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 .
DETAILED ACTION
This application is a divisional of 16/091,624, issued as US Patent No. 11,859,223, which is a 371 of PCT/US2017/026615.
The response filed on May 4, 2026 has been entered.
Election/Restrictions
Applicant’s election of the species of OptoINVRT3 circuit, which includes:
(1) PTEF1 →VP16-EL222 →Tcyc1. Constitutive expression of the light-sensitive transcription factor VP16-EL222 (which includes a LOV sensing domain). When exposed to blue light wavelengths, VP16-EL222 binds the C120 promoter to drive GAL80 expression.
(2) PC120 →GAL80. GAL80 under the light-inducible C120 promoter. When light is activated, repressor Gal80p is produced, which reduces Gal4p-driven transcription.
(3) PADH1 →GAL4-PSD. GAL4 is driven by the ADH1 promoter and is fused at its C- terminus to a photosensitive degron (PSD). The PSD is derived from the fusion of the phototropin1 LOV2 domain (V19L mutant) from Arabidopsis thaliana and a synthetic degradation sequence derived from murine ornithine decarboxylase (ODC). This degron causes Gal4p to be degraded in the light, providing a second layer of light-dependent repression.
(4) PGAL1 target gene. The output promoter, where the gene of interest (such as a enzyme, a GFP reporter, etc.) is placed under GAL1 promoter control.in the reply filed on May 4, 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.03(a)).
Status of Claims
Claims 1-14 are pending.
Claims 1-14 are under examination.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on November 6, 2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Specification/Abstract
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
In the instant case, the abstract is composed of 184 words.
Appropriate correction is requested.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1 and claims 2-14 depending therefrom are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention.
Claim 1 and claims 2-14 depending therefrom are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. The omitted steps are: Claim 1 recites only one step, delivering a time-varying dose. However, claim 1 recites “comprising the steps of:”. Clarification is requested.
Claim 1 and claims 2-14 depending therefrom are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The terms “overproduce” and “overproduction” in claim 1 are relative terms which renders the claim indefinite. The term “overproduce” and “overproduction” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is unclear as to what level of production of the chemical or protein is considered as overproduced.
Claims 9-10 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 9-10 recite the limitation “gene under..promoter”. The metes and bounds of this limitation in the context of the above claim are not clear. It is unclear how a gene is “under” a promoter. Clarification is requested.
Claim Rejections - 35 USC § 112(a)
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 1-14 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 written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In this case, the claims 1-4 have been broadly interpreted to encompass a method of enabling any cell or Saccharomyces cerevisiae to overproduce any chemical or protein, the method comprising the steps delivering a time-varying does or pulses of any one wavelength of the light to the cell and wherein the cell comprises any plurality of genes capable of being controlled with said any wavelength of light, wherein the overproduction of the any chemical or any protein is controlled by varying the dose of the any wavelength of light during fermentation or splitting fermentation into a growth and production phase having a different light schedule. Claims 5-14 further limit claim 1 to encompass a plurality of genes comprising (1) any first sequence comprising a nucleotide sequence that encodes any light-activated transcription factor or any derivative of LOV sensing domain to any first promoter or any constitutive first promoter, (2) any second sequence comprising any second promoter which can be activated by the light activated transcription factor encoded by the any first sequence and further comprising a nucleotide sequence that encodes a first metabolic enzyme and is required for cell growth, (3) any third sequence comprising any third promoter which can be activated by the any light-activated transcription factor and further comprising a nucleotide sequence that encodes any repressor or the repressor GAL80, (4) any fourth sequence comprising any fourth promoter or any galactose-inducible promoter via GAL4 which can be repressed by the any repressor encoded by the third sequence and further comprising a nucleotide sequence that encodes any second metabolic enzyme that drives the metabolic pathway to completion and/or any degron, (5) any gene under any first or second promoter or any third promoter configured to be activated when exposed to a first additional wavelength of light and not activated when not exposed to the first additional wavelength of light, and (6) further coupling the production of the chemical with a response from any biosensor or any protein cascade system that produces any measurable result in response to the chemical Therefore, the claims are drawn to a method of enabling a genus of cells or S. cerevisiae to produce a genus of chemicals or proteins by delivering a genus of time-varying dose of wavelengths to bi-directionally controlled the genus of genes encode a first, second, third, fourth, or fifth sequences having unknown structure and comprising the genus of first, second, third, and fourth promoters having unknown structure.
MPEP 2163 I. states that to “satisfy the written description requirement, a patent specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention.
