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 .
Status of the Claims
The status of the claims upon entry of the present amendment stands as follows:
Pending claims: 1-20
Withdrawn claims: None
Previously canceled claims: None
Newly canceled claims: None
Amended claims: None
New claims: None
Claims currently under consideration: 1-20
Currently rejected claims: 1-20
Allowed claims: None
Specification
The disclosure is objected to because of the following informalities:
In paragraph [012], “Aspergillus oryzae”, “Neurospora intermedia”, and “Neurospora crassa” should be italicized to conform with accepted taxonomic convention.
In paragraph [014], “Aspergillus oryzae” should be italicized to conform with accepted taxonomic convention.
In paragraphs [017] and [029], “alfafa” should read, “alfalfa”.
In paragraphs [017] and [029], “Neurospora crassa”, “Aspergillus oryzae”, and “Saccharomyces cerevisiae” should be italicized to conform with accepted taxonomic convention.
In paragraph [028], “Aspergillus”, in two places, should be italicized to conform with accepted taxonomic convention.
Appropriate correction is required.
Claim Objections
Claims 8-10 and 14-15 are objected to because of the following informalities:
In claim 8, “Aspergillus oryzae”, “Neurospora intermedia”, and “Neurospora crassa” should be italicized to conform with accepted taxonomic convention.
In claim 9, “Aspergillus oryzae” should be italicized to conform with accepted taxonomic convention.
In claim 10, “levels” should read, “level”.
In claim 14, “alfafa” should read, “alfalfa”.
In claim 15, “Neurospora crassa”, “Aspergillus oryzae”, “Saccharomyces”, and “cerevisiae” should be italicized to conform with accepted taxonomic convention.
In claim 15, it appears that “Saccharomyces or cerevisiae” should read “or Saccharomyces cerevisiae” per paragraph [029] of the instant specification.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claim 19 is rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Regarding claim 19, the term “inexpensive” is a relative term which renders the claim indefinite; what is inexpensive for one may not be inexpensive for another. The term “inexpensive” 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.
Additionally, the phrase “such as” renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
For purposes of examination, claim 19 is construed to be, “The composition of claim 6, wherein the fungus is growable on cellulosic biomass, molasses, or food waste.”
Claim Rejections - 35 USC § 102
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 the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-3 and 20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024).
Regarding claim 1, Elrod teaches a composition comprising an edible filamentous fungus genetically engineered to overexpress heme biosynthesis genes and/or genes encoding heme proteins to elevate heme levels above a corresponding non-engineered level – “The present Invention relates to methods of producing hemoproteins comprising (a) introducing into a filamentous fungal cell, which is capable of producing the hemoprotein, (i) one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme…(b) cultivating the filamentous fungal cell in a nutrient medium suitable for production of the hemoprotein and the heme biosynthetic enzymes; and (c) recovering the hemoprotein from the nutrient medium of the filamentous fungal cell” (Abstract). “In one aspect of the present invention, a hemoprotein is produced in higher amounts in a filamentous fungal cell by introducing into the filamentous fungal cell one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme encoded by the first nucleic acid sequence endogenous to the filamentous fungal cell” (col. 5, lines 13-20). “In a most preferred embodiment, the filamentous fungal host cell is…an Aspergillus oryzae cell.” (col. 11, lines 60-64). Aspergillus oryzae (a.k.a., koji) is well-known in the food art to be an edible filamentous fungus. Elrod exemplifies the invention in Example 17 by co-overexpression of hemA and hemB in Aspergillus oryzae resulting in increased peroxidase production (col. 23, line 62 – col. 25, line 21). “The hemA/hemB co-overexpression strains showed approximately a 4-fold average increase over non-engineered strains (SE22) and a 1.8-fold average increase over hemA overexpression strains (SE28).” (col. 24, lines 33-43). The results are validated to be due to increased heme in Example 20 (col. 28, lines 5-60).
Claim 1 is therefore anticipated by Elrod.
Regarding claim 2, Elrod teaches the composition of claim 1, wherein the genes include aminolevulinic acid synthase (ALAS), aminolevulinic acid dehydratase (ALAD), uroporphyrinogen decarboxylase (UROD), coproporphyrinogen oxidase (CPO), protoporphyrinogenidase (PPO), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS) and ferrochelatase (FC), or a subcombination thereof – Elrod exemplifies the invention in Example 17 by co-overexpression of hemA and hemB in Aspergillus oryzae resulting in increased peroxidase production (col. 23, line 62 – col. 25, line 21). HemA is aminolevulinic acid synthase (ALAS) (col. 13, line 21), and hemB is porphobilinogen synthase (col. 20, line 63), also called 5-aminolevulinic acid dehydratase (col. 1, lines 34-35), which is the claimed ALAD.
Claim 2 is therefore anticipated by Elrod.
