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 of claim 10, 13, 15, 16, 19, 20, in the paper of 12/29/2025, is acknowledged. Applicants' arguments filed on 12/29/2025, have been fully considered and are deemed to be persuasive to overcome some of the rejections previously applied. Rejections and/or objections not reiterated from previous office actions are hereby withdrawn. Claims 10, 11, 13, 15-22 are still at issue and are present for examination.
Claim Objections
Claims 20-22 are objected to because of the following informalities:
Claims 20-22 recites “Zr-FFZ1” which should be “ZrFFZ1”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 20-22 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
This rejection was stated in the previous office action as it applied to previous claims 20-22. In response to the rejection applicants have amended some of the claims and traverse the rejection as it applied to the newly amended claims.
The original rejection is repeated herein.
The invention appears to employ novel genetically engineered Kluryveromyces strains SD 98, SD1755 and JSS1397. Since the Kluryveromyces strains SD 98, SD1755 and JSS1397 are essential to the claimed invention, they must be obtainable by a repeatable method set forth in the specification or otherwise be readily available to the public. The Kluryveromyces strains SD 98, SD1755 and JSS1397 are not fully disclosed, nor have all the sequences required for their construction been shown to be publicly known and freely available. The enablement requirements of 35 U.S.C. 112 may be satisfied by a deposit of the Kluryveromyces strains SD 98, SD1755 and JSS1397. Accordingly, it is deemed that a deposit of the Kluryveromyces strains SD 98, SD1755 and JSS1397 should have been made in accordance with 37 CFR 1.801-1.809.
If a deposit was made under the terms of the Budapest Treaty, then an affidavit or declaration by applicants, or a statement by an attorney of record over his or her signature and registration number, stating that the specific strain has been deposited under the Budapest Treaty and that the strain will be available to the public under the conditions specified in 37 CFR 1.808, would satisfy the deposit requirement made herein.
If the deposit has not been made under the Budapest treaty, then in order to certify that the deposit meets the criteria set forth in 37 CFR 1.801-1.809, applicants may provide assurance or compliance by an affidavit or declaration, or by a statement by an attorney of record over his or her signature and registration number, showing that:
1. during the pendency of this application , access to the invention will be afforded to the Commissioner upon request;
2. upon granting of the patent the strain will be available to the public under the conditions specified in 37 CFR 1.808;
3. the deposit will be maintained in a public repository for a period of 30 years or 5 years after the last request or for the effective life of the patent, whichever is longer; and
4. the deposit will be replaced if it should ever become inviable.
Applicants Response
Applicants traverse the rejection on the on the basis that applicants submit that the strains have previously been described in previous US patent application publications and the strains are the result of well known molecular genetic techniques related to transformation protocols. Applicants submit that the rejection cannot be maintained because the three strains are publicly available or have been deposited under the terms of the Budapest Treaty.
Applicants complete argument is acknowledged and has been carefully considered, however, is not found persuasive for the reasons previously stated and repeated herein.
As stated previously, the invention appears to employ novel genetically engineered Kluryveromyces strains SD 98, SD1755 and JSS1397. Since the Kluryveromyces strains SD 98, SD1755 and JSS1397 are essential to the claimed invention, they must be obtainable by a repeatable method set forth in the specification or otherwise be readily available to the public. The Kluryveromyces strains SD 98, SD1755 and JSS1397 are not fully disclosed, nor have all the sequences required for their construction been shown to be publicly known and freely available. The enablement requirements of 35 U.S.C. 112 may be satisfied by a deposit of the Kluryveromyces strains SD 98, SD1755 and JSS1397. Accordingly, it is deemed that a deposit of the Kluryveromyces strains SD 98, SD1755 and JSS1397 should have been made in accordance with 37 CFR 1.801-1.809.
