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 .
Application Status and Species Election
Applicant’s amendment filed September 4, 2025, amending claims 1, 4, and 17-19, canceling claims 8-10, and adding new claims 29-38 are acknowledged. Claims 1-7 and 11-38 are pending. Claims 20-28 remain withdrawn from further consideration pursuant to 37 CFR l.142(b) as being drawn to nonelected groups, there being no allowable generic or linking claim.
The limitations in previously withdrawn claim 8 (now canceled) have been incorporated into claim 1. As amended, claim 1 requires the Fe-S cluster polypeptide to be CobG and the produced compound to be cobalamin. In the Response to the Restriction Requirement, Applicant elected the Fe-S cluster protein species IlvD and IspH (See Remarks filed 10-30-2024, page 20). In accordance with the species election, claim 8 was previously withdrawn (See Non-Final Office Action mailed 11-25-2024, page 2). Although the previously examined claims directed to the elected species IlvD and IspH (claims 9-10) were canceled, their limitations are now recited in new claims 29-32. Applicant’s remarks support this conclusion: “newly added claim 29 includes recitations substantially similar to the recitations of claims 1 and previously pending claim 9” (Remarks, page 36) and “newly added claim 32 includes recitations substantially similar to the recitations of claims 1 and previously pending claim 10” (Remarks, page 39). Thus, claims directed to the elected species are still present in the claim set and are pending. Because Applicant has not removed claims directed to the elected species, the Species Election still applies in the examination of the amended claims filed 9-4-2025. As a result, claims 1-7, 11-28 and 33-38 are now withdrawn from further consideration pursuant to 37 CFR l.142(b) as being drawn to nonelected species of Fe-S polypeptides, CobG, there being no allowable generic or linking claim.
Claims 29-32 are under examination.
Notes on Previous Rejections/Objections
In view of Applicant’s incorporation of a non-elected species into claim 1, claims that were previously examined are now withdrawn. However, for the purposes of compact prosecution, Examiner will comment on whether the claim rejections or objections made over the withdrawn claims would be maintained or overcome after a very cursory review.
The amendments to claims 1, 4 and 17 would overcome the previous claim objections.
While the amendment to claim 19 appears to overcome the rejection for improper Markush grouping, the amendments to claim 17-18 are not sufficient to remedy the improper Markush grouping. Claims 17 and 18 still recite compounds that don’t share a common structural similarity or common use. For instance, claims 17 and 18 still recite “hydrogen”, which is an atom or element, “Hydrogenase”, which is a large enzyme, “branched chain amino acids”, which is an entire genus of amino acids, and/or “quinoprotein”, which is a genus of three distinct groups of enzymes. There is nothing in the record or prior art that indicates there is a common structural similarity or use at least between hydrogen, Hydrogenase, quinoprotein, or branched chain amino acid.
Regarding claim 29, the inclusion of “wherein the at least one compound is selected from L-valine…” in addition to the limitations previously recited in claims 1 and 9 overcomes the § 103 rejection over Cheong in view of Tan, Akhtar and others.
Regarding claim 32, the inclusion of “wherein the at least one compound is selected from isoprenoids…” in addition to the limitations previously recited in claims 1 and 10 overcomes the § 103 rejection of Zhou, Kumaran, Giel and others. However, claim 32 is still obvious over the same prior art. The rejection of claim 32 below is updated to reflect the claim numbering and inclusion of “wherein the at least one compound is an isoprenoid”.
The NSDP patenting rejection is updated to reflect the new claim numbering.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Drawings
The drawings are objected to because the text of FIG 16A is still not sufficiently legible, and the text in FIGs 1, 12A, 12D, 12E, 13, 14A-B, 15, 16B, 17, 18, 19, 20, 21 and 23 of the Drawings filed 9-4-2025 are not sufficient to provide satisfactory reproduction characteristics. 37 CFR 1.84(l) states that “all drawings must be made by a process which will give them satisfactory reproduction characteristics. Every line, number, and letter must be durable, clean, black (except for color drawings), sufficiently dense and dark, and uniformly thick and well-defined.” In the instant case, the text in above FIGs is very small and of poor resolution, and/or light grey or otherwise not sufficiently dense and dark to permit satisfactory reproduction characteristics. Additionally, the letters over shading especially in FIG 15 are not sufficiently dark for reproduction.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections - New
Claims 29, 31 and 32 are objected to because of the following informalities:
Claims 29 and 32 recite "FE-S cluster" in lines 4 or 5, which should be changed to "Fe-S cluster".
