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 .
Withdrawal of Rejections
The response and amendments filed on 12/12/2025 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minor objections and/or minor rejections (i.e., formal matters), not explicitly restated here for brevity, have been withdrawn necessitated by Applicant’s formality correction and/or amendments. For the purposes of clarity of the record, the reasons for the Examiner’s withdrawal, and/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Briefly, the previous claim rejections under 35 U.S.C. 112(b) for indefiniteness have been withdrawn necessitated by Applicant’s amendments; however, new grounds of rejection have been set forth below. The previous claim rejections under 35 U.S.C. 112(a) for written description have been withdrawn necessitated by Applicant’s amendments.
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
Claims 1 and 6-7 are objected to because of the following informalities: these claims recite “…having sequence SEQ ID…”; however, this should read “…having SEQ ID…”. Appropriate correction is required.
New Grounds of Rejection Necessitated by Amendments
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.
Claim 16 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 (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 16 depends on cancelled claim 5; however, for the purposes of applying prior art, the Examiner has interpreted claim 16 to be dependent on independent claim 1. Therefore, the limitations of claim 1 are imported into claim 16.
Examiner’s Response to Arguments
Regarding Applicant’s arguments that all claims are clear and the 112(b) rejection should be withdrawn (remarks, page 8), as discussed above, all previous 112(b) rejections have been withdrawn necessitated by Applicant’s amendments. However, Applicant cancelled claim 5, which claim 16 was dependent on, thereby resulting in the new grounds of rejection set forth above.
New Grounds of Rejection Necessitated by Amendments
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 1-2 and 10-12 are rejected under 35 U.S.C. 103 as being unpatentable over Eilert (EP2743350; Date of Publication: June 18, 2014 – cited in the IDS filed on 11/28/2022 – previously cited) in view of Xie (CN105018403; Date of Publication: November 11, 2015 – cited in the IDS filed on 01/27/2025 – previously cited), Bathe (US2015/0353973; Date of Publication: December 10, 2015 – previously cited), Kim (WO2015/199396; Date of Publication: December 30, 2015 – previously cited), and Way (WO2010/141468; Date of Publication: December 9, 2010 – previously cited).
Eilert’s general disclosure relates to “a method for producing ectoine or a derivative thereof, using host cells containing at least one recombinant DNA sequence encoding each of one or more heterologous enzymes for biosynthesis of ectoine” (see, e.g., Eilert, [0001]). Moreover, Eilert discloses a method for synthesizing “ectoine or a derivative thereof, in particular 5-hydroxyectoine, independently of stress parameters, such as high salt conditions which are hostile conditions for production and subsequence downstream processing” (see, e.g., Eilert, [0010]).
Regarding claim 1 pertaining to ectABC expression, Eilert teaches SEQ ID NO: 1, which has 100% sequence identity to instant SEQ ID NO: 1 (see, e.g., Office Action Appendix). Eilert teaches SEQ ID NO: 2, which as 100% sequence identity to instant SEQ ID NO: 6 (see, e.g., Office Action Appendix). Eilert teaches SEQ ID NO: 3, which has 100% sequence identity to instant SEQ ID NO: 11 (see, e.g., Office Action Appendix).
However, Eilert does not teach: a microorganism for genetically modified for the production of ectoine, wherein the ectABC genes are heterologous to the microorganism (claim 1); or deletion of pykA and pykF genes (claim 1); or at least 50% reduction in citrate synthase enzyme activity as compared to an unmodified organism (claim 1); or 75% reduction in citrate synthase activity (claim 2); or wherein the microorganism belongs to the family of the bacteria Enterobacteriaceae, Clostridiaceae, Bacillaceae, Streptomycetaceae, or Corynebacteriaceae, or to the family of yeasts Saccharomycetaceae (claim 10); or wherein the Enterobacteriaceae bacterium is Escherichia coli or Klebsiella pneumoniae, the Clostridiaceae bacterium is Clostridium acetobutylicum, the Corynebacteriaceae bacterium is Corynebacterium glutamicum, or the Saccharomycetaceae yeast is Saccharomyces cerevisiae (claim 11); or wherein the Enterobacteriaceae bacterium is Escherichia coli (claim 12).
