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 .
Status of Claims
Receipt of Arguments/Remarks filed on 4/23/2026 is acknowledged. Claims 1-10, 13-16, 25-29, and 31 are pending. Claims 1, 4, 13, and 27 were amended. Claims 11-12, 17-24, 30, and 32 are canceled. Claims 13-16, 25-29, and 31 are withdrawn as being directed to a non-elected invention.
Withdrawn Objections
The objections to the drawings, specification, and claims 1, 4, and 5 are withdrawn in view of amendments.
Maintained rejections
Claim Rejections - 35 USC § 103
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, 3, and 5-10 are rejected under 35 U.S.C. 103 as being unpatentable over Bassalo et al., ACS synthetic biology; 5(7):561-8 (cited in IDS 3/13/2024) in view of Galzerani et al., WO 2018/071563; Marliere US 2014/0370565 A1; and Orishimo et al., US 2018/0245104 A1; and as evidenced by Rossoni et al., Applied and Environmental Microbiology; 81(7):2625-34 (cited in IDS 3/13/2024).
Regarding claim 1, Bassalo teaches a rapid and efficient method for importing heterologous genes into E. coli (Bassalo “Abstract”). Bassalo teaches a construct with 5' and 3' E. coli homology arms, with a PAM mutation, and a constitutive promoter (Bassalo pg. 562-563 Fig. 1-2). Bassalo teaches that this method allows for one-step, high efficiency integration of entire metabolic pathways into the E. coli genome in a high-throughput manner, facilitating metabolic engineering studies (Bassalo pg. 566 para. 2).
Regarding claim 3, Bassalo teaches a constitutive T7A1 promoter (Bassalo pg. 563 para. 2).
Regarding claim 5, Bassalo teaches that the PAM mutation is CAAA (Bassalo Supplemental Fig. 1).
Regarding claim 7, Bassalo teaches that the nucleic acid construct with E. coli homology arms and a PAM mutation for expression of genes in the isobutanol pathway is linear (Bassalo Fig. 4; pg. 565 para. 5).
Regarding claim 10, Bassalo teaches a vector comprising the nucleic acid sequence, i.e. a linear template or construct for transformation into E. coli (Bassalo Fig. 2; Fig. 4; pg. 566 “Plasmids and Cloning Methods”).
Bassalo does not teach that the nucleic acid sequence comprises an M3K gene with 90% identity to SEQ ID NO: 1 or an MVD gene with 90% identity to SEQ ID NO: 2, as recited in claim 1; that the MVD and M3K genes are operably linked as recited in claim 6; or that the M3K gene comprises the sequence of SEQ ID NO: 1 and the MVD gene comprises the sequence of SEQ ID NO: 2 as recited in claims 8 and 9.
Regarding claim 1, Galzerani teaches recombinant microorganisms having one or more biosynthesis pathways for the production of monoethylene glycol and isobutene (Galzerani pg. 2 para. 7). Galzerani teaches a nucleic acid sequence (plasmid) comprising mevalonate-3-kinase (M3K or 3HIV kinase) and mevalonate diphosphate decarboxylase (MVD or MDD) genes (Galzerani pg. 148-149 para. 546). Galzerani teaches a sequence according to SEQ ID NO: 116, which is 73.4% identical to instant SEQ ID NO: 1 (see sequence alignment in OA Appendix). Instant SEQ ID NO: 1 is a codon optimized M3K gene sequence. SEQ ID NO: 116 of Galzerani is 100% identical to instant SEQ ID NO: 3, which is the WT sequence of instant SEQ ID NO: 1 (see sequence alignment in OA Appendix). Galzerani teaches that the genes can be optimized for expression in E. coli (Galzerani pg. 148-149 para. 546) and that a skilled artisan would recognize that it is advantageous to optimize coding sequences to those preferred by a particular host in order to improve translation (Galzerani pg. 144 para. 531-532).
Regarding claim 6, Galzerani teaches that the M3K and MVD genes are operably linked, i.e. transcribed from the same promoter (Galzerani pg. 149 para. 546).
Regarding claim 10, Galzerani teaches a vector comprising the nucleic acid sequence, i.e. a plasmid harboring the isobutene route genes (Galzerani pg. 148 para. 546).
Galzerani does not teach M3K and MVD gene sequences with at least 90% identity to SEQ ID NO: 1 and SEQ ID NO: 2, as recited in claims 1, 8, and 9.