Applicants statement that the strains have been described in previous US patent application publications and the strains are the result of well-known molecular genetic techniques related to transformation protocols is not found persuasive in overcoming the rejection. Applicants statement that the strains the three strains are publicly available or have been deposited under the terms of the Budapest Treaty is not found persuasive without a showing of where the strains are publicly available or deposited as well as the statement of their public availability.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 claim(s) 10-13, 15-18 under 35 U.S.C. 103 as being unpatentable over Lee et al. (US 2014/0093901), Pina et al. (Microbiology 150, pp 2429-2433, 2004) and Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013) is withdrawn based upon applicants’ amendment of the claims and arguments presented in the paper of 3/17/2025.
Claim(s) 10, 11, 13, 15-19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (US 2014/0093901), Leandro et al. (Eukaryotic C ell, Vol 13, No. 1, pp 1371-1379, Nov 2014, see IDS of 5/8/2024) and Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013).
This rejection was stated in the previous office action as it applied to previous claims 10, 11, 13, 15-19. In response to the rejection applicants have amended the claims and traverse the rejection as it applied to the newly amended claims.
For applicants convenience the original rejection is repeated herein.
Lee et al. (US 2014/0093901) discloses a genetically engineered Kluyveromyces sp. yeast strain (a modified microorganism such as Kluyveromyces marxianus; paragraphs [0070]-[0071]; claims 1, 14, 15) that is capable of producing lactic acid (the microorganism may produce lactate; paragraph [0072]) from carbon source selected from glucose, fructose, sucrose or a mixture thereof (the carbon source may be glucose or fructose; claim 9) wherein the genetically engineered yeast comprises at least one heterologous gene (genes encoding enzymes are introduced into the microorganism; paragraph [0055]), rather than a heterologous DNA cassette, that confers production of a protein selected from a large group including a fructose importer (the introduced gene may have the function of D-fructose transport in via proton symport, and phosphofructokinase, among many other options; paragraph [0058]). Lee et al. does not specifically disclose a heterologous DNA cassette, and a protein functioning as a fructose importer.
Leandro et al. (Eukaryotic C ell, Vol 13, No. 1, pp 1371-1379, Nov 2014) teach cloning and expression of a high-capacity specific fructose facilitator ZrFfz1 from Zygosaccharomyces roux . Leandro et al. teach that the high-capacity specific fructose facilitator ZrFfz1 from Zygosaccharomyces roux is essential for fructophilic behavior of Zygosaccharomyces rouxii. Leandro et al. teach that ZrFfz1 from Zygosaccharomyces rouxii is a good candidate to improve the fructose fermentation performance of other yeast strains such as Saccharomyces cerevisiae.
Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) is a general review article that teaches transporter engineering in biomass utilization by yeast. Hara et al. teach a general review of yeast engineering and teach that biomass resources are attractive carbon sources for bioproduction because of their sustainability.
It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Lee et al. Kluyveromyces marxianus, to incorporate a heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. to improve lactate production from fructose as a carbon source. Leandro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein that increases import of fructose, and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate. The obvious genetically engineered Kluyveromyces marxianus, strain taught by Lee et al. with the incorporated heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. would be capable of consuming glucose and fructose as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013). Fonseca et al. evidence that Kluyveromyces marxianus is able to naturally uptake and utilize glucose. The obvious genetically engineered Kluyveromyces marxianus, strain taught by Lee et al. with the incorporated heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. would be capable of consuming all measurable glucose and fructose depending on how the measurable glucose and fructose was measured.
Lee et al. and Leandro et al., in combination, disclose the genetically engineered Kluyveromyces sp. yeast strain of Claim 1, but Lee et al. does not disclose wherein said fructose importer functions by facilitated diffusion. Leandro et al. discloses wherein said fructose importer functions by facilitated diffusion (plasmid encoding FFZ1, a Z. rouxii fructose-specific transporter (importer), which functions by facilitated diffusion; (see abstract; page 1371).
