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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 07/16/2026 has been entered.
This Office Action is in response to the Request for Continued Examination and Arguments in Remarks filed 07/16/2026.
Priority
Applicant’s claim to priority from US provisional Applications 61/973,181 filed 03/31/2014 and 62/058,230 filed 10/01/2014, as well as from PCT/US2015/023679 filed 03/31/2015, is hereby acknowledged.
Application Status
Claim 1 is cancelled. Claims 2-21 are pending. Claims 10-19 are still withdrawn. Therefore, claims 2-9 and 20-21 are under examination in this office action.
Any objection or rejection not reiterated herein has been overcome by amendments and/or arguments and is therefore withdrawn.
Applicant’s arguments have been thoroughly reviewed but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 07/16/2026 was filed after the mailing date of the Office Action on 04/22/2026. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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The following rejections are new rejections:
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The 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 non-obviousness.
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 2, 8 and 20 are rejected under 35 U.S.C. § 103 as being unpatentable over Elrod (Elrod, S.L. et al. US 6,261,827 B1; published Jul. 17, 2001; previously cited) and Mattoon (Mattoon, J.R. et al. US 5,824,511, published October 20, 1998), in view of Kwon (Kwon, S.J. et al. “High-level production of porphyrins in metabolically engineered Escherichia coli: Systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis” Applied and Environmental Microbiology, Vol. 69, No. 8 (2003), pp: 4875-4883; previously cited), Partow (Partow, S. et al. "Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae". PLoS ONE, Vol. 7, No.12 (2012), p: e52498 (1-12)), and Marx (Marx, H. et al. "Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris". Microbial Cell Factories, Vol. 7 (2008), p:23 (1-11)).
Regarding claims 2 and 8, Elrod teaches a recombinant yeast cell, expressing one or more heme biosynthetic enzymes and a hemoprotein (see abstract). Elrod teaches that the hemoprotein may be a globin (see column 3, lines 63-64).
Elrod teaches that the recombinant yeast may overexpress an endogenous (i.e. yeast) first nucleic acid encoding a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coporphyrinogen oxidase, a protoporphyrinogen oxidase and a ferrochelatase (see column 5, lines 26-36).
Elrod also teaches that the recombinant yeast may overexpress one or more second nucleic acids encoding a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coporphyrinogen oxidase, a protoporphyrinogen oxidase and a ferrochelatase (see column 7,lines 41-52).
Elrod also teaches that the nucleic acids encoding the recombinant heme biosynthesis enzymes are stably integrated into the recombinant yeast genome (see column 4, lines 39-45). Elrod teaches “at least one” or “one or more” heme biosynthesis enzymes to be overexpressed in the recombinant yeast.
Elrod also teaches that “when a nucleic acid sequence encoding one of the enzymes in the heme biosynthetic pathway is introduced into a filamentous fungal cell, one or more pathway intermediates in one or more preceding steps may become rate-limiting” (see column 11, lines 10-14).
Elrod teaches a preferred embodiment, i.e., the host being a filamentous fungal cell (see title, abstract and claims 1-17).
Elrod does not give specific on the “one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme” (see title and abstract; “Summary of the invention”, column 2, lines 22-48). Elrod only gives a specific designation for the enzymes selected in claims 7 to 16.
Mattoon teaches a method for increasing the production of a desired hemoprotein in which a heme biosynthetic pathway is deregulated to increase the quantity of the rate-limiting enzyme (see title and abstract).
Mattoon teaches the same heme biosynthetic pathway involving the eight enzymes claimed, named HEM1 (5-ALA synthase), HEM2 (5-ALA dehydratase), HEM3 (Porphobilinogen deaminase), HEM4 (Uroporphyrinogen synthetase), HEM6 (Uroporphyrinogen decarboxylase), HEM13 (Coproporphyrinogen oxidase), HEM14 (Protoporphyrinogen IX oxidase) and HEM15 (Ferrochelatase) (see Figure 1).
Mattoon teaches overexpression of two genes simultaneously from the pathway: the heme protein gene of interest, e.g., catalase T, together with HEM2 gene, a possible enzyme responsible for “bottleneck issue”. Mattoon teaches a series of transfections comprising two of the enzymes of the pathway simultaneously to check on “bottleneck’ issue resolution (see Figures 6 and 7). Mattoon states that the present invention permits investigation of other factors effecting the heme biosynthetic pathway by reducing interference from heme feedback inhibition within that pathway (see abstract). Mattoon also teaches specifically the transformation of a yeast cell with the HEM2 gene to overproduce ALA dehydratase (see column 2, lines 27-30).