Regarding claim 3, Elrod teaches the composition of claim 1, wherein the genes include aminolevulinic acid synthase (ALAS) and aminolevulinic acid dehydratase (ALAD) – Elrod exemplifies the invention in Example 17 by co-overexpression of hemA and hemB in Aspergillus oryzae resulting in increased peroxidase production (col. 23, line 62 – col. 25, line 21). HemA is aminolevulinic acid synthase (ALAS) (col. 13, line 21), and hemB is porphobilinogen synthase (col. 20, line 63), also called 5-aminolevulinic acid dehydratase (col. 1, lines 34-35), which is the claimed ALAD.
Claim 3 is therefore anticipated by Elrod.
Regarding claim 20, Elrod teaches a method of making an edible filamentous fungus, the method comprising genetically engineering the fungus to overexpress heme biosynthesis genes to elevate heme levels above a corresponding non-engineered level – “The present Invention relates to methods of producing hemoproteins comprising (a) introducing into a filamentous fungal cell, which is capable of producing the hemoprotein, (i) one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme…(b) cultivating the filamentous fungal cell in a nutrient medium suitable for production of the hemoprotein and the heme biosynthetic enzymes; and (c) recovering the hemoprotein from the nutrient medium of the filamentous fungal cell” (Abstract). “In one aspect of the present invention, a hemoprotein is produced in higher amounts in a filamentous fungal cell by introducing into the filamentous fungal cell one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme encoded by the first nucleic acid sequence endogenous to the filamentous fungal cell” (col. 5, lines 13-20). “In a most preferred embodiment, the filamentous fungal host cell is…an Aspergillus oryzae cell.” (col. 11, lines 60-64). Aspergillus oryzae (a.k.a., koji) is well-known in the food art to be an edible filamentous fungus. Elrod exemplifies the invention in Example 17 by co-overexpression of hemA and hemB in Aspergillus oryzae resulting in increased peroxidase production (col. 23, line 62 – col. 25, line 21). “The hemA/hemB co-overexpression strains showed approximately a 4-fold average increase over non-engineered strains (SE22) and a 1.8-fold average increase over hemA overexpression strains (SE28).” (col. 24, lines 33-43). The results are validated to be due to increased heme in Example 20 (col. 28, lines 5-60).
Claim 20 is therefore anticipated by Elrod.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 4-6, 8, and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024).
Regarding claim 4, Elrod teaches the composition of claim 1.
While Elrod suggests the use of a first nucleic acid sequence encoding a heme biosynthetic enzyme and one or more second nucleic acid sequences encoding a heme biosynthetic enzyme in a single embodiment (see Elrod claim 1), Elrod does not specifically disclose an embodiment of a composition wherein those genes include aminolevulinic acid synthase (ALAS), aminolevulinic acid dehydratase (ALAD), uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (CPO).
However, Elrod teaches, “The first nucleic acid sequence may be any filamentous fungal nucleic acid sequence encoding a heme biosynthetic enzyme selected from the group consisting of a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase…” (col. 5, lines 21-31). “In another aspect of the present invention, a hemoprotein is produced in higher amounts in a filamentous fungal cell by introducing into the filamentous fungal cell one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme. The second nucleic acid sequence may be any nucleic acid sequence encoding a heme biosynthetic enzyme selected from the group consisting of a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase.” (col. 7, line 36 – col. 8, line 62). Porphobilinogen synthase is also called 5-aminolevulinic acid dehydratase (col. 1 lines 34-35). Elrod therefore teaches that the first and one or more second amino acids may be the claimed ALAS, ALAD, UROD, and/or CPO.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to include ALAS, ALAD, UROD, and CPO in the composition with the motivation of producing higher amounts of hemoprotein. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches that the first and one or more second nucleic acid sequences encoding a heme biosynthetic enzyme may be a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase., and that a hemoprotein is produced in higher amounts by introducing into the filamentous fungal cell one or more copies of one or more such second nucleic acid sequences (col. 7, line 36 – col. 8, line 62). Porphobilinogen synthase is also called 5-aminolevulinic acid dehydratase (col. 1 lines 34-35), which is the claimed ALAD. The disclosure by Elrod of a first nucleic acid sequence comprising any one of the heme biosynthesis genes and one or more second nucleic acid sequences comprising any of the heme biosynthesis genes renders obvious any combinations of the heme biosynthesis genes, including which includes the claimed ALAS, ALAD, UROD, and CPO, as claimed.
Therefore, claim 4 is rendered obvious.
Regarding claim 5,
Elrod teaches the composition of claim 1.
While Elrod suggests the use of a first nucleic acid sequence encoding a heme biosynthetic enzyme and one or more second nucleic acid sequences encoding a heme biosynthetic enzyme in a single embodiment (see Elrod claim 1), Elrod does not specifically disclose an embodiment of a composition wherein those genes include aminolevulinic acid synthase (ALAS), aminolevulinic acid dehydratase (ALAD), uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (CPO) protoporphyrinogenidase (PPO), and porphobilinogen deaminase (PBGD).
