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 .
This second non-final rejection is being sent out to the Applicant in view of Applicant’s assertion that that the finality of the office action dated 1/26/2026 is improper. Applicant contends that the amendment of claim 1 to recite ‘an engineered bacterium comprising a genetic alteration in a native gene involved in phosphate solubilization selected from one or more of phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT, and gad; and wherein said genetic alteration in the gene involved in phosphate solubilization comprises an insertion of a regulatory element, codon optimization, codon randomization, a loss-of- function mutation, or a gene deletion; whereby said engineered bacterium solubilizes phosphates at a greater capacity or rate as compared to a non-engineered bacterium of the same species’ was foreseeable and should reasonable have been expected based on the rejections raised in the Office Action of August 6, 2025.
Applicants’ claims filed on 3/26/2026 are acknowledged. This listing of claims replaces all prior listings of claims in the application.
Claims 1-2, 10-11, 19, 21, 32, 50, 53-54, 139-147 are pending.
Claims 3-9, 12-18, 20, 22-31, 33-49, 51-52, 55-138 are canceled.
Withdrawn Rejections
The rejection of claims 2, 139 under 35 U.S.C. 102(a)(1) as being anticipated by Kim et al (2003, Current Microbiology, cited on PTO-892 dated 3/26/2026) {herein Kim} as evidenced by Anton et al (2016, genome announcements, cited on PTO-892 dated 3/26/2026) {herein Anton} and Liang et al (1999, JMB, cited on PTO-892 dated 3/26/2026) {herein Liang} are withdrawn in view of Applicant’s remarks dated 3/26/2026 that Kim does not anticipate and/or make obvious the recitation ‘wherein said engineered bacterium is a non-intergeneric engineered bacterium.’
Maintained Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
The rejection of claims 1, 10-11, 19, 32, 53-54, 140-144, 146 rejected under 35 U.S.C. 102(a)(1) as being anticipated by Kim et al (2003, Current Microbiology, cited on PTO-892 dated 3/26/2026) {herein Kim} as evidenced by Anton et al (2016, genome announcements, cited on PTO-892 dated 3/26/2026) {herein Anton} and Liang et al (1999, JMB, cited on PTO-892 dated 3/26/2026) {herein Liang} is maintained. See MPEP 2131.01 regarding multiple reference 102 rejections. The rejection has been modified in view of Applicant’s Remarks dated 3/26/2026 that Kim does not anticipate the recitation of a non-intergeneric engineered bacterium.
Previously presented claims 1, 10-11, 19, 32 are drawn to an engineered bacterium comprising a genetic alteration in a native gene involved in phosphate solubilization selected from one or more of phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT, and gad; and wherein said genetic alteration in the gene involved in phosphate solubilization comprises an insertion of a regulatory element, codon optimization, codon randomization, a loss-of- function mutation, or a gene deletion; whereby said engineered bacterium solubilizes phosphates at a greater capacity or rate as compared to a non-engineered bacterium of the same species.
Previously presented claims 53-54, 140-144, 146 are drawn to a method of producing an engineered bacterium with improved phosphate solubilization activity, the method comprising: a) altering a codon usage of a native coding sequence of at least one native gene involved in phosphate solubilization to yield a codon altered coding sequence involved in phosphate solubilization, wherein the at least one gene involved in phosphate solubilization is selected from one or more of phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT, and gad; b) functionally linking each codon altered coding sequence involved in phosphate solubilization to a heterologous constitutive promoter; and c) introducing each promoter and codon altered coding sequence involved in phosphate solubilization into a bacterium to produce the engineered bacterium, whereby said engineered bacterium has improved phosphate solubilization activity as compared to a non- engineered bacterium of the same species.
With respect to claims 1, 10-11, 19, 53, 144, Kim teaches a genetically engineered Escherichia coli strain (DH5 alpha) that lacks the ability to solubilize insoluble phosphate (page 458, column 2, para 1). Evidentiary reference of Anton is cited to demonstrate that E. coli DH5 alpha is deficient in PhoA (page 1, column 1).As such, absent evidence otherwise, it is the Examiner’s position that since said strain lacks the ability to solubilize insoluble phosphate, that said strain has a loss-of-function mutation. Said genetic alteration also includes a stop codon with a potential rho-independent termination signal downstream of pqqA involving a stem-loop structure in the mRNA followed by several U bases (page 458, column 2, para 4). Absent evidence otherwise, it is the Examiner’s position that said genetic modification constitutes that insertion of a regulatory element (stop codon). In addition, it is the Examiner’s position that the utilization of the optimized stop codon downstream of pqqA is the same as the recited ‘codon optimization’ within claims 19, 144 of the instant application as Kim teaches the optimization of mRNA by adding several UUU bases to form a rho-independent termination sequence for the expression of the pqq cluster from E. intermedium in E. coli (page 458, column 2, para 4). Furthermore, Kim, teaches in the presence of a plasmid expressing the pqq gene cluster (pqqA, B, C,D, E, and F), p7EB3, of Enterobacter intermedium there is an increase in the ability of the genetically engineered strain to solubilize phosphate compared to E. coli DH5-alpha without the pqq gene cluster (abstract and page 458, column 2, para 1, Fig. 4). Evidentiary reference of Liang is cited to demonstrate that the plasmid p7EB3 has a constitutive promoter (abstract). As such, it is the Examiner’s position that the p7EB3 promoter is a heterologous constitutive promoter as said promoter is not native to E. coli, upon which the plasmid is transformed into. Additionally, It is the Examiner’s position that the plasmid expressing the pqq gene cluster (pqqA, B, C,D, E, and F), taught by Kim, is a regulatory element inserted into E. coli as the plasmid is a genetic element that contains and functions as a regulatory system.