Mattoon teaches preferred yeast strains as hosts, i.e., Pichia, Hansenula, strains of Saccharomyces cerevisiae, Aspergillus and Neurospora (see column 2, lines 30-34).
Mattoon teaches that using their method, desired hemoproteins, such as hemoglobin, myoglobin and certain cytochromes and enzymes such as catalases and peroxidases can be produced in unprecedented quantities, up to 20% or 40% more than can otherwise be produced in unmodified hemoprotein-producing cells (see column 2, lines 34-40).
Elrod and Mattoon do not teach eight genes encoding recombinant heme biosynthesis enzymes integrated into a recombinant yeast cell simultaneously.
However, Kwon teaches expression of eight genes involved in heme biosynthetic pathways in different combinations and up to seven simultaneously, in a recombinant Escherichia coli (E. coli) cell (see title and Tables 1 and 2). Kwon teaches the assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host (see abstract).
Kwon teaches at first expression of five genes (hemA (ALAS), hemB (porphobilinogen synthase; ALA dehydratase), hemC (hydroxymethylbilane synthase; porphobilinogen deaminase), hemD (uroporphyrinogen III synthase) and hemE (uroporphyrinogen decarboxylase) (see page 4879, left column, first paragraph). Kwon teaches that the assembled heme pathway expressed in a recombinant E. coli, accumulated large amounts of porphyrins in the culture medium, with partial precipitation of porphyrins (same paragraph).
Kwon teaches systematic extension of the engineered heme biosynthetic pathway, adding hemF (oxygen-dependent coproporphyrinogen III oxidase), hemY (protoporphyrinogen oxidase or coproporphyrinogen oxidase) and hemH (ferrochelatase heme synthase) (see page 4880, left column).
Kwon teaches that the production level of porphyrins increased from 40% with overexpression of hemA alone or with hemB, to an additional 2-to-7-fold increase with overexpression of hemC and hemD. When adding hemABC with hemE, there was no observed change in production, however when adding hemF, there was an additional 70 to 80% increase in production of porphyrins. Although adding hemH to hemABCDEF did not significantly change the production/accumulation of heme, Kwon teaches expression of these seven genes simultaneously in E. coli (see pages 4880-4881, “Production levels” section). Kwon also teaches expression of hemABCDEFY simultaneously in E. coli (see Figure 2).
Kwon teaches optimization of the system, and also the development of a versatile system for tailored (over)production of major porphyrins that occur as intermediates in heme biosynthesis (see page 4881, left column, “Discussion” section).
Elrod, Mattoon and Kwon do not teach the integration of eight yeast genes encoding enzymes of the heme biosynthetic pathway into a yeast cell genome.
However, Partow teaches the reconstruction and evaluation of a synthetic pathway in Saccharomyces cerevisiae (S. cerevisiae) (see title and abstract).
Partow teaches a bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway integrated into the S. cerevisiae’s genome (see abstract).
Partow teaches that gene integration offers a stable manipulation without requirement of selective pressure provided through media, usually necessary for the maintenance of plasmids, said selective media increasing the costs of metabolite production (see “Introduction” section, page 1, right column, lines 20-23).
Partow teaches two integration sites on chromosome XVI of S. cerevisiae, YPRC∆15 and YPRCꞇ3 (see page 2, right column, lines 13-18).
Partow teaches Eight codon optimized MEP pathway genes organized on four synthetic fragment (see Figure 2A, and page 2, left column, “Genomic integration of MEP pathway genes” section). Therefore, Partow shows that expression and integration of multiple (eight) genes into a yeast genome is feasible.
Partow teaches that expressing all the genes encoding enzymes necessary in a biosynthetic pathways can avoid “bottleneck” issues; however, using bacterial genes out of their original context, without codon-optimization, and overexpressing all the genes encoding these enzymes may not be sufficient, leading to a non-functional pathway (page 2, left column, first paragraph). Partow concludes that proper physical interaction or compartmentalization is required for in vivo biogenesis, that could involve other bacterial proteins, and lead to activation of the pathway (see page 2, left column, second paragraph; page 9, left column, last paragraph of “Discussion” section).
Marx teaches Pichia pastoris as a host for recombinant protein expression (see title). Marx teaches that overexpression of a whole biosynthetic pathway is possible in Pichia pastoris (see title and abstract).