However, Elrod teaches, “The first nucleic acid sequence may be any filamentous fungal nucleic acid sequence encoding a heme biosynthetic enzyme selected from the group consisting of a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase…” (col. 5, lines 21-31). “In another aspect of the present invention, a hemoprotein is produced in higher amounts in a filamentous fungal cell by introducing into the filamentous fungal cell one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme. The second nucleic acid sequence may be any nucleic acid sequence encoding a heme biosynthetic enzyme selected from the group consisting of a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase.” (col. 7, line 36 – col. 8, line 62). Porphobilinogen synthase is also called 5-aminolevulinic acid dehydratase (col. 1 lines 34-35). Elrod therefore teaches that the first and one or more second amino acids may be the claimed ALAS, ALAD, UROD, CPO, PPO, and/or PBGD.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to include ALAS, ALAD, UROD, CPO, PPO, and PBGD in the composition with the motivation of producing higher amounts of hemoprotein. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches that the first and one or more second nucleic acid sequences encoding a heme biosynthetic enzyme may be a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase., and that a hemoprotein is produced in higher amounts by introducing into the filamentous fungal cell one or more copies of one or more such second nucleic acid sequences (col. 7, line 36 – col. 8, line 62). Porphobilinogen synthase is also called 5-aminolevulinic acid dehydratase (col. 1 lines 34-35), which is the claimed ALAD. The disclosure by Elrod of a first nucleic acid sequence comprising any one of the heme biosynthesis genes and one or more second nucleic acid sequences comprising any of the heme biosynthesis genes renders obvious any combinations of the heme biosynthesis genes, including which includes the claimed ALAS, ALAD, UROD, CPO, PPO, and PGBD, as claimed.
Therefore, claim 5 is rendered obvious.
Regarding claim 6,
Elrod teaches the composition of claim 1.
While Elrod suggests the use of a first nucleic acid sequence encoding a heme biosynthetic enzyme and one or more second nucleic acid sequences encoding a heme biosynthetic enzyme in a single embodiment (see Elrod claim 1), Elrod does not specifically disclose an embodiment of a composition wherein those genes include aminolevulinic acid synthase (ALAS), aminolevulinic acid dehydratase (ALAD), uroporphyrinogen decarboxylase (UROD) and coproporphyrinogen oxidase (CPO), protoporphyrinogenidase (PPO), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and ferrochelatase (FC).
However, Elrod teaches, “The first nucleic acid sequence may be any filamentous fungal nucleic acid sequence encoding a heme biosynthetic enzyme selected from the group consisting of a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase…” (col. 5, lines 21-31). “In another aspect of the present invention, a hemoprotein is produced in higher amounts in a filamentous fungal cell by introducing into the filamentous fungal cell one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme. The second nucleic acid sequence may be any nucleic acid sequence encoding a heme biosynthetic enzyme selected from the group consisting of a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase.” (col. 7, line 36 – col. 8, line 62). Porphobilinogen synthase is also called 5-aminolevulinic acid dehydratase (col. 1 lines 34-35). Elrod therefore teaches that the first and one or more second amino acids may be the claimed ALAS, ALAD, UROD, CPO, PPO, PGBD, UROS, and/or FC.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to include ALAS, ALAD, UROD, CPO, PPO, PGBD, UROS, and FC in the composition with the motivation of producing higher amounts of hemoprotein. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches that the first and one or more second nucleic acid sequences encoding a heme biosynthetic enzyme may be a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coproporphyrinogen oxidase, a protoporphyrinogen oxidase, and a ferrochelatase., and that a hemoprotein is produced in higher amounts by introducing into the filamentous fungal cell one or more copies of one or more such second nucleic acid sequences (col. 7, line 36 – col. 8, line 62). Porphobilinogen synthase is also called 5-aminolevulinic acid dehydratase (col. 1 lines 34-35), which is the claimed ALAD. The disclosure by Elrod of a first nucleic acid sequence comprising any one of the heme biosynthesis genes and one or more second nucleic acid sequences comprising any of the heme biosynthesis genes renders obvious any combinations of the heme biosynthesis genes, including which includes the claimed ALAS, ALAD, UROD, CPO, PPO, PGBD, UROS, and FC, as claimed.
Therefore, claim 6 is rendered obvious.
Regarding claim 8, Elrod teaches the composition of claim 6, wherein the fungus is selected from Aspergillus oryzae, Neurospora intermedia, and Neurospora crassa – “In a most preferred embodiment, the filamentous fungal host cell is an…Aspergillus oryzae cell.” (col. 11, lines 60-64). “In another most preferred embodiment, the filamentous fungal host cell is a Neurospora crassa cell.” (col. 12, lines 5-7).
MPEP § 2144.07 states, “The selection of a known material based on its suitability for its intended use support[s] a prima facie obviousness determination”. Therefore, where Elrod teaches that Aspergillus oryzae and Neurospora crassa are preferred fungi for the composition, selection of Aspergillus oryzae or Neurospora crassa as the fungus would have been prima facie obvious.
