a reasonable expectation of success of substituting pBAD with pibpA because they are both are promoters and Rodrigues taught the use of pibpA to control heterologous protein expression.
Regarding claim 17, since the combination of Rugbjerg, Champion, Rodrigues, Kim, and Tao teaches and/or suggests the actively recited process steps of claim 11, it is the Examiner’s position that the 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 required in claim 17 would inherently result from the method taught and/or suggested by the combination of Rugbjerg, Champion, Rodrigues, Kim, and Tao. Since the Office does not have the facilities for examining and comparing applicant’s method wherein said burden conferred by synthesis of said product has a fitness cost measured as a percent reduction in the exponent, the burden is on the applicant to show a novel or unobvious difference between the recited product and the recited method of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594. As a result, the combination of Rugbjerg, Champion, Rodrigues, and Kim as described above arrive at the invention of claim 17.
Consequently, the invention of claims 11-12 and 16-24 would have been obvious to one of ordinary skill in the art before the effective filing date.
Double Patenting Rejections
Claims 11-12, 17-19, 21, and 23-24 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 6-7, 9-11, 13, and 16 of copending Application No. 18/683,429 (hereafter “app’429”).
Regarding instant claim 11, claim 1 of app’429 recites a microbial production cell genetically engineered to synthesize a product, said microbial cell further comprising: a. a first essential gene operably linked to a first burden-sensing promoter, and b. a second essential gene operatively linked to a second burden-sensing promoter, wherein said first burden-sensing promoter is heterologous with respect to said first essential gene, and said second burden-sensing promoter is heterologous with respect to said second essential gene; wherein synthesis of the product confers a burden on said cell, and wherein expression of said first essential genes is up-regulated when said first burden- sensing promoter is induced by said burden relative to a basal level expression of said first essential gene when said first burden-sensing promoter is not induced, and expression of said second essential gene is up-regulated when said second burden-sensing promoter is induced by said burden relative to a basal level expression of said second essential gene when said second burden-sensing promoter is not induced. Claim 11 of app’429 recites a method of product biosynthesis comprising the steps of: i. providing at least one microbial production cell according to any one of claim 1, ii. introducing the at least one cell into a cultivation medium comprising substrate for production of said product, iii. recovering said product.
Regarding instant claim 12, claim 13 of app’429 recites a method of product biosynthesis according to claim 11 wherein the product is selected from among: an organic acid, terpenoid, isoprenoid, polyketide, alcohol, sugar, vitamin, aldehyde, carboxylic acid, fatty acid, amino acid, peptide, enzyme (such as an amylase, lipase, protease, barnase, 3-galactosidase, crystal protein, cutinase, PETase, laccase and carbohydrate active enzyme (such as a xylanase, lichenase, cellulase, lytic polysaccharide monooxoygenase, and pectase)), a therapeutic protein and a precursor thereof (such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal- and polyclonal-antibody, single-fragment antibody and nanobody),a protein naturally found in eggs (such as ovalbumin), a milk protein (such as casein, lactadherin, lactoferrin), secreted immunoglobulin A and G, secretory components. Claim 16 of app’429 recites the use of a microbial production cell according to any one of claim 1 for producing a biosynthetic product, such as organic acids, terpenoids, isoprenoids, polyketides, alcohols, sugars, vitamins, aldehydes, carboxylic acids, fatty acids, amino acids, peptides, enzymes (such as amylases, lipases, proteases, barnases, 3- galactosidases, crystal proteins, cutinases, PETases, laccases and carbohydrate active enzymes (such as xylanases, lichenases, cellulases, lytic polysaccharidemonooxoygenases, and pectases)), therapeutic proteins and precursors thereof (such as human growth hormone, insulin, glucagon-like peptide-1, monoclonal and polyclonal antibodies, single-fragment antibodies), proteins naturally found in eggs (such as ovalbumin), milk proteins (such as caseins, lactadherins, lactoferrin), secreted immunoglobulin A and G, secretory components, nanobodies.
Regarding instant claim 17, claim 2 of app’429 recites microbial production cell according to claim 1, wherein said burden conferred by synthesis of said product, when said first and second essential gene in the cell are operably linked to their native promoters, has a fitness cost measured as a percent reduction in the maximum exponential phase growth rate of the microbial production cell selected from among >5 %, >10%, >15%, >20%, >25%, >35% and >45 % measured relative to a corresponding non-producing microbial cell.