Marx teaches that P. pastoris is well established as a host system for production of heterologous proteins, most necessary tools for genetic manipulation are available (see page 2, left column, last paragraph).
Marx teaches that recently the genome sequence has been made available through commercial ERGO platform of Integrated Genomics, In. Thus, the basis for cell and metabolic engineering of P. pastoris has been significantly broadened (see page 2, right column, first paragraph). Marx teaches testing for rate-limiting steps in the pathways (see page 7, left column, second paragraph). Marx teaches that, since the P. pastoris genome sequence became available, they decided to proceed with overexpression of homologous P. pastoris genes instead of heterologous genes from S. cerevisiae (see page 7, “Identification of further key players within the P. pastoris riboflavin biosynthetic pathway” section, left column).
Marx teaches overexpression of five rate-limiting enzymes genes from the riboflavin pathway, and integration of the constructs into the HIS4 locus of P. pastoris GS115 strain (see page 7, “Identification of further key players within the P. pastoris riboflavin biosynthetic pathway” section, left column and right column, lines 4-8). Marx teaches six (6) genes as transgenes integrated within a P. pastoris strain (see Table 2).
Therefore, Marx teaches that P. pastoris can be used to express multiple homologous genes from a biosynthetic pathway with success (see table 4, page 9).
In sum, Elrod teaches one or more genes of the heme biosynthetic pathway to be integrated into a yeast genome for the production of a heme-protein. Mattoon teaches transforming two genes at a time from said biosynthetic pathway and teaches investigating other possible rate-limiting steps/enzymes. Kwon teaches that it is possible to transform a host with the genes of the enzymes claimed and reassemble the whole biosynthetic pathway using three plasmid systems. Partow teaches that integrating heterologous genes of interest is better for stability of the transgenes and for cost saving on selective media. Partow also teaches integration of eight genes encoding enzymes from a whole metabolic pathway into a host yeast cell’s genome is feasible.
Marx teaches that obtaining an active biosynthetic pathway using overexpression and integration of multiple (six) homologous genes in P. pastoris is feasible with success. Marx teaches that the choice of P. pastoris and homologous yeast genes is obvious due to recent advances in P. pastoris genome sequencing.
Therefore, it would have been obvious to one having ordinary skills in the art before the effective filing date of the claimed invention to have modified Elrod/Mattoon and introduced the eight genes HemABCDEFHY simultaneously in a cell and obtained eight stably integrated genes into the genome of a recombinant yeast to optimize production of a heme-containing protein. Since Kwon teaches that it is feasible to overexpress HemABCDEFY and HemABCDEFH simultaneously in recombinant E. coli, one with ordinary skills in the art could have express HemABCDEFHY in a yeast host cell, stably integrated within the genome, using the teachings of Partow and Marx. Since each one of the heme biosynthesis enzyme could be rate-limiting for the overexpression of a heme-containing protein, one motivated in optimizing a yeast system for overexpression of a heme-containing protein could have performed modifications to the biosynthetic pathway through routine optimization and trials as taught by Mattoon, Kwon, Partow and Marx, and introduced eight rate-limiting enzymes into a recombinant yeast, and more specifically in P. pastoris.
Since the claimed invention is merely a combination of old elements, and in the combination each element merely would have performed the same function as it did separately, one of ordinary skill in the art would have recognized that the results of the combination were predictable. KSR, 127 S.Ct. at 1740, 82 USPQ2d at 1396.
Regarding claims 8 and 20, Mattoon teaches a preferred host cell being Pichia (see column 2, line 31).
Marx also teaches Pichia pastoris as a host for recombinant protein expression (see title). Marx teaches that overexpression of a whole biosynthetic pathway is possible in Pichia pastoris (see title and abstract).
Marx teaches that P. pastoris is well established as a host system for production of heterologous proteins, most necessary tools for genetic manipulation are available, as well as genome sequence information (see page 2, left column, last paragraph, right column, first paragraph).
It would have been obvious to one with ordinary skills in the art before the effective filing date to have substituted the host cell taught by Elrod, Mattoon, Kwon and Partow, with a recombinant Pichia pastoris strain as taught by Marx. One motivated in increasing productivity and yield in the recombinant expression system previously used with success and for which genetic manipulation tools are available, could have performed this substitution with a reasonable expectation of success and arrived at the claimed invention.
