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 .
Applicant’s amendment to the claims filed on 10/3/2025 in response to the Non-Final Rejection mailed on 4/7/2025 is acknowledged. This listing of claims replaces all prior listings of claims in the application.
Claims 1, 3, 9, 14-15, 17-19, 21, 24, 26, 28, 30-35 are pending and examined on the merits.
Claims 30-35 are withdrawn from consideration pursuant to 37 CFR 1.142(b).
Claims 2, 4-8, 10-13, 16, 20, 22-23, 25, 27, 29 are cancelled.
Applicant’s remarks filed on 10/3/2025 in response to the Non-Final Rejection mailed on 4/7/2025 have been fully considered and are not deemed persuasive to overcome at least one of the rejections and/or objections as previously applied.
The text of those sections of Title 35 U.S. Code not included in the instant action can be found in the prior Office Action.
Maintained 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 rejection of claims 1, 3, 9, 14-15, 17-19, 21, 24, 26, 28 under 35 U.S.C. 103 as being unpatentable over Argyros et al. (Patent Application WO 2012/138942 Al: cited on IDS filed on 5/11/2021) {herein Argyros} in view of Hou et al. (2010, Available online February 2010. Applied and Environ. Micro., 10.1128/AEM.02040-09, cited on PTO-892 filed 4/1/2024 and IDS filed 5/11/2021) {herein Hou} and Rossum et al. (2016. Available online 23 March 2016. Metabolic Engineering., 10.1016/j.ymben.2016.03.006, cited of PTO-892 filed 4/1/2024) {herein Rossum} is maintained. The rejection has been modified to address Applicant’s ‘Response to Remarks.’
As amended, claims 1, 3, 9, 14-15, 17-19, 21, 24, 26 are drawn to a recombinant yeast host cell having (i) a first genetic modification comprising one or more first heterologous nucleic acid molecules encoding a pyruvate formate lyase in the recombinant yeast host cell; and (ii) a second genetic modification comprising a second heterologous nucleic acid molecule encoding a formate dehydrogenase; wherein, in the presence of a stressor, the recombinant yeast host cell produces more ethanol when compared to a control yeast host cell comprising (i) the first genetic modification but lacking (ii) the second genetic modification.
Previously presented claim 28 is drawn to a combination for fermenting a biomass, the combination comprising (i) a first recombinant yeast host cell having a first genetic comprising one or more first heterologous nucleic acid molecule encoding a pyruvate formate lyase in the recombinant yeast host cell, when compared to a corresponding native yeast host cell, and (ii) a second recombinant yeast host cell having a second genetic modification comprising a second heterologous nucleic acid molecule encoding a formate dehydrogenase.
With respect to claims 1, 28, Argyros teaches the engineering of a formate pathway in yeast (para 0355). Specifically, Argyros teaches pyruvate formate lyase (PFL) and formate dehydrogenase (FDH) can be engineered in cells to increase the theoretical yield of ethanol (para 0360). Argyros futher teaches said recombinant microorganism comprises one or more heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol (claim 1). Absent evidence otherwise, it is the Examiner’s position that said engineered yeast cells comprising PFL is a first genetic modification, as recited in claim 1 of the instant application. Said strain grows in the presence of acetic acid (para 0161). Evidentiary reference of Guaragnella is cited to demonstrate that acetic acid is a commonly known stressor of yeast cell cultures (abstract). As such, absent evidence otherwise, it is the Examiner’s position that the acetic acid taught by Argyros is a stressor.
With respect to claim 3, Argyros teaches a pyruvate formate lyase having at least 87.5% identity of SEQ ID NO: 30 to SEQ ID NO: 6 of the instant application (appendix A). PFL is from Bifidobacterium adolescentis PflA (para 0009).
With respect to claim 21, Argyros teaches the deletion of formate dehydrogenase from S. cervisiea expressing heterologous pyruvate formate lysase (para 0168).
With respect to claim 24, Argyros teaches a host strain of Saccharomyces (para 0506).
With respect to claim 26, Argyros teaches a host strain of Saccharomyces cerevisiae (para 0506).