Xie’s general disclosure relates to a genetically engineered bacterium for the production of ectoine (see, e.g., Xie, English Translation, abstract). Moreover, Xie discloses a genetically engineered Escherichia coli bacterium that expresses ectABC from Halomonas elongatus in order to produce ectoine by glucose fermentation and overcome the harsh reaction conditions and energy consumption of chemical synthesis (see, e.g., Xie, English Translation, abstract).
Regarding claims 1 and 10-12 pertaining to a microorganism genetically modified for the production of ectoine, Xie teaches a genetically engineered Escherichia coli that includes the ectABC genes derived from Halomonas elongatus (see, e.g., Xie, English Translation, abstract). Therefore, the ectABC genes are heterologous to the microorganism.
Bathe’s general disclosure relates to “a process for the fermentative production of L-amino acids using microorganisms of the Enterobacteriaceae family” (see, e.g., Bathe, [0001]). Furthermore, Bathe discloses that the microorganism harbors an attenuated proP gene encoding the ProP symporter (see, e.g., Bathe, [0001]), wherein, the ProP symporter is responsible for absorption of ectoine (see, e.g., Bathe, [0010]).
Regarding claim 1 pertaining to deletion of pykA and pykF genes, Bathe teaches that the pykA and pykF genes may be eliminated (i.e., deleted) in E. coli (see, e.g., Bathe, [0229], [0237]-[0238]). Additionally, Bathe teaches that deletion of pykA and pykF modulates the expression of L-amino acids, which, in response, modulates the expression of ProP, which catalyzes the absorption of ectoine within the microorganism (see, e.g., Bathe, [0010], [0229]).
Kim’s general disclosure relates to using Escherichia coli to produce O-acetyl hemoserine in high yield (see, e.g., Kim, English Translation, “Description”, pg. 1). Furthermore, Kim discloses that O-acetyl homoserine may be used as methionine precursors (see, e.g., Kim, English Translation, pg. 2).
Regarding claims 1 and 2 pertaining to a reduction in citrate synthase enzyme activity, Kim teaches the inactivation of the citrate synthase enzyme (see, e.g., Kim, English Translation, Description, pgs. 2, 4 & Example 1, pg. 7). One of ordinary skill in the art would readily understand that deletion of citrate synthase would result in a reduction in enzyme activity that is at least 75% or more.
Way’s general disclosure relates to “metabolic engineering of cells for the enhanced production of a cellular product” (see, e.g., Way, abstract). Furthermore, Way discloses that the metabolism of a host cell is “altered so that the synthesis and/or secretion of a desired product is enhanced. In a further embodiment, the desired product is obtained from the cell or the medium” (see, e.g., Way, pg. 2, lines 10-13).
Regarding claim 1 regarding SEQ ID NO: 18 and the gltA gene, Way teaches SEQ ID NO: 20, which encodes the gltA gene, and has 100% sequence identity to instant SEQ ID NO: 18 (see, e.g., Office Action Appendix).
It would have been first obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to express the heterologous genes ectA, ectB, and ectC, as taught by Eilert, within a genetically modified microorganism for the production of ectoine, as taught by Xie. One would have been motivated to do so because Xie teaches that expression of the heterologous ectABC genes within a genetically modified microorganism for the production of ectoine allows for the stain to “overcome the harsh reaction conditions and energy consumption of chemical synthesis” and “overcomes the deficiencies such as complicated process and high production” (see, e.g., Xie, English Translation, abstract). Furthermore, Eilert teaches that production of ectoine through heterologous expression of ectABC allows for the possibility of ectoine synthesis independent of “stress parameters, such as high salt concentrations, which are hostile conditions for production and subsequent downstream processing” (see, e.g., Eilert, [0010]). Therefore, based on the teachings of Eilert and Xie, it would have been obvious to produce a genetically modified microorganism that expresses the heterologous ectA, ectB, and ectC genes because this would allow of expression and production of ectoine without the use of high-stress conditions, such as high salinity. One would have expected success because Eilert and Xie both teach production of ectoine by microorganisms.