Regarding claim 1, Marliere teaches production of 1,3-dienes through a biological process (Marliere pg. 1 para. 1-8). Marliere teaches a mevalonate diphosphate decarboxylase sequence from Picrophilus torridus according to SEQ ID NO: 21, which is 99.8% identical to instant SEQ ID NO: 1, corresponding to a codon-optimized sequence (Marliere pg. 3 para. 41; see sequence alignment in OA Appendix). It is established in the art that the mevalonate diphosphate decarboxylase homolog from Picrophilus torridus actually functions as a mevalonate-3-kinase, M3K, which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate (see Rossoni, Abstract). Thus, Marliere teaches an M3K gene sequence having at least 90% identity to instant SEQ ID NO: 1.
Regarding claim 8, the sequence taught by Marliere is 99.8% identical to instant SEQ ID NO: 1, with 973 matches and 2 mismatches (see sequence alignment in OA Appendix). Given the minimal differences between these two sequences and the known function of the P. torridus MVD gene product as an M3K, it would have been obvious to a skilled artisan to utilize an M3K gene comprising the sequence according to instant SEQ ID NO: 1.
Regarding claim 1, Orishimo teaches a method of producing olefins using diphosphomevalonate decarboxylase variants (Orishimo pg. 1 para. 1). Orishimo teaches Saccharomyces-derived MVD, having a sequence according to SEQ ID NO: 3, which is codon optimized for expression in E. coli (Orishimo pg. 11 para. 140). SEQ ID NO: 3 of Orishimo is 97.1% identical to instant SEQ ID NO: 2 (see sequence alignment in OA Appendix).
Regarding claim 9, the sequence taught by Orishimo is 97.1% identical to instant SEQ ID NO: 2, having 1157 matches and 34 mismatches (see sequence alignment in OA Appendix). Many of the mismatches are at positions which can be codon optimized in instant SEQ ID NO: 2 according to the instant specification pp. 26-27, Table 2, including positions 69, 156, 249, 300, 333, 369, 597, 690, 699, 720, 846, 1062 (see sequence alignment in OA Appendix). Further, as the gene taught by Orishimo encodes a protein having the same activity as instant SEQ ID NO: 2, mevalonate diphosphate decarboxylase activity, it would have been obvious to a skilled artisan to utilize an MVD gene comprising the sequence according to instant SEQ ID NO: 2.
It would have been obvious to a skilled artisan, before the effective filing date, to modify the nucleic acid construct of Bassalo to include M3K and MVD genes having sequences according to SEQ ID NOs: 1 and 2. Bassalo teaches that the system utilizing E. coli homology regions and a PAM mutation can be used to efficiently integrate entire metabolic pathways in E. coli in an efficient manner, with an example directed to a nucleic acid construct comprising multiple genes in the isobutanol pathway (Bassalo Abstract; Fig. 4). A skilled artisan would therefore recognize that the M3K and MVD genes from a different metabolic pathway, the mevalonate pathway, could be integrated in such a nucleic acid construct. As plasmid constructs for the expression of these genes in E. coli are known in the art as taught by Galzerani, and the sequences are known as taught by Marliere and Orishimo, a skilled artisan would have found it obvious to incorporate M3K and MVD genes with SEQ ID NOs: 1 and 2 into a nucleic acid construct as taught by Bassalo.
A skilled artisan would have found it obvious to use M3K and MVD gene sequences comprising SEQ ID NOs: 1 and 2, as these sequences are codon-optimized for expression in E. coli, and Galzerani teaches that it is beneficial to codon optimize the gene sequences for efficient expression. As codon-optimization is established in the art, and codon-optimized M3K and MVD sequences having 97% or more identity to SEQ ID NOs: 1 and 2 are known in the art as set forth above, it would have been obvious to arrive at instant SEQ ID NOs: 1 and 2.
A person of ordinary skill in the art would have been motivated to modify the teachings of Bassalo and create a construct having M3K and MVD genes expressed because products of the mevalonate pathway such as isobutene have industrial importance, being used in fuel additives, rubber, and specialty chemicals, and current methods of producing isobutene and monoethylene glycol rely on fossil fuels, making it of interest to develop environmentally friendly and efficient methods of producing such products (Galzerani pg. 1-2 para. 4-5). As the nucleic acid constructs taught by Bassalo allow for efficient and scalable integration of metabolic pathways into E. coli, a skilled artisan would have been motivated to produce a construct with the homology regions and PAM mutation taught by Bassalo that includes M3K and MVD genes, given the industrial relevance of the mevalonate pathway products.
