Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
DETAILED ACTION
Claims 1, 3, 6-12 and 16-17 are pending.
Applicant amended claim 1 and added new limitation “wherein there is a difference among expression levels of the first gene, the second gene, and the third gene in the at least one selected strain of the plurality of yeast transformants”.
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.
Claims 1, 3, 6-8, 12 and 16-17 remain rejected under 35 U.S.C. 103 as being unpatentable over Safak (Turkish Electronic Journal of Biotechnology 1 (2002): 11-17, of record in Office Correspondence mailed on 02/14/2024) in view of Li (Journal of Industrial Microbiology and Biotechnology 44.4-5 (2017): 605-612, hereinafter “Li”, of record in Office Correspondence mailed on 06/20/2023), Petrasovits (Plant Biotechnology Journal 10.5 (2012): 569-578, hereinafter “Petrasovits”), Pi (Scientific Reports 8.1 (2018): 1-10, hereinafter “Pi”, of record in Office Correspondence mailed on 06/20/2023), and Yen (Journal of the Taiwan Institute of Chemical Engineers 97 (2019): 80-87, of record in Office Correspondence mailed on 08/30/2024, hereinafter “Yen”).
Regarding claim 1, Safak teaches yeasts such as Rhodotorula glutinis accumulate small amount of polyhydroxybutyrate PHB and teaches eukaryotic production of PHB through genetic engineering is being pursued as a potentially inexpensive way to produce PHB (Abstract, page 12 para. 2).
Safak does not teach transforming a plurality of vectors into an oleaginous yeast of Rhodotorula glutinis to obtain a yeast transformant, wherein each of the plurality of vectors contains a single polyhydroxybutyrate biosynthesis related gene selected from a group consisting of a first gene, a second gene and a third gene, and at least one of the plurality of vectors contains the first gene, at least one of the plurality of vectors contains the second gene, and at least one of the plurality of vectors contains the third gene, wherein all of the plurality of vectors have a same promoter, and the first gene is driven by the same promoter and capable of encoding acetyl-CoA C-acetyltransferase, the second gene is driven by the same promoter and capable of encoding NADPH-dependent acetoacetyl-CoA reductase, and the third gene is driven by the same promoter and capable of encoding PHA synthase; screening the yeast transformant, and obtaining a yeast transformant inserted with the first gene from the at least one of the plurality of vectors, the second gene from the at least one of the plurality of vectors, and the third gene from the at least one of the plurality of vectors, and capable of expressing a protein of the first gene, a protein of the second gene, or a protein of the third gene; and cultivating the yeast transformant with glucose, crude glycerol, or a combination thereof, to obtain the polyhydroxybutyrate.
However, Li teaches engineering an oleaginous yeast, Yarrowia lipolytica, to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway (Abstract, page 606, left column, paragraph 2). Li teaches 3 vectors, pQK1-phaA comprising phaA (i.e. acetyl-CoA C-acetyltransferase), pQK3-phaB comprising phaB (i.e. NADPH-dependent acetoacetyl-CoA reductase), pMT15-phaC vector comprising phaC (i.e. PHA synthase) (Table 1). Li teaches assembling the transcription units (promoter-gene-terminator) from pQK1-phaA, pQK3-phaB, and pMT15-phaC to create vector pMT15-CAB and transforming the yeast with the linearizing pMT15-CAB, where the PHB operon is then integrated in the yeast genome (page 607 left column para. 1 and 2). Li teaches selecting the transformants (i.e. plurality of yeast) on defined medium, validating the successfully engineered colonies by PCR amplification of the integrated gene, and teaches cultivating the engineered strain and collecting the cells to measure the intracellular PHB content (i.e. extract from the cells of each strain) (page 607, left column, paragraphs 1-3, right column para. 2) (i.e., cultivating the plurality of yeast transformants, collecting cells of each strain of yeast transformant, analyzing PHB accumulation in the plurality of extracts). Li teaches the transformed yeast is capable of accumulating intracellular PHB biopolymers and teaches quantitative real-time PCR was performed to study the transcriptional level of the phaC gene (page 609 para “Strain engineering”). Li teaches cultivating the transformed yeast in medium supplemented with glucose (page 607 para. “Yeast transformation”).