Regarding instant claim 18, claim 3 of app’429 recites a microbial production cell according to claim 1, wherein the cell is:
a bacterium belonging to a genus selected from among 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.
Regarding instant claim 19, claim 6 of app’429 recites a microbial production cell according to claim 1, wherein the first and/or second essential gene is a non-conditional essential gene.
Regarding instant claim 21, claim 7 of app’429 recites a microbial production cell according to claim 1, wherein the first and/or second essential gene is operably linked to a synthetic RBS whose sequence is selected to modify the translational strength of the first and/or second essential gene independent of induction of said first and/or second burden-sensing promoter, respectively.
Regarding instant claim 23, claim 9 of app’429 recites a microbial production cell according to claim 1, wherein 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 first and second essential genes operably linked to said first and second burden-sensing promoters, respectively.
Regarding instant claim 24, claim 10 of app’429 recites a microbial production cell according to claim 1, wherein 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 first and second essential genes operably linked to said first and second burden-sensing promoters, respectively.
For the reasons stated herein, claims 11-12, 17-19, 21, and 23-24 of this application are unpatentable over claims 1-3, 6-7, 9-11, 13, and 16 of app’429.
Claim 20 is newly provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 6-7, 9-11, 13, and 16 of app’429 in view of Rugbjerg.
Regarding claim 20, the claims of app’429 do not explicitly recite wherein the microbial production cell is a bacterial and the essential gene is an E. coli gene or operon selected from among folP-glmM, glmM, mur1, asd, thyA, usA, rpoD, nusG, rpsU, accD, degS, fldA, ftsN, hfl, lolA, mraY, mreD, murA, murB, murF, nadD, rplV and rpsG or a homologue thereof.
Rugbjerg teaches that microbial cells engineered for chemical synthesis and natural extraction frequently exhibit reduced cell fitness due to the loads of production (p. 2347, col 1, para 1; meeting claim limitations of independent claim 11). Production loads typically arise from metabolic depletions in addition to toxicities from pathway intermediates and end products (p. 2347, col 1, para 1).
Rugbjerg teaches the synthetically addicted Escherichia coli (abbreviated E. coli) production cells limit the metabolic burden associated with high-yield bioproduction of metabolites (Abstract; p. 2347, col 2, box labeled “Significance”; p. 2358, col 2, para 2). The E. coli engineered to produce mevalonic acid utilize a product-responsive pBAD promoter that controls the expression of two essential genes (folp and glmM) (Abstract; p. 2348, col 1, para 2 – p. 2350, col 1, para 1; p. 2352, col 1, para 2; Figure 2 – see image below).
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Rugbjerg teaches that AraC can act as both a repressor and an activator as well as that L-arabinose induces AraC (p. 2350, col 1, para 5; p. 2348, col 2, para 2). The precursor cells employing L-arabinose sensitive AraC developed by Rugbjerg were cultured in the presence of the AraC inducer L-arabinose (p. 2347, col, 2 para 2; p. 2353, col 1, para 2). Rugbjerg then modified the AraC to be mevalonic acid-responsive (AraCmev) (p. 2348, col 2, para 3). An ordinary artisan before the effective filing date would have immediately interpreted the production systems depicted in Figure 2, using the arabinose-responsive AraC or the mevalonic acid-responsive biosensor AraCmev, used by Rugbjerg activate the essential genes in the system.
Rugbjerg teaches the mevalonic product-addicted production organism retains high-yield (>95%) mevalonic acid production through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, at which time the control nonaddicted strain exhibits severely diminished production (<5%) (Abstract; p. 2349, col 1, para 2 - p. 2349, col 2, para 2; Figure 3; Table 3; Table S4).
In view of the combination of claims 1-3, 6-7, 9-11, 13, and 16 of app’429 and Rugbjerg, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to substitute the first essential gene in the microbial production cell of the claims of app’429 for folP-glmM taught by Rugbjerg, thereby arriving at the invention of instant claim 20.
An ordinary artisan would have been motivated and would have had a reasonable expectation of success of substituting the first essential gene in the microbial production cell of app’429 for folP-glmM because Rugbjerg taught the regulated expression of folP-glmM in Escherichia coli engineered to produce a product of interest allowed the high production of the product of interest over 95 generations whereas control cells exhibited severely diminished production.