MPEP 2163. II.A.3.(a) sates that “Possession may be shown in many ways. For example, possession may be shown by describing an actual reduction to practice of the claimed invention. Possession may also be shown by a clear depiction of the invention in detailed drawings or in structural chemical formulas which permit a person skilled in the art to clearly recognize that inventor had possession of the claimed invention. An adequate written description of the invention may be shown by any description of sufficient, relevant, identifying characteristics so long as a person skilled in the art would recognize that the inventor had possession of the claimed invention.
According to MPEP 2163.II.A.3.(a).ii), “Satisfactory disclosure of a ‘representative number’ depends on whether one of skill in the art would recognize that the applicant was in possession of the necessary common attributes or features possessed by the members of the genus in view of the species disclosed. For inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus…Instead, the disclosure must adequately reflect the structural diversity of the claimed genus, either through the disclosure of sufficient species that are ‘representative of the full variety or scope of the genus,’ or by the establishment of ‘a reasonable structure-function correlation.’"
The recitation of “first sequence.”, “first promoter”, “second sequence..”, “second promoter”, “third sequence..”, “third promoter”, “fourth sequence..”, “fourth promoter”, “light-activated transcription factor”, “light-oxygen voltage (LOV) sensing domain” and “cell” fails to provide a sufficient description of the genus of cell of the claimed method as it merely describes the functional features of the genus without providing any definition of the structural features of the species within the genus. The specification does not specifically define any of the species that fall within the genus. The specification does not define any structural features commonly possessed by members of the genus that distinguish them from others. One skilled in the art therefore cannot, as one can do with a fully described genus, visualize or recognize the identity of the members of the genus.
Gardner (US 2014/0325692 – form PTO-1449) discloses a method of controlling gene expression in eukaryotic cells using a blue light inducible gene expression system comprising of the light transcriptional factor EL-222 operably linked to VP-16 that binds to the promoter PC120 (Figures 1A and 1B). Chua (WO 2015/020649 – form PTO-1449) discloses using the GAL regulon comprising of GAL80 and GAL4 to activate gene expression or inactivate gene expression (abstract, Figure 5 and pages 3 and 30-33, page 52 and page 87, lines 1-13). However, a method of controlling any genes encoding metabolic enzymes using any light-activated transcription factors, repressors, and promoters to enable any cell to overproduce any chemical or protein was not known in the art.
The specification is limited to the disclosure of a method of using a blue light inducible gene expression system VP16-EL222, which is configured into OptoEXP, OptoINVRT1, OptoINVRT2, and OptoINVRT3 as described in Supplementary Table 3 at page 40 and S. cerevisiae comprising said OptoEXP, OptoINVRT1, OptoINVRT2, and/ or OptoINVRT3 for production of ethanol. While MPEP 2163 acknowledges that in certain situations “one species adequately supports a genus,” it also acknowledges that “[f]or inventions in an unpredictable art, adequate written description of a genus which embraces widely variant species cannot be achieved by disclosing only one species within the genus.” In view of the widely variant species encompassed by the genus, a single species of a blue light inducible gene expression system VP16-EL222 is not enough and does not constitute a representative number of species to describe the cells used in the claimed method. Therefore, the specification fails to describe a representative species of the claimed cells.
The claimed invention requires a defined set of first-fourth sequences and first-fourth promoters that are bi-directionally controlled via a light-activated transcription factors and cells. Although the specification discloses exemplary light-activated transcription factors, first-fifth sequences, and first-fourth promoters, a “laundry list” disclosure of every possible moiety does not necessarily constitute a written description of every species in a genus because it would not “reasonably lead” those skilled in the art to any particular species, see Fujikawa v. Wattanasin, 93 F.3d 1559, 1571, 39 USPQ2d 1895, 1905 (Fed. Cir. 1996) or MPEP 2163. While the exemplary light-activated transcription factors, first-fifth sequences, and first-fourth promoter were known in the art, this knowledge alone would not allow one level of skill in the art to immediately envisage the claimed genus. Therefore, the level of skill and knowledge in the art is such that one of ordinary skill would not be able to identify without further testing which combination of light-activated transcription factors, first-fourth sequences, and first-fourth promoter to use in the claimed method.
Given this lack of description of the representative species encompassed by the genus of the claims, the specification fails to sufficiently describe the claimed invention in such full, clear, concise, and exact terms that a skilled artisan would recognize that applicants were in possession of the inventions of claims 1-14.