Claim 31 is missing a space in line 6 between “SEQ ID No.:177” and “(origin: Corynebacterium glutamicum)”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 30 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, 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. This is a new rejection necessitated by amendment.
Claim 30 is indefinite for two reasons.
First, the claim recites “wherein said additional transgenes or endogenous genes further encode one or more of:” and then in each of (a) and (b), the claim recites “an IlvN [IlvNbis] polypeptide… together with IlvB catalyzes the conversion of pyruvate…” It is unclear if only the IlvN or IlvNbis polypeptide is required in options (a) and (b) or if IlvB is also required to be encoded in the cell if IlvN and IlvNbis are encoded in the cell.
Second, each of (a), (b), (c), and (d) recite “such as a polypeptide with an amnio acid sequence having 80, 85, 90, 95 or 100% sequence identity to [SEQ ID NOs].” The phrase "such as" renders the claim indefinite because it is unclear whether the limitations following the phrase (i.e., the sequence identity to SEQ ID NOs) are part of the claimed invention. See MPEP § 2173.05(d).
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 29-30 are rejected under 35 U.S.C. 103 as being unpatentable over Park (Park et al., PNAS (2007), 104: 7797-7802) in view of Tan (Tan et al., Biochem. J. (2009), 420: 463-472, of record), Flint (US 20120064561 A1), Akhtar (Akhtar et al., Appl Microbial Biotechnol (2008) 78: 853-862, of record) and Giel (Giel et al., Molecular Microbiology (2013), 87: 478-492, of record). This is a new rejection necessitated by amendment.
Regarding claims 29-30, Park teaches metabolic engineering of E. coli (i.e., a prokaryotic cell) for the production of L-valine (title). Park teaches dihydroxy acid dehydratase encoded by the IlvD gene is involved in L-valine biosynthesis (page 7798, last ¶; Fig 1). Park teaches cloning the IlvD gene into a plasmid and transformed into E. coli (i.e., the prokaryotic cell comprising a transgene encoding IlvD) (page 7799, ¶1). Park teaches amplification of the IlvD gene in E. coli increased the production L-valine (page 7799, ¶1). Park teaches also co-expressing additional copies of IlvB, IlvN and IlvC (wherein said additional transgenes encode an IlvC polypeptide, an IlvE polypeptide, and further encode an IlvN polypeptide together with ah IlvB polypeptide) (page 7799, ¶1).
Park does not teach that IlvD is a Fe-S polypeptide. Park does not teach the cells also comprise a mutant IscR with an increased apoprotein: holoprotein ratio.
Tan teaches the biosynthetic pathway of branched amino acids, including valine, in E. coli requires the dihydroxy-acid dehydratase enzyme IlvD (page 465, ¶4). Tan teaches IlvD is an Fe-S cluster polypeptide (page 465, ¶4). Tan teaches that the Fe-S cluster donor polypeptide, IscA is required for full IlvD activity (page 465, ¶3 through page 466, ¶1; Fig 2A).
Flint teaches increasing expression of dihydroxy acid dehydratase (DHAD) polypeptides (i.e., IlvD) in a recombinant host ([0004]). Flint teaches increasing the activity of the DHAD polypeptides by increasing Fe-S cluster biosynthesis activity of the cell ([0004]). Flint teaches that high levels of DHAD activity are desired for increased production from biosynthetic pathways, but that the biosynthesis of Fe-S clusters must also be increased where the Fe-S cluster protein is expressed ([0008]). Flint teaches that the E. coli IscA can be upregulated to increase flux in an Fe-S cluster biosynthesis pathway and improve the activity of Fe-S proteins like DHAD ([0125], [0132]; Table 9).
