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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/27/2025 has been entered.
Withdrawal of Rejections
The response and amendments filed on 10/27/2025 are acknowledged. Any previously applied minor objections and/or minor rejections (i.e., formal matters), not explicitly restated here for brevity, have been withdrawn necessitated by Applicant’s formality corrections and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, or maintaining, if applicable, of the substantive or essential claim rejections are detailed directly below and/or in the Examiner’s Response to Arguments section.
Briefly, the previous claim rejections under 35 U.S.C. 112(b) for indefiniteness have been withdrawn necessitated by Applicant’s amendments; however, new grounds of rejection are set forth below. The previous claim rejections under 35 U.S.C. 103 for obviousness have been withdrawn necessitated by Applicant’s amendments; however, new grounds of rejection are set forth below.
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set presently being applied to the instant application.
Claim Objections
Claim 54 is objected to because of the following informalities: “E. coli” should be spelled out completely upon first use and should instead be “Escherichia coli”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b), Indefiniteness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 54, 56-58, 61-62, 65-70, and 107-114 are 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 (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 54 recites “a suitable culture medium”; however, what constitutes “a suitable culture medium” is not defined in the instant Specification. Moreover, a “suitable culture medium” is considered a relative term and one of ordinary skill in the art would not readily understand that this means in light of the specification (see, e.g., MPEP 2173.05(b)). The instant specification merely states that the culture medium, in certain embodiments, comprises glucose (see, e.g., instant specification, pg. 6, line 26). Moreover, Example 13 describes growth of bacterial isolates in LB broth containing 1% glucuronate, 100 µg/ml carbenicillin and 50 µg/ml kanamycin (see, e.g., instant specification, Example 13, pg. 48, lines 9-10; however, this is not described to be a ”suitable culture medium”; therefore, it is unclear if this is what constitutes a “suitable culture medium”, or if something else comprises a “suitable culture medium”. For the purposes of applying prior art, the Examiner has interpreted “a suitable culture medium” to be any culture medium that Escherichia coli grows in.
Claims 56-58, 61-62, 65-70, and 107-114 are included in this rejection for depending on independent claim 54 and failing to rectify the noted deficiencies.
Claim Rejections - 35 USC § 103, Obviousness
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 54, 61-62, 110, and 112-114 are rejected under 35 U.S.C. 103 as being unpatentable over Miyake (US 2005/0054042; Date of Publication: March 10, 2005 – previously cited) in view of Sutter (Key Enzymes of the Semiphosphorylative Entner-Doudoroff Pathway in the Haloarchaeon Haloferax volcanii: Characterization of Glucose Dehydrogenase, Gluconate Dehydratase, and 2-Keto-3-Deoxy-6-Phosphogluconate Aldolase; 2016 – newly cited), Liang (Comparison of individual component deletions in a glucose-specific phosphotransferase system revealed their different applications; 2015 – newly cited), Bausch (Sequence analysis of the GntII (subsidiary) system for gluconate metabolism reveals a novel pathway for L-idonic acid catabolism in Escherichia coli; 1998 – newly cited), and Diaz (Deletion of four genes in Escherichia coli enables preferential consumption of xylose and secretion of glucose; 2019 – newly cited).
Miyake’s general disclosure relates to “a novel gluconate dehydratase capable of efficiently producing a 2-keto-3-deoxyaldonic acid from aldonic acid” (see, e.g., Miyake, [0002]). Moreover, Miyake discloses “a base sequence encoding the gluconate dehydratase, a plasmid containing the base sequence, and a cell into which a host cell is transformed with the plasmid. The base sequence is advantageously used in productions of the gluconate dehydratase and of 2-keto-3-deoxyaldonic acids using the gluconate dehydratase” (see, e.g., Miyake, [0008]). Additionally, Miyake discloses an E. coli host cell with a plasmid containing a gluconate dehydratase gene derived from Achromobacter xylosoxidans (see, e.g., Miyake, [0017]-[0025]).
Regarding claim 54 pertaining to a method of producing 2-keto-3-deoxy gluconate (KDG), Miyake teaches in Examples 6-8 culturing an Escherichia coli host cell transformed with a gluconate dehydratase from Achromobacter xylosoxidans in a LB liquid medium containing 50 µg/mL of ampicillin at 37oC overnight (see, e.g., Miyake, Examples 6-8, [0076]-[0081]). Moreover, Miyake teaches that the encoded gluconate dehydratase enzyme (see, e.g., Miyake, SEQ ID NO: 2) has 95.9% sequence identity to SEQ ID NO: 1, as taught in the instant application (see, e.g., Office Action Appendix). Miyake teaches that expression of gluconate dehydratase results in expression of KDG (see, e.g., Miyake, [0033]); therefore, one of ordinary skill in the art would readily assume that the production of KDG would occur during the culturing process. Moreover, it would be inherent that expression of the heterologous gluconate dehydratase in the E. coli cell would result in production of KDG during the culturing process. Miyake teaches the same method of producing KDG through expression of a heterologous gluconate dehydratase enzyme with at least 95% identity to instant SEQ ID NO: 1; therefore, this would inherently lead to production of KDG by the E. coli cell during processing.
Regarding claims 61-62 pertaining to purifying KDG from cell culture and supernatant, Miyake teaches that the KDG was derived from Achromobacter xylosoxidans (grown in culture) by crushing the cells with an ultrasonic cell crusher to prepare a crude enzyme liquid (see, e.g., Miyake, [0067]). Miyake further teaches that the supernatant of the crude enzyme liquid was filtered through an ultrafilter membrane, which allows for purification (see, e.g., Miyake, [0068]). Therefore, the KDG, which was derived from cell culture, was purified by passing the supernatant of the crude enzyme liquid through an ultrafilter membrane for purification.