Claims 1-14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a method of using a blue light inducible gene expression system VP16-EL222, which is configured into OptoEXP, OptoINVRT1, OptoINVRT2, and OptoINVRT3 as described in Supplementary Table 3 at page 40 and S. cerevisiae comprising said OptoEXP, OptoINVRT1, OptoINVRT2, and/ or OptoINVRT3 for production of ethanol, does not reasonably provide enablement for a method of enabling any cell or S. cerevisiae to produce any chemical or protein by delivering any time-varying dose of any wavelengths to bi-directionally controlled any first, second, third, fourth, and fifth sequences having unknown structure and comprising any first, second, third, and fourth promoters having unknown structure. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Factors to be considered in determining whether undue experimentation is required are summarized in In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988). They include (1) the quantity of experimentation necessary, (2) the amount of direction or guidance presented, (3) the presence or absence of working examples, (4) the nature of the invention, (5) the state of the prior art, (6) the relative skill of those in the art, (7) the predictability or unpredictability of the art, and (8) the breadth of the claims.
The breadth of the claims.
MPEP 2111.01 states that ''[d]uring examination, the claims must be interpreted as broadly as their terms reasonably allow.'' In this case, the claims 1-4 have been broadly interpreted to encompass a method of enabling any cell or Saccharomyces cerevisiae to overproduce any chemical or protein, the method comprising the steps delivering a time-varying does or pulses of any one wavelength of the light to the cell and wherein the cell comprises any plurality of genes capable of being controlled with said any wavelength of light, wherein the overproduction of the any chemical or any protein is controlled by varying the dose of the any wavelength of light during fermentation or splitting fermentation into a growth and production phase having a different light schedule. Claims 5-14 further limit claim 1 to encompass a plurality of genes comprising (1) any first sequence comprising a nucleotide sequence that encodes any light-activated transcription factor or any derivative of LOV sensing domain to any first promoter or any constitutive first promoter, (2) any second sequence comprising any second promoter which can be activated by the light activated transcription factor encoded by the any first sequence and further comprising a nucleotide sequence that encodes a first metabolic enzyme and is required for cell growth, (3) any third sequence comprising any third promoter which can be activated by the any light-activated transcription factor and further comprising a nucleotide sequence that encodes any repressor or the repressor GAL80, (4) any fourth sequence comprising any fourth promoter or any galactose-inducible promoter via GAL4 which can be repressed by the any repressor encoded by the third sequence and further comprising a nucleotide sequence that encodes any second metabolic enzyme that drives the metabolic pathway to completion and/or any degron, (5) any gene under any first or second promoter or any third promoter configured to be activated when exposed to a first additional wavelength of light and not activated when not exposed to the first additional wavelength of light, and (6) further coupling the production of the chemical with a response from any biosensor or any protein cascade system that produces any measurable result in response to the chemical. Therefore, the claims are drawn to a method of enabling any cell or S. cerevisiae to produce any chemical or protein by delivering any time-varying dose of any wavelengths to bi-directionally controlled any first, second, third, fourth, and fifth sequences having unknown structure and comprising any first, second, third, and fourth promoters having unknown structure.
The claims are not commensurate with the enablement provided by the disclosure with regard to the extremely large number of cells, light inducible expression systems, and wavelengths of light to control the expression of any gene. In the instant case, the specification is limited to the teaching of a method of using a blue light inducible gene expression system VP16-EL222, which is configured into OptoEXP, OptoINVRT1, OptoINVRT2, and OptoINVRT3 as described in Supplementary Table 3 at page 40 and S. cerevisiae comprising said OptoEXP, OptoINVRT1, OptoINVRT2, and/ or OptoINVRT3 for production of ethanol.
The quantity of experimentation required to practice the claimed invention based on the teachings of the specification.
While the skilled artisan can transform cells with various polynucleotides using well-known and widely used techniques in the art, the amount of experimentation required is not routine due to the fact that the number of species encompassed by the claims is extremely large.
In the absence of: (a) rational and predictable scheme for bi-directionally controlling any gens in any cell or Saccharomyces cerevisiae using any wavelength of light, and (b) a correlation between structure and the activity of controlling activation/inactivation/expression with any light in any cell or Saccharomyces cerevisiae, the specification provides insufficient guidance as to which of the essentially infinite possible choices is likely to be successful. One of skill in the art would have to test these infinite possible cells, genes, and light inducible promoters/expression systems to determine which combination is effective in bi-directionally controlling the recited polynucleotides. While enablement is not precluded by the necessity for routine screening, if a large amount of screening is required, as is the case herein, the specification must provide a reasonable amount of guidance which respect to the direction in which the experimentation should proceed so that a reasonable number of species can be selected for testing. In view of the fact that such guidance has not been provided in the instant specification, it would require undue experimentation to enable the full scope of the claims.