It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Lee et al. to incorporate a heterologous DNA cassette carrying a facilitated diffusion fructose importer, as taught by Leandro et al., as Lee et al. discloses a genetically modified yeast cell transformed to include a heterologous protein to improve lactate production from fructose as a carbon source, Leandro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein that increases import of fructose via facilitated diffusion, and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate in high-fructose media where facilitated diffusion is highly efficient. It would have been further obvious to a person of ordinary skill in the art, before the effective filing date, to express phosphofructokinase as suggested by Lee et al. to phosphorylate the imported fructose for further metabolism (claim 15).
Lee et al. and Leandro et al., in combination, disclose the genetically engineered Kluyveromyces sp. yeast strain of Claims 1-2, but Lee et al. does not disclose wherein said fructose importer is encoded by an exogenous FFZ1 gene from Zygosaccharomyces rouxii. Leandro et al. discloses wherein said fructose importer is encoded by an exogenous FFZ1 gene. It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Lee et al. to incorporate a heterologous DNA cassette carrying FFZ1 from Zygosaccharomyces rouxii, as taught by Leandro et al., as Lee et al. discloses a genetically modified yeast cell transformed to include a heterologous protein to improve lactate production from fructose as a carbon source, Leandro et al. discloses that ZrFfz1 from Zygosaccharomyces rouxii is a good candidate to improve the fructose fermentation performance of other yeast strains and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate in high-fructose media where facilitated diffusion is highly efficient. It would have been further obvious to a person of ordinary skill in the art, before the effective filing date, to culture the obvious Kluyveromyces strain in liquid culture medium to produce lactic acid (lactate). It would have been further obvious to a person of ordinary skill in the art, before the effective filing date, to culture the obvious Kluyveromyces strain in liquid culture medium to produce lactic acid (lactate) in an amount to completely consume fructose during fermentation (claim 19). The obvious genetically engineered Kluyveromyces Marxianus, strain taught by Lee et al. with the incorporated heterologous DNA cassette carrying a fructose importer from Zygosaccharomyces rouxii, as taught by Leandro et al., would be capable of consuming all glucose and fructose at a rate of at least 1.25 g L-1 hr-1, as evidenced by Leandro et al. (Figure 3 and supporting text) and Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013). Fonseca et al. evidence that Kluyveromyces marxianus is able to naturally uptake and utilize glucose at a rate of at least 1.25 g L-1 hr-1.
The expectation of success would have been high based upon the high level of skill in the art of yeast genetic engineering as exemplified by Lee et al., Leandro et al. and Hara et al. who teach all the methodology necessary to genetically transform Kluyveromyces with a heterologous DNA cassette encoding the FFZ1 fructose transporter from Zygosaccharomyces rouxii.
Applicants Response
Applicants continue to traverse the rejection as previously argued by applicants referring to applicants previously submitted responses. Applicants complete traversal of the above rejection for the reasons stated previously and herein are not found persuasive for the reasons stated previously and for those reasons repeated herein.
Applicants request for a technical response to each technical point is respectfully requested including (a) why the Office maintains that a donor gene from one genus of yeast would necessarily be functional in yeasts of a different genus and (b) which Applicant evidence of non- functional expression of the ZbFFZ 1 when transferred to a yeast with a different genetic background was not considered to weigh toward non-obviousness of the inventors' discovery that ZrFFZ I genes when transferred across a similar barrier surprisingly was highly functional.
Applicants submit that the Examiner’s consideration that ZrFfZl from Z. rouxii to be a good candidate to improve fructose fermentation performance assumes functionality of the ZrFfZl gene which applicants submit is not supported by the cited art. Applicants further submit that in improving fructose fermentation in Kluyveromyces one of ordinary skill in the art would not have been limited to selection of the ZrFfZ1 gene as there are thousands of other yeast genes involved in fructose uptake and metabolism. Applicants submit that absent hindsight, the Office has not established any reason for one to have selected ZrFfZ I instead any other potential fructose metabolism genes. Applicants submit that this deficiency in selecting an appropriate gene to enhance fructose fermentation is further emphasized by the lack of success and teaching away in expression of the ZbFFZ 1 in Kluveronyces as discussed above.