Claim 8 is therefore rendered obvious.
Regarding claim 13, Elrod teaches the composition of claim 6, wherein the fungus is engineered to express a hemoglobin protein, which acts as a sink for elevated heme inside the fungus – Elrod teaches, “The present Invention relates to methods of producing hemoproteins…” (Abstract). “"Hemoprotein" is defined herein as any member of a group of proteins containing heme as a prosthetic group. The hemoprotein may be a globin, a cytochrome, an oxidoreductase, or any other protein containing a heme as a prosthetic group. Heme-containing globins include hemoglobin and myoglobin.” (col. 3, lines 55-60). “The methods of the present invention may further comprise introducing one or more copies of one or more third nucleic acid sequences encoding the hemoprotein into the filamentous fungal cell.” (col. 11, lines 26-34).
MPEP § 2144.07 states, “The selection of a known material based on its suitability for its intended use support[s] a prima facie obviousness determination”. Therefore, where Elrod teaches that hemoglobin is a suitable hemoprotein for which to increase expression, selection of a hemoglobin would have been prima facie obvious.The phrase, “which acts as a sink for elevated heme protein inside the fungus” is a statement of intended use for the expressed hemoglobin protein. A statement with regard to intended use is not further limiting insofar as the structure of the product is concerned. In order to patentably distinguish the claimed invention from the prior art, a claimed intended use must result in a structural difference between the claimed invention and the prior art. See MPEP § 2111.02(II). In the present case, there is no difference between the composition wherein the fungus is engineered to expressed a hemoglobin protein of the prior art and the composition of the claimed invention. Furthermore, Elrod teaches, “The conversion of an apoprotein into a hemoprotein depends on the availability of heme provided by the heme biosynthetic pathway. The apoprotein form of the hemoprotein combines with heme to produce the active hemoprotein which acquires a conformation which makes the hemoprotein more stable against proteolytic attack than the apoprotein. If the amount of heme produced by a microorganism is less relative to the amount of the apoprotein produced, the apoprotein will accumulate and undergo proteolytic degradation lowering the yield of the active hemoprotein.” (col. 1, lines 58-67). Therefore, it stands to reason that in a filamentous fungus engineered to increase heme levels and to overexpress a hemoprotein (e.g., hemoglobin), the hemoprotein would act as a sink for the increased levels of heme.
Claim 13 is therefore rendered obvious.
Claim 7 is rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 6 above, and further in view of Franken et al. (Franken, A. C., Lokman, B. C., Ram, et al. (2011). Heme biosynthesis and its regulation: towards understanding and improvement of heme biosynthesis in filamentous fungi. Applied microbiology and biotechnology, 91(3), 447-460. https://doi.org/ 10.1007/s00253-011-3391-3).
Regarding claim 7, Elrod teaches the composition of claim 6, wherein the genes include aminolevulinic acid synthase (ALAS) comprising a Heme Regulatory Motif (HRM) – “FIG. 3 shows the nucleotide and deduced amino acid sequences of an Aspergillus oryzae 5-aminolevulinic acid synthase [i.e., ALAS] gene (SEQ ID NOS: 1 and 2, respectively)…The two conserved putative HRM motifs are boxed…” (col. 2, lines 54-61).
Elrod does not discuss a mutant of the HRM.
However, Elrod provides, “The conversion of an apoprotein into a hemoprotein depends on the availability of heme provided by the heme biosynthesis pathway…If the amount of heme produced by a microorganism is less relative to the amount of the apoprotein produced, the apoprotein will accumulate and undergo proteolytic degradation lowering the yield of the active hemoprotein.” (col. 1, lines 58-67). Elrod further teaches, “The methods of the present invention may further comprise introducing one or more copies of one or more third nucleic acid sequences encoding the hemoprotein into the filamentous fungal cell.” (col. 11, lines 26-34). Therefore, Elrod teaches the need for increasing the amount of heme produced to accommodate an increase in hemoprotein produced by introducing hemoprotein sequences.
Additionally, regarding the regulation of ALAS, Franken teaches that the HRMs are responsible for the rate limiting character of the enzyme by feedback inhibition of heme at the posttranslational level. Excess intracellular heme is highly toxic to cells and is therefore tightly controlled at low levels. Due to the binding of heme, translocation of the newly synthesized peptide into the mitochondria is hindered and results in lower amounts of ALAS present in the matrix. This negative feedback is abolished when the cysteine residue in the HRM is either mutated to serine or deleted (p. 450, col. 2, ¶ 3). Therefore, Franken teaches that heme may be elevated by mutating the ALAS heme regulatory motif to remove the negative feedback of heme biosynthesis.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify Elrod with the teachings of Franken to mutate the HRM of the ALAS sequence to remove the negative feedback of heme biosynthesis at the point of ALAS, thereby increasing the heme levels to accommodate increased amounts of hemoprotein expression. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Franken teaches that heme may be elevated by mutating the ALAS heme regulatory motif cysteine to serine or by deleting the cysteine (p. 450, col. 2, ¶ 3).