With respect to claims 54, 140-143, it is known by those of ordinary skill in the art that E. coli and E. intermedium are members of the same species, both phylogenetically and genetically. As such, it is the position that the pqq cluster from E. intermedium and the host bacterium, E. coli, result in an improved engineered bacterium as Kim teaches said engineered bacterium exhibits improved phosphate solubilization (page 460, column 2, para 2).
With respect to claims 32, 146, since the art teaches the structure of an engineered bacterium that is able to solubilize phosphates at a greater rate, compared to non-engineered bacterium of the same species (Kim: abstract), it is the Examiners interpretation that the engineered bacterium taught by Kim would necessarily fix nitrogen. Especially since Kim teaches the pqq cluster that is transformed into E. coli is derived from E. intermedium which is a grass rhizosphere bacterium (abstract).
For the reasons stated herein, the teachings of Kim anticipates claims 1, 10-11, 19, 32, 53-54, 140-144, 146.
RESPONSE TO REMARKS: Applicant's remarks filed on 3/26/2026 have been fully considered by the Examiner, but they are found to be not persuasive for the reasons already of record and the reasons set forth below. Beginning on page 1 of Applicant’s Remarks, Applicant, in summary, contends that the finality of the office action dated 1/26/2026 is improper. Applicant contends that the amendment of claim 1 to recite ‘an engineered bacterium comprising a genetic alteration in a native gene involved in phosphate solubilization selected from one or more of phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT, and gad; and wherein said genetic alteration in the gene involved in phosphate solubilization comprises an insertion of a regulatory element, codon optimization, codon randomization, a loss-of- function mutation, or a gene deletion; whereby said engineered bacterium solubilizes phosphates at a greater capacity or rate as compared to a non-engineered bacterium of the same species’ was foreseeable and should reasonable have been expected based on the rejections raised in the Office Action of August 6, 2025.
Examiner appreciates Applicant’s remark’s dated 3/2/2026. Examiner contends that amending the claims to recite ‘…one or more of phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT,and gad…’ sufficiently broadens the claim language by removing the recitation ‘… and a gene encoding a homolog of a protein encoded by phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT, or gad…’
Applicant contends that Kim does not anticipate an engineered bacterium comprising a genetic alteration in a native gene involved in phosphate solubilization, wherein the at least one gene involved in phosphate solubilization is selected from one or more of phoA, phoC, phoD, napA, napD, napE, acpA, appA, phy, pqqA, pqqB, pqqC, pqqD, pqqE, gcd, gabY, gntT, and gad.’
Examiner contends that Kim teaches a genetically engineered Escherichia coli strain (DH5 alpha) that lacks the ability to solubilize insoluble phosphate (page 458, column 2, para 1). Evidentiary reference of Anton is cited to demonstrate that E. coli DH5 alpha is deficient in PhoA (page 1, column 1). As such, absent evidence otherwise, it is the Examiner’s position that since said strain lacks the ability to solubilize insoluble phosphate, that said strain has a loss-of-function mutation. Said genetic alteration also includes a stop codon with a potential rho-independent termination signal downstream of pqqA involving a stem-loop structure in the mRNA followed by several U bases (page 458, column 2, para 4). Absent evidence otherwise, it is the Examiner’s position that said genetic modification constitutes an insertion of a regulatory element (stop codon).
Maintained Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The rejection of claims 21, 50, 145, 147 under 35 U.S.C. 103 as being unpatentable over Kim et al (2003, Current Microbiology, cited on PTO-892 dated 3/26/2026) {herein Kim} as applied to claims 1, 10-11, 19, 32, 53-54, 140-144, 146 as evidenced by Anton et al (2016, genome announcements, cited on PTO-892 dated 3/26/2026) {herein Anton}, Liang et al (1999, JMB, cited on PTO-892 dated 3/26/2026) {herein Liang} and Shahab (2009, ispub, cited on PTO-892 dated 3/26/2026) {herein Shahab} is maintained. The rejection has been modified in view of Applicant’s Remarks dated 3/26/2026 that Kim does not anticipate of make obvious the recitation of a non-intergeneric engineered bacterium.
Previously presented claim 21 is drawn to the engineered bacterium of claim 1, wherein said genetic alteration in a gene involved in phosphate solubilization comprises a reduction in gene function, loss-of-function mutation, or a gene deletion as compared to a non-engineered bacterium of the same species, wherein the gene involved in phosphate solubilization is selected from gntT or gad.