The state of prior art, the relative skill of those in the art, and predictability or unpredictability of the art.
The specification does not provide guidance as to (1) cells other than S. cerevisiae to bi-directionally control any polynucleotide with any light, (2) which combinations cells, light inducible promoters/expression systems, promoters, polynucleotides, and light to use in order to bi-directionally control any polynucleotides, and (3) the general tolerance and flexibility of cells, light inducible promoters/expression systems, promoters, polynucleotides, and light to bi-directionally controlling the polynucleotides.
Gardner (US 2014/0325692 – form PTO-1449) discloses a method of controlling gene expression in eukaryotic cells using a blue light inducible gene expression system comprising of the light transcriptional factor EL-222 operably linked to VP-16 that binds to the promoter PC120 (Figures 1A and 1B). Chua (WO 2015/020649 – form PTO-1449) discloses using the GAL regulon comprising of GAL80 and GAL4 to activate gene expression or inactivate gene expression (abstract, Figure 5 and pages 3 and 30-33, page 52 and page 87, lines 1-13). However, a method of controlling any genes encoding metabolic enzymes using any light-activated transcription factors, repressors, and promoters to enable any cell to overproduce any chemical or protein was not known in the art.
The amount of direction or guidance presented and the existence of working examples.
The specification is limited to the teaching of a method of using a blue light inducible gene expression system VP16-EL222, which is configured into OptoEXP, OptoINVRT1, OptoINVRT2, and OptoINVRT3 as described in Supplementary Table 3 at page 40 and S. cerevisiae comprising said OptoEXP, OptoINVRT1, OptoINVRT2, and/ or OptoINVRT3 for production of ethanol. However, the speciation fails to provide any information as to the structural elements required to bi-directionally control any polynucleotides in any cell or Saccharomyces cerevisiae.
Thus, in view of the overly broad scope of the claims, the lack of guidance and working examples provided in the specification, the high level of unpredictability of the prior art in regard to structural changes and their effect on function and the lack of knowledge about a correlation between structure and function, an undue experimentation would be necessary one having ordinary skill in the art to make and use the claimed invention in a manner reasonably correlated with the scope of the claims. The scope of the claims must bear a reasonable correlation with the scope of enablement (In re Fisher, 166 USPQ 19 24 (CCPA 1970)). Without sufficient guidance, determination of polypeptides having the desired biological characteristics recited in the claim are unpredictable and the experimentation left to those skilled in the art is unnecessarily, and improperly, extensive and undue. See In re Wands 858 F.2d 731, 8 USPQ2nd 1400 (Fed. Cir, 1988).
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 of this title, 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.
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 1-6 are rejected under 35 U.S.C. 103 as being unpatentable over Gardner (US 2014/0325692 – form PTO-1449), Tokuhiro (Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene Appl Microbiol Biotechnol (2009) 82:883–890. – form PTO-1449), and Da Silva (FEMS Yeast Res. 2012 Mar; 12(2):197-214. – form PTO-1449).
Gardner discloses that light induced genes have advantages over chemically induced genes in that light is inexpensive to apply, easily delivered, and does not interfere with cellular pathways ([0061]-[0062]). Gardner discloses controlling gene expression in eukaryotic cells using a blue light inducible gene expression system comprising of the light transcriptional factor EL-222 operably linked to VP-16 that binds to the promoter PC120 (Figures 1A and 1B).
Regarding claims 1 and 5-6, Gardner discloses a method of overproduction of a protein of interest by using a yeast cell comprising of genes controlled bi-directionally with blue light: a first polynucleotide comprising VP16-EL222/EL222-VP16 and a second polynucleotide comprising Pc120 (C1-2 20bp sequence) linked to a gene of interest, wherein said gene is turned on (transcribed) when exposed to blue light and turned off when not exposed to blue light (Figure 1A, [0071]-[0077], and [108]). VP16-EL222/EL222-VP16, which is derived from LOV sensing domain, reads on the “first sequence” recited in claims 5-6, a nucleotide sequence encoding a light-activated transcription factor that binds to a promoter (Pc120) initiating transcription. Pc120 (C1-2 20bp sequence) reads on the “second sequence” recited in claim 5, a promoter which can be activated by the light-activated transcription factor VP16-EL222/EL222-VP16. Gardner discloses that the light responsive DNA-binding protein/light-activated transcription factor (such as VP16-EL222) regulates expression of a gene of interest operably linked to the DNA-binding site/promoter (such Pc120) recognized by said light responsive DNA-binding protein/light-activated transcription factor ([0074). Gardner discloses that the protein encode by the gene of interest can be a reporter protein such as GFP, industrial enzymes, biofuel production enzymes, or a regulatory protein ([0077]).