Claim 7 is therefore rendered obvious.
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 6 above, and further in view of He et al. (He, B., Tu, Y., Jiang, C., Zhang, Z., Li, Y., & Zeng, B. (2019). Functional Genomics of Aspergillus oryzae: Strategies and Progress. Microorganisms, 7(4), 103. https://doi.org/10.3390/microorganisms7040103).
Regarding claim 9, Elrod teaches the composition of claim 6.
Elrod also teaches, “In a most preferred embodiment, the filamentous fungal host cell is an…Aspergillus oryzae cell.” (col. 11, lines 60-64).
Elrod does not discuss that the fungus is Aspergillus oryzae NSAR1 or Aspergillus oryzae RIB40.
However, He teaches the use of Aspergillus oryzae as a common filamentous fungus model organism strain due to its small genome size and ease of sequencing, and that the Aspergillus oryzae RIB40 genome was sequenced in 2005 (p. 2, ¶ 1). He also teaches that the screening of transformants with auxotrophic markers is convenient and efficient, based on selective culture media (p. 3, ¶ 2), and discloses that several studies have used Aspergillus oryzae RIB40 auxotrophs for research (p. 3, ¶ 2 and Table 2). Therefore, He teaches that Aspergillus oryzae RIB40 is a known and commonly used strain of Aspergillus oryzae.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the Aspergillus oryzae disclosed by Elrod with the Aspergillus oryzae RIB40 taught by He by simple substitution of one known element for another to obtain predictable results. See MPEP § 2143(I)(B). First, Elrod discloses that Aspergillus oryzae is a preferred host cell. Elrod does not require a specific strain of Aspergillus oryzae. As such, any Aspergillus oryzae strain is deemed to be suitable. He teaches that Aspergillus oryzae RIB40 is a known and commonly used strain of Aspergillus oryzae. Therefore, one of ordinary skill in the art could have substituted the Aspergillus oryzae of Elrod for Aspergillus oryzae RIB40 to obtain the predictable result of providing a suitable host strain of Aspergillus oryzae for subsequent genetic modification.
Claim 9 is therefore rendered obvious.
Claims 10-12 are rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 6 above, and further in view of Chang et al. (US 2009/0098626 A1).
Regarding claims 10-12, Elrod teaches the composition of claim 6.
Elrod teaches, “The conversion of an apoprotein into a hemoprotein depends on the availability of heme provided by the heme biosynthesis pathway…If the amount of heme produced by a microorganism is less relative to the amount of the apoprotein produced, the apoprotein will accumulate and undergo proteolytic degradation lowering the yield of the active hemoprotein.” (col. 1, lines 58-67). Elrod further teaches, “introducing into a filamentous fungal cell (i) one or more first control sequences capable of directing the expression of a heme biosynthetic enzyme encoded by a first nucleic acid sequence endogenous to the filamentous fungal cell, wherein the one or more of the first control sequences are operably linked to the first nucleic acid sequence; and/or (ii) one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme” (Abstract). Elrod teaches, “In a most preferred embodiment, the filamentous fungal host cell is an…Aspergillus oryzae cell.” (col. 11, lines 60-64). The first nucleic acid sequence may be any filamentous fungal nucleic acid sequence encoding a heme biosynthetic enzyme, including 5-aminolevulinic acid synthase (ALAS) (col. 5, lines21-31). Therefore, Elrod teaches the need for increasing the amount of heme produced to accommodate an increase in hemoprotein, and teaches modulating the heme biosynthetic pathway at ALAS.
Elrod does not discuss that the fungus provides an increased heme levels at least 5-fold above the non-engineered background strain (re: claim 10), at least 10-fold above the non-engineered background strain (re: claim 11), and at least 15-fold above the non-engineered background strain (re: claim 12).
However, Chang teaches increasing heme production in a host cell by at least about 5-fold, at least about 10-fold and at least about 15-fold – “In some embodiments, a subject genetically modified host cell comprises one or more additional genetic modifications that provide for enhanced heme production, e.g., to achieve an… at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold, or greater, increase in heme production, compared to a host cell that does not comprise the one or more additional genetic modifications.” ([0170]). The host cell may be a filamentous fungus, including Aspergillus oryzae ([0145]). Chang teaches increasing heme production by increasing the level of GTR reductase expression ([0171]) and/or activity ([0172]) and/or overexpressing ALA synthase ([0174]) and/or increasing its activity ([0175]). See also Figure 13.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify Elrod with the teachings of Chang to increase the heme production in the host cell by at least about 5-fold, at least about 10-fold, or at least about 15-fold by increasing the activity and/or expression of ALAS and/or GTR reductase. One of ordinary skill in the art would have been motivated to increase the heme production in order to provide sufficient heme for increased amounts of apoprotein such that an increased amount of hemoprotein is produced by the host cell. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches increasing heme biosynthesis at the level of ALAS, and Chang teaches increasing the expression and/or activity of ALAS and/or GTR reductase can achieve an increase in heme levels of at least about 5-fold, at least about 10-fold and at least about 15-fold.