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 a genetically modified microorganism that produces ectoine, as taught by Eilert, wherein the pykA and pykF genes are deleted from the microorganism, as taught by Bathe. One would have been motivated to do so because Bathe teaches that deletion of pykA and pykF modulates the expression of L-amino acids, which, in response, modulates the expression of ProP, which catalyzes the absorption of ectoine within the microorganism (see, e.g., Bathe, [0010], [0229]). Furthermore, Eilert teaches alternative methods of producing ectoine in yeast cells, wherein high stress conditions, such as high salinity, are not required (see, e.g., Eilert, [0014]). Therefore, based on the teachings of Bathe, it would have been obvious to produce a genetically modified microorganism that does not express the pykA and pykF genes because this would allow for the expression and production of ectoine without the use of high-stress conditions, such as high salinity. One would have expected success because Eilert and Bathe both teach ectoine production and genetically modified microorganisms.
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 a genetically modified microorganism that produces ectoine and absorbs ectoine, as taught by Eilert and Bathe, respectively wherein the microorganism has a reduction in citrate synthase, as taught by Kim, and wherein the citrate synthase enzyme is encoded by the gene gltA, as taught by Way. One would have been motivated to do so because Kim teaches that when citrate synthase is decreased, O-acetyl homoserine production is increased, which can lead to an increase in methionine, such as L-methionine synthesis (see, e.g., Kim, English Translation, Description, pg. 2). Moreover, from the teachings of Bathe, it is known in the art that L-amino acids modulate the expression of ProP, which catalyzes the absorption of ectoine within the microorganism (see, e.g., Bathe, [0010], [0229]). Furthermore, Eilert teaches that synthesis of 5-hydroxyectoine is based on the synthesis of amino acids, specifically from the aspartate family (see, e.g., Eilert, [0047]). Additionally, Eilert teaches alternative methods of producing ectoine in yeast cells, wherein high stress conditions, such as high salinity, are not required (see, e.g., Eilert, [0014]). Therefore, based on the teachings of Eilert, Kim, and Bathe, it would have been obvious to produce a genetically modified microorganism that has decreased citrate synthase activity because the decreased citrate synthase activity promotes the expression of L-methionine, which can modulate the expression of ProP, and further catalyzes the absorption of ectoine. One would have expected success because Eilert and Kim both teach genetically modified microorganisms.
Claims 4 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Eilert, Xie, Bathe, Kim, and Way as applied to claims 1-2 and 10-12 above, and further in view of Figge (WO2011/073738; Date of Publication: June 23, 2011 – previously cited).
The combined teachings of Eilert, Xie, Bathe, Kim, and Way, herein referred to as modified-Eilert-Xie-Bathe-Kim-Way, are discussed above as it pertains to a modified microorganism for production of ectoine.
Regarding claims 4 and 16 pertaining to the genetically modified microorganism of claim 1, modified-Eilert-Xie-Bathe-Kim-Way teaches a genetically engineered Escherichia coli that includes the ectABC genes derived from Halomonas elongatus (see, e.g., Xie, English Translation, abstract). Therefore, the ectABC genes are heterologous to the microorganism.
Regarding claims 4 and 16 pertaining to the gltA gene, modified-Eilert-Xie-Bathe-Kim-Way teaches SEQ ID NO: 20, which encodes the gltA gene, and has 100% sequence identity to instant SEQ ID NO: 18 (see, e.g., Office Action Appendix).
Regarding claim 16 pertaining to the promoter, modified-Eilert-Xie-Bathe-Kim-Way teaches the trc promoter (see, e.g., Xie, English Translation, abstract).
However, modified-Eilert-Xie-Bathe-Kim-Way does not teach: wherein citrate synthase enzyme activity is reduced by placing the gltA gene encoding the citrate synthase under the control of promoter PgltA or a heterologous inducible promoter (claim 4); or wherein expression of the gltA gene is under the control of a trc promoter (claim 16).
Figge’s general disclosure relates to the use of heterologous inducible promoters in the production of methionine by fermentation in a modified microorganism (see, e.g., Figge, abstract). Furthermore, Figge teaches the expression of L-methionine (see, e.g., Figge, pg. 1, lines 17-24), which can be used to modulate the expression of ProP, and further catalyzes the absorption of ectoine (see, e.g., Bathe, [0010], [0229]).
Regarding claims 4 and 16 pertaining to the heterologous inducible promoter, Figge teaches heterologous inducible promoters, such as the trc promoter (see, e.g., Figge, Example 1), or the lac promoter (see, e.g., Figge, pg. 5, lines 3-4). Furthermore, Figge teaches that “the fact that the inducible promoter is not the native promoter of the gene and was introduced in a way to control, at least partially, the level of expression of the gene that is operably linked to it. The activity of an inducible promoter is induced by the presence or absence of biotic or abiotic factors. Expression of genes can be turned on or off, according to the needs of the man skilled in the art. These promoters might be chemically-regulated (in presence of tetracycline, hormones, etc) or physically-regulated, especially by heat or light” (see, e.g., Figge, pg. 4, lines 5-11).
