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 Terminal Disclaimer
Applicant’s amendments filed March 2, 2026, amending claims 1-5 and 7-10 and canceling claim 6 is acknowledged. Claims 1-5 and 7-29 are pending. Claims 11-29 remain withdrawn from further consideration by the examiner, 37 CFR 1.142(b), as being drawn to a non-elected invention (claims 20-29) or a non-elected species (claims 11-19).
Claims 1-5 and 7-10 are under examination.
The terminal disclaimer filed on March 2, 2026 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of any patent granted on US Application 18607210 has been approved.
Withdrawn Rejections
The amendments to the claims overcome the 112(b) rejection and objections to the claims. The terminal disclaimer overcomes the nonstatutory double patenting rejection. The amendment to claim 1 requiring the second variation to be in “one or more genes in a glutamine conversion pathway… that regulate fitness” overcomes the 112(a) rejection of the previous office action. However, the claims are still rejected for lack of sufficient written description of the reasons recited below.
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.
Priority
As indicated in the previous office action, the first evidence of support of introducing a second variation to increase fitness that was lost upon a first variation is the PCT application PCT /US2019/039528 (filed June 27, 2019). As such, the effective filing date for at least claims 1-5 and 7-10 is June 27, 2019.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-5 and 7-10 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new rejection necessitated by amendment.
MPEP 2163.II.A3.(a).(i) states, “whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention.”
For claims drawn to a genus, MPEP 2163.II.A3.(a).(ii) states, “written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species” where “representative number of species' means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.”
Claim 1 recites a modified plant-associated microbe that comprises at least two genetic variations – 1 in a nitrogen fixation or assimilation gene/pathway and 1 in a gene in a glutaminase conversion pathway involved in the microbe’s fitness. As such claim 1 recites several large genera: 1) a plant-associated microbe, 2) a first genetic variation in nitrogen fixing or assimilation that, presumably, should result in increasing nitrogen fixing activity of the microbe, and 3) a second genetic variation in a “glutamine conversion pathway… that regulate[s] fitness” that, together with the first genetic variation results in both increased nitrogen fixing activity of the microbe and also at least the same fitness/growth/reproduction rate of the wild-type microbe. Although the first and second genera are sufficiently described in the specification and the prior art (see paragraphs 13-14 below), the combination of the first genus and second genus with the third genus of variations in a glutamine conversion pathway that has the recited function of having both increased nitrogen fixation and wild type fitness.
The genus of microbes that are either naturally nitrogen fixing or can be engineered to be capable of fixing nitrogen is large and evolutionarily and physiologically diverse. “Microbes” encompass both prokaryotic bacteria and archaea and also eukaryotic fungi and protists (See Specification [0136]). Regarding bacteria, Sharma explains that nitrogen-fixing bacteria are found associated with plants and free-living in the rhizosphere and aquatic ecosystems (Sharma et al., Diversity and Evolution of Nitrogen-fixing bacteria. N. K. Singh et al. (eds.), Sustainable Agriculture Reviews 60., published 2023, pages 95-120; Section 5.2.1). Nitrogen-fixing bacteria are dispersed throughout bacterial phyla (page 99, ¶1). Free-living bacteria that are associated with plant roots are found in 6 different bacteria families (page 99, ¶2). Even the most well-studied group of nitrogen-fixing bacteria that form symbiotic relationships with plants do not belong to a single phylogenetic branch (Section 5.2.2). Regarding nitrogen-fixing archaea, Mowafry indicates that archaea use a variety of reductive N-cycle reactions, including both assimilation and N2 fixation, nitrate respiration and denitrification (Nitrogen-fixing archaea and sustainable agriculture." Nitrogen Fixing Bacteria: Sustainable Growth of Non-legumes. Singapore: Springer Nature Singapore, 2022. 115-126; Section 6.3.1). Importantly, Mowafry states that Archaea’s nitrogen metabolism is far less well understood in comparison to that of bacteria (Section 6.3.1). Additionally, Mowafry states that diverse archaeal phyla are represented in the soil and rhizosphere environments (Section 6.4). Regarding eukaryotic organisms, although most eukaryotic organisms obtain nitrogen through symbiotic relationships, there is at least one eukaryote that has nitrogen-fixing organelles (Rong et al., Trends in Biotechnology (2024), 42: P946-948). Finally, microbes that are not naturally capable of fixing nitrogen can be engineered to express nitrogen fixing machinery, for instance nif operons, resulting in microbes that can fix nitrogen. See e.g., Li and Chen, ChemBioChem (2020), 21: 1717-1722. Taken together, the genus of microbes that can naturally fix nitrogen or be engineered to fix nitrogen is extremely vast and diverse.