Akhtar teaches that bacteria have three Fe-S cluster assembly operons, of which the ISC operon is considered as providing the house-keeping role of Fe-S cofactor assembly and insertion (page 853, ¶2). Akhtar teaches that Fe-S assembly and incorporation was a limiting factor for the majority of recombinant Fe-S proteins (page 854, ¶1). Akhtar teaches that plasmid-based ISC operon overexpression enhances the ability of E. coli to produce Fe-S clusters and enhances the activity of Fe-S polypeptides (page 854, ¶1-2). Akhtar teaches that although deletion of the ISC repressor, IscR, provides enhanced expression of the isc operon, IscR deletion effects the expression of 35 additional genes (page 854, ¶3).
Giel teaches that the isc operon is responsible for the majority of Fe-S cluster biogenesis in E. coli (page 478, ¶1). Giel teaches the transcription of the isc operon is repressed by the isc Repressor, IscR (page 478, ¶1). Giel teaches that IscR itself is an Fe-S cluster protein that binds to and represses the Isc Promoter (Pisc) when IscR is in its halo-form bound with 2 Fe-S clusters (page 479, ¶3). Giel teaches (a) an IscR mutants with alanine substitutions at one or more C92, C98, C104 residues cannot bind Fe-S clusters and therefore remains in the apo-form (i.e., wherein the mutant IscR has an increased apoprotein : holoprotein ratio in the cell) (page 481, ¶3). Giel teaches that IscR mutants with either one or all three alanine substitutions in C92, C98 and C104 are incapable of repressing the Pisc (Fig 3). Giel teaches that previous studies showed that the lscR-C92A/C98A/C104A mutant was functional at other promoters, that only require the apo-IscR polypeptide for regulation (page 482, ¶1).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included Giel's lscR-C92A/C98A/C104A in Park’s E. coli cells with the llvBNCED transgenes. It would have amounted to the simple combination of known elements by known means to yield predictable results. Tan demonstrates and Flint teaches that IlvD, a protein with dihydroxy-acid dehydratase activity, is an Fe-S polypeptide. Akhtar’s teachings provide predictability that increasing ISC operon expression would increase the activity of IlvD by providing additional Fe-S clusters. Additionally, Flint teaches that when Fe-S cluster polypeptides are heterologously expressed in a cell, flux through the Fe-S biogenesis pathway should also be increased to increase the activity of the Fe-S cluster polypeptides in the cell. As such the skilled artisan would predict that alternative means to increase ISC expression would likewise increase IlvD activity. Giel demonstrates one such alternative mean for increasing ISC operon expression - expressing lscR-C92A/C98A/C104A in cells. Since Akhtar teaches that for most Fe-S proteins, Fe-S assembly and loading are the rate limiting steps in their activity, the skilled artisan would predict that Fe-S assembly/loading is also the rate limiting step in the L-valine pathway in E. coli. As such, the skilled artisan would predict that providing lscR-C92A/C98A/C104A to the cells would increase flux through the L-valine resulting in enhanced L-valine production. The skilled artisan would have been motivated to use the lscR-C92A/C98A/C104A mutant instead of deleting IscR as taught in Akhtar, because both Akhtar and Giel teach that IscR regulates other operons in addition to the ISC operon. Using lscR-C92A/C98A/C104A would not disrupt non-ISC operon expression. The skilled artisan would also be motivated to use the lscR-C92A/C98A/C104A rather than plasmid-expressed ISC operon because expressing a single IscR transgene is simpler than expressing six transgenes (iscS, iscU, iscA, hscB, hscA and fdx). The skilled artisan would have been motivated to increase expression of the Fs-S biosynthetic machinery using lscR-C92A/C98A/C104A, for the purpose of increasing Fe-S cluster polypeptide activity in cells thereby increasing flux through the L-valine biosynthetic pathway.