Applicants submit that the Examiners assertion that the consumption rate of fructose for Kluveronyces transformed with ZrFfZl would meet the limitations of 1.25 g/L/hr required by the claims based on prior art systems is inaccurate as, those of ordinary skill in the art would have consider this rate to be unpredictable, for example due to expression barriers for a heterologous protein in Kluyveromyices or other incompatibilities.
Applicants further submit additional arguments against the references D1, D2, D3, D4 and D5 individually.
Applicants continue to submit that in response to the Examiner’s statement that D2 teach that ZrFfz1 from Zygosaccharomices rouxii is a good candidate to improve the fructose fermentation performance of other yeast strains such as Saccharomyces cerevisiae, while acknowledgeable, is not found persuasive to applicants on the basis that applicants submit that every yeast might present different challenges when it is used as host cell for heterogenous expression. Applicants submit that there is no evidence in this work about the heterogenous expression of Zrfzzi or Zrffz2 genes in S. cerevisiae.
Applicants submit that Pina et al. (2004) suggested that ZbFfz1 is a transporter specifically for fructose and that it can be easily realized that it would be a good candidate to improve the fructose fermentation in other yeast such as S. cerevisiae. Applicants submit that in fact, they already did heterologous express the ZbFFZ1 gene in S. cerevisiae as shown in Figure 1 thus providing satisfactory evidence for heterologous expression in S. cerevisiae.
Applicants submit however, in the present invention applicants show that it is not similar to K. marxianus, as applicants submit data to suggest that ZbFFZ1 is not consistently expressed in K. marxianus using different expression constructs referring to applicants submitted Figures 3 and 5 results and discussion.
Applicants submit that based upon the above, the results show that there was a dramatic difference in performance between the two cassettes designed to install FFZ1 from Z. rouxii into K. marxianus.
Applicants submit that based upon the above applicants submit that claim 10 contains inventive steps over the basis of D! in combination with D2, D3, D4 and D5.
Applicants amendment of the claims, applicants complete argument, as well as the previous Declaration under 37 CFR 1.132 by one of the inventors, Phatthanon Prasitchoke, is acknowledged and has been carefully considered, however, is not found persuasive for the reasons presented previously and for those reasons repeated herein.
In response to applicants request for a technical response to each technical point, these have been made previously and throughout the previous rejection and in response to applicants traversal. In response to applicants point (a) why the Office maintains that a donor gene from one genus of yeast would necessarily be functional in yeasts of a different genus, this is not found persuasive. The office has not maintained that a donor gene from one genus of yeast would necessarily be functional in yeasts of a different genus. However in view of what appears to be a point being presented by applicants, applicants are reminded that in the instant case, "Obviousness does not require absolute predictability of success." Id. at 903, 7 USPQ2d at 1681.
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In response to applicants submission that the Examiner has not established a reasonable expectation of success for the invention as now claimed, is not found persuasive on the basis that as previously stated, Lee et al. and Leandro et al., in combination, disclose the genetically engineered Kluyveromyces sp. yeast strain of the Claims, but Lee et al. does not disclose wherein said fructose importer is encoded by an exogenous FFZ1 gene from Zygosaccharomyces rouxii. Leandro et al. discloses wherein said fructose importer is encoded by an exogenous FFZ1 gene. It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Lee et al. to incorporate a heterologous DNA cassette carrying FFZ1 from Zygosaccharomyces rouxii, as taught by Leandro et al., as Lee et al. discloses a genetically modified yeast cell transformed to include a heterologous protein to improve lactate production from fructose as a carbon source, Leandro et al. discloses that ZrFfz1 from Zygosaccharomyces rouxii is a good candidate to improve the fructose fermentation performance of other yeast strains and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate in high-fructose media where facilitated diffusion is highly efficient. It would have been further obvious to a person of ordinary skill in the art, before the effective filing date, to culture the obvious Kluyveromyces strain in liquid culture medium to produce lactic acid (lactate). The obvious genetically engineered Kluyveromyces marxianus, strain taught by Lee et al. with the incorporated heterologous DNA cassette carrying a fructose importer from Zygosaccharomyces rouxii, as taught by Leandro et al., would be capable of consuming all glucose and fructose at a rate of at least 1.25 g L-1 hr-1, as evidenced by Leandro et al. (Figure 3 and supporting text) and Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013). Fonseca et al. evidence that Kluyveromyces marxianus is able to naturally uptake and utilize glucose at a rate of at least 1.25 g L-1 hr-1.