Response to Arguments
Applicant’s arguments with respect to claim 2 rejection under 35 U.S.C. §103 have been considered but are moot because the new ground of rejection does not rely on the same combination of references applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Applicant argues on page 6 of 16 of Remarks filed 07/16/2026 that “there must be some suggestion or motivation, either in the references themselves or in the knowledge generally available to one of ordinary sill in the art, to modify the reference or to combine reference teachings”, “the teaching or suggestion to make the claimed combination must be found in the prior art, not in Applicant’s disclosure” and that “there must be a reasonable expectation of success”.
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the combination of Elrod, Mattoon, Kwon, Partow and Marx teaches on the status of the prior art before the effective filing date of the claimed invention. Elrod, Mattoon, Partow and Marx clearly teach that there is a “bottleneck” in the production of heme-proteins, due to rate-limiting enzymes in the heme biosynthetic pathways. To resolve this “bottleneck” issue, Mattoon clearly teaches the expression of a second enzyme to the heme-protein of interest. Mattoon, Partow and Marx also teach searching for the enzyme responsible for “bottleneck” issue by overexpressing the enzymes in combination. Kwon and Partow teach combining multiple genes from a biosynthetic pathway and trying to “reassemble” the pathway in a functional way. Marx teaches success for reassembly of a biosynthetic pathway by overexpressing genes homologous to the yeast. Partow teaches the advantages of integrating the genes in the yeast genomes rather than using plasmid-based systems. Marx teaches the advantages of using P. pastoris because of availability of genome sequences. Therefore, there is a reasonable expectation of success.
Regarding Applicant’s arguments on page 7 of 16, against the references individually referring to “a filamentous fungal cell” (Elrod), “Escherichia coli” (Kwon), in response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
The combination of references points to the preferred use of a yeast cell as a host and more specifically Pichia pastoris.
Regarding Applicant’s argument on pages 7 and 8 of Remarks against “Kwon” for using a three-plasmid system, the use of Kwon in the rejection is to show the state of the art in prior art, about the genetic manipulation of heme biosynthetic pathway genes. Kwon was used to indicate the extent of knowledge about the problems to be addressed and overcome, i.e., finding a host cell and troubleshooting the rate-limiting steps and finding a method of reassembly of a whole biosynthetic pathway in a recombinant host cell. Kwon teaches that the skill level in the art is high, and all the enzymes claimed are known and reassembly of the whole biosynthetic pathway was considered as a solution for production of heme-proteins in a recombinant host cell.
Elrod, Partow and Marx teaches integrating the transgenes into the host genome, because it is more stable and cost-effective, according to Partow. Marx teaches the logic of using Pichia pastoris, since the genome sequence is known. Partow teaches the set-backs of using heterologous, non-codon optimized genes. Marx teaches the logic in using yeast genes, and specifically homologous genes.
Therefore, one with ordinary skills in the art would favor using a yeast and stably integrated transgenes for overexpression of biosynthetic pathway genes.
Claims 5, 6 and 7 are rejected under 35 U.S.C. § 103 as being unpatentable over (Elrod, S.L. et al. US 6,261,827 B1; published Jul. 17, 2001; previously cited) and Mattoon (Mattoon, J.R. et al. US 5,824,511, published October 20, 1998), in view of Kwon (Kwon, S.J. et al. “High-level production of porphyrins in metabolically engineered Escherichia coli: Systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis” Applied and Environmental Microbiology, Vol. 69, No. 8 (2003), pp: 4875-4883; previously cited), Partow (Partow, S. et al. "Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae". PLoS ONE, Vol. 7, No.12 (2012), p: e52498 (1-12)), and Marx (Marx, H. et al. "Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris". Microbial Cell Factories, Vol. 7 (2008), p:23 (1-11)), as applied to claim 2 above, and in further view of Brown (Brown, P. et al. WO 2013/010042 A1; published Jan. 17, 2013; previously cited).
It is noted that the combination of references Elrod, Mattoon, Kwon, Partow and Marx renders elements of claim 2 obvious. The rejection of claim 2 is described above.
Regarding claim 5, Elrod teaches that the heme-containing protein may be a myoglobin (see column 3, lines 65-66). Elrod teaches a myoglobin as the heme-containing protein (column 3, line 66); however, Elrod does not teach a mammalian myoglobin. Elrod does not specifically teach a mammalian myoglobin. However, Elrod teaches genes from different sources: Saccharomyces cerevisiae, Escherichia coli, Bacillus subtilis, Aspergillus nidulans, Rhodobacter capsulans, Bovine or Human sources for enzymes in the heme metabolic pathways (see column 8). Elrod teaches that the hemoprotein can be native to the filamentous fungal cell (see column 74, claim 17) or can be foreign to the filamentous fungal cell (see column 75, claim 18).