Claim 31 is rejected under 35 U.S.C. 103 as being unpatentable over Park (Park et al., PNAS (2007), 104: 7797-7802), Tan (Tan et al., Biochem. J. (2009), 420: 463-472, of record), Flint (US 20120064561 A1), Akhtar (Akhtar et al., Appl Microbial Biotechnol (2008) 78: 853-862, of record) and Giel (Giel et al., Molecular Microbiology (2013), 87: 478-492, of record) as applied to claims 29-30 above, and further in view of Genbank (WP_116324143.1, dihydroxy-acid dehydratase [Escherichia coli], https://www.ncbi.nlm.nih.gov/protein/1452085069, published 8-21-2018, [retrieved 9-23-2025]). This is a new rejection necessitated by amendment.
The teachings of Park, Tan, Flint, Akhtar and Giel are recited above and applied as for claims 29-30. Park describes the IlvD gene as “amplified” when additionally expressed in E. coli from a plasmid (page 7799, ¶1), indicating that the endogenous E. coli ilvD gene that encodes the endogenous E. coli IlvD polypeptide was expressed as a transgene.
Park, Tan, Flint, Akhtar and Giel do not teach the amino acid sequence of the IlvD polypeptide.
Genbank teaches the amino acid sequence of dihydroxy-acid dehydratase (DHAD) from Escherichia coli (page 1), which is 99.8% identical to SEQ ID NO 172 (See OA Appendix, pages 2-3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have specifically used the DHAD amino acid sequence taught in Genbank in the cell rendered obvious above. It would have amounted to using a known IlvD enzyme sequence in place of a generic one by known genes to yield predictable results. The skilled artisan would have expected that Genbank’s DHAD amino acid sequence could be expressed in the obvious cell because Park teaches expressing the E. coli IlvD polypeptide from a plasmid, and Genbank teaches the DHAD is the wildtype DHAD protein sequence. The skilled artisan would have been motivated to do so since the sequence in Genbank is the accepted IlvD/DHAD amino acid sequence.
Claim 32 is rejected under 35 U.S.C. 103 as being unpatentable over Zhou (Zhou et al., Journal of Biotechnology (2017), 248: 1–8; of record), as evidenced by Sigma (95098, https://www.sigmaaldrich.com/US/en/product/sigma/95098, [retrieved November 18, 2024]; of record), in view of Kumaran (US 20180245103 A1, published August 30, 2018; of record) and Giel (Giel et al., Molecular Microbiology (2013), 87: 478-492; of record). This is a new rejection, updated to reflect the new claim numbering and added limitations.
Regarding claim 32, Zhou teaches IspG and IspH catalyze the last two reactions in the MEP pathway, which is used to produce the isoprenoids (page 1, ¶1; Fig 1). Zhou teaches that IspG and IspH are Fe-S cluster polypeptides (page 2, ¶2). Zhou teaches IspH catalyzes the reduction of HMBPP (1-hydroxy-2-methyl-butenyl- 4-diphosphate) (Fig 1). As evidenced by Sigma, HMBPP is equivalent to 4-Hydroxy-3-methylbut-2-enyl diphosphate (page 1); therefore, IspH is an Fe-S polypeptide with 4-Hydroxy-3-methylbut-2-enyl diphosphate reductase activity. Zhou teaches genetically modified E. coli bacterial cells (i.e., prokaryotic cells) comprising (b) a transgene encoding IspH and IspG under the control of the lac promoter (Sections 2.1 and 2.2; Fig 2A). Zhou also teaches the transgene also encodes IspC (Fig 2), which has 1-deoxy-D-xylulose-5-phosphate reductoisomerase activity (Fig 1, legend). Zhou teaches the production of protoilludene (i.e., an isoprenoid) resulting from transgenic IspG/IspH activity (Fig 2B). Zhou teaches that IspG is a rate limiting step in the MEP pathway (page 5, ¶2). Zhou teaches the ISC operon is involved in the maturation process of IspG/H (page 2, ¶2). Zhou teaches that previous reports showed that co-overexpression of the ISC operon from a transgene and IspG/H increases the functional formation of IspG/H (page 2, ¶2).