The expectation of success would have been high based upon the high level of skill in the art of yeast genetic engineering as exemplified by Lee et al., Leandro et al. and Hara et al. who teach all the methodology necessary to genetically transform Kluyveromyces with a heterologous DNA cassette encoding the FFZ1 fructose transporter from Zygosaccharomyces rouxii.
In response to applicants submission that those skilled in the art of molecular yeast genetics understood the challenges of constructing useful genetically-engineered strains especially those associated with transformation of yeasts with heterologous genes and these include overcoming problems with heterologous expression of a gene from one yeast clade in a different yeast clade to produce a functional protein, is not found persuasive, on the basis that while there may be challenges in expressing heterologous proteins, the level of skill in the art is high that these challenges are routinely overcome by the art.
In response to applicants submission that these include expression barriers such as inoperability of promoters, enhancers or other regulatory genes for a heterologous gene which can result in the gene being poorly transcribed and translated or not transcribed or translated at all, these are all things that the level of skill in the art is able to overcome.
In response to applicants submission of which Applicant evidence of non- functional expression of the ZbFFZ 1 when transferred to a yeast with a different genetic background was not considered to weigh toward non-obviousness of the inventors' discovery that ZrFFZ I genes when transferred across a similar barrier surprisingly was highly functional, this was stated previously that applicants submission of the lack of success of specific expression constructs is not found persuasive in overcoming the rejection for the reasons previously made of record. As stated previously, one of skill in the art would know and understand that different expression constructs would have different levels of expression based upon numerous factors including but not limited to promoter strength, insertion point as well as other cis and trans factors.
In response to applicants submission that in improving fructose fermentation in Kluyveromyces one of ordinary skill in the art would not have been limited to selection of the ZrFfZ1 gene as there are thousands of other yeast genes involved in fructose uptake and metabolism, this is not found persuasive for the reasons previously made of record. As previously stated and repeated above, it would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Lee et al. Kluyveromyces marxianus, to incorporate a heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. to improve lactate production from fructose as a carbon source. Leandro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein that increases import of fructose, and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate. The obvious genetically engineered Kluyveromyces marxianus, strain taught by Lee et al. with the incorporated heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. would be capable of consuming glucose and fructose as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013). Fonseca et al. evidence that Kluyveromyces marxianus is able to naturally uptake and utilize glucose. The obvious genetically engineered Kluyveromyces marxianus, strain taught by Lee et al. with the incorporated heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. would be capable of consuming all measurable glucose and fructose depending on how the measurable glucose and fructose was measured.
In response to applicant's arguments against the references D1, D2, D3, D4 and D5individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
In response to applicants submission that there is no evidence in this work about the heterogenous expression of Zrfzzi or Zrffz2 genes in S. cerevisiae, this is not found persuasive for the reasons and evidence presented previously. Further applicants submission that Pina et al. (2004) show heterologous expression of the ZbFFZ1 gene in S. cerevisiae as shown in Figure 1 provides satisfactory evidence for heterologous expression in S. cerevisiae. While applicants submit this is not K. marxianus, this is acknowledged, however, this is evidence for heterologous expression in a different yeast strain.
Thus claim(s) 10, 11, 13, 15-19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Lee et al. (US 2014/0093901), Leandro et al. (Eukaryotic C ell, Vol 13, No. 1, pp 1371-1379, Nov 2014, see IDS of 5/8/2024) and Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013).