Mattoon teaches myoglobin as a desired hemoprotein (see column 1, line 41; column 2, lines 35, 55; column 6, line 51; column 7, line 32; column 12, claim 13). Mattoon also teaches human hemoproteins (see column 1, lines 29-31).
As Elrod, Mattoon does not specifically teach a mammalian myoglobin either. The combination of references Elrod, Mattoon, Kwon, Partow and Marx does not render a mammalian myoglobin obvious.
However, Brown also teaches an animal-source of myoglobin ( see ¶ [0051]-[0052]) that can be expressed in a non-animal source such as a yeast or bacteria (¶ [00140], [00142], [00223]-[00224]). Brown teaches an equine heart muscle myoglobin (see ¶ [00258]).
It would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have substituted a myoglobin protein taught by Elrod and Mattoon, to a myoglobin protein from a mammal source as taught by Brown. Both proteins have the same function in the system. One motivated in overproducing a mammal protein for use in a mammalian animal model or cell system, could have performed this substitution with a reasonable expectation of success, and arrived at the claimed invention.
Regarding claims 6 and 7, the combination Elrod, Mattoon, Kwon, Partow and Marx teaches the elements of claim 2, however the combination of reference does not teach the recombinant expression of leghemoglobin, wherein the leghemoglobin is from Glycine max, as in claims 6 and 7.
However, Brown teaches the recombinant expression of Soybean (Glycine max) leghemoglobin in a genetically modified yeast or bacteria as a non-animal source of isolated leghemoglobin protein (see ¶ [00223]-[00224], [00261], [00378], [00381]).
In KSR Int 'l v. Teleflex, the Supreme Court, indicated that “The principles underlying [earlier] cases are instructive when the question is whether a patent claiming the combination of elements of prior art is obvious. When a work is available in one field of endeavor, design incentives and other market forces can prompt variations of it, either in the same field or a different one. If a person of ordinary skill can implement a predictable variation, § 103 likely bars its patentability.” KSR Int'l v. Teleflex lnc., 127 S. Ct. 1727, 1740 (2007).
Applying the KSR standard of obviousness to Elrod, Mattoon, Kwon, Partow, Marx and Brown, it is concluded that the combination of the references represents a combination of known elements which yield the predictable result. At the time of invention, a practitioner could have combined the teachings of Elrod, Mattoon and Kwon expressing multiple heme biosynthesis enzymes in a host cell, with teachings from Brown, using a Glycine max leghemoglobin as the heme-containing protein or a mammalian myoglobin. As a result, the predictable result of designing a genetically modified yeast expressing at least eight heme biosynthesis enzymes gene, overexpressing a heme-containing protein, i.e. a Glycine max leghemoglobin or a mammalian myoglobin, would be achieved. Such a combination is merely a "predictable use of prior art elements according to their established functions." KSR Int’l 7, 127 S. Ct. at 1740.
Response to Arguments
Applicant's arguments filed 07/16/2026 have been fully considered but they are not persuasive.
Applicant argues on page 10 of 16 of Remarks that there is no “integration” taught by the combination of references.
In response, Partow teaches that gene integration offers a stable manipulation without requirement of selective pressure provided through media, usually necessary for the maintenance of plasmids, said selective media increasing the costs of metabolite production (see “Introduction” section, page 1, right column, lines 20-23). Partow specifically teaches homologous recombination, integrating the transgene into the host genome (see Figure 2A).
Marx also teaches integration into the HIS4 locus of P. pastoris (page 7, right column, lines 7-8).
Therefore, the combination of references Elrod, Mattoon, Kwon, Partow and Marx teaches stably integrating the transgenes into the host genome.
Claims 3, 4, 9 and 21 are rejected under 35 U.S.C. § 103 as being
unpatentable over Elrod (Elrod, S.L. et al. US 6,261,827 B1; published Jul. 17, 2001; previously cited), and Mattoon (Mattoon, J.R. et al. US 5,824,511, published October 20, 1998), in view of Kwon (Kwon, S.J. et al. “High-level production of porphyrins in metabolically engineered Escherichia coli: Systematic extension of a pathway assembled from overexpressed genes involved in heme biosynthesis” Applied and Environmental Microbiology, Vol. 69, No. 8 (2003), pp: 4875-4883; previously cited), Partow (Partow, S. et al. "Reconstruction and evaluation of the synthetic bacterial MEP pathway in Saccharomyces cerevisiae". PLoS ONE, Vol. 7, No.12 (2012), p: e52498 (1-12)), Marx (Marx, H. et al. "Overexpression of the riboflavin biosynthetic pathway in Pichia pastoris". Microbial Cell Factories, Vol. 7 (2008), p:23 (1-11)), and Kim (Kim, S-J et al. Journal of Microbiology and Biotechnology, Vol. 19 (2009), pp: 966-971; previously cited).