In addition, Petrasovits teaches one strategy to increase PHB production is to increase the amount of recombinant protein in the host organism (page 569 right column last para.). Petrasovits teaches using same strong promoter for the transcription of phaA, phaB, and phaC genes such as pMBXS165, pMBXS166 and pMBXS167 which are individual constructs with phbA, phbB and phaC, respectively, each driven by the same rUbi promoter (page 571 left column “Use of strong promoters in transgene expression cassettes”, Table 2 legend) (i.e., all of the plurality of vectors have a same promoter, and the first gene is driven by the same promoter and capable of encoding acetyl-CoA C-acetyltransferase, the second gene is driven by the same promoter and capable of encoding NADPH-dependent acetoacetyl-CoA reductase, and the third gene is driven by the same promoter and capable of encoding PHA synthase).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method taught Safak by engineering Rhodotorula glutinis to accumulate more PHB as suggested by Safak, by transforming the yeast with the genes of the PHB biosynthesis pathway, screening the yeast transformant, obtaining a transformant inserted with the genes, and cultivating the yeast transformant as suggested by Li, and using the same promoter to control transcription of each of the genes, as suggested by Petrasovits. One of ordinary skill in the art would be motivated to do so in order to successfully engineer Rhodotorula glutinis for increase in PHB production, ensure robust and strong transcription of the PHB synthesis genes, and successfully screen and isolated yeast cells with high PHB production. Since Safak, Li, and Petrasovits all teach a desire for increasing PHB production, there is a reasonable expectation of success in combining features of the methods taught in these references.
Safak does not teach transforming the yeast with a plurality of vectors.
However, Pi teaches a method of transforming multiple genes into Rhodotorula glutinis genome and teaches each gene was integrated into a vector with a different marker and the DNA were then transformed into the yeast (page 2 last paragraph, Table 1). Pi also teaches screening several transformants and assaying single colonies of transformants (page 8 para. 2-4).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method taught by Safak by cloning each gene in a plasmid and transforming each plasmid into the yeast as suggested by Pi. One of ordinary skills in the art would be motivated to do so since Pi teaches a successful method of introducing multiple genes into Rhodotorula glutinis.
Safak does not teach cultivating the strain with a carbon source of crude glycerol in a concentration of 30-60 g/L.
Pi teaches using 2% glycerol as a carbon source (page 7 para. 2).
However, Yen teaches crude glycerol was found conclusively to have the potential for being utilized as an efficient way to obtain lipid accumulation in oleaginous R. glutinis (Abstract) and teaches the concentration of crude glycerol is 60 g/L (page 81 para. 2.1).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method taught by Safak using 60 g/L of crude glycerol as a carbon source as suggested by Yen. One of ordinary skill in the art would be motivated to do so since this carbon source leads to lipid accumulation in oleaginous R. glutinis.
The limitation “wherein there is a difference among expression levels of the first gene, the second gene, and the third gene in the at least one selected strain of the plurality of yeast transformants, and the PHB accumulation of the at least one selected strain is the highest” is considered as the result of the recited active steps. The claim scope is not limited by claim language that suggest or makes optional but does not require steps to be performed. A whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited. See MPEP 2111.04.
Regarding claim 3, Safak teaches the yeast producing PHB is Rhodotorula glutinis (Abstract) and Pi teaches using R. glutinis BCRC 22360 (page 7 “Strains, media, and growth conditions”).
Regarding claim 6, Li teaches 3 vectors, pQK1-phaA comprising phaA (i.e. acetyl-CoA C-acetyltransferase), pQK3-phaB comprising phaB (i.e. NADPH-dependent acetoacetyl-CoA reductase), pMT15-phaC vector comprising phaC (i.e. PHA synthase) (Table 1). Li teaches assembling the transcription units (promoter-gene-terminator) from pQK1-phaA, pQK3-phaB, and pMT15-phaC to create vector pMT15-CAB and transforming the yeast with the linearizing pMT15-CAB, where the PHB operon is then integrated in the yeast genome (page 607 left column para. 1 and 2).
Regarding claim 7, Li teaches the PHB pathway genes were cloned in a plasmid (pMT15-CAB Table 1). The plasmid was then linearized and transformed into the yeast (page 607, left column, para. 2). One of ordinary skill in the art would have been motivated to transform the yeast with linearized plasmid comprising the PHB pathway genes in order to successfully integrate the genes in the yeast’s genome.