Regarding claim 4, Gardner discloses using light pulses ([0067]).
Regarding claim 6, the light-activated transcription factor VP16-EL222/EL222-VP16 is derived from the LOV sensing domain ([0008] and Figure 1A).
Gardner does not disclose using a constitutive promoter as the first promoter and bidirectional expression of PDC1 under growth phase and expression of LDH under production phase.
Tokuhiro summarizes past metabolic engineering approaches in reducing ethanol production and increasing lactate production via fermentation by S. cerevisiae. Tokuhiro discloses that yields of lactate production in recombinant S. cerevisiae expressing heterologous lactate dehydrogenase is low because the strong tendency for ethanol to be competitively produced from pyruvate (page 884, left column). Pyruvate decarboxylases encoded by pdc1, pdc5, and pdc6 were targets of inactivation because decarboxylation of pyruvate is the first step to ethanol from pyruvate. Single pdc1 or pdc5 mutants improved lactate yields but a large amount of ethanol was still produced. However, the triple inactivation of pdc1, pdc5, and pdc6 has impaired growth on glucose (page 884, left column).
Regarding 2, Tokuhiro discloses fermentation with two-stage batch culture (page 887, 1st paragraph).
Regarding claim 3, Tokuhiro discloses providing a growth medium and refreshing the growth medium (page 887, 1st paragraph).
Regarding claim 5, PDC and LDH are metabolic enzymes since they are involved in the metabolism of pyruvate and PDC and LDH compete for the same metabolite, pyruvate (Figure 1). Tokuhiro discloses expression of LDH enables a cell to overproduce lactate dehydrogenase to an amount greater than what is produced by a wild-type cell (abstract and page 884).
Regarding claim 6, PDC is essential for cell growth, as disclosed by Tokuhiro (page 884, left column).
Regarding claims 1 and 5, Da Silva discloses well known promoters and terminators for expression of genes in S. cerevisiae. Da Silva also discloses that bi-directional promoters are very useful for metabolic engineering in S. cerevisiae (page 200, right column). Da Silva discloses the combination of PTEF1 and TCYC1 (page 204, left column). Da Silva also discloses the constitutive promoters, such as PADH1 (page 204, right column).
Therefore, in combining the teachings of Gardner, Tokuhiro, and Da Silva, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to adapt the light-inducible expression system of Gardner for increasing production of lactate in S. cerevisiae by (A) inducing expression of PDC1 upon exposure to blue light during the growth phase and (B) not inducing expression of PDC1 during the production phase by expressing in an S. cerevisiae Δpdc1Δpdc5Δpdc6 a light induced expression system comprising (1) a first sequence comprising PTEF1-EL222-VP16-TCYC1 (light-activated transcription factor), (2) a second sequence comprising PC120-PDC1 (OptoEXP-PDC1) (promoter activated by said light-activated transcription factor and a polynucleotide encoding the metabolic enzyme PDC1), and (3) an additional sequence comprising a PTEF1-LDH. One having ordinary skill in the art would have been motivated to express PDC1 during the growth phase and not express PDC1 during the production phase in order for S. cerevisiae Δpdc1Δpdc5Δpdc to grow during the growth phase and direct carbon and cellular energy toward lactate production and away from ethanol production during the production phase. The motivation of using S. cerevisiae Δpdc1Δpdc5Δpdc is to reduce production of ethanol and increase production of lactate. The motivation of using a light inducible gene expression system of Gardner over chemically induced genes in that light is inexpensive to apply, easily delivered and does not interfere with cellular pathways. One having ordinary skill in the art would have had a reasonable expectation of success since Gardner discloses a light inducible expression system for inducing/not inducing expression of a gene and Tokuhiro discloses Δpdc1Δpdc5Δpdc S. cerevisiae and a lactate producing S. cerevisiae expressing LDH. The rationale supporting that the claims would have been obvious is that a method of enhancing a particular class of devices (bidirectionally turning on and off genes using a light inducible expression system) has been made part of the ordinary capabilities of one skilled in the art based upon the teaching of such improvement in other situations. One of ordinary skill in the art would have been capable of applying this known method of enhancement to a “base” device (expression of genes) in the prior art and the results would have been predictable to one of ordinary skill in the art.