Regarding claim 110 pertaining to the pH, Miyake teaches that the “pH of the reaction is set in a range in which the gluconate dehydratase can maintain the activity, and preferably in the range of 7 to 9” (see, e.g., Miyake, [0046]).
Regarding claims 113-114 pertaining to the culturing temperature, Miyake teaches an Escherichia coli host cell transformed with a gluconate dehydratase from Achromobacter xylosoxidans in a LB liquid medium containing 50 µg/mL of ampicillin was cultured at 37oC overnight (see, e.g., Miyake, Examples 6-8, [0076]-[0081]).
However, Miyake does not teach: genetic modifications resulting in decrease or elimination of: (A) glucose assimilation; (B) gluconate-6-phosphate production from gluconate; and (C) glucose-6-phosphate production from glucose (claim 54(ii)).
Liang’s general disclosure relates to studying the effect of a single glucose-specific phosphotransferase system mutation on cell growth and substrate consumption by knocking out the genes involved in the phosphotransfer cascade of the glucose-specific phosphotransferase system (see, e.g., Liang, abstract). Moreover, Liang discloses “Numerous studies have used approaches including inactivating the PTSGlc components for eliminating CCR in E. coli7,8. The ptsG gene has been frequently used as the first target because it separates from the other three genes and it encodes a component that locates the final step in the phosphotransfer cascade of the PTSGlc (Fig. 1). Deleting the ptsG gene in E. coli reduced the specific growth rate to approximately 85% of that of the parent strain9 and resulted in the simultaneous assimilation of glucose and xylose10–12. Deleting another target in the PTSGlc, the entire ptsHI-crr operon, blocked the two common steps involved in the PEP: carbohydrate phosphotransfer cascades and resulted in a limited capacity for transporting glucose” (see, e.g., Liang, Introduction, pg. 2). Furthermore, Liang discloses “To investigate the effect of the inactivation of each component on substrate consumption and cell growth, we deleted the corresponding genes in the PTSGlc of wild-type E. coli W3110, generating E. coli W3110I, W3110H, W3110C, and W3110G (Table 1). Cultivating these mutants in the presence of substrate glucose resulted in reduced glucose consumption and retarded growth under aerobic condition (Fig. 2a,b)” (see, e.g., Liang, Results and Discussion, pg. 3).
Regarding claims 54 and 112 pertaining to genetic modifications resulting in decrease or elimination of glucose assimilation, Liang teaches the W3110I, W3110H, and W3110C E. coli mutants, which have deletions in ptsI, ptsH, and crr, respectively (see, e.g., Liang, Table 1, pg. 3). Liang teaches that “Cultivating these mutants in the presence of substrate glucose resulted in reduced glucose consumption and retarded growth under aerobic condition (Fig. 2a,b)” (see, e.g., Liang, Results and Discussion, pg. 3).
Bausch’s general disclosure relates to “Genomic sequence analysis of the region known to contain genes of the GntII system led to a hypothesis which was tested biochemically and confirmed: the GntII system encodes a pathway for catabolism of L-idonic acid in which D-gluconate is an intermediate. The genes have been named accordingly: the idnK gene, encoding a thermosensitive gluconate kinase, is monocistronic and transcribed divergently from the idnD-idnO-idnT-idnR operon, which encodes L-idonate 5-dehydrogenase, 5-keto-D-gluconate 5-reductase, an L-idonate transporter, and an L-idonate regulatory protein, respectively” (see, e.g., Bausch, abstract). Moreover, Bausch discloses “E. coli K-12 strains are able to utilize L-idonate as the sole carbon and energy source, and as predicted, the ability of idnD, idnK, idnR, and edd mutants to grow on L-idonate is altered” (see, e.g., Bausch, abstract).
Regarding claims 54 and 112 pertaining to genetic modifications resulting in decrease or elimination of gluconate-6-phosphate production from gluconate, Bausch teaches the production of gntK and idnK E. coli mutants (see, e.g., Bausch, “Construction of mutants”, pg. 3705& Table 2, pg. 3705 & Results, pg. 3707). Moreover, Bausch teaches that “A secondary mutation of idnK (gntV) eliminated the subsidiary gluconate kinase activity” (see, e.g., Bausch, Discussion, pg. 3708). Additionally, Bausch teaches “E. coli NP202, a gntK deletion mutant, is able to grow on D-gluconate” (see, e.g., Bausch, Discussion, pg. 3709).
Diaz’s general disclosure relates to an “engineered an E. coli strain, which we have named X2G, that not only exhibits a reversed substrate preference for xylose over glucose, but also demonstrates an unusual ability to produce significant amounts of glucose. We obtained this nonintuitive phenotype by deleting four genes in upper central metabolism: ptsI, glk, pfkA, and zwf, which respectively encode Enzyme I of the phosphotransferase system, glucokinase, the dominant isozyme of phosphofructokinase, and glucose-6-phosphate dehydrogenase. The deletion of ptsI and glk blocks glucose uptake in E. coli, while the deletion of pfkA and zwf prevents the reassimilation of carbons through glycolysis and the oxidative pentose phosphate pathway, respectively” (see, e.g., Diaz, abstract).
Regarding claims 54 and 112 pertaining to genetic modifications resulting in decrease or elimination of glucose-6-phosphate production from glucose, Diaz teaches an E. coli mutant with glk deleted (see, e.g., Diaz, abstract). Moreover, Diaz teaches that “The deletion of ptsI and glk blocks glucose uptake in E. coli” (see, e.g., Diaz, abstract).