Regarding the phrase, “as assessed by LC-MS”, the means by which a parameter is measured does not render the claimed composition patentably distinct from a prior art composition.
Claims 10-12 are therefore rendered obvious.
Claims 14 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 13 above, and further in view of Fraser et al. (US 2017/0342132 A1, cited on the IDS filed on 1 July 2024).
Regarding claim 14, Elrod teaches the composition of claim 13.
Elrod teaches that the composition comprises medium containing the filamentous fungal cell and secreted hemoprotein in that the method comprises “recovering the hemoprotein from the nutrient medium of the filamentous fungal cell” (Abstract). Elrod teaches that “[t]he methods of the present invention may further comprise introducing one or more copies of one or more third nucleic acid sequences encoding the hemoprotein into the filamentous fungal cell.” (col. 11, lines 26-34). The hemoprotein may be a...hemoglobin (col. 3, lines 55-60). Elrod teaches that the hemoprotein may be native or foreign to the filamentous fungal cell (col. 4, lines 16-17).
Elrod does not discuss that the hemoglobin protein is from cow, pig, chicken, salmon or tuna.
However, in an invention relating to producing heme-containing polypeptide, Fraser teaches methods and compositions for the expression and secretion of heme-containing polypeptides (Abstract). Fraser teaches a cell comprising an exogenous nucleic acid molecule comprising, in the 5′ to 3′ direction, a promoter sequence operably linked to a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding a heme-containing polypeptide. (claim 1). The nucleic acid encoding a heme-containing polypeptide is leghemoglobin (claim 9). “In some instances, the sequence (amino acid and/or nucleic acid) of a leghemoglobin can be a plant leghemoglobin sequence. Various legumes species and their varieties, for example, Soybean… Alfalfa…” ([0055]). Fraser teaches that “codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.” ([0048]).
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the hemoglobin disclosed by Elrod with the leghemoglobin from soybean or alfalfa disclosed by Fraser by simple substitution of one known element for another to obtain predictable results. See MPEP § 2143(I)(B). First, Elrod discloses introducing one or more copies of the nucleic acid sequence encoding the hemoprotein into the filamentous fungal cell, that the hemoprotein may be hemoglobin, and that the hemoprotein may be native or foreign to the host cell. Elrod does not require a specific hemoglobin. As such, any hemoglobin is deemed to be suitable. Fraser discloses leghemoglobin from soybean or alfalfa. Therefore, one of ordinary skill in the art could have substituted the general hemoglobin of Elrod for a soy or alfalfa leghemoglobin by known molecular biology methods to obtain the predictable result of providing additional hemoprotein to the filamentous fungal cell. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Fraser teaches that codons can be optimized for expression in a particular species (e.g., bacteria or fungus), and Elrod teaches expression in filamentous fungus.
Claim 14 is therefore rendered obvious.
Regarding claim 16, Elrod teaches the composition of claim 13.
Elrod teaches that the composition comprises medium containing the filamentous fungal cell and secreted hemoprotein in that the method comprises “recovering the hemoprotein from the nutrient medium of the filamentous fungal cell” (Abstract). Elrod teaches that “[t]he methods of the present invention may further comprise introducing one or more copies of one or more third nucleic acid sequences encoding the hemoprotein into the filamentous fungal cell.” (col. 11, lines 26-34). The hemoprotein may be a...hemoglobin (col. 3, lines 55-60). Elrod teaches that the hemoprotein may be native or foreign to the filamentous fungal cell (col. 4, lines 16-17).
Elrod does not discuss that the hemoglobin protein is from cow, pig, chicken, salmon or tuna.
However, in an invention relating to producing heme-containing polypeptide, Fraser teaches methods and compositions for the expression and secretion of heme-containing polypeptides (Abstract). Fraser teaches a cell comprising an exogenous nucleic acid molecule comprising, in the 5′ to 3′ direction, a promoter sequence operably linked to a nucleic acid encoding a signal peptide operably linked to a nucleic acid encoding a heme-containing polypeptide. (claim 1). The heme-containing polypeptide can be a hemoglobin (claim 8). “[T]he sequence (amino acid and/or nucleic acid) of a heme-containing polypeptide can be from a non-plant organism, such as from animals (e.g., a cow, pig, dog, rat, or horse), fish, archaea, protists, bacteria, fungus, eubacteria, metazoa, or yeast. ([0056]). Fraser teaches that “codons in the coding sequence for a given enzyme can be modified such that optimal expression in a particular species (e.g., bacteria or fungus) is obtained, using appropriate codon bias tables for that species.” ([0048]).