The genus of genetic variations that result in increased nitrogen fixation or assimilation is also diverse. The Specification teaches that modifications can be made in nif cluster genes (i.e., nifA, nifB, nifC. .. nifZ), genes encoding NtrC or NtrB, genes in the gin cluster, as well as ammonia transporters or permeases ([0123]). Types of genetic variations include missense mutations leading to proteins with single amino acid changes, gene deletions, and promoter changes to alter expression in any of the genes. Despite the large number of possible variations, changes that would increase a microbe's ability to fix or assimilate nitrogen are predictable based on the art. Batista reviews the start of the art for manipulating nitrogen regulation in diazotrophic (i.e., nitrogen fixing) bacteria around the effective filing date of the claimed invention (Batista and Dixon, Biochemical Society Transactions (2019), 47: 603-614). The pathways for nitrogen regulation in a few model diazotrophs have been characterized (See Fig 1 and discussion therein). The enzymes required for nitrogen fixation (e.g., nitrogenase) are encoded by the nif operon, which is activated by the NifA transcription factor, which in turn is negatively regulated by NifL and GlnK (Figs 1-2). The nif operon is also regulated by glutamine synthase (GS) through its production of glutamine, which causes de-uridylation of PII, thereby inactivating NifA and repressing expression from the nif operon (Fig 1). In some nitrogen-fixing bacteria like Paenibacillus riograndensis, glutamine synthase also plays a more direct role in nif repression by promoting the binding of the Gin repressor (GlnR) to the nif promoter (Fernandes et al., FEBS Journal (2017), 284: 903-918, Fig 8, Table 3; page 912, ¶1- 3). Nitrogen metabolic pathway regulation is complex even beyond the aforementioned regulatory mechanisms as it is also regulated by oxygen availability and ATP state (See e.g., Bautista, page 604-608). However, if the nitrogen metabolic pathways and their regulatory mechanisms are characterized in a nitrogen-fixing species, the skilled artisan should be able to predict what effect a genetic mutation would have on expression of the nif operon. For instance, knocking out the repressor nifL in several diazotroph species results in mutants having increased nitrogen fixation under high nitrogen conditions as expected (page 610, ¶3). Additionally, it is predictable that entire nif operons and their regulatory machinery can be introduced into non-fixing bacteria or the model yeast Saccharomyces cerevisiae to promote nitrogen fixation (See Li and Chen above).
The third genus - a second genetic variation in a glutamine conversion pathway - could encompasses any change in any coding or noncoding region that plays a role in “glutamine conversion” of any gene in a nitrogen-fixing microbe that has been genetically altered in a large number of ways as described in the previous paragraph. In diazotrophs, there are two "glutamine conversion" enzymes – glutamate synthase (GOGAT) and L-glutamine aminohydrolase, also known as glutaminase, both of which result in the production of glutamate from glutamine. However, a “pathway” is not limited to a single reaction and reasonably encompasses proteins that function upstream and downstream of GOGAT and glutaminase reactions, and also encompass genes involved in the regulation of GOGAT and glutaminase expression. GOGAT appears to function downstream of GS by using glutamine as a substrate and upstream of nitrogenase as it provides glutamate as a product, and is therefore intimately involved in the entire nitrogen fixation pathway and glutamine sensing (Bastista, Fig 1). Additionally, since GOGAT and glutaminase are integral in the production of glutamate, which is an amino acid required for all proteins and which feeds into the TCA cycle through formation of alpha-ketoglutarate, a “gene within a glutamine conversion pathway” is in actuality quite broad. The claimed function of the variation in the glutamine conversion pathway – maintaining wildtype fitness levels of a microbe in the context of the microbe having increased nitrogen fixation capabilities - is also highly variable because fitness depends on the environment in which the microbe lives. Traits that provide a higher fitness in one environment, say ideal controlled conditions of a lab, may not confer a competitive advantage in the rhizosphere or in the relatively anaerobic conditions of a plant nodule.
For the reasons that follow it is not predictable what genetic change in glutamine conversion pathway would maintain at least wild type fitness in the genus of microbes that also have increased nitrogen fixing capabilities and in the genus of environments for which fitness of the modified and wildtype microbes can be measured.