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 genetically modified microorganism expressing ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the microorganism expresses the gltA gene under control of a heterologous inducible promoter, as taught by Figge. One would have been motivated to do so because Figge teaches that the use of heterologous inducible promoters can results in increased, or decreased, production of the gene it regulates (see, e.g., Figge, pg. 4, lines 5-12). Furthermore, Figge teaches that “the fact that the inducible promoter is not the native promoter of the gene and was introduced in a way to control, at least partially, the level of expression of the gene that is operably linked to it. The activity of an inducible promoter is induced by the presence or absence of biotic or abiotic factors. Expression of genes can be turned on or off, according to the needs of the man skilled in the art. These promoters might be chemically-regulated (in presence of tetracycline, hormones, etc) or physically-regulated, especially by heat or light” (see, e.g., Figge, pg. 4, lines 5-11). Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Figge, it would have been obvious to link expression of the citrate synthase gltA gene to a heterologous inducible promoter, because this may result in decreased expression of the gltA gene. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Figge both teach production of genetically modified microorganisms.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Eilert, Xie, Bathe, Kim, and Way as applied to claims 1-2 and 10-12 above, and further in view of Lynch (US2018/0312887; Date of Publication: November 1, 2018 – herein referred to as “Lynch 2018”) and Lynch (U.S. Patent No. 8,809,027; Date of Publication: August 19, 2014 – herein referred to as “Lynch 2014” – previously cited).
The teachings of Eilert, Xie, Bathe, Kim, and Way, herein referred to as modified-Eilert-Xie-Bathe-Kim-Way, are discussed above as it pertains to a modified microorganism for the production of ectoine.
However, modified-Eilert-Xie-Bathe-Kim-Way does not teach: deletion of the gene ppc and overexpression of the gene pck encoding a phosphoenolpyruvate carboxykinase having SEQ ID NO: 29 (claim 6).
Lynch 2018’s general disclosure relates to engineered bacterial strains to increase utilization of malonyl-CoA production of a chemical product (see, e.g., Lynch 2018, abstract).
Regarding claim 6 pertaining to deletion of the ppc gene, Lynch 2018 teaches that the ppc gene encoding a phosphoenolpyruvate carboxylase is deleted in E. coli (see, e.g., Lynch 2018, [0701]).
Lynch 2014’s general disclosure relates to microorganism compositions that comprise combinations of genetic modifications that increase oxaloacetate alpha-decarboxylase enzymatic activity (see, e.g., Lynch 2014, abstract).
Regarding claim 6 pertaining to SEQ ID NO: 29, Lynch 2014 teaches SEQ ID NO: 8, which has 100% sequence identity to instant SEQ ID NO: 29 (see, e.g., Office Action Appendix).
Regarding claim 6 pertaining to overexpression of the pck gene, Lynch 2014 teaches that genetic modifications can be made to the pck gene to increase overall expression of these genes and increase protein function in a microorganism cell (see, e.g., Lynch 2014, [0133]).
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 a genetically modified microorganism for the production of ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the genetically modified microorganism has a deletion in the ppc gene, as taught by Lynch 2018. One would have been motivated to do so because Lynch 2018 teaches that the ppc gene is linked to oxaloacetate production, which is made within the citric acid cycle (see, e.g., Lynch 2018, [0701]), and one of ordinary skill in the art would readily understand that oxaloacetate is used to produce ectoine. Furthermore, Lynch 2018 teaches “Genetic modifications to reduce flux through the TCA cycle can include genetic modifications to key steps such as those aimed to reduce activity or expression of the citrate synthase enzyme encoded by the gltA gene, alternatively enzymes that lead to oxaloacetate production can be reduced or eliminated such as phosphoenolpyruvate carboxylase (such as encoded by the ppc gene) or phosphoenolpyruvate carboxykinase such as encoded by the pck genes. Alternatively genetic modifications may be introduced to cause temperature sensitive (“ts”) activity in these enzymes (such as in the genes gltA, ppc, pck) to low activity at a permissive temperature such as 30 degrees Celsius, but no activity at a nonpermissive temperature such as 37 degrees Celsius” (see, e.g., Lynch 2018, [0701]). Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Lynch 2018, it would have been obvious to delete the ppc gene within a genetically modified microorganism for ectoine production. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Lynch 2018 both teach the production of genetically modified microorganisms.