Sutter’s general disclosure relates to the degradation of glucose by gluconate dehydratase via the Entner-Doudoroff Pathway in Haloferax volvanii (see, e.g., Sutter, abstract). Moreover, Sutter discloses that glucose is converted to KDG via glucose dehydrogenase and gluconate dehydratase (see, e.g., Sutter, Introduction, pg. 2251). Additionally, Sutter discloses identification of HVO_1488, a gene that encodes metabolic gluconate dehydratase enzyme, and determines that this enzyme is involved in glucose degradation (see, e.g., Sutter, Results, pg. 2256).
Regarding claims 54 pertaining to production of KDG, Sutter teaches that gluconate is converted to KDG via gluconate dehydratase (see, e.g., Sutter, Introduction, pg. 2251). Based on the teachings of Sutter, one of ordinary skill in the art would have understood to add gluconate to a cell culture, wherein the recombinant cell is expressing gluconate dehydratase, in order to produce KDG (see, e.g., Sutter, Figure 1, pg. 2252). Additionally, one of ordinary skill in the art would have understood to add glucose to a cell culture, wherein the recombinant cell is expressing both glucose dehydrogenase and gluconate dehydratase, in order to produce KDG (see, e.g., Sutter, Figure 1, pg. 2252).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Miyake’s recombinant E. coli cell, wherein the cell comprises deletions in the ptsI, ptsH, and crr genes resulting in elimination of glucose assimilation, as taught by Liang. One would have been motivated to do so because Liang teaches that cultivating E. coli mutants with deletions in the ptsI, ptsH, and crr genes “in the presence of substrate glucose resulted in reduced glucose consumption and retarded growth under aerobic condition (Fig. 2a,b)” (see, e.g., Liang, Results and Discussion, pg. 3). Additionally, Liang teaches “Deleting another target in the PTSGlc, the entire ptsHI-crr operon, blocked the
two common steps involved in the PEP: carbohydrate phosphotransfer cascades and resulted in a limited capacity for transporting glucose” (see, e.g., Liang, Introduction, pg. 2); therefore, one of ordinary skill in the art would readily understand that glucose assimilation is eliminated in these mutants with deletions in the ptsI, ptsH, and crr genes. Moreover, Miyake teaches expression of a novel gluconate dehydratase from Achromobacter xylosoxidans in E. coli, wherein the gluconate dehydratase has “thermostability and storage stability as to be industrially used in practice and capable of efficiently producing a 2-keto-3-deoxyaldonic acid” (see, e.g., Miyake, [0007]). Furthermore, Miyake teaches the addition of D-gluconate to E. coli cells expressing the novel gluconate dehydratase from Achromobacter xylosoxidans in order to produce KDG (see, e.g., Miyake, Example 2, [0067]). Therefore, based on the teachings of Miyake and Liang, it would have been obvious to eliminate glucose assimilation in the recombinant E. coli cell by deleting the ptsI, ptsH, and crr genes because this results in gluconate being taken up by the E. coli cell and utilized to produce KDG. One would have expected success because Miyake and Liang both teach E. coli transport systems for different carbon sources, as well as components within the Entner-Doudoroff pathway.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Miyake’s recombinant E. coli cell, wherein the cell comprises deletions in the gntK and idnK genes resulting in elimination of gluconate kinase activity, as taught by Bausch. One would have been motivated to do so because Bausch teaches “IdnK catalyzes an ATP-dependent phosphorylation of D-gluconate to form 6-phosphogluconate, which is metabolized further via the Entner-Doudoroff pathway” (see, e.g., Bausch, abstract). Furthermore, Bausch teaches that gntK is part of the GntII system, which codes for a thermoresistant gluconate kinase (see, e.g., Bausch, Introduction, pg. 3704). Moreover, Miyake teaches the addition of D-gluconate to E. coli cells expressing the novel gluconate dehydratase from Achromobacter xylosoxidans in order to produce KDG (see, e.g., Miyake, Example 2, [0067]). Therefore, based on the teachings of Miyake and Bausch, it would have been obvious to eliminate gluconate-6-phosphate production from gluconate through deletion of the gntK and idnK genes because this would result in production of KDG from gluconate instead of the gluconate being phosphorylated and turned in gluconate-6-phosphate. One would have expected success because Miyake and Bausch both teach gluconate metabolism in E. coli.
It would have been thirdly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Miyake’s recombinant E. coli cell, wherein the cell comprises deletions in the ptsI and glk genes resulting in elimination of glucose uptake by E. coli, as taught by Diaz. One would have been motivated to do so because Diaz teaches “that deletion of just two genes involved in the phosphorylation of glucose to glucose 6-phosphate
(G6P), namely ptsI and glk, is sufficient to block glucose utilization” (see, e.g., Diaz, Introduction, pg. 168); therefore, one of ordinary skill in the art would readily understand that since glucose utilization is blocked, gluconate would be utilized by E. coli when administered. Moreover, Miyake teaches the addition of D-gluconate to E. coli cells expressing the novel gluconate dehydratase from Achromobacter xylosoxidans in order to produce KDG (see, e.g., Miyake, Example 2, [0067]). Therefore, based on the teachings of Miyake and Diaz, it would have been obvious to eliminate glucose-6-phosphate production from glucose by blocking glucose uptake through deletion of the ptsI and glk genes so that the E. coli cell is forced to uptake gluconate in order to produce KDG. One would have expected success because Miyake and Diaz both teach E. coli transport systems for different carbon sources.