Therefore, the above references render claims 1-6 and prima facie obvious.
Claims 1-13 are rejected under 35 U.S.C. 103 as being unpatentable over Gardner (US 2014/0325692 – form PTO-1449), Tokuhiro (Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene Appl Microbiol Biotechnol (2009) 82:883–890. – form PTO-1449), Chua (WO 2015/020649 – form PTO-1449), and Da Silva (FEMS Yeast Res. 2012 Mar; 12(2):197-214. – form PTO-1449).
Gardner discloses that light induced genes have advantages over chemically induced genes in that light is inexpensive to apply, easily delivered, and does not interfere with cellular pathways ([0061]-[0062]). Gardner discloses controlling gene expression in eukaryotic cells using a blue light inducible gene expression system comprising of the light transcriptional factor EL-222 operably linked to VP-16 that binds to the promoter PC120 (Figures 1A and 1B).
Regarding claims 1, 5-6, and 9-10, Gardner discloses a method of overproduction of a protein of interest by using a yeast cell comprising of genes controlled bi-directionally with blue light: a first polynucleotide comprising VP16-EL222/EL222-VP16 and a second polynucleotide comprising Pc120 (C1-2 20bp sequence) linked to a gene of interest, wherein said gene is turned on (transcribed) when exposed to blue light and turned off when not exposed to blue light (Figure 1A, [0071]-[0077], and [108]). VP16-EL222/EL222-VP16, which is derived from LOV sensing domain, reads on the “first sequence” recited in claims 5-6 and 9, a nucleotide sequence encoding a light-activated transcription factor that binds to a promoter (Pc120) initiating transcription. Pc120 (C1-2 20bp sequence) reads on the “second sequence” recited in claims 5 and 9, a promoter which can be activated by the light-activated transcription factor VP16-EL222/EL222-VP16. Gardner discloses that the light responsive DNA-binding protein/light-activated transcription factor (such as VP16-EL222) regulates expression of a gene of interest operably linked to the DNA-binding site/promoter (such Pc120) recognized by said light responsive DNA-binding protein/light-activated transcription factor ([0074). Gardner discloses that the protein encode by the gene of interest can be a reporter protein such as GFP, industrial enzymes, biofuel production enzymes, or a regulatory protein ([0077]).
Regarding claim 4, Gardner discloses using light pulses ([0067]).
Regarding claim 6, the light-activated transcription factor VP16-EL222/EL222-VP16 is derived from the LOV sensing domain ([0008] and Figure 1A).
The difference between the method of Gardner and the instant claims is that Gardner does not disclose bidirectional expression of PDC1 and LDH nor the third and fourth sequences.
Tokuhiro summarizes past metabolic engineering approaches in reducing ethanol production and increasing lactate production by S. cerevisiae. Tokuhiro discloses that yields of lactate production in recombinant S. cerevisiae expressing heterologous lactate dehydrogenase is low because the strong tendency for ethanol to be competitively produced from pyruvate (page 884, left column). Pyruvate decarboxylases encoded by pdc1, pdc5, and pdc6 were targets of inactivation because decarboxylation of pyruvate is the first step to ethanol from pyruvate. Single pdc1 or pdc5 mutants improved lactate yields but a large amount of ethanol was still produced. However, the triple inactivation of pdc1, pdc5, and pdc6 has impaired growth on glucose (page 884, left column).
Regarding 2, Tokuhiro discloses fermentation with two-stage batch culture (page 887, 1st paragraph).
Regarding claim 3, Tokuhiro discloses providing a growth medium and refreshing the growth medium (page 887, 1st paragraph).
Regarding claims 5 and 7-8, PDC and LDH are metabolic enzymes since they are involved in the metabolism of pyruvate and PDC and LDH compete for the same metabolite, pyruvate (Figure 1). Tokuhiro discloses expression of LDH enables a cell to overproduce lactate dehydrogenase to an amount greater than what is produced by a wild-type cell (abstract and page 884).
Regarding claim 6, PDC is essential for cell growth, as disclosed by Tokuhiro (page 884, left column).
Chua discloses using a combination of the GAL regulon and a maltose switch to control genes that are expressed/not expressed during the growth phase and not expressed/expressed production phase: not expressing an enzyme involved in the formation of a product of interest during the growth phase and expressing said enzyme during the production phase to increase yield of the product (page 2, line 28 through page 3, line 2).