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 a genetically modified microorganism that produces ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the genetically modified microorganism overexpresses the gene pck which encodes a phosphoenolpyruvate carboxykinase, as taught by Lynch 2014. One would have been motivated to do so because Lynch 2014 teaches that the phosphoenolpyruvate carboxykinase is linked to oxaloacetate production (see, e.g., Lynch 2014, [132]), which one of ordinary skill in the art would understand is made within the citric acid cycle, and used to produce ectoine. Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Lynch 2014, it would have been obvious to overexpress the pck gene within a genetically modified microorganism for ectoine production. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Lynch 2014 both teach the production of genetically modified microorganisms.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Eilert, Xie, Bathe, Kim, and Way as applied to claims 1-2 and 10-12 above, and further in view of Ikeda (US2011/0312042; Date of Publication: December 22, 2011) and Baldenius (EP2706118; Date of Publication: March 12, 2014 – previously cited).
The teachings of Eilert, Xie, Bathe, Kim, and Way, herein referred to as modified-Eilert-Xie-Bathe-Kim-Way, are discussed above as it pertains to a modified microorganism for the production of ectoine.
Regarding claim 7 pertaining to overexpression, modified-Eilert-Xie-Bathe-Kim-Way teaches methods of overexpression, which include a “widespread method for achieving overexpression is the process of chromosomal gene amplification. In this method, at least one additional copy of the polynucleotide of interest is inserted into the chromosome of a bacterium” (see, e.g., Bathe, [0219]). Additionally, modified-Eilert-Xie-Bathe-Kim-Way teaches “A further method for achieving overexpression comprises linking the respective gene or allele in a functional manner (operably linked) to a promoter or an expression cassette” (see, e.g., Bath, [0220]).
However, modified-Eilert-Xie-Bathe-Kim-Way does not teach: overexpression of an aspartate transaminase having at least 80% sequence identity with SEQ ID NO: 35 and a glutamate dehydrogenase having SEQ ID NO: 39 (claim 7).
Ikeda’s general disclosure relates to a process for producing an amino acid using a microorganism in which aspartate aminotransferase activity is modified (see, e.g., Ikeda, abstract). Moreover, Ikeda teaches “overexpressing the aspC gene of a microorganism of the genus Escherichia, the L-amino acid productivity can be improved ” (see, e.g., Ikeda, [0003]).
Regarding claim 7 pertaining to SEQ ID NO: 35, Ikeda teaches SEQ ID NO: 2, which has 100% sequence identity to instant SEQ ID NO: 35 (see, e.g., Office Action Appendix).
Baldenius’ general disclosure relates to “a novel enzymatically catalyzed method for the production of aliphatic primary amines” (see, e.g., Baldenius, abstract). Furthermore, Baldenius discloses a three-enzyme catalytic cycle” that “requires no external source of reducing equivalents because the reduced cofactor is formed during alcohol oxidation. Only catalytic amounts of the redox nicotinamide cofactor and of the compound that transfers the amine group are needed. Furthermore, the cycle requires no external amino acid, since the amino acid is formed by a reaction of the ketoacid with ammonia, using the reduced cofactor” (see, e.g., Baldenius, [0006]).
Regarding claim 7 pertaining to SEQ ID NO: 39, Baldenius teaches SEQ ID NO: 49, which has 100% sequence identity to instant SEQ ID NO: 39 (see, e.g., Office Action Appendix).
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 a genetically modified microorganism for ectoine production, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the genetically modified microorganism overexpresses an aspartate transaminase, as taught by Ikeda. One would have been motivated to do so because Ikeda teaches that aspartate aminotransferase expression affects L-amino acids production (see, e.g., Ikeda, [0011]). Additionally, Ikeda teaches that “overexpressing the aspC gene of a microorganism of the genus Escherichia, the L-amino acid productivity can be improved” (see, e.g., Ikeda, [0003]). Moreover, modified-Eilert-Xie-Bathe-Kim-Way teaches that L-amino acids modulates the expression of ProP, which catalyzes the absorption of ectoine within the microorganism (see, e.g., Bathe, [0010], [0229]). Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Ikeda, it would have been obvious to produce a microorganism wherein the aspartate transaminase is overexpressed. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Ikeda both teach production of genetically modified microorganisms.