It would have been fourthly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce Miyake’s gluconate dehydratase in cell culture, wherein the gluconate dehydratase is used to produce KDG through addition of gluconate as the substrate, as taught inherent taught by Miyake and taught by Sutter. One would have been motivated to do so because Sutter teaches that gluconate is converted to KDG via gluconate dehydratase (see, e.g., Sutter, Introduction, pg. 2251); therefore, one of ordinary skill in the art would understand that addition of gluconate to a culture that recombinant produces gluconate dehydratase would result in KDG production. Furthermore, Miyake teaches the enzymatic production of KDG outside of cell culture through the addition of sodium D-gluconate to gluconate dehydratase produced by Achromobacter xylosoxidans (see, e.g., Miyake, [0066], [0067], Example 2). Furthermore, Miyake teaches the same method of producing a recombinant E. coli cell that expresses a gluconate dehydratase by Achromobacter xylosoxidans in order to produce KDG (see, e.g., Miyake, abstract); therefore, this would inherently lead to the production of KDG in cell culture when the cell is given glucose or gluconate. Therefore, based on the teachings of Miyake and Sutter, it would have been obvious to produce KDG in the same cell culture used to produce gluconate dehydratase by merely adding glucose or gluconate into the culture. One would have expected success because Miyake and Sutter both teach production of KDG from gluconate dehydratase.
Claim 56 is rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Gupta (Project Report Codon Optimization; 2003 – previously cited) and Gustafsson (Condon bias and heterologous protein expression; 2004 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: wherein the gene encoding a heterologous gluconate dehydratase comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 4 (claim 56).
Regarding claim 56 pertaining to a heterologous gluconate dehydratase gene, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches SEQ ID NO: 1 (see, e.g., Miyake, [0021], [0037]), which has 58.5% sequence identity to SEQ ID NO: 4, as taught in the instant application. Gupta teaches basic rationales for optimizing codons for specific living organisms (see, e.g., Gupta at §§ 2.1, 4.1, 4.2, 4.3) and identifies that circa 2003 web-based software was available for converting yeast and human codons (id. at §5). Gustafsson identifies that a “common strategy" to improve expression of heterologous proteins in a host is to alter rare codons in a gene to reflect codon usage of a host without modifying the amino acid sequence of the encoded protein (see, e.g., Gustafsson et al. at 348 col II §”Results from Codon Optimization”, Table 1 on 349). Therefore, based on the teachings of Gupta and Gustafsson, it is routine to one of ordinary skill in the art to use different nucleic acid sequences to express a protein in a different organism (i.e., codon optimization). Additionally, the nucleic acid encoding the protein would be optimized for expression in a particular organism. Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz’s SEQ ID NO: 1 could be optimized so that the codons are different compared to instant SEQ ID NO: 4, but the amino acid sequence is the same.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a host cell containing SEQ ID NO: 1, as taught by modified-Miyake-Sutter-Liang-Bausch-Diaz, for expression of gluconate dehydratase. Modified-Miyake-Sutter-Liang-Bausch-Diaz’s SEQ ID NO: 1 (see, e.g., Miyake, [0021], [0037]) has 58.5% sequence identity to instant SEQ ID NO: 4; however, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close (see, e.g., MPEP 2144.05(I)). One would have been motived to do so because modified-Miyake-Sutter teaches that gluconate dehydratase is utilized to produce KDG (see, e.g., Miyake, abstract). Moreover, based on the teachings of Gupta and Gustafsson, it would have been obvious to optimize the codons within modified-Miyake-Sutter’s SEQ ID NO: 1 in order to produce instant SEQ ID NO: 4, wherein the gluconate dehydratase protein sequence is the same, but the nucleic acid sequence is different. One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz teaches a gluconate dehydratase gene (SEQ ID NO: 1) that can be expressed in a host cell to produce KDG.
Claims 57 and 67 are rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Pouyssegur (Genetic Control of the 2-keto-3-deoxy-d-gluconate metabolism in Escherichia coli K-12: kdg regulon; 1974 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
Regarding claim 57 pertaining to culture medium comprising glucose, modified-Miyake-Sutter teaches the use of glucose as a carbon and energy source in cell culture (see, e.g., Sutter, Materials and Methods, pg. 2253).
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: wherein the culture medium comprises glycerol (claim 57).
Pouyssegur’s general disclosure relates to “the chromosomal location of the regulatory gene (kdgR) and of the presumptive structural gene for the KDG kinase (kdgK)” (see, e.g., Pouyssegur, Introduction, pgs. 641-642). Moreover, Pouyssegur discloses a KDG transport system for transporting KDG into the cell against a concentration gradient (see, e.g., Pouyssegur, Introduction, pgs. 641). Additionally, Pouyssegur discloses the growth of KDG mutants in glycerol medium supplemented with glycuronate or galacturonate as carbon sources (see, e.g., Pouyssegur, Results, pg. 643).
Regarding claim 57 pertaining to the culture medium comprising glycerol, Pouyssegur teaches that glycerol minimal medium, and other mediums containing glycerol, were used to grow bacterial strains that generate KDG (see, e.g., Pouyssegur, Methods, “Genetic techniques”, pg. 642).
Regarding claim 67 pertaining to a recombinant cell comprising one or more genetic modifications resulting in decrease or elimination of 2-keto-3-deoxy-D-gluconate (KDG) phosphorylation, Pouyssegur teaches a kdgK E. coli mutant (see, e.g., Pouyssegur, Bacterial strains, pg. 642 & Table 1). Moreover, Pouyssegur teaches that the KDG kinaseless mutant (kdgK) has loss of kinase activity and “large amounts of KDG were excreted in the medium when these mutants were grown on glycerol supplemented with either glucuronate or galacturonate” (see, e.g., Pouyssegur, Results, pg. 643).
