NOVEL ENZYMES FOR THE PRODUCTION OF E-COPALOL

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 . Priority This application claims benefit of priority to Provisional Application 63/222,597 filed 07/16/2021 and is also a 371 of PCT/US2022/073759 filed 07/15/2022. Support for the instant claims can be found in the Provisional Application, therefore, the effective filing date of the claimed invention for the purposes of applying prior art is 07/16/2021. Drawings The Drawings filed 08/06/2026 are accepted by the Examiner. Claim Status In the reply filed 08/06/2026, Applicant amended claims 1, 7 and 12 and canceled claim 11. Claims 3-4, 10 and 13-16 remain withdrawn as they are not encompassed by Applicant’s elections. Claims 1-10 and 12-16 are currently pending. Claims 3-4, 10 and 13-16 are withdrawn. Claims 1-2, 5-9 and 12 are under examination. 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 1-2, 5-9 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Schalk et al. (WO 2020011883 A1, 01/16/2020) (IDS Reference of 03/17/2025) in view of UniProt (A0A7U2EY07_PHANO, 06/02/2021) (IDS Reference of 03/17/2025). Regarding claims 1-2 and 12, Schalk et al. disclose a method for synthesizing terpene compounds, including copalol (See entire document, Abstract). More specifically, Schalk et al. disclose the production of copalol in Saccharomyces cerevisiae cells, reading on the elected cell/strain, using a copalyl-pyrophosphate phosphatase and different alcohol dehydrogenases (Page 83, Example 9). For the production of copalol, multiple genes were cDNA optimized for expression in yeast, including the geranylgeranyl-pyrophosphate (GGPP) synthase CrtE from Pantoea agglomerans, the copalyl-pyrophosphate (CPP) synthase SmCPS2 from Salvia miltiorrhiza, the copalyl-pyrophosphate phosphatase TalVeTPP from Talaromyces verruculosus and different alcohol dehydrogenases were expressed in engineered S. cerevisiae cells with increased levels of endogenous farnesyl-diphosphate (Page 83, Example 9). The utilized TalVeTPP from Talaromyces verruculosus was predicted to be a member of the protein tyrosine phosphatase family because it contains the signatures of protein tyrosine phosphatase family proteins (Page 75, Lines 21-31). Regarding TalVeTPP, Schalk et al. disclose the polypeptides of the invention having terpenyl diphosphate phosphatase activity, converting CPP to copalol, are members of the Protein tyrosine phosphatase family, in particular, the Y_phosphatase3 family having the Pfam ID number PF13350 (Page 11, Lines 7-9). To determine if the prediction was correct, Schalk et al. integrated TalVeTPP into a host cell, cultivated the cell and analyzed the products for the presence of copalol (Page 76, Lines 24-27). The host cells with the integrated TalVeTPP were able to effectively express copalol (Page 76, Lines 30-31). Thus, TalVeTPP was shown to be able to enzymatically convert copalyl-diphosphate pyrophosphatase (CPP) to copalol and can be used to produce copalol in engineered cells (Page 78, Lines 18-21). Additionally, all of the enzymes discussed above are from microorganisms other than S. cerevisiae, which are integrated into the genome of the host cell (Page 15, Lines 26-28), thus, they are all heterologous nucleic acids encoding an amino acid sequence. Schalk et al. do not disclose the genetically modified host cell comprises an enzyme with an amino acid sequence that is at least 90% identical to or is the sequence of instant SEQ ID NO: 3. However, UniProt discloses A0A7U2EY07_PHANO, a tyrosine specific protein phosphatase domain-containing protein (Top of Page 1) belonging to Pfam PF13350 and the Y_phosphatase3 family (Top of Page 4). A0A7U2EY07_PHANO disclosed by UniProt shares 100% sequence identity to instant SEQ ID NO: 3, the elected amino acid sequence. A sequence alignment is provided below wherein Qy is instant SEQ ID NO: 3 and Db is A0A7U2EY07_PHANO disclosed by UniProt. PNG media_image1.png 144 894 media_image1.png Greyscale PNG media_image2.png 582 630 media_image2.png Greyscale Thus, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized the tyrosine specific protein phosphatase domain-containing protein A0A7U2EY07_PHANO, belonging to Pfam PF13350 and the Y_phosphatase3 family, as the enzyme for converting CPP to copalol in the host cell of Schalk et al. because it was a known tyrosine specific protein phosphatase domain-containing protein as taught by UniProt and would be expected, absent evidence to the contrary, to have the same function and effect as other similarly annotated proteins, motivated by the desire to effectively produce copalol as it amounts to simple substitution of one known element for another to obtain predictable results. Exemplary rationales that may support a conclusion of obviousness include simple substitution of one known element for another to obtain predictable results. See MPEP 2143(I)(B). Regarding claims 5-6, as discussed above, Schalk et al. disclose the production of copalol in Saccharomyces cerevisiae cells using a copalyl-pyrophosphate phosphatase and different alcohol dehydrogenases wherein the genes encoding for the GGPP synthase CrtE, the copalyl-pyrophosphate synthase SmCPS2, the copalyl-pyrophosphate phosphate TalVeTPP and different alcohol dehydrogenases were expressed in engineered S. cerevisiae cells with increased levels of endogenous farnesyl-diphosphate (Page 83, Example 9). As Schalk et al. disclose SmCPS2 is involved in the production of copalol, SmCPS2 reads on an enzyme of a pathway for making E-copalol. The SmCPS2 from Salvia miltiorrhiza, reading on a heterologous nucleic acid, is SEQ ID NO: 63 (Page 83, Example 9). SEQ ID NO: 63 of Schalk et al. is a nucleic acid sequence and SEQ ID NO: 64 of Schalk et al. is the