Applicant has described a single example of further genetically modifying a microbe that has been genetically modified for increased nitrogen fixation (Example 7, [0657]-[0661]). In Example 7, a wildtype Klebsiella variicola strain was genetically modified by deletion of nifL and incorporation of a constitutively active glnE allele ([0661]). This modified strain had increased ammonia excretion (i.e., increase nitrogen fixation), but reduced ability to colonize roots (Figs 26-28). Upon further modification of the gene encoding glutaminase (glsA2), the DnifL, glnEAR, glsA2::Prm1.2 modified microbe maintained the ability to fix nitrogen at higher rates and regained the wildtype efficiency of root colonization (Figs 26-28, [0661], Table 33). It is not entirely clear how the glsA2 gene was modified. The Specification teaches that the cspE promoter - presumably a cold shock-responsive promoter - replaced native glsA2 promoter ([0661]). However, Table 33 and Table 32 (page 312) indicate that the native glsA2 promoter was replaced with a promoter called "Prm1.2", which Table 32 on page 312 states is the promoter of the constitutively expressed infC gene. In any event, it appears that increased and/or constitutive expression of the glsA2 gene can restore the ability to colonize roots of the DnifL, glnEAR mutant. However, this example does not disclose if other microbes were in the soil during the colonization assay to determine relative fitness values. Additionally, this example does not address fitness in other environments such as a field test. Therefore, Applicant has described a single genetic variation combination - increased or constitutive glsA2 expression in a DnifL, glnEAR background - in a single bacterial species in a single controlled environment with a single plant species with undisclosed abiotic and biotic variables, that is capable of at least wild type fitness levels and increased nitrogen fixation. This single example does not sufficiently represent the breadth of variation of the claimed genus.
Batista states "The complex regulatory circuits described [for nitrogen regulation] ensure that components required for the biosynthesis and activity of nitrogenase are expressed only under demanding physiological conditions. They also ensure that regulation of nitrogen fixation and ammonia assimilation are intertwined, so that fixed nitrogen becomes readily available to support bacterial growth instead of being altruistic released to the environment ... Ideally, these metabolic perturbations should result in the excretion of a major proportion of the ammonia produced by nitrogenase while maintaining assimilation rates at sufficient levels to support bacterial fitness." (page 610, ¶2). However, Batista stresses that attempts at removing the regulatory inputs into nif expression are unstable, “potentially due to the energetic penalty associated with constitutive synthesis of high levels of nitrogenase.” Additionally, Batista states that often strains demonstrating increased ammonia secretion in the controlled conditions of a greenhouse are not capable of contributing to nitrogen nutrition of plants in field conditions (page 611, ¶1). Batista states "diazotrophic strains expressing nitrogenase constitutively are likely to encounter severe fitness penalties in competitive soil environments" (page 611, ¶1). Batista does not give any insight into genetic solutions to prevent or circumvent the fitness reduction.
Most prior art references disclosing diazotroph mutants that have increased nitrogen fixation do not disclose growth assays of the mutants in any condition or in comparison to wildtype (See e.g., US 20180002243 A1, Fig 4 demonstrates increased nitrogenase activity, Table 5 only demonstrates colonization rate of wild type strains). Additionally, in an earlier attempt by Applicant to engineer plant-associated microbes for increased nitrogen fixation, Temme states “A 50 fold difference in colonization was observed between PBC6.38 (DnifL::Prm1glnEAR1) and PBC6.94 (DnifL::Prm1glnEAR1, DamtB). This difference could be an indication that PBC6.94 has reduced fitness in the rhizosphere relative to PBC6.38 as a result of high levels of fixation and excretion” (US 20190039964 A1; [0376]). This suggests that Applicants realize the fitness cost of increased nitrogen fixation.
Although art was identified that discloses diazotrophic mutants having increased nitrogen fixation and lower growth rate (i.e., lower fitness in bacterial culture), the art did not identify genetic means to recover growth rates to wild type levels (See e.g., Grant Thesis (2018), Oxford University, Fig 6-23: effect of reduced GS expression). In 2017, Ambrosio reported the diazotroph A. vinelandii harboring an inducible glnA (i.e., encodes glutamine synthase (GS)) combined with a nifL knockout and measured ammonia output and cell growth (Ambrosio et al., Metabolic Engineering (2017), 40: 59-68; Figs 8-9). However, it is not clear what GS-induction level correlates to "wildtype" GS levels. Additionally, wildtype A. vinelandii cells were never grown in the same assay as genetically modified strains, so it is not clear what the relative fitness levels are of the wildtype, single modified, and double modified strains. In 2024, five years after the effective filing date of the claimed invention, another report by Ambrosio demonstrated that when engineered microbes have highest levels of nitrogen fixation, their growth rates were lowest (Ambrosio et al., Applied Microbiology and Biotechnology (2024) 108:378, pages 1-16; Figs 2-3). In competition assays, wildtype strains outcompeted strains that had deregulated nif expression (Fig 6). Again, Ambrosio provides no insight of how to increase the fitness of the engineered strains using genetic means. Additionally, Han reported that the non-diazotrophic bacteria E. coli, engineered with a heterologous nif operon, had reduced growth rate compared to the wild type E. coli (Han et al., J. Microbiol. Biotechnol. (2015), 25: 1339-1348). This result makes sense based on Batista’s teaching that nitrogen fixation is an energetically expensive endeavor. Thus, based on the prior art it was not predictable what combination of genetic variations would result in maintained wild type fitness and increased nitrogen fixation of microbial strains.