It would have been first obvious before the effective filing date of the claimed invention to grow modified-Miyake-Sutter-Liang-Bausch-Diaz’s recombinant cell that is grown in glucose and expressing gluconate dehydratase, wherein the cell is also grown in glycerol, as taught by Pouyssegur, respectively. One would have been motivated to do so because Pouyssegur teaches that bacterial strains can produce KDG when grown on glycerol media (see, e.g., Pouyssegur, Results, pg. 644). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz and Pouyssegur teach that glucose and glycerol, respectively, can be used as carbon sources, during bacterial culturing, to produce KDG (see, e.g., Sutter, Materials and Methods, pg. 2253) (see, e.g., Pouyssegur, Methods, “Genetic techniques”, pg. 642). Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz and Pouyssegur, it would have been obvious to grow recombinant cells expressing gluconate dehydratase in culture medium comprising glucose and glycerol because glucose and glycerol can be used as carbon and energy sources to produce KDG. One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Pouyssegur both teach KDG production.
It would have been secondly obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Miyake-Sutter-Liang-Bausch-Diaz’s recombinant E. coli cell, wherein the cell has a deletion in the kdgK gene resulting in elimination of KEG phosphorylation, as taught by Pouyssegur. One would have been motivated to do so because Pouyssegur teaches that the KDG kinaseless mutant (kdgK) has loss of kinase activity and “large amounts of KDG were excreted in the medium when these mutants were grown on glycerol supplemented with either glucuronate or galacturonate” (see, e.g., Pouyssegur, Results, pg. 643). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches “KDG is phosphorylated by KDG kinase (KDGK) to generate 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is cleaved to pyruvate and glyceraldehyde-3-phosphate (GAP) by a bifunctional KDG/KDPG aldolase (KDPGA) that catalyzes the cleavage of both KDG and KDPG (see, e.g., Sutter, Introduction, pgs. 2251-2252). Furthermore, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches a semiphosphorylative pathway, wherein glucose and gluconate can be utilized to produce KDG, and wherein KDG is phosphorylated to KDPG by KdgK (see, e.g., Sutter, Figure 1, pg. 2252). Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz and Pouyssegur, it would have been obvious to eliminate the phosphorylation of KDG by deletion of the kdgK gene because this would inhibit phosphorylation of KDG to KDPG which would result in increased production of KDG though elimination of downstream processes within the cell. One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Pouyssegur both teach production of KDG from gluconate and/or glucose.
Claims 58 and 111 are rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Matsubara (One-step synthesis of 2-keto-3-deoxy-d-gluconate by biocatalytic dehydration of d-gluconate; 2014 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
Regarding claim 111 pertaining to glucose as the substrate, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches glucose as a substrate for KDG production (see, e.g., Sutter, Introduction, pg. 2251). Furthermore, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches that glucose can be converted to gluconate via glucose dehydrogenase, and the gluconate produced can be converted to KDG via gluconate dehydratase (see, e.g., Sutter, Figure 1).
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: wherein the yield of KDG is at least 60% calculated as the ratio of the weight of KDG produced to the weight of substrate consumed (claim 58).
Matsubara’s general disclosure relates to “utilization of dehydratases for 2-keto-3-deoxy sugar acid syntheses” (see, e.g., Matsubara, abstract). Moreover, Matsubara discloses “a straightforward and highly economic one-step biocatalytic synthesis procedure of 2-keto-3-deoxy-d-gluconate (KDG) from d-gluconate using recombinant gluconate dehydratase (GAD) from the hyperthermophilic crenarchaeon Thermoproteus tenax. This method is highly advantageous to KDG production schemes described so far for several reasons: (i) the d-gluconate is completely converted to stereochemically pure D-KDG without side-product formation, (ii) the final KDG yield is approximately 90%, (iii) the newly developed quantitative and qualitative” (see, e.g., Matsubara, abstract).
Regarding claim 58 pertaining to the yield of KDG, Matsubara teaches that the final KDG yield is approximately 90% (see, e.g., Matsubara, abstract). Matsubara teaches that yield is calculated by conversion of “compound A” (i.e., substrate consumed) into “compound B” (i.e., KDG produced) (see, e.g., Matsubara, Section 2.3, pg. 73). Moreover, Matsubara teaches the kinetics of gluconate dehydratase-catalyzed conversion of gluconate into KDG (see, e.g., Matsubara, Figure 6); therefore, one of ordinary skill in the art would recognize that this correlates to calculating the ratio of KDG produced to gluconate consumed.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce modified-Miyake-Sutter-Liang-Bausch-Diaz’s KDG, wherein the yield of KDG is approximately 90%, as taught by Matsubara. One would have been motivated to do so because Matsubara teaches a resource-efficient, one-step synthesis of KDG without side product formation because a high number of different reaction steps leads to increased waste compounds and decreases yields (see, e.g., Matsubara, abstract & Introduction, pg. 69). Furthermore, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches the conversion of gluconate into KDG via gluconate dehydratase in cell culture (see, e.g., Miyake, [0017]-[0025], [0033]). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches the production of a novel gluconate dehydratase that can efficiently produce KDG due to its stability at high temperatures (see, e.g., Miyake, [0010]). Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz and Matsubara, it would have been obvious to produce KDG at a yield of approximately 90% because both modified-Miyake-Sutter-Liang-Bausch-Diaz and Matsubara teach methods of increasing the efficiency of the gluconate dehydratase and the conversion reaction in order to efficiently produce KDG. One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Matsubara both teach KDG production.