Regarding claims 7-8 and 10, Chua discloses CEN.PK2 S. cerevisiae (ΔGAL80 and constitutive promoter-GAL4) comprising (1) pMAL-GAL80 and (2) pGAL1-GFP or pathway genes, wherein the in the presence of the chemical inducer maltose, GPF or pathway genes are not expressed (due to the expression of GAL80 that prevents GAL4 activation of the GAL1 promoter) and in the absence of the chemical inducer maltose, GFP or pathway genes are expressed (abstract, Figure 5 and pages 3 and 30-33, page 52 and page 87, lines 1-13). pMAL-GAL80 is similar to the “third sequence” recited in claims 7-8 of the instant application in that it is comprised of a promoter linked to a repressor, GAL80. pGAL1-GFP/pathway genes is similar to the “fourth sequence” recited in claims 7-8 of the instant application in that pGAL1 is a promoter that is repressed by GAL80 and the GFP/pathway genes is a nucleotide sequence encodes for a metabolic enzyme. Chua also discloses S. cerevisiae comprising pMAL-GFP, wherein in the presence of the chemical inducer, GFP is expressed and in the absence of the chemical inducer, GFP is not expressed. pMAL-GPF is similar to the “second sequence” recited in claims 7-8 of the instant application in that pMAL is a promoter activated by maltose Chua discloses using pGAL1 as the GAL4 activated promoter (page 32, line 16-25). Chua discloses expressing GAL4 under the control of a constitute promoter (page 32, lines 16-21).
Regarding claims 9-10, the maltose/Gal regulon switch of Chua is similar to the light/Gal regulon switch of the claimed invention in that a chemical/light induces expression of a first enzyme and does not induce expression a second enzyme due to the activated repressor (GAL80) and in the absence of the chemical/light does not induce expression of said first enzyme and induces expression of the second enzyme due to repression of GAL80, which allows for GAL4 activation of the GAL1 promoter.
Regarding claims 1, 5, and 7-10, Da Silva discloses well known promoters and terminators for expression of genes in S. cerevisiae. Da Silva also discloses that bi-directional promoters are very useful for metabolic engineering in S. cerevisiae (page 200, right column). Da Silva discloses the combination of PTEF1 and TCYC1 (page 204, left column). Da Silva also discloses the constitutive promoters, such as PADH1 (page 204, right column).
Therefore, in combining the teachings of Gardner, Tokuhiro, Chua and Da Silva, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to adapt the light-inducible expression system of Gardner for increasing production of lactate in S. cerevisiae by (A) inducing expression of PDC1 and not inducing expression of LDH1 upon exposure to blue light during the growth phase and (B) not inducing expression of PDC1 and inducing expression of LDH1 during the production phase by expressing in an S. cerevisiae ΔGAL80 Δpdc1Δpdc5Δpdc6 a light induced expression system comprising (1) a first sequence comprising PTEF1-EL222-VP16-TCYC1 (light-activated transcription factor), (2) a second sequence comprising PC120-PDC1 (OptoEXP-PDC1) (promoter activated by said light-activated transcription factor and a polynucleotide encoding the metabolic enzyme PDC1), (3) a third sequence comprising PC120-GAL80 (OptoINVRT1-LDH) (a promoter activated by said light-activated transcription factor and a polynucleotide encoding the repressor GAL80), and (4) a fourth sequence comprising PADH1-GAL4, PTEF1-VP16-EL222, and PGAL1-LDH (promoter that is repressed by GAL80 and a polynucleotide encoding the second metabolic enzyme LDH), wherein (i) the first gene, PDC1 gene, is turned “on” when exposed to blue light and turned “off” when not exposed to said light and (ii) the second gene, LDH gene, is turned “off” when exposed to blue light (due to the expression of GAL80 that prevents GAL4 activation of the GAL1 promoter) and turned “on” when not exposed to said light (since GAL80 is not expressed in the absence of said light), which reads on claims 9-10, (iii) rephrase the growth medium, and (iv) monitor the production of lactate production via a protein cascade system and vary the light schedule, which reads on 11-13. One having ordinary skill in the art would have been motivated to express PDC1 during the growth phase and not express PDC1 during the production phase in order for S. cerevisiae Δpdc1Δpdc5Δpdc to grow during the growth phase and direct carbon and cellular energy toward lactate production and away from ethanol production during the production phase, which reads on claims 2-4. One having ordinary skill in the art would have been motivated to not express LDH during the growth phase and express LDH during the production phase in order for S. cerevisiae Δpdc1Δpdc5Δpdc to direct carbon and cellular energy toward lactate production