Previously presented claim 50 is drawn to a method of increasing an amount of soluble phosphate in soil, the method comprising contacting soil comprising insoluble phosphate with the engineered bacterium of claim 1.
Previously presented claim 145 is drawn to the engineered bacterium of claim 2, wherein said genetic alteration in a gene involved in phosphate solubilization comprises a reduction in gene function, a loss-of-function mutation, or a gene deletion as compared to a non-engineered bacterium of the same species, wherein the gene involved in phosphate solubilization is selected from gntT or gad.
Previously presented claim 147 is drawn to a method of increasing an amount of soluble phosphate in soil, the method comprising contacting soil comprising insoluble phosphate with the engineered bacterium of claim 2.
The teachings of Kim as applied to claims 1, 10-11, 19, 32, 53-54, 140-144, 146 are set forth in the 102a1 rejection above.
With respect to claims 50, 147, Kim teaches E. intermedium, of which the pqq cluster was derived, is a soil microbe that possesses a strong ability to solubilize insoluble phosphate from the rhizosphere of grass (page 457, column 1, para 1). The enzymes involved are glucose dehydrogenase, gluconic acid dehydrogenase, and 2-ketogluconic acid dehydrogenase (page 457, column 1, para 2 and page 457, column 2, para 1). The genetic construct taught by Kim produces said enzymes (abstract). Evidentiary reference of Shahab is cited to demonstrate that gluconate permease (gntT) is a membrane transporter of gluconate into the cell, of which is native to E. coli (fig 4). Furthermore, Kim teaches E. coli lacks the ability to synthesize gluconate dehydrogenase, thereby resulting in an influx of gluconate in culture which also results in an increase in soluble phosphate concentration (page 460, column 1, para 1).
However, Kim does not teach wherein the gene involved in phosphate solubilization is selected from gntT or gad (claims 21, 145). Kim does not teach a method of increasing an amount of soluble phosphate in soil, the method comprising contacting soil comprising insoluble phosphate with the engineered bacterium of claim 1 (claims 50, 147).
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to try adding the engineered bacterium taught by Kim with insoluble phosphate because doing so would increase the amount of soluble phosphate. Additionally, it would be obvious to one of ordinary skill in the art to modify the engineered bacterium taught by Kim by reducing or eliminating the expression of gntT as doing so would result in an increase in extracellular gluconate, thereby resulting in an increase in soluble phosphate.
One of ordinary skill in the art would have had a reasonable expectation of success, a reasonable level of predictability, and would be motivated to contact the engineered bacterium taught by Kim with soil because doing so would increase the amount of soluble phosphate in the soil. Furthermore, one would be motivated to do so since Kim teaches said engineered bacterium that expresses the pqq cluster from E. intermedium activates endogenous glucose dehydrogenase to permit gluconic acid secretion that solubilizes the insoluble phosphate (abstract). Therefore, one of ordinary skill in the art would expect similar results if the engineered bacterium were in soil with insoluble phosphate.
Additionally, one of ordinary skill in the art would have had a reasonable expectation of success, a reasonable level of predictability, and would be motivated try mutating the engineered bacterium taught by Kim to reduce or eliminate the expression of gntT because the gntT gene encodes a gluconate transporter, responsible for moving gluconate from the bacterial periplasm or environment into the cell for further metabolism. It is the Examiner’s position that deleting gntT in phosphate-solubilizing bacteria would block or reduce the uptake of gluconate into the cell, thereby forcing the secreted gluconate to remain in the surrounding environment, where it can actively chelate metal ions (like Ca²⁺, Fe³⁺, Al³⁺) and lower the pH to solubilize insoluble mineral phosphates. As such, resulting in increased extracellular gluconate concentration and enhanced phosphate solubilization activity in the bacterial surroundings. Therefore there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Applicant’s remarks filed on 11/6/2025 have been fully considered; however, they are rendered not persuasive in view of the rejection set forth above, which is necessitated by Applicants’ amendment to the claims. Examiner contends that in view of Kim, it would have been obvious to one of ordinary skill in the art to try adding the engineered bacterium, taught by Kim, with insoluble phosphate as doing so would increase the amount of soluble phosphate. Additionally, it would be obvious to one of ordinary skill in the art to modify the engineered bacterium taught by Kim by reducing or eliminating the expression of gntT as doing so would result in an increase in extracellular gluconate, thereby resulting in an increase in soluble phosphate.
Allowable Subject Matter
Claims 2, 139 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims, as Kim does not anticipate a non-intergeneric engineered bacterium. Furthermore, said limitation is not obvious over the prior art.
Conclusion
Status of the claims
Claims 1, 10-11, 19, 21, 32, 50, 53-54, 140-147 are pending.
Claims 3-9, 12-18, 20, 22-31, 33-49, 51-52, 55-138 are canceled.
Claims 2 and 139 are objected to.
Claims 1-2, 10-11, 19, 21, 32, 50, 53-54, 139-147 are rejected.
No claims are in condition for allowance.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERICA NICOLE JONES-FOSTER whose telephone number is (571)270-0360. The examiner can normally be reached mf 7:30a - 4:30p.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/MANJUNATH N RAO/Supervisory Patent Examiner, Art Unit 1656
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