Regarding claims 3 and 9, Elrod teaches a recombinant yeast cell, expressing one or more heme biosynthetic enzymes and a hemoprotein (see abstract). Elrod teaches that the hemoprotein may be a globin (see column 3, lines 63-64).
Elrod teaches that the recombinant yeast may overexpress an endogenous (i.e. yeast) first nucleic acid encoding a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coporphyrinogen oxidase, a protoporphyrinogen oxidase and a ferrochelatase (see column 5, lines 26-36).
Elrod also teaches that the recombinant yeast may overexpress one or more second nucleic acids encoding a 5-aminolevulinic acid synthase, a porphobilinogen synthase, a porphobilinogen deaminase, an uroporphyrinogen synthase, an uroporphyrinogen decarboxylase, a coporphyrinogen oxidase, a protoporphyrinogen oxidase and a ferrochelatase (see column 7,lines 41-52).
Elrod also teaches that the nucleic acids encoding the recombinant heme biosynthesis enzymes are stably integrated into the recombinant yeast genome (see column 4, lines 39-45). Elrod teaches “at least one” or “one or more” heme biosynthesis enzymes to be overexpressed in the recombinant yeast.
Elrod also teaches that “when a nucleic acid sequence encoding one of the enzymes in the heme biosynthetic pathway is introduced into a filamentous fungal cell, one or more pathway intermediates in one or more preceding steps may become rate-limiting” (see column 11, lines 10-14).
Elrod teaches a preferred embodiment, i.e., the host being a filamentous fungal cell (see title, abstract and claims 1-17).
Elrod does not give specific on the “one or more copies of one or more second nucleic acid sequences encoding a heme biosynthetic enzyme” (see title and abstract; “Summary of the invention”, column 2, lines 22-48). Elrod only gives a specific designation for the enzymes selected in claims 7 to 16.
Mattoon teaches a method for increasing the production of a desired hemoprotein in which a heme biosynthetic pathway is deregulated to increase the quantity of the rate-limiting enzyme (see title and abstract).
Mattoon teaches the same heme biosynthetic pathway involving the eight enzymes claimed, named HEM1 (5-ALA synthase), HEM2 (5-ALA dehydratase), HEM3 (Porphobilinogen deaminase), HEM4 (Uroporphyrinogen synthetase), HEM6 (Uroporphyrinogen decarboxylase), HEM13 (Coproporphyrinogen oxidase), HEM14 (Protoporphyrinogen IX oxidase) and HEM15 (Ferrochelatase) (see Figure 1).
Mattoon teaches overexpression of two genes simultaneously from the pathway: the heme protein gene of interest, e.g., catalase T, together with HEM2 gene, a possible enzyme responsible for “bottleneck” issue. Mattoon teaches a series of transfections comprising two of the enzymes of the pathway simultaneously to check on “bottleneck’ issue resolution (see Figures 6 and 7). Mattoon states that the present invention permits investigation of other factors effecting the heme biosynthetic pathway by reducing interference from heme feedback inhibition within that pathway (see abstract). Mattoon also teaches specifically the transformation of a yeast cell with the HEM2 gene to overproduce ALA dehydratase (see column 2, lines 27-30).
Mattoon teaches preferred yeast strains as hosts, i.e., Pichia, Hansenula, strains of Saccharomyces cerevisiae, Aspergillus and Neurospora (see column 2, lines 30-34).
Mattoon teaches that using their method, desired hemoproteins, such as hemoglobin, myoglobin and certain cytochromes and enzymes such as catalases and peroxidases can be produced in unprecedented quantities, up to 20% or 40% more than can otherwise be produced in unmodified hemoprotein-producing cells (see column 2, lines 34-40).
Elrod and Mattoon do not teach eight genes encoding recombinant heme biosynthesis enzymes integrated into a recombinant yeast cell simultaneously.