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 a genetically modified microorganism for ectoine production, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the genetically modified microorganism overexpresses a glutamate dehydrogenase, as taught by Baldenius. One would have been motivated to do so because Baldenius teaches an alpha amino acid dehydrogenase (see, e.g., Baldenius, claims 4, 9), wherein glutamate dehydrogenase converts glutamate into α-ketoglutarate, and wherein α-ketoglutarate is part of the citric acid cycle. Moreover, as previously mentioned, the citric acid cycle results in production of oxaloacetate, which is used to produce ectoine synthesis. Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Baldenius, it would have been obvious to produce a genetically modified microorganism, wherein the microorganism overexpresses glutamate dehydrogenase. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Baldenius both teach the production of genetically modified microorganisms.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Eilert, Xie, Bathe, Kim, and Way as applied to claims 1-2 and 10-12 above, and further in view of Kakuda (Construction of Pta-Ack Pathway Deletion Mutants of Escherichia coli and Characteristic Growth Profiles of the Mutants in a Rich Medium; 1994 – previously cited).
The teachings of Eilert, Xie, Bathe, Kim, and Way, herein referred to as modified-Eilert-Xie-Bathe-Kim-Way, are discussed above as it pertains to a modified microorganism for the production of ectoine.
However, modified-Eilert-Xie-Bathe-Kim-Way does not teach: a deletion of the gene ackA-pta (claim 8).
Kakuda’s general disclosure relates to construction of Pta-Ack pathway deletion mutants in E. coli to study acetate production and reuse (see, e.g., Kakuda, abstract). Furthermore, Kakuda discloses that the Pta-Ack pathway in E. coli is important for carbon influx (see, e.g., Kakuda, Discussion, pg. 2234).
Regarding claim 8 pertaining to the ackA-pta gene deletion, Kakuda teaches the construction of a deletion mutant comprising a deletion of the ackA-pta gene (see, e.g., Kakuda, “Construction of mutants as to the Pta-Ack pathway, pg. 2233).
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 genetically modified microorganism for ectoine production, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the genetically modified microorganism has a ackA-pta gene deletion, as taught by Kakuda. One would have been motivated to do so because Kakuda teaches that the pathway encoded by the ackA-pta gene is responsible for acetate production and reuse, wherein the lack of this pathway triggers the activity of acetyl-CoA synthetase (see, e.g., Kakuda, Introduction, pg. 2232), which is responsible for acetyl-CoA that can be used within the citric acid cycle for production of oxaloacetate and further downstream production of ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way. Furthermore, Kakuda teaches that the Pta-Ack pathway is important for carbon influx (see, e.g., Kakuda, Discussion, pg. 2234), which one of ordinary skill in the art would understand is important for ectoine synthesis. Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Kakuda, it would have been obvious to produce a genetically modified microorganism, wherein the microorganism has a deletion in the gene ackA-pta. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Kakuda both teach production of genetically modified microorganisms.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Eilert, Xie, Bathe, Kim, and Way as applied to claims 1-2 and 10-12 above, and further in view of Zeng (EP3470512; Date of Publication: April 17, 2019 – previously cited).
The teachings of Eilert, Bathe, Kim, and Way, herein referred to as modified-Eilert-Xie-Bathe-Kim-Way, are discussed above as it pertains to a modified microorganism for the production of ectoine.
Regarding claim 9 pertaining to overexpression, modified-Eilert-Xie-Bathe-Kim-Way teaches methods of overexpression, which include a “widespread method for achieving overexpression is the process of chromosomal gene amplification. In this method, at least one additional copy of the polynucleotide of interest is inserted into the chromosome of a bacterium” (see, e.g., Bathe, [0219]). Additionally, modified-Eilert-Xie-Bathe-Kim-Way teaches “A further method for achieving overexpression comprises linking the respective gene or allele in a functional manner (operably linked) to a promoter or an expression cassette” (see, e.g., Bath, [0220]).
However, modified-Eilert-Xie-Bathe-Kim-Way does not teach: wherein the microorganism has been genetically modified to be able to utilize sucrose as a carbon source, and wherein said microorganism further comprises the overexpression of: the heterologous cscBKAR gene of E. coli EC3132, or the heterologous scrKYARgenes of Salmonella sp. (claim 9).