However, Argyros does not teach the recombinant yeast host cell of claims 1, 28 of a second genetic modification comprising a second heterologous nucleic acid molecule encoding a formate dehydrogenase (claims 1, 28). Argyros does not teach the product of claim 9, wherein the formate dehydrogenase: - is FDH1; - uses NAD+ as a primary cofactor; - has the amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 1 or 5, uses NADP+ as a primary cofactor; and/or - has the amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 3, 4, 21, 23, 25, 26 or 27 (claim 9). Argyros does not teach the recombinant yeast host cell of claim 14, wherein the second heterologous nucleic acid molecule further comprises a nucleic acid sequence encoding a mitochondrial target sequence operatively associated with the nucleic acid sequence encoding the polypeptide having formate dehydrogenase activity (claim 14). Argyros does not teach the recombinant yeast host cell of claim 14, wherein the mitochondrial target sequence: - is a CYB2 gene; and/or- has the amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 11 (claim 15). Argyros does not teach the recombinant yeast host cell of claim 1,wherein the second heterologous nucleic acid molecule further comprises a promoter operatively associated with the nucleic acid sequence encoding the formate dehydrogenase activity (claim 17). Argyros does not teach the recombinant yeast host cell of claim 17, wherein the promoter comprises at least one of tef2p, ssalp, adhlp, cdcl9p, tpilp, cyclp, pgklp, tdh2p, eno2p, hxt3p, qcr8p, tdhlp, tdh3p or hor7p (claim 18). Argyros does not teach the recombinant yeast host cell of claim 1, wherein the recombinant yeast host cell expresses a native formate dehydrogenase (FDH) gene (claim 19).
With respect to claims 1, 28, Hou teaches the FDH1 (formate dehydrogenase) gene from S. cerevisiae CEN.PK113-7D strain was amplified and expressed in CEN.PK1001-71C (fdh1Δ fdh2Δ ura3-52) under the control of a strong constitutive promoter and a high-copy number plasmid (page 853, column 2, paragraph 6). pTEF-CYB2-FDH1 was utilized in experimentation to synthetically express FDH1 (page 853, column 1, paragraph 6). Absent evidence otherwise, it is the Examiner’s position that the engineered yeast strain that expresses heterologous FDH1 is the same as a ‘ second genetic modification comprising a second heterologous nucleic acid molecule encoding a formate dehydrogenase,’ as recited in the instant application claims 1, 28.
With respect to claim 14, Hou teaches the fusing of mitochondrial gene CYB2 to formate dehydrogenase in a recombinant yeast host cell (abstract).
With respect to claim 15, Hou teaches the mitochondrial signal sequence of Cyb2 was efficient in directing FDH1 into the mitochondria (page 853, column 2, paragraph 6). Examiner is interpreting the mitochondria signal sequence as being equivalent to the mitochondrial target sequence as in claim 15 of the instant application.
With respect to claims 17-19 Hou teaches a TEF promoter controls the expression of the nucleic acid FDH1 from S. cervisiae (page 852, table 2). The strain CEN.PK1001-71C [MATa SUC2 MAL8c ura3-52 FDH1(41,1091)::loxP-Kan-loxP FDH2(41,1091)::loxp-kan-loxp] was transformed with pTEF-CYB2-FDH (page 853, column 1, paragraph 2). Utilizing the broadest reasonable interpretation, Examiner is interpreting the strain utilized in experimentation has retained its native expression since the host strain does not indicate a deletion of FDH1.
However, Hou does not teach the product of claim 9, wherein the formate dehydrogenase: - is FDH1; - uses NAD+ as a primary cofactor; - has the amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 1 or 5, uses NADP+ as a primary cofactor; and/or - has the amino acid sequence having at least 70% identity to the amino acid sequence of SEQ ID NO: 2, 3, 4, 21, 23, 25, 26 or 27 (claim 9).
With respect to claim 9, Rossum teaches protein engineering has yielded FDH enzymes that use NADP+ as a cofactor (page 103, column 2, paragraph 1).
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to apply the teachings of Argyros et al of the engineering of heterologous pyruvate formate lyase (PFL) in yeast to increase the theoretical yield of ethanol (para 0360 and claim 1) or combine the teachings of Hou and Rossum because Rossum teaches protein engineering has yielded FDH enzymes that use NADP+ as a cofactor (page 103, column 2, paragraph 1). While Hou teaches an engineered yeast strain that expresses heterologous FDH1 (page 8532, table 2 and page 853, column 1, para 2) with a TEF promoter (page 852, table 2) and the fusing of mitochondrial gene CYB2 to formate dehydrogenase in a recombinant yeast host cell (abstract).