Claim 65 is rejected under 35 U.S.C. 103 as being unpatentable Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Doring (US Patent No. 7,858,775; Date of Publication: December 28, 2010 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: the method further comprising converting KDG to a 2’-deoxynucleoside or a precursor thereof (claim 65).
Doring’s general disclosure relates to “a process for preparing 2’-deoxynucleoside compounds or 2’-deoxynucleoside precursors using KDG” (see, e.g., Doring, abstract). Moreover, Doring discloses “2'-deoxynucleosides and their analogues are used as a starting material for synthesis or drug formulation in production of an antiviral, anticancer or antisense agent” (see, e.g., Doring, pg. 2, col. 1, lines 22-28). Additionally, Doring discloses that an “aspect of the invention is a convenient and cost-effective method for preparing KDG or its salts to be used in the above methods. This method starts either from D-gluconate or from D-glucosaminate through the use of recombinant enzymes. The invention provides a novel nucleotide sequence encoding a polypeptide having D-gluconate dehydratase activity and a nucleotide sequence encoding a polypeptide having D-glucosaminate deaminase activity” (see, e.g., Doring, pg. 3, col. 3, lines 1-6).
Regarding claim 65 pertaining to converting KDG to 2’-deoxynucleoside or precursors thereof, Doring teaching preparing 2’-deoxynucleoside compounds or 2’-deoxynucleoside precursors using KDG (see, e.g., Doring, abstract). Doring teaches that conversion of KDG to 2’-deoxynucleoside or precursors thereof is a cost effective method of producing 2’-deoxynucleoside and its precursors (see, e.g., Doring, [0021]), and allows the reaction to not be dependent on unreliable natural sources (see, e.g., Doring, [0012]). Furthermore, Doring teaches that “2'-deoxynucleosides and their analogues are used as a starting material for synthesis or drug formulation in production of an antiviral, anticancer or antisense agent” (see, e.g., Doring, abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to convert modified-Miyake-Sutter-Liang-Bausch-Diaz’s KDG into 2’-deoxynucleoside compounds or 2’-deoxynucleoside precursors, as taught by Doring. One would have been motivated to do so because Doring teaches that “2'-deoxynucleosides and their analogues are used as a starting material for synthesis or drug formulation in production of an antiviral, anticancer or antisense agent” (see, e.g., Doring, abstract). Furthermore, Doring teaches that conversion of KDG to 2’-deoxynucleoside or precursors thereof is a cost effective method of producing 2’-deoxynucleoside and its precursors (see, e.g., Doring, [0021]), and allows the reaction to not be dependent on unreliable natural sources (see, e.g., Doring, [0012]). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches the production of KDG from glucose and gluconate via gluconate dehydrogenase and glucose dehydrogenase (see, e.g., Miyake, [0066], [0067], Example 2) (see, e.g., Sutter, Introduction); therefore, one of ordinary skill in the art would recognize that the downstream product (i.e., KDG) produced from modified-Miyake-Sutter-Liang-Bausch-Diaz’s methods can be used to produce 2’-deoxynucleoside compounds and 2’-deoxynucleoside precursors, as taught by Doring. Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz and Doring, it would have been obvious to produce 2’-deoxynucleoside compounds and 2’-deoxynucleoside precursors from KDG this production method is cost effective and the KDG products (i.e., 2’-deoxynucleoside compounds and 2’-deoxynucleoside precursors) can be used in drug formulations. One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Doring both teach KDG synthesis.
Claim 66 is rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Binder (US 2018/0057897; Date of Publication: March, 1, 2018 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: the method further comprising converting KDG to 5-hydroxymethyl-2-furoic acid (HMFA) and/or furan dicarboxylic acid (FDCA) (claim 66).
Binder’s general disclosure relates to “a method of preparing a furan derivative comprising the steps of (a) converting a monosaccharide to provide a keto-intermediate product; and (b) dehydrating the keto-intermediate product to provide a furan derivative; wherein the keto-intermediate product is pre-disposed to forming keto-furanose tautomers in solution” (see, e.g., Binder, abstract). Moreover, Binder discloses “conversion of glucose to furandicarboxylic acid through 2-keto-3-deoxygluconic acid as a keto-sugar intermediate” (see, e.g., Binder, Figure 1, [0006]).
Regarding claim 66 pertaining to converting KDG to HMFA and/or FDCA, Binder teaches that KDG may be dehydrated by acid catalysis into HMFA (see, e.g., Binder, [0028] & Figure 1). Moreover, Binder teaches that the production of HMFA from KDG does not rely on fructose as an intermediate, which is advantageous because fructose is not as common and the pathways are not as efficient (see, e.g., Binder, [0002]-[0003]).
It would have been obvious before the effective filing date of the claimed invention to convert modified-Miyake-Sutter-Liang-Bausch-Diaz’s KDG into HMFA, as taught by Binder. One would have been motivated to do so because Binder teaches that KDG is one of the most common 2-keto sugars to prefer furanose tautomeric forms; therefore, KDG can be used to produce HMFA instead of glucose (see, e.g., Binder, [0011], [0015], [0028]). Additionally, Binder teaches that the production of HMFA from KDG does not rely on fructose as an intermediate, which is advantageous because fructose is not as common and the pathways are not as efficient (see, e.g., Binder, [0002]-[0003]). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches the production of KDG from glucose and gluconate via gluconate dehydrogenase and glucose dehydrogenase (see, e.g., Miyake, [0066], [0067], Example 2) (see, e.g., Sutter, Introduction); therefore, one of ordinary skill in the art would recognize that the downstream product (i.e., KDG) produced from modified-Miyake-Sutter-Liang-Bausch-Diaz’s methods can be used to produce HMFA, as taught by Binder. Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz and Binder, it would have been obvious to produce HMFA from KDG because production via this pathway does not involve fructose, which is advantageous because it allows the production of HMFA from KDG to be more efficient (see, e.g., Binder, [0002]-[0003]). One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Binder both teach KDG production.