Although Zhou teaches that increased expression of the ISC operon increases the functional formation of IspG and IspH, Zhou does not teach using a mutant IscR with an increased apoprotein : holoprotein ratio in the cell to cause the increased expression.
Kumaran also teaches the MEP biosynthetic pathway (Fig 1). Kumaran teaches IspG and IspH are Fe-S enzymes ([0008]). Kumaran teaches that increasing the availability or activity of Fe-S cluster proteins by altering expression of the isc operon ([0008]). Kumaran teaches that IscR represses transcription of the isc operon ([0045]). Kumaran teaches that inactivating the IscR gene results in improved Fe-S cluster enzyme performance ([0045]) and increased isoprenoid production (Fig 6, [0112]). Kumaran teaches one such way to inactivate the IscR gene is by “amino acid mutation” ([0045]).
Giel teaches that the isc operon is responsible for the majority of Fe-S cluster biogenesis in E. coli (page 478, ¶1). Giel teaches the transcription of the isc operon is repressed by the isc Repressor, IscR (page 478, ¶1). Giel teaches that IscR itself is an Fe-S cluster protein that binds to and represses the Isc Promoter (Pisc) when IscR is in its holo-form bound to 2 Fs-S clusters (page 479, ¶3). Giel teaches (a) an IscR mutants with alanine substitutions at one or more C92, C98, C104 residues cannot bind Fe-S clusters and therefore remains in the apo-form (i.e., wherein the mutant IscR has an increased apoprotein : holoprotein ratio in the cell) (page 481, ¶3). Giel teaches that IscR mutants with either one or all three alanine substitutions in C92, C98 and C104 are incapable of repressing the Pisc (Fig 3). Giel teaches that previous studies showed that the IscR-C92A/C98A/C104A mutant was functional at other promoters, that only require the apo-IscR polypeptide for regulation (page 482, ¶1).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included Giel’s IscR-C92A/C98A/C104A in Zhou’s E. coli cells with the IspH transgene. It would have amounted to the simple combination of known elements by known means to yield predictable results. Zhou teaches that co-expression of the ISC operon from a transgene increases holo-IspH formation. Kumaran teaches that increased ISC operon expression via IscR deletion increases flux through the MEP pathway. As such, the art recognized that there are multiple means to increase ISC expression and the skilled artisan would predict that alternative means to increase ISC expression would likewise increase holo-IspH formation and isoprenoid production. Giel demonstrates one such alternative mean for increasing ISC operon expression – expressing IscR-C92A/C98A/C104A in cells. Additionally, since Zhou teaches that the two reductases, IspG and IspH, are rate limiting steps in the isoprenoid synthesis, providing IscR-C92A/C98A/C104A in the cells would predictably increase flux through the MEP pathway resulting in enhanced isoprenoid production. The skilled artisan would have been motivated to use the IscR-C92A/C98A/C104A mutant instead of overexpression of a recombinant ISC operon, because 1) Zhou teaches that using a recombinant ISC operon only resulted in modest increase in a MEP pathway, 2) expressing a single IscR transgene is simpler than expressing six transgenes (iscS, iscU, iscA, hscB, hscA and fdx), and 3) Kumaran suggests that IscR can be inactivated by amino acid mutation to increase ISC expression. The skilled artisan would have been motivated to increase expression of the Fe-S biosynthetic machinery using IscR-C92A/C98A/C104A, for the purpose of increasing Fe-S cluster polypeptide activity in cells thereby increasing flux through the MEP pathway.
Response to Arguments - §103
Applicant’s arguments in regards to §103 rejections on pages 26-35 are directed to amended claim 1, reciting an unelected Fe-S polypeptide, CobG. Because claim 1 and its dependent claims are withdrawn as drawn to an unelected species, Applicant’s arguments on pages 26-35 are moot.