One of ordinary skill in the art would be motivated to either use the teachings of Argyros et al. by itself or combine the teachings of Hou and Rossum because Hou provides the motivation for Argyros to utilize heterologous formate dehydrogenase linked to a mitochondrial targeting sequence (CYB2) (second genetic modification) with a TEF promoter as doing so stimulates the co-metabolism of glucose in the mitochondria of yeast cells for fermentation into ethanol (page 854, column 1, paragraph 1) with the TEF promoter serving as a strong, constitutive driver for high-level FDH expression. Whereas, Rossum provides the motivation for Argyros, to utilize heterologous FDH that uses NADP+ as opposed to NADPH as a cofactor because doing so has been shown to enable growth of said engineered yeast strains under microaerobic conditions (page 103, column 2, para 3). While Argyros conceptualizes the engineering of heterologous pyruvate formate lyase (PFL) and formate dehydrogenase (FDH) in yeast increases the theoretical yield of ethanol (para 0360 and claim 1). One of ordinary skill in the art would have a reasonable expectation of success that linking a mitochondrial targeting sequence to TEF-FDH along with the heterologous expression of PFL would result in the increased production of ethanol when compared to a recombinant control yeast host cell comprising the first genetic modification (PLF) but lacking the second genetic modification (TEF-FDH-linked CYB), as the heterologous expression of TEF-FDH linked to the mitochondrial targeting sequence CYB (second genetic modification) would allow for the transport and oxidation of NADPH within the mitochondria by mitochondrial dehydrogenase, thereby substantially increasing the production of ethanol as Hou teaches increased cytosolic NADPH decreases ethanol production (page 851, column 2, para 2). Additionally, One of ordinary skill in the art would have a reasonable expectation of success that expressing NADP+ as opposed to NADPH within said recombinant cells because it would allow for growth in oxygen limiting environments in the presence of the Applicant recited PFL. Thereby, resulting in a less stringent growth conditions and ease of culturing.
One of skill in the art would have a reasonable expectation of success to make and use the claimed recombinant yeast cell because Argyros provides the basic recombinant cell and its uses and methods of making it. Whereas Hou teaches the fusing of mitochondrial gene CYB2 to TEF-formate dehydrogenase in a recombinant yeast host cell (abstract). While Rossum teaches protein engineering has yielded FDH enzymes that use NADP+ as a cofactor (page 103, column 2, paragraph 1). Therefore, there would be a reasonable expectation of success to arrive at the above invention. Therefore, the above invention would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
RESPONSE TO REMARKS: Beginning on p. 6 of Applicant’s remarks, Applicant contends that Argyros teaches away from the present claims. Applicant contends that Argyros teaches away from the heterologous expression of formate dehydrogenase as described in Hou as a solution to increase ethanol production. Applicant contends that Argyros only discloses downregulation or inactivation of native formate dehydrogenase genes (see paragraphs [0363] and [0364]). Applicant contends that Argyros indicates that to increase the theoretical yield of ethanol, two enzymes could be engineered: pyruvate formate lyase (PFL) and formate dehydrogenase (FDH) (referencing Figure 1). Applicant contends that Argyros' teachings to be limited to reduction or disruption of the expression of native formate dehydrogenase gene(s) or deletion of said genes.
This argument is found to be not persuasive in view of the modified rejection set forth. Examiner contends that Argyos in-fact conceptualizes the instant application claim 1 of a recombinant yeast host cell having (i) a first genetic modification comprising one or more first heterologous nucleic acid molecules encoding a pyruvate formate lyase in the recombinant yeast host cell; and (ii) a second genetic modification comprising a second heterologous nucleic acid molecule encoding a formate dehydrogenase in the presence of a stressor.’ Whereas Argyros in view of Hou teaches said construct. Examiner contends that Argyros deleting FDH from the construct does not teach away from the heterologous expression of FDH as said deletion is of the native FDH gene, not a heterologous FDH gene. Examiner contends that Hou provides the motivation for Argyros to utilize the heterologous TEF-FDH-linked CYB taught by Hou as said construct increases the production of ethanol due to the decease of cytosolic NADPH (page 851, column 2, para 2). Additionally, Argyros provides conceptualization of the engineering of heterologous pyruvate formate lyase (PFL) and formate dehydrogenase (FDH) in yeast increases the theoretical yield of ethanol (para 0360 and claim 1), as opposed to teaching away from said construct.
Applicant contends that one of ordinary skill in the art would understand that increasing both pyruvate formate lyase activity and formate dehydrogenase activity by heterologous expression of corresponding enzymes is not contradictory to the teaching of Argyros as a solution to increase ethanol production.
This argument is found to be not persuasive in view of the modified rejection set forth. Examiner is unsure how Applicant came to that finding. Especially since Argyros provides the conceptualization of the engineering of heterologous pyruvate formate lyase (PFL) and formate dehydrogenase (FDH) in yeast increases the theoretical yield of ethanol (para 0360 and claim 1), as opposed to teaching away from said construct.