However, Kwon teaches expression of eight genes involved in heme biosynthetic pathways in different combinations and up to seven simultaneously, in a recombinant Escherichia coli (E. coli) cell (see title and Tables 1 and 2). Kwon teaches the assembly of the entire heme biosynthetic pathway in a three-plasmid system and overexpression of the corresponding genes with Escherichia coli as a host (see abstract).
Kwon teaches at first expression of five genes (hemA (ALAS), hemB (porphobilinogen synthase; ALA dehydratase), hemC (hydroxymethylbilane synthase; porphobilinogen deaminase), hemD (uroporphyrinogen III synthase) and hemE (uroporphyrinogen decarboxylase) (see page 4879, left column, first paragraph). Kwon teaches that the assembled heme pathway expressed in a recombinant E. coli, accumulated large amounts of porphyrins in the culture medium, with partial precipitation of porphyrins (same paragraph).
Kwon teaches systematic extension of the engineered heme biosynthetic pathway, adding hemF (oxygen-dependent coproporphyrinogen III oxidase), hemY (protoporphyrinogen oxidase or coproporphyrinogen oxidase) and hemH (ferrochelatase heme synthase) (see page 4880, left column).
Kwon teaches that the production level of porphyrins increased from 40% with overexpression of hemA alone or with hemB, to an additional 2-to-7-fold increase with overexpression of hemC and hemD. When adding hemABC with hemE, there was no observed change in production, however when adding hemF, there was an additional 70 to 80% increase in production of porphyrins. Although adding hemH to hemABCDEF did not significantly change the production/accumulation of heme, Kwon teaches expression of these seven genes simultaneously in E. coli (see pages 4880-4881, “Production levels” section). Kwon also teaches expression of hemABCDEFY simultaneously in E. coli (see Figure 2).
Kwon teaches optimization of the system, and also the development of a versatile system for tailored (over)production of major porphyrins that occur as intermediates in heme biosynthesis (see page 4881, left column, “Discussion” section).
Elrod, Mattoon and Kwon do not teach the integration of eight yeast genes encoding enzymes of the heme biosynthetic pathway into a yeast cell genome.
However, Partow teaches the reconstruction and evaluation of a synthetic pathway in Saccharomyces cerevisiae (S. cerevisiae) (see title and abstract).
Partow teaches a bacterial 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway integrated into the S. cerevisiae’s genome (see abstract).
Partow teaches that gene integration offers a stable manipulation without requirement of selective pressure provided through media, usually necessary for the maintenance of plasmids, said selective media increasing the costs of metabolite production (see “Introduction” section, page 1, right column, lines 20-23).
Partow teaches two integration sites on chromosome XVI of S. cerevisiae, YPRC∆15 and YPRCꞇ3 (see page 2, right column, lines 13-18).
Partow teaches Eight codon optimized MEP pathway genes organized on four synthetic fragment (see Figure 2A, and page 2, left column, “Genomic integration of MEP pathway genes” section). Therefore, Partow shows that expression and integration of multiple (eight) genes into a yeast genome is feasible.
Partow teaches that expressing all the genes encoding enzymes necessary in a biosynthetic pathways can avoid “bottleneck” issues; however, using bacterial genes out of their original context, without codon-optimization, and overexpressing all the genes encoding these enzymes may not be sufficient, leading to a non-functional pathway (page 2, left column, first paragraph). Partow concludes that proper physical interaction or compartmentalization is required for in vivo biogenesis, that could involve other bacterial proteins, and lead to activation of the pathway (see page 2, left column, second paragraph; page 9, left column, last paragraph of “Discussion” section).
Marx teaches Pichia pastoris as a host for recombinant protein expression (see title). Marx teaches that overexpression of a whole biosynthetic pathway is possible in Pichia pastoris (see title and abstract).
Marx teaches that P. pastoris is well established as a host system for production of heterologous proteins, most necessary tools for genetic manipulation are available (see page 2, left column, last paragraph).
Marx teaches that recently the genome sequence has been made available through commercial ERGO platform of Integrated Genomics, In. Thus, the basis for cell and metabolic engineering of P. pastoris has been significantly broadened (see page 2, right column, first paragraph). Marx teaches testing for rate-limiting steps in the pathways (see page 7, left column, second paragraph). Marx teaches that, since the P. pastoris genome sequence became available, they decided to proceed with overexpression of homologous P. pastoris genes instead of heterologous genes from S. cerevisiae (see page 7, “Identification of further key players within the P. pastoris riboflavin biosynthetic pathway” section, left column).