Applicant’s arguments on pages 36-38 in regards to the §103 rejections over Cheong in view of Tan, Akhtar and Giel are moot because the previous §103 rejection over Cheong in view of others is withdrawn. However, to the extent Applicant’s arguments can be applied to the current §103 rejection over Park in view of Tan, Flint, Akhtar and Giel, they are addressed as follows.
Applicant argues that Cheong does not recognize IlvD as an Fe-S cluster protein (page 36, ¶5). Examiner agrees and likewise, Park does not teach that IlvD is an Fe-S cluster protein either. However, this argument is not persuasive even as applied to Park because it is well known in the art that IlvD, which encodes a DHAD, is an Fe-S cluster protein as evidenced by Tan and Flint.
Applicant points out that 1) Tan does not suggest modification of the ISC regulatory elements, 2) Akhtar only teaches deletion of IscR or overexpression of Isc Operon, and 3) Giel does not apply the IscR mutants in any production system or suggest using it to enhance IlvD branched chain amino acid synthesis (Remarks, page 37, ¶1-3). Applicant then argues that the prior art references could not be combined with a likelihood of success of increased branched amino acid output because the cited references provide no evidence that the combination would work, would outperform known strategies, and ignores the toxicity and growth defects associated with IscR deletions/overexpression (through page 38, ¶2). These arguments have been fully considered but are not persuasive. First, the arguments are merely conclusive statement without evidence. MPEP 716.01(c) makes clear that arguments of counsel cannot take the place of evidence in the record. Additionally, the argument relies on features that are not claimed, namely outperformance of known strategies. Finally, the rejection does not rely on “IscR deletion” or “Isc overexpression”. The rejection states that it would have been obvious to express the apo-forming IscR mutant in cells. Additionally, the rejection above provides the predictability for increased valine synthesis. The prior art shows that 1) increased IlvD expression increases valine production and with 2) increased ISC operon expression results in higher Fe-S cluster protein activity, of which IlvD is a known Fe-S proteins. Flint even suggests that increasing the biogenesis of Fe-S clusters is needed in the cell if the activity of the transgenic Fe-S proteins are to be increased. As such, it was entirely predictable that Giel’s apo-IscR mutant would increase Fe-S cluster production, and as a result provide IlvD with Fe-S clusters, thereby increasing the flux through the branched chain amino acid biosynthetic pathway.
Applicant provides evidence from Example 6 and FIGs 16-17 in the present Application of expressing IscR (H107Y) in combination if IlvD leads to significant improvement in the production of valine and exhibits higher optical density compared to wild type strains. It is not clear if Applicant is attempting to argue that the claimed invention has unexpected results. However, it is noted that unexpected results must be commiserate in scope with the claimed invention. The claims recite any mutant IscR polypeptide that has increased apoprotein : holoprotein ratio in the cell, which represents a genus of proteins, including Giel’s IscR-C92A/C98A/C104A mutant, and not just the IscR H107Y mutant. It is noted that Giel does not disclose any growth defects of bacteria harboring the IscR-C92A/C98A/C104A mutant. In view of the teachings of Akhtar and Giel regarding the regulation of IscA by IscR noted in the rejection above, the skilled artisan would have expected that the production of L-valine would increase by increasing IscA expression to provide additional Fe-S clusters to the increased expression of IlvD.