Claims 68-69 are rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Ajikumar (US 2011/0189717; Date of Publication: August 4, 2011 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: wherein the recombinant cell further comprises genetic modifications resulting in: (b) increased production of isopentenyl pyrophosphate (IPP) and/or dimethylallyl pyrophosphate (DMAPP) from KDG (claim 68); or wherein the genetic modifications resulting in increased production of: (c) IPP or DMAPP from KDG comprises increasing expression of enzymes involved in the non-mevalonate (MEP) pathway (claim 69).
Ajikumar’s general disclosure relates to “recombinant expression of a taxadiene synthase enzyme and a geranylgeranyl diphosphate synthase (GGPPS) enzyme in cells and the production of terpenoids” (see, e.g., Ajikumar, abstract). Moreover, Ajikumar discloses “The upstream mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathways can produce the two common building blocks, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), from which Taxol and other isoprenoid compounds are formed” (see, e.g., Ajikumar, [0007]). Additionally, Ajikumar discloses “the optimal balancing between the upstream, IPP-forming pathway with the downstream terpenoid pathway of taxadiene synthesis. This is achieved by grouping the nine-enzyme pathway into two modules--a four-gene, upstream, native (MEP) pathway module and a two-gene, downstream, heterologous pathway to taxadiene (FIG. 1). Using this basic configuration, parameters such as the effect of plasmid copy number on cell physiology, gene order and promoter strength in an expression cassette, and chromosomal integration are evaluated with respect to their effect on taxadiene production” (see, e.g., Ajikumar, [0009]).
Regarding claims 68-69 pertaining to increased IPP and/or DMAPP production, Ajikumar teaches “in some embodiments, a cell that overexpresses one or more components of the non-mevalonate (MEP) pathway is used, at least in part, to amplify isopentyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), substrates of GGPPS. In some embodiments, overexpression of one or more components of the non-mevalonate (MEP) pathway is achieved by increasing the copy number of one or more components of the non-mevalonate (MEP) pathway. For example, copy numbers of components at rate-limiting steps in the MEP pathway such as (dxs, ispD, ispF, idi) can be amplified, such as by additional episomal expression” (see, e.g., Ajikumar, [0064]). Therefore, “overexpression of one or more components of the non-mevalonate (MEP) pathway”, as taught by Ajikumar, could include increasing expression of enzymes involved in the MEP pathway.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to genetically manipulate modified-Miyake-Sutter-Liang-Bausch-Diaz’s recombinant cell that produces KDG, in order to increase IPP and DMAPP production from KDG, as taught by Ajikumar. One would have been motivated to do so because Ajikumar teaches that increasing IPP and DMAPP from KDG can enhance terpenoid production (see, e.g., Ajikumar, [0020], [0049]). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches that glucose and gluconate can be used to produce KDG within the Entner-Doudoroff pathway (see, e.g., Sutter, Fig. 1). Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz and Ajikumar, it would have been obvious to utilize the Entner-Doudoroff pathway to produce KDG from glucose and/or gluconate because the KDG can be utilized to produce IPP and DMAPP. One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Ajikumar both teach metabolic pathways that can utilize KDG.
Claim 70 is rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, Diaz, and Ajikumar as applied to claims 54, 61-62, 68-69, 110, and 112-114 above, and further in view of Jiang (Extraction and Analysis of Terpenes/Terpenoids; 2016 – previously cited).
The teachings of Miyake, Sutter, Liang, Bausch, Diaz, and Ajikumar herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz-Ajikumar, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
However, modified-Miyake-Sutter-Liang-Bausch-Diaz-Ajikumar does not teach: wherein the method further comprising a step of purifying from cell culture: (c) terpenoids (claim 70).
Jiang’s general disclosure relates to “protocols for extraction of terpenes/terpenoids from various plant sources” (see, e.g., Jiang, abstract). Moreover, Jiang discloses extraction of “terpenes/terpenoids with various levels of chemical decoration, from the relative small, nonpolar, volatile hydrocarbons to substantially large molecules with greater physical complexity due to their chemical modifications” (see, e.g., Jiang, abstract).
Regarding claim 70 pertaining to purifying terpenoids from cell culture, Jiang teaches purification of terpenoids (see, e.g., Jiang, “Larger scale silica chromatography purification”, pg. 5).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to purify terpenoids, as taught by Jiang, from modified-Miyake-Sutter-Liang-Bausch-Diaz-Ajikumar’s KDG. One would have been motivated to do so because Jiang teaches that terpenoids “have garnered much attention because of their important physiological roles (i.e. hormones, aliphatic membrane anchors, maintaining membrane structure), ecological roles” (see, e.g., Jiang, Introduction, pg. 1) and can be used as “flavors, fragrances, high grade lubricant, biofuels, agricultural chemicals and medicines” (see, e.g., Jiang, abstract). Moreover, Jiang teaches that terpenoids can be produced from IPPs (see, e.g., Jiang, Introduction, pg. 1), and modified-Miyake-Sutter-Liang-Bausch-Diaz-Ajikumar teaches that KDG can be used to produce IPPs. Therefore, based on the teachings of modified-Miyake-Sutter-Liang-Bausch-Diaz-Ajikumar and Jiang, it would have been obvious to purify terpenoids from the cell culture because terpenoids can be produced from KDG with IPP as the intermediate. One would have been motivated to do so because modified-Miyake-Sutter-Liang-Bausch-Diaz-Ajikumar and Jiang both teach metabolic pathways that can utilize KDG.