Marx teaches overexpression of five rate-limiting enzymes genes from the riboflavin pathway, and integration of the constructs into the HIS4 locus of P. pastoris GS115 strain (see page 7, “Identification of further key players within the P. pastoris riboflavin biosynthetic pathway” section, left column and right column, lines 4-8). Marx teaches six (6) genes as transgenes integrated within a P. pastoris strain (see Table 2).
Therefore, Marx teaches that P. pastoris can be used to express multiple homologous genes from a biosynthetic pathway with success (see table 4, page 9).
In sum, Elrod teaches one or more genes of the heme biosynthetic pathway to be integrated into a yeast genome for the production of a heme-protein. Mattoon teaches transforming two genes at a time from said biosynthetic pathway and teaches investigating other possible rate-limiting steps/enzymes. Kwon teaches that it is possible to transform a host with the genes of the enzymes claimed and reassemble the whole biosynthetic pathway using three plasmid systems. Partow teaches that integrating heterologous genes of interest is better for stability of the transgenes and for cost saving on selective media. Partow also teaches integration of eight genes encoding enzymes from a whole metabolic pathway into a host yeast cell’s genome is feasible.
Marx teaches that obtaining an active biosynthetic pathway using overexpression and integration of multiple (six) homologous genes in P. pastoris is feasible with success. Marx teaches that the choice of P. pastoris and homologous yeast genes is obvious due to recent advances in P. pastoris genome sequencing.
Therefore, it would have been obvious to one having ordinary skills in the art before the effective filing date of the claimed invention to have modified Elrod/Mattoon and introduced the eight genes HemABCDEFHY simultaneously in a cell and obtained eight stably integrated genes into the genome of a recombinant yeast to optimize production of a heme-containing protein. Since Kwon teaches that it is feasible to overexpress HemABCDEFY and HemABCDEFH simultaneously in recombinant E. coli, one with ordinary skills in the art could have express HemABCDEFHY in a yeast host cell, stably integrated within the genome, using the teachings of Partow and Marx. Since each one of the heme biosynthesis enzyme could be rate-limiting for the overexpression of a heme-containing protein, one motivated in optimizing a yeast system for overexpression of a heme-containing protein could have performed modifications to the biosynthetic pathway through routine optimization and trials as taught by Mattoon, Kwon, Partow and Marx, and introduced eight rate-limiting enzymes into a recombinant yeast, and more specifically in P. pastoris.
Since the claimed invention is merely a combination of old elements, and in the combination each element merely would have performed the same function as it did separately, one of ordinary skill in the art would have recognized that the results of the combination were predictable. KSR, 127 S.Ct. at 1740, 82 USPQ2d at 1396.
Regarding claims 3, 4, 9 and 21, The combination of references Elrod, Mattoon, Kwon, Partow and Marx does teach Pichia pastoris yeast cell; however, the combination of references does not teach a recombinant heme-containing protein expressed by a methanol inducible promoter, or wherein the promoter is pAOX1 promoter, as in claims 3 and 4.
However, Kim teaches the use of a methanol inducible, i.e. pAOX1, referred to as pPICZαA, in the expression vector of a recombinant peroxidase (see title, abstract and figures 1A and 2).
Kim also teaches a recombinant Pichia pastoris yeast cell (see page 967, left column, lines 4-5).
Kim teaches that “[I]ndustrial applications of a fungal peroxidase require an efficient and economical production system” and that “[t]he methylotrophic yeast Pichia pastoris is well known for being effective in producing recombinant genetic material when facilitated by including a gene coding for a foreign protein behind the promoter of the AOX1 gene normally needed for methanol utilization, such that high quantities of foreign protein (10-100 times more than S. cerevisiae) can be expressed” (see page 966, right column, lines 7-18).
It would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention to have substituted the control sequence in the vector used by Elrod/Mattoon modified by Kwon, Partow and Marx, with the methanol-inducible promoter taught by Kim. One motivated in using an endogenous/homologous sequence to the Pichia pastoris yeast, as taught by Marx, and an endogenous methanol-inducible promoter to increase production of an exogenous protein as taught by Kim, could have performed this substitution with a reasonable expectation of success, and would have arrived at the claimed invention.
Response to Arguments
Applicant’s arguments with respect to claims 3, 4, 8, 9 and 21 under 35 U.S.C.§
103 have been considered but are moot because the new grounds of rejections do not rely on the same combination of references applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Conclusion
No claim is allowed.
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/A.D./Examiner, Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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