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 .
Election/Restrictions
Applicant's election with traverse of claims 1-10 in the reply filed on 8/7/2025 is acknowledged. The traversal is on the grounds that Jensen teaches a two-step fermentation process with each step in a different organism, whereas the current invention teaches M3K and MVD in a single vector, and therefore the instant invention makes a contribution over the prior art. This is not found persuasive because, as set forth in the rejection under 35 U.S.C. § 103 below, the nucleic acid sequence of claim 1 is obvious in view of the prior art, and thus does not constitute a special technical feature.
The requirement for restriction is therefore maintained, and claims 1-10 are being examined on the merits.
Claims 13-16, 25-29, and 31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 8/7/2025.
Priority
This application is a 371 of PCT/US2021/027510 (4/15/2021) which claims benefit of 63/010,409 (4/15/2020) as reflected in the filing receipt issued on 8/28/2025.
Information Disclosure Statement
The information disclosure statements (IDS) filed on 3/17/2023, 3/13/2024, and 1/6/2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Drawings
The drawings are objected to because Figures 3, 4, 8, and 9 refer to color to differentiate drawing elements, and this is difficult to interpret in the black and white drawings. It is requested that Applicant label figure elements in a different way and/or provide higher quality drawings to differentiate elements in the drawing.
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.
Specification
The disclosure is objected to because of the following informalities:
In the “Brief Description of the Drawings” section, pp. 2-3, the Figures are incorrectly numbered. Paragraph 17 of page 3 recites “Figure 1 shows solvent-induced dysregulation”; however, this should instead be “Figure 3”. Similarly, the Figure labels are incorrect in paragraphs 18-25. Appropriate correction is required.
Claim Objections
Claims 1 and 5 are objected to because of the following informalities: the acronym “PAM” is not defined in the claims. When an acronym is used in a claim set, it should be defined the first time it appears in the claims. For the purposes of examination, the term “PAM” is interpreted to mean “protospacer adjacent motif”.
Claims 1 and 4 are objected to because the claims recite microorganism names (E. coli) which should be italicized.
Appropriate correction is required.
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 factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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 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 an 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.
Conclusion
Claims 1-10 are rejected.
No claims are allowed.
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
/JENNIFER M.H. TICHY/Primary Examiner, Art Unit 1653