A skilled artisan would have a reasonable expectation of success in making this modification to achieve a nucleic acid sequence with E. coli homology regions, a PAM mutation, constitutive promoter, and M3K and MVD genes, as Bassalo teaches such constructs having different metabolic pathway genes, and further teaches that the technique utilizing homology regions and a PAM mutation is amenable to use with genes in various metabolic pathways.
Claim 2 is rejected under 35 U.S.C. 103 as being unpatentable over Bassalo in view of Galzerani, Marliere, and Orishimo and as evidenced by Rossoni; as applied to claims 1, 3, and 5-10 above, and further in view of Ye et al., US 2013/0217008 A1 (cited in IDS 3/17/2023).
Bassalo, Galzerani, Marliere, and Orishimo teach the nucleic acid sequence of claim 1 as set forth above. Bassalo teaches that the nucleic acid sequence comprises a constitutive promoter (Bassalo pg. 563 para. 2). These references do not teach that the constitutive promoter is a 16s rRNA promoter, as recited in claim 2.
Regarding claim 2, Ye teaches the use of 16S rDNA (i.e. the DNA sequence that encodes 16s rRNA) promoters in a nucleic acid sequence for heterologous gene expression (Ye pg. 1 para. 12). Ye teaches that the 16S rDNA promoter results in high levels of gene expression when introduced into a host cell such as E. coli (Ye pg. 3 para. 33). Ye teaches that strong promoters such as 16S rRNA promoters are beneficial when producing genetically engineered bacteria for commercial, chemical, industrial, or pharmaceutical purposes (Ye pg. 1 para. 10).
It would have been obvious to a skilled artisan, before the effective filing date, to utilize a 16S rRNA constitutive promoter in the construct as taught by Bassalo. Bassalo teaches the use of a constitutive promoter, T7A1, as discussed above. It would have been obvious to a skilled artisan that a different constitutive promoter, such as a 16S rRNA promoter as taught by Ye, could be substituted in place of the T7A1 constitutive promoter. Thus, it is considered prima facie obvious to use a 16S rRNA promoter in place of the constitutive promoter taught by Bassalo, as this would be a simple substitution of one known element for another, with a reasonable expectation of obtaining predictable results, i.e. strong expression of the gene when introduced into E. coli.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Bassalo in view of Galzerani, Marliere, and Orishimo and as evidenced by Rossoni; as applied to claims 1, 3, and 5-10 above, and further in view of Handel et al., Antimicrobial agents and chemotherapy; 60(3):1319-27.
Bassalo, Galzerani, Marliere, and Orishimo teach the nucleic acid sequence of claim 1 as set forth above. Bassalo teaches E. coli homology arms using E. coli strain BW25113 (Bassalo pg. 566 “Strains and Culture Media”). These references do not teach homology regions homologous to E. coli strain MG1655 as recited in claim 4.
Regarding claim 4, Handel teaches that E. coli strains BW25113 and MG1655 are derived from the W1485 strain background and are therefore closely related (Handel pg. 1320 “Bacterial strains, growth media, antibiotics, and MIC measurement”).
Given the close similarity of these strains as taught by Handel, it would have been obvious to a skilled artisan to use E. coli strain MG1655 in place of BW25113 in the nucleic acid sequence of Bassalo, as these strains are highly genetically similar and have similar properties. Thus, it is considered prima facie obvious to use E. coli strain MG1655 in place of BW25113 as this would be a simple substitution of one known element for another, with a reasonable expectation of obtaining predictable results given the similarity of the two strains.
Response to Arguments
Applicant's arguments filed 4/23/2026 have been fully considered but they are not persuasive.
Applicant argues that there would have been no motivation to combine Bassalo with Galzerani, Marliere, and Orishimo, as evidenced by Rossoni, because Bassalo teaches integration of heterologous constructs comprising genes in the isobutanol pathway, not the isobutene pathway, and provides no teaching or suggestion regarding M3K and MVD genes. Applicant argues that the number of genes involved in metabolic pathways is large and the selection of M3K and MVD from all the possible options, as well as expressing them on a single construct, is a highly specific combination not suggested by the art. Applicant argues that the selection of M3K and MVD from countless metabolic pathways requires impermissible hindsight and there would have been no motivation to combine the MVD/M3K pathway described in Galzerani, Marliere, and Orishimo with the construct of Bassalo.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007).