Applicant argues that the subject matter of claim 29 is not a simple substitution of one regulatory element for another. Applicant again states that the claimed combination achieves a result not predicable from the cited references because the toxicity and growth defects associated with IscR deletion or unbalanced Isc overexpression means that there is not a reasonable expectation of success (Remarks, page 38, ¶4). These arguments have been fully considered but are not persuasive. First, the rejection of record is not based on a simple substitution of elements, but on a combination of elements. Second, the rejection is based on increased apo-IscR mutant expression in the cell, which is the claimed invention. The combination of elements would not be predicted to cause “IscR deletion” since an IscR polyprotein will still be present in the cells for regulating non-ISC genes. Additionally, it is not clear what Applicant is arguing by stating “unbalanced Isc overexpression”; however, it is noted that the entire Isc operon would be expressed, and as such there would not be an “unbalanced” level of one isc gene versus another. Additionally, Akhtar demonstrates in Fig 1, that providing additional copies of the Isc operon does not lead to cell toxicity (Fig 1a, pIsc). As such, the skilled artisan would predict that overall increases in the entire Isc operon would not be toxic to the cells. Finally, the claims do not recite a specific IscR mutant. If Applicant believes that the H107Y IscR mutant provides unexpected results in terms of cell growth or compound output compared to other apo-forming IscR, Applicant should explain why the H107Y IscR mutant functioned greater than expected from other apo-forming IscR mutants and/or recite the specific IscR mutant in the claims.
Applicant’s arguments on pages 39-40 address the §103 rejection over Zhou, Kumaran and Giel of former claim 10 and are addressed below as they apply to new claim 32.
Applicant points out that 1) Zhou does not teach IscR mutants or use with the MEP pathway, 2) Kumaran only teaches deletion of IscR, and 3) Giel only focuses on the repression mechanics and does not connect the IscR mutants to isoprenoid production (Remarks, page 39, ¶5). These arguments have been fully considered but are not persuasive because Applicant is attacking 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). Thus, Applicant’s arguments are not directed to the merits of the obviousness rejection.
Applicant provides evidence from Example 7 in the present Application of expressing IspG/H in IscR mutant strains increased the production of IPP and DMAPP (i.e., isoprenoids), and argues that the increased IPP and DMAPP production was not predictable from the cited art (Remarks, page 40, ¶2). This argument was fully considered but is not persuasive because the features upon which applicant relies (i.e., production of IPP and DMAPP) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993). Claim 32 encompasses an increase in production of any isoprenoid. Zhou teaches increased protoilludene, an isoprenoid, when both IspG/H genes and iscA transgenes are expressed, which would predictably increase activity of both IspG/H and IscA in the cell. Because Giel teaches that IscA activity increases when the apo-form IscR triple C[Wingdings font/0xE0]A mutant is expressed in the cell, the skilled artisan would predict that IscR mutant expression would likewise increase protoilludene production when IspG/H are recombinantly expressed in bacteria.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 29-32 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-18 of U.S. Patent No. 11,851,461 in view of Xu (Xu et al., ChemBioChem (2008), 9: 2355-2362; of record) and Giel (Molecular Microbiology (2013), 87: 478-492; of record). Claims 29-31 are rejected further in view of Park (Park et al., PNAS (2007), 104: 7797-7802), Tan (Tan et al., Biochem. J. (2009), 420: 463-472; of record), Flint (US 20120064561 A1), and Akhtar (Akhtar et al., Appl Microbiol Biotechnol (2008) 78: 853–862; of record). Claim 31 is rejected further in view of Genbank (WP_116324143.1, dihydroxy-acid dehydratase [Escherichia coli], https://www.ncbi.nlm.nih.gov/protein/1452085069, published 8-21-2018, [retrieved 9-23-2025]). Claim 32 is rejected further in view of Zhou (Zhou et al., Journal of Biotechnology (2017), 248: 1–8; of record) and Kumaran (US 20180245103 A1, published August 30, 2018; of record). This is a new rejection necessitated by amendment.