Zeng’s general disclosure relates to “a genetically modified microorganism for the production of a derivative of 4-hydroxy-2-ketobutyrate” (see, e.g., Zeng, abstract). Furthermore, Zeng discloses that “the genetically modified microorganism of the invention is also modified to stimulate the flux in the oxaloacetate biosynthesis pathway; this result can be achieved by increasing the level of expression of phosphoenolpyruvate carboxylase, encoded by the ppc gene or by increasing the level of expression of pyruvate carboxylase, encoded by the gene pyc” (see, e.g., Zeng. [0074]). Moreover, Zeng discloses “Another way to stimulate the flux into homoserine biosynthesis pathway is to increase the expression of aspartate aminotransferase encoded by aspC gene” (see, e.g., Zeng, [0078]).
Regarding claim 9 pertaining to the utilization of sucrose, Zeng teaches the modification of a microorganism to express the scrKYABR genes from Salmonella, and the cscBKAR genes grom E. coli EC3132, which encodes a sucrose transport system; therefore, allowing the microorganism to uptake and utilize sucrose as a carbon source (see, e.g., Zeng, [0086]-[0087]).
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 genetically modified microorganism for the production of ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way, wherein the genetically modified microorganism has overexpression of the scrKYABR genes from Salmonella, or the cscBKAR genes grom E. coli EC3132, as taught by Zeng. One would have been motivated to do so because Zeng teaches that scrKYABR and cscBKAR both include genes for sucrose transport (see, e.g., Zeng, [0086]-[0087]), wherein one of ordinary skill in the art would readily understand that sucrose can be used as a carbon source for subsequent downstream production of ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way. Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way and Zeng, it would have been obvious to produce a genetically modified microorganism wherein the microorganism expresses scrKYABR or cscBKAR. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way and Zeng both teach production of genetically modified microorganisms.
Claims 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Eilert, Xie, Bathe, Kim, Way, Ikeda and Baldenius as applied to claims 1-2, 7, and 10-12 above, and further in view of Kakuda (Construction of Pta-Ack Pathway Deletion Mutants of Escherichia coli and Characteristic Growth Profiles of the Mutants in a Rich Medium; 1994 – previously cited).
The teachings of Eilert, Xie, Bathe, Kim, Way, Ikeda, and Baldenius, herein referred to as modified-Eilert-Xie-Bathe-Kim-Way-Ikeda-Baldenius, are discussed above as it pertains to a modified microorganism for the production of ectoine.
Regarding claim 18 pertaining to the microorganism belonging to the family of the bacteria Enterobacteriaceae, modified-Eilert-Xie-Bathe-Kim-Way-Ikeda-Baldenius teaches a genetically engineered Escherichia coli (see, e.g., Xie, English Translation, abstract).
However, modified-Eilert-Xie-Bathe-Kim-Way-Ikeda-Baldenius does not teach: a deletion of the ackA-pta gene (claim 17).
Kakuda’s general disclosure was previously discussed above. Moreover, the disclosure relates to construction of Pta-Ack pathway deletion mutants in E. coli to study acetate production and reuse (see, e.g., Kakuda, abstract). Moreover, Kakuda teaches that the Pta-Ack pathway is important for carbon influx (see, e.g., Kakuda, Discussion, pg. 2234)
Regarding claim 17 pertaining to the ackA-pta gene deletion, Kakuda teaches the construction of a deletion mutant comprising a deletion of the ackA-pta gene (see, e.g., Kakuda, “Construction of mutants as to the Pta-Ack pathway, pg. 2233).
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 genetically modified microorganism for ectoine production, as taught by modified-Eilert-Xie-Bathe-Kim-Way-WIkeda-Baldenius, wherein the genetically modified microorganism has a ackA-pta gene deletion, as taught by Kakuda. One would have been motivated to do so because Kakuda teaches that the pathway encoded by the ackA-pta gene is responsible for acetate production and reuse, wherein the lack of this pathway triggers the activity of acetyl-CoA synthetase (see, e.g., Kakuda, Introduction, pg. 2232), which is responsible for acetyl-CoA that can be used within the citric acid cycle for production of oxaloacetate and further downstream production of ectoine, as taught by modified-Eilert-Xie-Bathe-Kim-Way-Ikeda-Baldenius. Furthermore, Kakuda teaches that the Pta-Ack pathway is important for carbon influx (see, e.g., Kakuda, Discussion, pg. 2234), which one of ordinary skill in the art would understand is important for ectoine synthesis. Therefore, based on the teachings of modified-Eilert-Xie-Bathe-Kim-Way-Ikeda-Baldenius and Kakuda, it would have been obvious to produce a genetically modified microorganism, wherein the microorganism has a deletion in the gene ackA-pta. One would have expected success because modified-Eilert-Xie-Bathe-Kim-Way-Ikeda-Baldenius and Kakuda both teach production of genetically modified microorganisms.