For the reasons stated herein, claim 20 of this application is unpatentable over
the combination of claims 1-3, 6-7, 9-11, 13, and 16 of app’429 and Rugbjerg.
Claim 22 is newly provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-3, 6-7, 9-11, 13, and 16 of app’429 in view of Rugbjerg, Champion, Rodrigues, Kim, and Tao.
Regarding claim 22, claims 1-3, 6-7, 9-11, 13, and 16 of app’429 do not recite wherein the promoter is a σ factor regulated promoter.
Champion teaches E. coli cells producing human growth hormone (hGH) increase expression of inclusion body binding protein A (IbpA) six-fold (Abstract; p. 1366, col 1, para 2; p. 1370, col 1, para 2; Figures 1-2). IbpA is a heat shock protein shown to bind insoluble heterologous proteins that accumulate in the cytoplasm (p. 1370, col 1, para 2). Since 10% of recombinant hGH typically accumulates as a cytoplasmic precursor, this accumulation is likely to trigger overexpression of IbpA (p. 1370, col 1, para 2). Champion teaches recombinant hGH is an FDA approved replacement for endogenous growth hormone for patients with growth hormone deficiency (p. 1365, col 2, para 2 – p. 1366, col 1, para 1).
Kim teaches high-level expression of recombinant human growth hormone (hGH) in E. coli leads to the formation of insoluble aggregates as inclusion bodies (Abstract). Kim teaches cloning the human growth hormone (hGH) gene into an IPTG inducible pET-28a plasmid and recombinantly expressing hGH in E. coli transformed with the plasmid (p. 1, col 2, para 3 – p. 2, col 1, para 1). Kim teaches a method to recover and purify active recombinant human growth hormone (hGH) expressed in E. coli (Abstract; p. 1, col 1, para 1 - p. 1, col 2, para 2; p. 2, col 1, para 3 – p. 2, col 2, para 1).
Rodrigues teaches the use of an ibpA promoter (hereafter “pibpA”) to induce gene expression in E. coli (Abstract; p. 46, col 1, para 1). Rodrigues also teaches production of heterologous proteins causes metabolic stress (p. 47, col 1, para 1).
Tao teaches wherein the expression of ibpA is regulated by the σ32 protein (p. 8, col 1, para 2).
In view of the combination of claims 1-3, 6-7, 9-11, 13, and 16 of app’429 and the teachings of Rugbjerg, Champion, Rodrigues, Kim, and Tao, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the first promoter that regulates expression of the essential folP-glmM taught by the combination of app’429 and Rugbjerg with the ibpA promoter (pibpA) taught by Rodrigues, use the modified pibpA-folP-glmM system in the E. coli that Kim taught to overexpress and recover recombinant hGH, thereby arriving at the invention of instant claim 22.
An ordinary artisan would have been motivated to and would have had a reasonable expectation of success producing and recovering hGH recombinantly expressed in E. coli using an IPTG inducible pET-28a plasmid as taught by Kim because Champion taught recombinant hGH is an approved therapy for growth hormone deficiency. An ordinary artisan would have been motivated to and would have had a reasonable expectation of success modifying the first promoter that regulates expression of the essential folP-glmM taught by the combination of app’429 and Rugbjerg with the ibpA promoter (pibpA) taught by Rodrigues, use the modified pibpA-folP-glmM system in the E. coli that Kim taught to overexpress and recover recombinant hGH in order to maintain high expression of hGH over many generations. Champion taught the heat shock protein ibpA binds insoluble heterologous proteins in the cytoplasm, hGH generating E. coli cells exhibit increased ibpA levels, and that recombinant expression of hGH accumulation in the cytoplasm is likely to trigger the overexpression ibpA. A simple substation of the promtoer in the folP-glmM system taught by the combination of app’429 and Rugbjerg for the ibpA promoter (pibpA) and inclusion of the operon in the hGH producing cell of Kim would permit regulation the expression of the folP and glmM based on the burden associated with the accumulation of recombinant hGH and its propensity to aggregate, which would resultingly increase the productive lifespan of the cell and allow high-capacity production of hGH over many generations. Tao taught the expression of ibpA is regulated by the σ32 protein.