Patented claim 1 recites a genetically modified bacterium wherein the bacterium has an enhanced production of biotin or lipoic acid or thiamine; wherein said bacterium comprises: (a) a genetically modified endogenous iscR gene encoding a mutant IscR polypeptide, wherein the amino acid sequence of said mutant IscR polypeptide has at least 80% amino acid sequence identity to a sequence selected from the group consisting of SEQ ID No: 2, 4, 6, 8, 10, 12 and 14, and wherein said amino acid sequence has at least one amino acid substitution selected from the group consisting of: i) L15X, C92X, C98X, C104X, and H 107X; wherein X is any amino acid other than the corresponding amino acid residue in SEQ ID No.: 2, 4, 6, 8, 10, 12 and 14; and b.) at least one transgene encoding a polypeptide selected from the group consisting of: ii) a polypeptide having biotin synthase activity (EC 2.8.1.6). Patented claim 2 recites a genetically modified bacterium according to claim 1, wherein said at least one amino acid substitution in said mutant IscR polypeptide is selected from the group consisting of: b) C92X, wherein X is any one of Y, A, M, F and W; c) C98X, wherein X is any one of A, V, I, L, F and W; and d) C104X, wherein X is any one of A V, I, L, F and W. Patented claim 8 recites A genetically modified bacterium according to claim 1, wherein said bacterium is a genus of bacterium selected from the group consisting of Escherichia.
The patented claims do not recite the transgene is an Fe-S polypeptide, or that the transgene is IspG, IspH or IlvD. The patented claims do not recite additional genes in the isoprenoid or branched amino acids pathways encoded by a transgene. The patented claims do not recite the production of isoprenoids or L-valine.
Xu teaches that Biotin Synthase (BioB) is an Fe-S polypeptide that attains its Fe-S cluster from IscA (page 2356, ¶2).
The teachings of Giel are recited above in paragraphs 31 and 42 and incorporated here.
The teachings of Park are recited above in paragraph 27 and incorporated here.
The teachings of Tan are recited above in paragraph 28 and incorporated here.
The teachings of Flint are recited above in paragraph 29 and incorporated here.
The teachings of Akhtar are recited above in paragraph 30 and incorporated here.
The teachings of Zhou are recited above in paragraph 39 and incorporated here.
The teachings of Kumaran are recited above in paragraph 41 and incorporated here.
Regarding claims 29-30 and 32, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have replaced the transgene encoding a Biotin Synthase polypeptide in the patented cells with a transgene encoding either IlvD or IspH for the purpose of producing L-valine or isoprenoids, respectively. It would have amounted to the simple substitution of one known Fe-S polypeptide for another by known means to yield predictable results. Based on the teachings of Xu, Giel, Flint, Akhtar and Kumaran, it was understood in the art that the patented claims are to increase the expression from the ISC operon thereby increasing the activity and functionality of the patented Fe-S polypeptide to increase flux through the biosynthetic pathway. Because Akhtar teaches that Fe-S cluster assembly and loading of Fe-S polypeptides is often a rate limiting step, the skilled artisan would have been motivated to make additional bacterial strains harboring the patented and Giel’s IscR-C92A/C98A/C104A mutant with various other known Fe-S polypeptides. The skilled artisan would have predicted that transgenes encoding IspH and/or IlvD along with IlvBNCE and IspC, respectively, could be swapped in for the transgene encoding biotin synthase because 1) Zhou and Park teach introducing the genes into E. coli cells, and 2) the genes are genetically encoded and genetic manipulation of E. coli is well established in the art as evidenced by all the cited references. The increased production of isoprenoids and/or L-valine would have also been a predictable outcome of replacing transgenes encoding IspH and/or IlvD because Zhou and Park teach those compounds are the product of the MEP and branched amino acid biosynthesis pathway, respectively.
Regarding claim 31, the teachings of Genbank are recited above in paragraph 36. It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have specifically used the DHAD amino acid sequence taught in Genbank in place of the patented BioB polypeptide. It would have amounted to using a known IlvD enzyme sequence in place of the BioB Fe-S cluster protein to yield predictable results. The skilled artisan would have expected that Genbank’s DHAD amino acid sequence could be used in patented method because Park teaches expressing the E. coli IlvD polypeptide from a plasmid, and Genbank teaches the DHAD is the wildtype DHAD protein sequence. The skilled artisan would have been motivated to do so since the sequence in Genbank is the accepted IlvD/DHAD amino acid sequence.
Response to Arguments – Double Patenting
Applicant does not present arguments traversing the nonstatutory double patenting rejection in the previous office action.
Conclusion
No claims are allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
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/CATHERINE KONOPKA/Examiner, Art Unit 1635