Applicant further contends that Hou does not teach that heterologous expression of formate dehydrogenase leads to increased ethanol production. Applicant contends that Hou only shows restoration of glycerol and/or ethanol production by reintroducing formate dehydrogenase activity in a background strain (identified as F-REF) that previously had disabled formate metabolism and that were unable to produce glycerol and/or ethanol.
This argument is found to be not persuasive in view of the modified rejection set forth. Examiner contends that Hou teaches increased cytosolic NADPH decreases ethanol production (page 851, column 2, para 2). Examiner contends that the heterologous expression of TEF-FDH linked to the mitochondrial targeting sequence CYB (second genetic modification) would allow for the transport and oxidation of NADPH within the mitochondria by mitochondrial dehydrogenase, thereby increasing the production of ethanol as Hou teaches increased cytosolic NADPH decreases ethanol production (page 851, column 2, para 2).
Applicant contends that Rossum does not cure the deficiencies of Argyros and Hou.
This argument is found to be not persuasive in view of the modified rejection set forth. Examiner contends that Rossum provides the motivation for Argyros to utilize heterologous FDH that uses NADP+ as opposed to NADPH as a cofactor because doing so has been shown to enable growth of said engineered yeast strains under microaerobic conditions (page 103, column 2, para 3).
Conclusion
Status of claims
Claims 1, 3, 9, 14-15, 17-19, 21, 24, 26, 28, 30-35 are pending.
Claims 30-35 are withdrawn from consideration pursuant to 37 CFR 1.142(b).
Claims 2, 4-8, 10-13, 16, 20, 22-23, 25, 27, 29 are cancelled.
Claims 1, 3, 9, 14-15, 17-19, 21, 24, 26, 28, 30-35 are rejected.
No claims are in condition for allowance.
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 extension fee 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 ERICA NICOLE JONES-FOSTER whose telephone number is (571)270-0360. The examiner can normally be reached mf 7:30a - 4:30p.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Manjunath Rao can be reached at 571-272-0939. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/ERICA NICOLE JONES-FOSTER/Examiner, Art Unit 1656
/PAUL J HOLLAND/Primary Examiner, Art Unit 1656
Appendix A
Argyros vs SEQ ID NO: 6
Query Match 87.5%; Score 1367; Length 293;
Best Local Similarity 86.4%;
Matches 248; Conservative 21; Mismatches 18; Indels 0; Gaps 0;
Qy 6 FRSTTRHMLRDSKDYVNQTLMGGLSGFESPIGLDRLDRIKALKSGDIGFVHSWDINTSVD 65
||:||||||::|| | :|||||||||||||||||| ||: ||||||||||||||||||||
Db 7 FRTTTRHMLKESKTYASQTLMGGLSGFESPIGLDRRDRLSALKSGDIGFVHSWDINTSVD 66
Qy 66 GPGTRMTVFMSGCPLRCQYCQNPDTWKMRDGKPVYYEAMVKKIERYADLFKATGGGITFS 125
||||||||||||||||||||||||||||||||||| :||:||::|| |||||| ||||||
Db 67 GPGTRMTVFMSGCPLRCQYCQNPDTWKMRDGKPVYLDAMIKKVDRYKDLFKATHGGITFS 126
Qy 126 GGESMMQPAFVSRVFHAAKQMGVHTCLDTSGFLGASYTDDMVDDIDLCLLDVKSGDEETY 185
|||||||||||||||||||:||||||||||||| :|||:|::|||||||||||||||||
Db 127 GGESMMQPAFVSRVFHAAKEMGVHTCLDTSGFLNTNYTDEMLEDIDLCLLDVKSGDEETY 186
Qy 186 HKVTGGILQPTIDFGQRLAKAGKKIWVRFVLVPGLTSSEENVENVAKICETFGDALEHID 245
|||||| ||||||||||||||||||||||||||||| |||||||||||||:||||:||||
Db 187 HKVTGGTLQPTIDFGQRLAKAGKKIWVRFVLVPGLTDSEENVENVAKICESFGDAVEHID 246
Qy 246 VLPFHQLGRPKWHMLNIPYPLEDQKGPSAAMKQRVVEQFQSHGFTVY 292
|| |||||||||| | ||||||:||||:|| ::|| ||: ||||||
Db 247 VLGFHQLGRPKWHELRIPYPLENQKGPNAATRERVTNQFKDHGFTVY 293