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the hemoglobin disclosed by Elrod with the hemoglobin from cow or pig disclosed by Fraser by simple substitution of one known element for another to obtain predictable results. See MPEP § 2143(I)(B). First, Elrod discloses introducing one or more copies of the nucleic acid sequence encoding the hemoprotein into the filamentous fungal cell, that the hemoprotein may be hemoglobin, and that the hemoprotein may be native or foreign to the host cell. Elrod does not require a specific hemoglobin. As such, any hemoglobin is deemed to be suitable. Fraser discloses hemoglobin from cow or pig. Therefore, one of ordinary skill in the art could have substituted the general hemoglobin of Elrod for a cow or pig hemoglobin by known molecular biology methods to obtain the predictable result of providing additional hemoprotein to the filamentous fungal cell. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Fraser teaches that codons can be optimized for expression in a particular species (e.g., bacteria or fungus), and Elrod teaches expression in filamentous fungus.
Claim 16 is therefore rendered obvious.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 13 above, and further in view of Hoogewijs et al. (Hoogewijs, D., Dewilde, S., Vierstraete, A., Moens, L., & Vinogradov, S. N. (2012). A phylogenetic analysis of the globins in fungi. PLoS One, 7(2), e31856. https://doi.org/10.1371/journal.pone.0031856).
Regarding claim 15, Elrod teaches the composition of claim 13.
Elrod teaches that “[t]he methods of the present invention may further comprise introducing one or more copies of one or more third nucleic acid sequences encoding the hemoprotein into the filamentous fungal cell.” (col. 11, lines 26-34). The hemoprotein may be a...hemoglobin (col. 3, lines 55-60). Elrod teaches that the hemoprotein may be native or foreign to the filamentous fungal cell (col. 4, lines 16-17). Elrod teaches, “In a most preferred embodiment, the filamentous fungal host cell is an…Aspergillus oryzae cell.” (col. 11, lines 60-64).
Elrod does not discuss that the hemoglobin protein is a flavohemoglobin from Neurospora crassa, Aspergillus oryzae, or Saccharomyces cerevisiae.
However, Hoogewijs teaches that Aspergillus oryzae possesses two flavohemoglobins, FHb1 and FHb2. (p. 10, col. 2, ¶ 2), and Saccharomyces cerevisiae also possesses flavohemoglobins (p. 1, col. 1, ¶ 1).
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to substitute the hemoglobin disclosed by Elrod with the flavohemoglobin disclosed by Hoogewijs by simple substitution of one known element for another to obtain predictable results. See MPEP § 2143(I)(B). First, Elrod discloses introducing one or more copies of the nucleic acid sequence encoding the hemoprotein into the filamentous fungal cell, that the hemoprotein may be hemoglobin, that the hemoprotein may be native or foreign to the host cell, and that Aspergillus oryzae is a preferred host cell. Elrod does not require a specific hemoglobin. As such, any hemoglobin is deemed to be suitable. Hoogeweijs discloses flavohemoglobins from Aspergillus oryzae and Saccharomyces cerevisiae. Therefore, one of ordinary skill in the art could have substituted the general hemoglobin of Elrod for an Aspergillus oryzae or Saccharomyces cerevisiae flavohemoglobin by known molecular biology methods to obtain the predictable result of providing additional hemoprotein to the filamentous fungal cell.
Claim 15 is therefore rendered obvious.
Claims 17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 6 above, and further in view of Fraser et al. (US 2017/0342132 A1, cited on the IDS filed on 1 July 2024) and Martinko (Martinko, K. (2021, May 14). This Plant-Based Meat and Seafood Is Made from Koji, an Umami-Packed Fungus. Treehugger. https://www.treehugger.com/plant-based-meat-seafood-made-from-koji-5184831).
Regarding claim 17, Elrod teaches the composition of claim 6.
Elrod teaches producing hemoprotein in a filamentous fungal host cell (Abstract). The hemoprotein need not be secreted (col. 5, lines 1-3). Elrod teaches, “In a most preferred embodiment, the filamentous fungal host cell is an…Aspergillus oryzae cell.” (col. 11, lines 60-64).
Elrod does not discuss that the composition is engineered with flavor components sufficient to mimic flavors and textures of a meat selected from beef, chicken or pork.
However, Fraser teaches combining a heme-containing polypeptide with a meat consumable. In some embodiments, the meat consumable comprises a replica selected from the group consisting of: a fat replica, a muscle replica, and a connective tissue replica, or any combination thereof ([0018]). “A composition can comprise a meat consumable and a host cell (e.g., bacterium). A host cell of the composition can be the host cell from which the polypeptide was expressed and/or secreted.” ([0194]). Fraser teaches, “The equivalent meat product can be derived from any animal. Non-limiting examples of animals used to derive the equivalent meat product include farmed animals such as, e.g., cattle, sheep, pig, chicken…” ([0197]).