Claim(s) 10, 11, 13, 15-19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Porro et al. (US 2003/0032152), Lee et al. (US 2014/0093901), Leandro et al. (Eukaryotic C ell, Vol 13, No. 1, pp 1371-1379, Nov 2014, see IDS of 5/8/2024) and Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013).
This rejection was stated in the previous office action as it applied to previous claims 10, 11, 13, 15-19. In response to the rejection applicants have amended the claims and traverse the rejection as it applied to the newly amended claims.
For applicants convenience the original rejection is repeated herein.
Porro et al. (US 2003/0032152) discloses a genetically engineered yeast strains including Kluyveromyces lactis and Zygosaccharomyces bailii paragraphs (claims 13, 15 and supporting text) that are capable of producing lactic acid from carbon source selected from glucose, fructose, sucrose or a mixture thereof wherein the genetically engineered yeast comprises at least one heterologous gene encoding lactic dehydrogenase (LDH) (see claim 1 and supporting text). Porro et al. teaches that it has been found that production of lactic acid can be obtained by metabolically modified yeasts belonging to the genera Kluyveromyces, Saccharomyces, Torulaspora and Zygosaccharomyces. Porro et al. further teach that yeast cells transformed with a heterologous LDH gene and overexpressing a lactate transporter, such as the JEN1 gene encoding the lactate transporter of S. cerevisiae (paragraph [0050].
Porro et al. does not specifically disclose a heterologous DNA cassette, and a protein functioning as a fructose importer.
Lee et al. (US 2014/0093901) discloses a genetically engineered Kluyveromyces sp. yeast strain (a modified microorganism such as Kluyveromyces marxianus; paragraphs [0070]-[0071]; claims 1, 14, 15) that is capable of producing lactic acid (the microorganism may produce lactate; paragraph [0072]) from carbon source selected from glucose, fructose, sucrose or a mixture thereof (the carbon source may be glucose or fructose; claim 9) wherein the genetically engineered yeast comprises at least one heterologous gene (genes encoding enzymes are introduced into the microorganism; paragraph [0055]), rather than a heterologous DNA cassette, that confers production of a protein selected from a large group including a fructose importer (the introduced gene may have the function of D-fructose transport in via proton symport, and phosphofructokinase, among many other options; paragraph [0058]). Lee et al. does not specifically disclose a heterologous DNA cassette, and a protein functioning as a fructose importer.
Leandro et al. (Eukaryotic C ell, Vol 13, No. 1, pp 1371-1379, Nov 2014) teach cloning and expression of a high-capacity specific fructose facilitator ZrFfz1 from Zygosaccharomyces roux . Leandro et al. teach that the high-capacity specific fructose facilitator ZrFfz1 from Zygosaccharomyces roux is essential for fructophilic behavior of Zygosaccharomyces rouxii. Leandro et al. teach that ZrFfz1 from Zygosaccharomyces rouxii is a good candidate to improve the fructose fermentation performance of other yeast strains such as Saccharomyces cerevisiae.
Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) is a general review article that teaches transporter engineering in biomass utilization by yeast. Hara et al. teach a general review of yeast engineering and teach that biomass resources are attractive carbon sources for bioproduction because of their sustainability.
It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Porro et al. Kluyveromyces, to incorporate a heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. to improve lactate production from fructose as a carbon source. Leandro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein that increases import of fructose, and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate. The obvious genetically engineered Kluyveromyces strain taught by Porro et al. with the incorporated heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. would be capable of consuming glucose and fructose as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013). Fonseca et al. evidence that Kluyveromyces marxianus is able to naturally uptake and utilize glucose. The obvious genetically engineered Kluyveromyces strain taught by Porro et al. with the incorporated heterologous DNA cassette carrying a fructose importer, as taught by Leandro et al. would be capable of consuming all measurable glucose and fructose depending on how the measurable glucose and fructose was measured.