amino acid sequence encoded by SEQ ID NO: 63 of Schalk et al. SEQ ID NO: 64 of Schalk et al. shares 100% sequence identity to instant SEQ ID NO: 43. An alignment is provided below wherein Qy is instant SEQ ID NO: 43 and Db is SEQ ID NO: 64 of Schalk et al. PNG media_image3.png 142 530 media_image3.png Greyscale PNG media_image4.png 996 634 media_image4.png Greyscale PNG media_image5.png 156 636 media_image5.png Greyscale Regarding claims 7-8, as discussed above, Schalk et al. disclose the engineered S. cerevisiae cells have increased levels of endogenous farnesyl-diphosphate (FPP) (Page 83, Example 9). Schalk et al. further disclose to increase the level of endogenous FPP in the S. cerevisiae cells, an extra copy of all yeast endogenous genes involved in the mevalonate pathway, ERG10 to ERG20, were integrated into the genome of the specific S. cerevisiae strain utilized, CEN.PK2-1C (Page 84, Lines 7-9). It is noted the instant Specification states “the term ‘heterologous’ refers to what is not normally found in nature” and “the term ‘heterologous compound’ refers to the production of a compound by a cell that does not normally produce the compound, or to the production of a compound at a level not normally produced by the cell” (Specification, Page 6, Lines 9-12). Thus, the inclusion of an extra copy of ERG20 being integrated into the genome of the S. cerevisiae strain reads on heterologous because the extra copy is not what is normally found in nature. Additionally, Schalk et al. disclose ERG20 converts isopentenyl-diphosphate (IPP) and dimethylallyl-diphosphate (DMAPP) into FPP (Page 74, Lines 14-16), thus, ERG20 reads on an enzyme capable of converting IPP and DMAPP into FPP. Regarding claim 9, Schalk et al. disclose the coding sequence of TalVeTPP which was codon optimized for its expression in S. cerevisiae was under the control of the bidirectional yeast promoter, GAL10/GAL1 (Page 87, Lines 16-18). The GAL10/GAL1 promoter reads on a single transcriptional regulator. 35 USC § 103 - Response to Arguments Applicant's arguments filed 08/06/2026 have been fully considered but they are not persuasive. Applicant argues the cited references do not establish that A0A7U2EY07_PHANO was known to convert CPP to copalol or that a person of ordinary skill in the art would have reasonably expected A0A7U2EY07_PHANO to perform that function in Schalk’s engineered cell because Schalk does not establish that proteins are predictably interchangeable because they contain a PF13350 domain (Middle of Page 7). Applicant states Schalk tested 8 sequences with a PF13350 domain and all of the candidates produced some copalol but titers varied substantially (Bottom of Page 7). Applicant further states UniProt only identifies A0A7U2EY07_PHANO as a predicted protein (Top of Page 8). The Examiner respectfully disagrees. It is the Examiner’s position that based upon Schalk testing 8 different sequences, all of which contained a PF13350 domain, and all of the sequences resulting in the production of at least some copalol, one would expect other proteins with a PF13350 domain to do the same. It is noted the claims are drawn to a genetically modified cell that is capable of producing E-copalol, not a cell that does produce E-copalol, much less a cell that produces a specific amount. The cell must only be capable of producing E-copalol. As the cell of Schalk produced some copalol with 8 different sequences that all have PF13350, it would be obvious to utilize another sequence with a PF13350 domain in the cell of Schalk with a reasonable expectation of success that a cell capable of producing E-copalol would be created. Further, as discussed above, UniProt specifically discloses A0A7U2EY07_PHANO has a Pfam PF13350 domain, therefore, it would be expected the protein would have the same function as other proteins with a PF13350 domain. Applicant further argues an unexpected and improved result with the A0A7U2EY07 protein (instant SEQ ID NO: 3) relative to the production of TalVeTPP in Schalk. Applicant points to Table 1 and Figure 2 to support the unexpected effect (Bottom of Page 8 – Page 9). It is the Examiner’s position that while an Table 1 could potentially show an unexpected result, however, the claims are not commensurate in scope with the showing. The closest prior art utilizes TalVeTPP which is shown in Table 1 of the instant disclosure. TalVeTPP produced 1.00 E-copalol (no unit of measure given) while Pn. TPP which is encoded by instant SEQ ID NO: 3 produced 4.25 E-copalol. The remaining phosphatase enzymes produced 2.06 or less E-copalol. It is acknowledged that instant SEQ ID NO: 3 resulted in the production of significantly more E-copalol. However, the claims are drawn to any sequence including SEQ ID NOS: 3, 6, 9, 12, 15 or 18. Thus, the claims are not commensurate in scope with a potential unexpected result because the only phosphatase enyzyme that could potentially show an unexpected result is SEQ ID NO: 3 yet the claims are directed to SEQ ID NOS: 3, 6, 9, 12, 15 or 18. It is also noted the standard deviation is not included in Figure 2 which is needed when determining an unexpected result. Conclusion Claims 1-2, 5-9 and 12 are rejected. No claims are allowed. 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. Any inquiry concerning this communication or earlier communications from the examiner should be directed to ASHLEY T WHITE whose telephone number is (571)272-0683. The examiner can normally be reached Monday-Friday 8:30-5:00 EST. Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. 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If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000. /A.T.W./Examiner, Art Unit 1653 /SHARMILA G LANDAU/Supervisory Patent Examiner, Art Unit 1653