Additionally, Martinko teaches faux meat products made from koji (p. 4, ¶ 2). Koji is Aspergillus oryzae (p. 4, ¶ 4). The fungi are grown in fermentation vats, and plant-based fats and flavors are added in to make the final product. (p. 4, ¶ 3). A koji superprotein bacon is disclosed (p. 4, Image). Bacon is a pork product.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify Elrod with the teachings of Fraser and Martinko to add flavor components sufficient to mimic the flavors and textures of pork bacon. Where Elrod teaches producing hemoproteins in engineered Aspergillus oryzae, and where Fraser teaches adding the hemoproteins and host cell to a meat replica to replicate beef, pork, or chicken, and where Martinko teaches adding flavors to Aspergillus oryzae to produce meat replicas including a koji bacon replica, one of ordinary skill in the art would have been motivated to add flavors to mimic bacon to the engineered Aspergillus oryzae to produce the koji bacon with added hemoprotein. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches the engineered Aspergillus oryzae, Fraser teaches adding hemoprotein and the host cells to meat replicas, and Martinko teaches producing a bacon replica from Aspergillus oryzae and added flavors.
Claim 17 is therefore rendered obvious.
Regarding claim 18, Elrod teaches the composition of claim 6.
Elrod teaches producing hemoprotein in a filamentous fungal host cell (Abstract). The hemoprotein need not be secreted (col. 5, lines 1-3). Elrod teaches, “In a most preferred embodiment, the filamentous fungal host cell is an…Aspergillus oryzae cell.” (col. 11, lines 60-64).
Elrod does not discuss that the composition is formatted as a mimic or substitute of a meat product selected from ground beef, beef steak, bacon and sausage.
However, Fraser teaches combining a heme-containing polypeptide with a meat consumable. In some embodiments, the meat consumable comprises a replica selected from the group consisting of: a fat replica, a muscle replica, and a connective tissue replica, or any combination thereof ([0018]). “A composition can comprise a meat consumable and a host cell (e.g., bacterium). A host cell of the composition can be the host cell from which the polypeptide was expressed and/or secreted.” ([0194]). Fraser teaches, “The equivalent meat product can be derived from any animal. Non-limiting examples of animals used to derive the equivalent meat product include farmed animals such as, e.g., cattle, sheep, pig, chicken…” ([0197]).
Additionally, Martinko teaches faux meat products made from koji (p. 4, ¶ 2). Koji is Aspergillus oryzae (p. 4, ¶ 4). The fungi are grown in fermentation vats, and plant-based fats and flavors are added in to make the final product. (p. 4, ¶ 3). A koji superprotein bacon is disclosed (p. 4, Image). Bacon is a pork product.
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify Elrod with the teachings of Fraser and Martinko to add produce a mimic of pork bacon. Where Elrod teaches producing hemoproteins in engineered Aspergillus oryzae, and where Fraser teaches adding the hemoproteins and host cell to a meat replica to replicate beef, pork, or chicken, and where Martinko teaches using Aspergillus oryzae to produce meat replicas including a koji bacon replica, one of ordinary skill in the art would have been motivated to use the engineered Aspergillus oryzae to produce the koji bacon with added hemoprotein. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches the engineered Aspergillus oryzae, Fraser teaches adding hemoprotein and the host cells to meat replicas, and Martinko teaches producing a bacon replica from Aspergillus oryzae.
Claim 18 is therefore rendered obvious.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Elrod et al. (US 6,100,057 A, cited on the IDS filed on 1 July 2024) as applied to claim 6 above, and further in view of Kozubal et al. (US 2021/0017486 A1, cited on the IDS filed on 1 July 2024).
Regarding claim 19, Elrod teaches the composition of claim 6.
Elrod does not discuss that the fungus is growable on cellulosic biomass, molasses or food waste.
However, Kozubal teaches “[a] novel method of growing fungi is disclosed which uses an engineered artificial media and produces high density filamentous fungi biomats that can be harvested with a minimum of processing…resulting in lowered fungus cultivation costs…” (Abstract). Kozubal teaches that the artificial medium can also be in the form of a liquid covering a solid carbon source, such as a lignocellulosic feedstock ([0079]). Kozubal also teaches that waste streams including molasses and fibers such as cellulose can be used as carbon sources for the artificial media ([0080]). Kozubal further teaches that filamentous fungi including Aspergillus oryzae are useful as host cells for recombinant protein production and expression platforms, resulting in useful products expressed in the biomass and/or a filamentous fungi biomat ([0107]).
It would have been obvious for one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify Elrod with the teachings of Kozubal to grow the filamentous fungal cells on the artificial medium comprising lignocellulose feedstock, cellulose, and/or molasses in order to reduce fungus cultivation costs. One of ordinary skill in the art would have had a reasonable expectation of success in doing so because Elrod teaches the use of filamentous fungi, including Aspergillus oryzae (col. 11, lines 60-64) to increase hemoprotein production (Abstract), and Kozubal teaches a culture medium comprising cellulosic material and/or molasses that can be used to grow Aspergillus oryzae biomass and/or biomats.
Claim 19 is therefore rendered obvious.
Conclusion
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/JAMES P. SHELLHAMMER/Examiner, Art Unit 1793
/EMILY M LE/Supervisory Patent Examiner, Art Unit 1793