Porro et al. and Leandro et al., in combination, disclose the genetically engineered Kluyveromyces sp. yeast strain of Claim 1, but Porro et al. does not disclose wherein said fructose importer functions by facilitated diffusion. Leandro et al. discloses wherein said fructose importer functions by facilitated diffusion (plasmid encoding FFZ1, a Z. rouxii fructose-specific transporter (importer), which functions by facilitated diffusion; (see abstract; page 1371).
It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Porro et al. to incorporate a heterologous DNA cassette carrying a facilitated diffusion fructose importer, as taught by Leandro et al., as Porro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein to improve lactate production from fructose as a carbon source, Leandro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein that increases import of fructose via facilitated diffusion, and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate in high-fructose media where facilitated diffusion is highly efficient. It would have been further obvious to a person of ordinary skill in the art, before the effective filing date, to express phosphofructokinase in the above obvious strain, as suggested by Lee et al. to phosphorylate the imported fructose for further metabolism (claim 15).
Porro et al. and Leandro et al., in combination, disclose the genetically engineered Kluyveromyces sp. yeast strain of the Claims, but Porro et al. does not disclose wherein said fructose importer is encoded by an exogenous FFZ1 gene from Zygosaccharomyces rouxii. Leandro et al. discloses wherein said fructose importer is encoded by an exogenous FFZ1 gene. It would have been obvious to a person of ordinary skill in the art, before the effective filing date, to have modified the genetically engineered yeast strain as disclosed by Porro et al. to incorporate a heterologous DNA cassette carrying FFZ1 from Zygosaccharomyces rouxii, as taught by Leandro et al., as Porro et al. discloses a genetically modified yeast cell transformed to include a heterologous protein to improve lactate production from fructose as a carbon source, Leandro et al. discloses that ZrFfz1 from Zygosaccharomyces rouxii is a good candidate to improve the fructose fermentation performance of other yeast strains and this combination would provide increased ability to utilize fructose as a carbohydrate nutrient in production of lactate in high-fructose media where facilitated diffusion is highly efficient. It would have been further obvious to a person of ordinary skill in the art, before the effective filing date, to culture the obvious Kluyveromyces strain in liquid culture medium to produce lactic acid (lactate). The obvious genetically engineered Kluyveromyces marxianus, strain taught by Porro et al. with the incorporated heterologous DNA cassette carrying a fructose importer from Zygosaccharomyces rouxii, as taught by Leandro et al., would be capable of consuming all measurable glucose and fructose at a rate of at least 1.25 g L-1 hr-1, as evidenced by Leandro et al. (Figure 3 and supporting text) and Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013). Fonseca et al. evidence that Kluyveromyces marxianus is able to naturally uptake and utilize glucose at a rate of at least 1.25 g L-1 hr-1.
The expectation of success would have been high based upon the high level of skill in the art of yeast genetic engineering as exemplified by Porro et al. , Lee et al., Leandro et al. and Hara et al. who teach all the methodology necessary to genetically transform Kluyveromyces with a heterologous DNA cassette encoding the FFZ1 fructose transporter from Zygosaccharomyces rouxii.
It is noted that applicants arguments and Declaration filed on 8/28/2025 and 12/29/2025 have been considered to the extent that they apply to the above rejection. Applicants arguments and Declaration are not persuasive in overcoming this rejection as discussed above.
Thus claim(s) 10, 11, 13, 15-19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Porro et al. (US 2003/0032152), Lee et al. (US 2014/0093901), Leandro et al. (Eukaryotic C ell, Vol 13, No. 1, pp 1371-1379, Nov 2014, see IDS of 5/8/2024) and Hara et al. (FEMS Yeast Research, Vol 17, No. 7, pp 1-14, 2017) as evidenced by Fonseca et al. (Appl. Microbiol. Biotechnol. Vol 97, pp 5055-5067, 2013).
Remarks
No claim is allowed.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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rgh
2/25/2026
/RICHARD G HUTSON/Primary Examiner, Art Unit 1652