Claims 107-109 are rejected under 35 U.S.C. 103 as being unpatentable over Miyake, Sutter, Liang, Bausch, and Diaz as applied to claims 54, 61-62, 110, and 112-114 above, and further in view of Kambourakis (US 2017/0106414; Date of Publication: April 17, 2014 – newly cited).
The teachings of Miyake, Sutter, Liang, Bausch, and Diaz, herein referred to as modified-Miyake-Sutter-Liang-Bausch-Diaz, are discussed above as it pertains to production of KDG in cell culture via expression of gluconate dehydratase.
However, modified-Miyake-Sutter-Liang-Bausch-Diaz does not teach: wherein the recombinant cell comprises a gene encoding a heterologous gluconate dehydratase enzyme having at least 99% sequence identity to SEQ ID NO: 1 (claim 107); or wherein the recombinant cell comprises a gene encoding a heterologous gluconate dehydratase enzyme having at least 98% sequence identity to SEQ ID NO: 1 (claim 108); or wherein the recombinant cell comprises a gene encoding a heterologous gluconate dehydratase enzyme having at least 100% sequence identity to SEQ ID NO: 1 (claim 109).
Kambourakis’ general disclosure relates to “ methods for producing a product of one or more enzymatic pathways” wherein “the methods also involve the use of engineered enzymes that perform reactions with high specificity and efficiency. Additional products that can be produce include metabolic products such as, but not limited to, guluronic acid, L-iduronic acid, idaric acid, glucaric acid. Any of the products can be produced using glucose as a substrate or using any intermediate in any of the methods or pathways of the invention” (see, e.g., Kambourakis, abstract). Moreover, Kambourakis discloses the use of gluconate dehydratase for dehydration of gluconic acid to dehydrogluconic acid (see, e.g., Kambourakis, [0061]).
Regarding claim 108 pertaining to the gluconate dehydratase enzyme, Kambourakis teaches a gluconate dehydrogenase corresponding to SEQ ID NO: 35 that has 98.9% sequence identity to instant SEQ ID NO: 1 (see, e.g., Kambourakis, [0089] & Office Action Appendix).
Regarding claims 107 and 109 pertaining to the gluconate dehydratase enzyme, Kambourakis teaches a gluconate dehydrogenase corresponding to SEQ ID NO: 35 that has 98.9% sequence identity to instant SEQ ID NO: 1 (see, e.g., Kambourakis, [0089] & Office Action Appendix). The 98.9% sequence identity of Kamboruakis’ SEQ ID NO: 35 is approaching the instantly claimed sequence identity of 99% and 100%; therefore, absent any criticality of the claimed sequence identity, the instantly claimed invention is prima facie obvious in view of Kambourakis (see, e.g., MPEP 2144.05)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to produce a modified-Miyake-Sutter-Liang-Bausch-Diaz’s recombinant cell expressing a heterologous gluconate dehydratase, wherein the gluconate dehydratase corresponds to Kambourakis’ gluconate dehydratase encoded by SEQ ID NO: 35. One would have been motivated to do so because Kambourakis teaches recombinantly expressing gluconate dehydratase in E. coli cells (see, e.g., Kambourakis, [0123]), in order to produce 3-dehydro-gluconic acid (see, e.g., Kambourakis, [0087]). Moreover, modified-Miyake-Sutter-Liang-Bausch-Diaz teaches the production of KDG from glucose and gluconate via gluconate dehydrogenase and glucose dehydrogenase (see, e.g., Miyake, [0066], [0067], Example 2) (see, e.g., Sutter, Introduction); therefore, one of ordinary skill in the art would recognize that the downstream product (i.e., KDG) produced from modified-Miyake-Sutter-Liang-Bausch-Diaz’s methods can be used to produce various chemicals and derivatives, such as 3-dehydro-gluconic acid, as taught by Kambourakis (see, e.g., Kambourakis, [0087]). One would have expected success because modified-Miyake-Sutter-Liang-Bausch-Diaz and Kambourakis both teach recombinantly expressing a heterologous gluconate dehydrogenase.
Examiner’s Response to Arguments
Applicant's arguments filed 10/27/2025 have been fully considered but they are not persuasive.
Applicant has traversed the previous 35 U.S.C. 103 rejections in view of Miyake and Sutter in light of the amendment of claim 54 which introduces new limitations pertaining to genetic modifications (remarks, pages 8-10). As discussed above, all rejections have been withdrawn and new grounds of rejections are presented due to Applicant’s amendment on 10/27/2025. While Miyake and Sutter are relied upon in the above-presented rejection, it is not relied upon for teaching the genetic modifications. As such, Applicant’s arguments regarding the teachings of Miyake and Sutter are moot.
Regarding Applicant’s argument pertaining to the sequence identity of SEQ ID NO: 1 compared to Kamourakis (remarks, page 11), the Examiner has uploaded a new Office Action Appendix showing that SEQ ID NO: 35, as taught by Kamourakis, has 98.9% sequence identity to instant SEQ ID NO: 1 (see, e.g., Office Action Appendix). Regarding Applicant’s arguments pertaining to sequence identity, query match and sequence identity are synonymous. Query match measure the percentage of identical amino acids at corresponding positions between two aligned biological sequences, which is what sequence identity is.
Conclusion
Claims 54, 56-58, 61-62, 65-70, and 107-114 are rejected.
No claims are allowed.
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653