For the reasons stated herein, claim 22 of this application is unpatentable over the combination of claims 1-3, 6-7, 9-11, 13, and 16 of app’429, Rugbjerg, Champion, Rodrigues, Kim, and Tao.
Applicant’s Arguments and Examiner’s Response
ARGUMENT: Applicants submit AraC blocks access of RNA polymerases to the heterologous promoter (e.g., PBAD) controlling the downstream gene and thereby
blocks transcription.
RESPONSE TO REMARKS: Applicant’s arguments are not found persuasive. Rugbjerg teaches that AraC can act as both a repressor and an activator as well as that L-arabinose induces AraC (p. 2350, col 1, para 5; p. 2348, col 2, para 2). The precursor cells employing L-arabinose sensitive AraC developed by Rugbjerg were cultured in the presence of the AraC inducer L-arabinose (p. 2347, col, 2 para 2; p. 2353, col 1, para 2). Rugbjerg then modified the AraC to a mevalonic acid-responsive AraCmev (p. 2348, col 2, para 3). An ordinary artisan would immediately interpret the production systems depicted in Figure 2, using the arabinose-responsive AraC or the mevalonic acid-responsive biosensor AraCmev, used by Rugbjerg activate the essential genes in the system.
ARGUMENT: Applicants submit that the secondary references (e.g., Champion, Rodrigues, and Kim) do not relate to the teachings of Rugbjerg as well as an ordinary artisan would not have been motivated to substitute pibpA -for pBAD. Applicants submit the teachings of Rodrigues suggest that 1) the use of pibpA would inherently impose a basal fitness cost on the production cells due to misfolding of the protein because of an elevated induction temperature and 2) the elevated induction temperature regulation would be incompatible with industrial fermentation, citing to the declaration under 37 CFR 1.132 to support these arguments.
RESPONSE TO REMARKS: Applicant’s arguments and declaration under 37 CFR 1.132 are not found persuasive. For the reasons set forth in the modified and new rejection above, it would have been obvious to one of ordinary skill in the art before the effective filing date to modify the promoter regulating the expression of two essential genes (folp and glmM) coli taught by Rugbjerg by substituting the pBAD for pibpA and using said operon in the hGH producing E. coli cell taught by Kim to improve the long-term production hGH. The increased productive lifespan of the modified cells would be desirable to obtain hGH to treat patients with hGH deficiency.
Furthermore, Rodrigues does not teach wherein the induction of pibpA requires high temperatures, but rather as it uses the well-known in the prior art condition of elevated temperatures to induce pibpA. However, Champion taught E. coli cells producing human growth hormone (hGH) increase the expression of inclusion body binding protein A (IbpA) six-fold (Abstract; p. 1366, col 1, para 2; p. 1370, col 1, para 2; Figures 1-2). Since 10% of recombinant hGH typically accumulates as a cytoplasmic precursor, this accumulation is likely to trigger overexpression of IbpA (p. 1370, col 1, para 2). The system taught and/or suggested by the combination of Rugbjerg, Champion, Rodrigues, Kim, and Tao would utilize the inherent burden of the aggregated proteins in the form of inclusion bodies and the concomitant induced expression of IbpA to regulate the expression of the essential genes (folp and glmM) in order to improve the long-term production of hGH by operably linking the expression of the essential genes to the pibpA. The induction culture conditions used by Kim to improve the solubility of hGH in the E. coli cells (e.g., a lower induction temperature of 16 °C or reduced IPTG concentration), while it maintains efficient expression does not solubilize all hGH in the cells (p. 3, col 1, para 1 - p. 4, col 2, para 1; Figure 1). Therefore, the presence of insoluble hGH in the method of Kim would still be expected to induce to induce pibpA, there is no elevated induction temperature that would increase protein misfolding which would inherently result in an increased burden or fitness cost, nor is there an elevated temperature regulation incompatible with industrial fermentation.
Conclusion
No claims are currently allowed for the reasons as stated above. Applicants must
respond to the objections/rejections in this Office action to be fully responsive in
prosecution.
The instant Office Action is non-final.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to SCOTT E. MULDER whose telephone number is (571)272-2372. The examiner can normally be reached Monday - Friday 7:30 AM - 3:30 PM.
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/SCOTT E. MULDER/Examiner, Art Unit 1656
/David Steadman/Primary Examiner, Art Unit 1656
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