There is nothing in the prior art to suggest that genetically modifying the expression or genes encoding glutaminase or GOGAT would increase the fitness of a diazotrophic microbe. In fact, prior art references that attempt to modify the expression of glutaminase or GOGAT found that doing so reduced the growth rate (i.e., fitness in a lab setting). Ambrosio states that modifying the GS-GOGAT pathway has been difficult to modify in the diazotrophic bacteria A. vinelandii because it relies exclusively on the GS-GOGAT pathway for ammonium assimilation (Ambrosio et al., Metabolic Engineering (2017), 40: 59-68; of record, page 155). Huerta-Saquero overexpressed glutaminase A in the diazotroph Rhizobium etli and found that such overexpression reduced the growth rate of the bacterium compared to a wildtype strain (Huerta-Saquero et al., Biochimica et Biophysica Acta (2004), 1673: 201-207; Fig 2). Given that there is no evidence in the prior art that modifying glutamine conversion pathways increases the fitness of a nitrogen-fixing bacteria, it was not predictable what combination of nitrogen fixation changes and “glutamine conversion” changes, and in specific diazotrophic species would have resulted in both increased nitrogen fixation and wild type of higher fitness of the genetically modified diazotroph.
In conclusion, given 1) the lack of representative examples in the Specification of combinations of genetic variations resulting in maintained wild type fitness and increased nitrogen fixation of microbial strains to support the full scope of genetic variations in the full scope of diazotrophic microbes with the full scope of genetic variations that increase nitrogen fixation, and 2) the lack of predictability in the art of which genetic variation combinations could allow strains with constitutive/increased nif expression to compete with or outcompete wildtype microbes in controlled or field environments, the skilled artisan would reasonably conclude that Applicant did not possess the full scope of the claimed invention.
Dependent claims
Claims 2-4 and 8-10 do not limit the genera of microbial species, the first genetic modification, or the second genetic modification and therefore lack sufficient written description for the reasons described above for claim 1.
Claim 5 recites a long list of genes in which first genetic variation is introduced. The list includes most, if not all, genes known to be involved in fixing nitrogen or regulation of the nif operon. As such the genus of first genetic variations is not substantially smaller than the genus of first genetic variations discussed above for claim 1.
Claims 7 limits the second genetic modification to one in the glsA gene, which encodes for glutaminase A. Applicant’s working example specifically increased expression of glsA in the DnifL, glnEAR, background of Klebsiella variicola. However, given Huerta-Saquero’s teaching that overexpressed glutaminase A in another diazotroph reduced the growth rate of the bacterium compared to a wildtype strain (Huerta-Saquero, Fig 2), it is not predictable that overexpression of glsA in other diazotroph species or in combination of other means to increase nitrogen fixation would increase growth rate to wild type levels. Although Applicant discloses a standard operating procedure on how to discover and/or screen for genetic combinations that increase both nitrogen fixation and growth rate (Examples 1 and 7), the court found in that screening assays are not sufficient to provide adequate written description for an invention because they are merely a wish or plan for obtaining the claimed invention. Rochester v. Searle, 358 F.3d 916, Fed Cir., 2004. “As we held in Lilly, “[a]n adequate written description of a DNA … ‘requires a precise definition, such as by structure, formula, chemical name, or physical properties,' not a mere wish or plan for obtaining the claimed chemical invention.” 119 F.3d at 1566 (quoting Fiers, 984 F.2d at 1171). As such, the skilled artisan would have concluded that Applicant did not possess the genus of combinations of 1) the genus of species with 2) the genus of genetic modifications in glsA with 3) the genus of genetic variations in nitrogen fixation.
Response to Arguments
Applicant argues that the instant Specification teaches that modification of a glutaminase gene in the glutamine conversion pathway improves fitness and discloses methods of iterative stacking genetic variations in the nitrogen fixation pathways and glutamine conversion pathways (Remarks, page 11). This argument has been fully considered but is not persuasive. The claims are directed to genetically modified microbes and not to methods for discovering/screening them. Applicant has only described a single genetically modified plant-associated diazotroph that has increased nitrogen fixing capabilities and wild type fitness – the Klebsiella DnifL, glnEAR variant with increased/constitutive expression of glsA. Based on the prior art cited above, it was not predictable what other genetic combinations would result in increased nitrogen fixing capabilities and wild type fitness. Although Applicant teaches in Example 1 a method for screening additional genetic combinations through iterative genetic manipulation, as indicated in the rejection above screening assays are not sufficient to provide adequate written description for an invention because they are merely a wish or plan for obtaining the claimed microbial invention.
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/Primary Examiner, Art Unit 1635