In this case, Bassalo teaches a system that is designed for high efficiency integration of entire metabolic pathways in E. coli, (Bassalo Abstract), and states that entire pathways are easily integrated and tested in a single day in stable strains that are directly amenable to further engineering and/or fermentation studies (Bassalo p. 566 para. 2). Thus, a skilled artisan would have been motivated to use an efficient system for integration of metabolic pathways for integration of M3K and MVD in E. coli. Given the teachings of Galzerani that M3K and MVD are co-expressed for isobutene production and industrially relevant, a skilled artisan would have been motivated to select these two genes for co-expression and identify efficient methods of expressing these genes in E. coli, such as the system taught by Bassalo that is designed for efficient integration of metabolic pathways.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). Expression of MVD and M3K on a single construct is suggested by the art, as Galzerani teaches co-expressing MVD and M3K in E. coli for isobutene production, and it is therefore considered that choosing these genes for expression and the decision to co-express them on a single construct does not require hindsight reconstruction.
Applicant argues that a skilled artisan would not have been motivated to select M3K and MVD with sequences having 90% identity to SEQ ID NOs: 1 and 2. Applicant argues that Marliere teaches a sequence with 99.8% identity to SEQ ID NO: 1, but characterizes the enzyme as a disphosphomevalonate decarboxylase, not a kinase, and a person of ordinary skill in the art would not have been motivated to use this gene as a kinase in a construct for isobutene production.
In response to this argument, it is noted that the structure of the claimed nucleic acid sequence as recited in claim 1 requires a gene with a sequence that is 90% identical to SEQ ID NO: 1. The sequence taught by Marliere is 99.8% identical to SEQ ID NO: 1, and thus has this same structure. Further, given its known function as a mevalonate-3-kinase as evidenced by Rossoni, a skilled artisan would have found it obvious to incorporate a gene having the sequence taught by Marliere in a construct for isobutene production as claimed. Despite the characterization of the gene as a decarboxylase rather than a kinase by Marliere, a skilled artisan would have been aware that this gene product also functions as a mevalonate-3-kinase based on established teachings in the prior art, i.e. Rossoni.
Applicant argues that a skilled artisan would not have been motivated to alter Galzerani’s established and functional plasmid based inducible expression system to instead chromosomally integrate M3K and MVD in a constitutive expression system as taught by Bassalo.
In response to this argument, it is noted that the construct of Bassalo is designed for one-step, high efficiency integration of entire metabolic pathways into the E. coli genome in a high-throughput manner, facilitating metabolic engineering studies (Bassalo pg. 566 para. 2), and that Bassalo teaches that chromosomal integration of heterologous constructs provides better long-term stability and reduced cell to cell variability in copy number and expression levels, making such strains more suitable for industrial-scale processes or downstream engineering (Bassalo p. 561 para. 1). Given the benefits of the chromosomal integration system taught by Bassalo for expression of metabolic pathways in E. coli, a skilled artisan would have been motivated to use such a system in place of the plasmid-based inducible expression system of Galzerani, thus arriving at a nucleic acid construct as claimed.
Applicant argues that there would be no reasonable expectation of success because the genes in the isobutene pathway differ from the isobutanol pathway genes taught by Bassalo and there is no evidence that constitutive expression of MVD and M3K would result in functional isobutene production.
In response to this argument, the system of Bassalo is designed to be adaptable for the integration of entire metabolic pathways in E. coli, and thus it could reasonably be expected that a metabolic pathway such as the mevalonate pathway, which has successfully been expressed in E. coli, could be integrated into a construct as taught by Bassalo. Further, it is noted that the instant claims are directed to a nucleic acid sequence with a structure comprising homology regions, a constitutive promoter, and M3K/MVD genes. The production of isobutene is not required by the claims. Thus, a skilled artisan would have a reasonable expectation of success in arriving at a nucleic acid construct as claimed.
Conclusion
Claims 1-10 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to EMILY F EIX whose telephone number is (571)270-0808. The examiner can normally be reached M-F 8am-5pm ET.
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/EMILY F EIX/Examiner, Art Unit 1653
/SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653
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