and away from ethanol production during the production phase. The motivation of using S. cerevisiae Δpdc1Δpdc5Δpdc is to reduce production of ethanol and increase production of lactate. The motivation of using a light inducible gene expression system of Gardner over chemically induced genes in that light is inexpensive to apply, easily delivered and does not interfere with cellular pathways. The motivation of incorporating the GAL regulon switch disclosed by Chua into the light inducible gene expression system of Gardner is the ability to turn on a first gene and at the same time turn off a second gene upon exposure to light and turn off the first gene and at the same time turn on the second gene in the absence of light, thereby allowing expression of the first gene essential for growth during the growth phase and expression of the second gene necessary for the production phase. In order to incorporate the GAL regulon switch into the light inducible gene expression system of Gardner, it would have been obvious to one having ordinary skill in the art to replace the maltose responsive promoter pMAL of pMAL-GAL80 of Chua with the light activated promoter of PC120 to arrive at PC120-GAL80 of the “third sequence” recited in claims 7-8 and replace the GFP/pathway genes of pGAL1-GFP/pathway genes of Chua with LDH to arrive at pGAL1-LDH of the “fourth sequence” recited in claims26-27 of the instant application. One having ordinary skill in the art would have had a reasonable expectation of success since Gardner discloses a light inducible expression system for inducing/not inducing expression of a gene, Chua discloses using a gene inducer and a regulon for bidirectional expression of genes, and Tokuhiro discloses Δpdc1Δpdc5Δpdc S. cerevisiae and a lactate producing S. cerevisiae expressing LDH. The rationale supporting that the claims would have been obvious is that a method of enhancing a particular class of devices (bidirectionally turning on and off genes using a combination of light inducible expression system and GAL regulon) has been made part of the ordinary capabilities of one skilled in the art based upon the teaching of such improvement in other situations. One of ordinary skill in the art would have been capable of applying this known method of enhancement (light inducible expression of genes in combination with a GAL regulon) to a “base” device (light or chemically inducible expression of genes) in the prior art and the results would have been predictable to one of ordinary skill in the art.
Therefore, the above references render claims 1-13 prima facie obvious.
Claim(s) 14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Claims 1-13 are rejected under 35 U.S.C. 103 as being unpatentable over Gardner (US 2014/0325692 – form PTO-1449), Tokuhiro (Double mutation of the PDC1 and ADH1 genes improves lactate production in the yeast Saccharomyces cerevisiae expressing the bovine lactate dehydrogenase gene Appl Microbiol Biotechnol (2009) 82:883–890. – form PTO-1449), Chua (WO 2015/020649 – form PTO-1449), and Da Silva (FEMS Yeast Res. 2012 Mar; 12(2):197-214. – form PTO-1449) as applied to claims 1-13 above, and further in view of Renicke (A LOV2 domain-based optogenetic tool to control protein degradation and cellular function. Chem Biol. 2013 Apr 18;20(4):619-26 – form PTO-1449).
The combined teachings of Gardner, Tokuhiro, Chua and Da Silva does not disclose a degron domain, which is a light-induced degron domain.
Regarding claim 14, Renicke discloses a photosensitive degron (PSD), which is derived from Arabidopsis thaliana LOV2 (atLOV2) domain and a degradation signal derived from murine ornithine decarboxylase (ODC) (abstract and page 620, 1st paragraph). Renicke discloses that blue-light irradiation of AtLOV2 results in activation of the ODC-like degron and leads to proteasomal degradation of the whole construct in S. cerevisiae (abstract, page 620, 1st paragraph, and page 625, 1st paragraph).
Therefore, in combining the teachings of Gardner, Tokuhiro, Chua, Da Silva, and Renicke, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to attach the PSD of Renicke to PADH1-GAL4. One having ordinary skill in the art would have been motivated to do so in order to further ensure repression of Gal4p. One having ordinary skill in the art would have had a reasonable expectation of success since Renicke teaches AtLOV2 results in activation of the ODC-like degron and leads to proteasomal degradation of the whole construct in S. cerevisiae.
Therefore, the above references render claims 1-14 prima facie obvious.
Conclusion
Claims 1-14 are pending.
Claims 1-14 are rejected.
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/YONG D PAK/Primary Examiner, Art Unit 1652
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