Examiner’s Response to Arguments
Applicant's arguments filed 12/12/2025 have been fully considered but they are not persuasive.
Regarding Applicant’s argument that Bathe and Kim are not dedicated to ectoine production (remarks, page 10), this argument is not persuasive for multiple reasons:
First, Bathe and Kim were not relied upon to teach the limitation of a microorganism modified for ectoine synthesis. As discussed above, Eilert and Xie were relied upon to teach the limitation(s) pertaining to ectoine synthesis. Eilert teaches “a method for producing ectoine or a derivative thereof, using host cells containing at least one recombinant DNA sequence encoding each of one or more heterologous enzymes for biosynthesis of ectoine” (see, e.g., Eilert, [0001]). Xie teaches a genetically engineered Escherichia coli bacterium that expresses ectABC from Halomonas elongatus in order to produce ectoine by glucose fermentation and overcome the harsh reaction conditions. Therefore, Eilert and Xie were relied upon for teaching a modified microorganism for ectoine synthesis.
Secondly, Bathe was relied upon for teaching the limitation regarding the deletion of pykA and pykF genes. Bathe teaches that the pykA and pykF genes may be eliminated (i.e., deleted) in E. coli (see, e.g., Bathe, [0229], [0237]-[0238]). Additionally, Bathe teaches that deletion of pykA and pykF modulates the expression of L-amino acids, which, in response, modulates the expression of ProP, which catalyzes the absorption of ectoine within the microorganism (see, e.g., Bathe, [0010], [0229]). Therefore, Bathe is analogous art to the instantly claimed invention because Bathe teaches genes that are responsible for modulating expression of proteins that are responsible for ectoine absorption within cells.
Thirdly, Kim was relied upon for teaching a reduction in citrate synthase enzyme activity. Kim teaches Kim teaches the inactivation of the citrate synthase enzyme (see, e.g., Kim, English Translation, Description, pgs. 2, 4 & Example 1, pg. 7). One of ordinary skill in the art would readily understand that deletion of citrate synthase would result in a reduction in enzyme activity that is at least 75% or more. Furthermore, Kim teaches using Escherichia coli to produce O-acetyl hemoserine in high yield (see, e.g., Kim, English Translation, “Description”, pg. 1). Furthermore, Kim discloses that O-acetyl homoserine may be used as methionine precursors (see, e.g., Kim, English Translation, pg. 2). Moreover, based on the teachings set forth in Eilert and Bathe, as discussed above, it is known in the art that L-amino acids modulate the expression of ProP, which catalyzes the absorption of ectoine within the microorganism (see, e.g., Bathe, [0010], [0229]). Furthermore, Eilert teaches that synthesis of 5-hydroxyectoine is based on the synthesis of amino acids, specifically from the aspartate family (see, e.g., Eilert, [0047]). Additionally, Eilert teaches alternative methods of producing ectoine in yeast cells, wherein high stress conditions, such as high salinity, are not required (see, e.g., Eilert, [0014]). Therefore, Kim is analogous art to the claimed invention based on the teachings of Eilert, Bathe, and Kim.
Fourthly, in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). As discussed above, Kim and Bathe were relied upon for the limitations regarding deletion of pykA and pykF genes, and a reduction in citrate synthase enzyme activity. Meanwhile, Eilert and Xie were relied upon for the limitation regarding a modified microorganism for ectoine production. Therefore, Applicant’s argument that Bathe and Kim don’t teach ectoine production is not persuasive because Bathe and Kim were not relied upon in the rejection above to teach these limitations.
Conclusion
Claims 1-2, 4, 6-12, and 16-18 are rejected.
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Correspondence Information
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/NATALIE IANNUZO/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653