Regarding claim 8, Li teaches extracted genomic DNA from the engineered yeast had PHB pathway genes which was confirmed by PCR (page 607, left column, para. 2). One of ordinary skill in the art would have been motivated to screen the yeast’s DNA to ensure the genes are successfully integrated in the genome.
Regarding claim 12, Li teaches, for PHB production, the engineered Y. lipolytica strain was cultivated in flasks (page 607, left column, last paragraph) and the fermentation was also carried out under aerobic conditions (page 610, left column, last paragraph). One of ordinary skill in the art would be motivated to grow the yeast to ensure the proliferation of the desired yeast and higher production of PHB.
Regarding claim 16, Pi teaches R. glutinis can grow in sea water (Abstract).
Regarding claim 17, Li teaches the successful engineered colonies were validated by screening the yeast transformant for phaA (i.e. acetyl-CoA C-acetyltransferase), phaB (i.e. NADPH-dependent acetoacetyl-CoA reductase), and phaC (i.e. PHA synthase) (page 607 para. “yeast transformation”).
Claims 9-11 remain rejected under 35 U.S.C. 103 as being unpatentable over Safak, Li, Petrasovits, Pi, and Yen as applied to claim 1 above, and further in view of Sandstrom (AMB Express 5 (2015): 1-9), of record in Office Correspondence mailed on 06/20/2023) and as evidenced by Appendix A, Appendix B, and Appendix C (Sequence alignments, of record in Office Correspondence mailed on 06/20/2023).
Regarding claims 9-11, Li teaches PHB biosynthetic genes phaA, phaB, and phaC were cloned from the genomic DNA of Ralstonia eutropha (page 606, right column, para. 3) and teaches the successful engineered colonies were validated by PCR amplification of the integrated gene, using extracted genomic DNA as template (page 607, left column, para. 2).
Li does not teach the primers set forth in SEQ ID NO: 4-9.
However, Sandstrom teaches engineering S. cerevisiae for the production of PHB by introducing in the cells PHB pathway genes phaA, phaB1, and phaC1 from C. necator (also known as Ralstonia eutropha as shown in UniProt P14611), which were codon optimized towards S. cerevisiae and deposited with the following accession numbers; phaA [GenBank:KP681582], phaB1 [GenBank:KP681583], and phaC1 [GenBank:KP681584] (page 2, right column, section titled “PHB pathway expression vector construction”). SEQ ID NO:4 and the reverse-complement of SEQ ID NO:5 align 100% with phaA [GenBank:KP681582] (see Appendix A), SEQ ID NO:6 and the reverse-complement of SEQ ID NO:7 align 100% with phaB1 [GenBank:KP681583] (see Appendix B), and SEQ ID NO: 8 and the reverse-complement of SEQ ID NO: 9 align 100% with phaC1 [GenBank:KP681584] (see Appendix C).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further modify the method taught by Safak by using the codon optimized genes taught by Sandstrom, and by using primers that would anneal to these synthetic genes with a reasonable expectation of success. One of ordinary skill in the art would be motivated to do so in order to use genes with codon optimized for use in yeast thus ensuring optimal translation and expression of the proteins, and to ensure the use of primers that are identical to their target genes, leading to an effective annealing and amplification of the target by PCR.
Response to Arguments
Applicant's arguments filed 06/30/2025 have been fully considered but they are not persuasive.
Applicant argues Petrasovits teaches three individual constructs with same promoter or a single megaconstruct and argues one of ordinary skill in the art would also be informed by Petrasovits of the potential advantages of using a single megaconstruct over multiple individual constructs.
In response to the argument, the rationale for the obviousness rejection in view of Petrasovits is whether a person of ordinary skill in the art would be motivated to express all genes using the same promoter.
Li teaches each gene was expressed using a different promoter. However, Petrasovits teaches each gene can be expressed using the same strong promoter and teaches in Figure 2a that the plants transformed with constructs where the strong promoter is the same had an increase in PHB content after 6 months, whether it is a single construct or a megaconstruct (R-Ubi S and R-Ubi M in Figure 2). Thus, a person of ordinary skill in the art would understand that the genes under the same strong promoter can be successfully expressed and PHB produced, and would have a reasonable expectation of success. Pi teaches a method of transforming multiple genes into Rhodotorula glutinis genome, the same yeast taught by Safak, by using multiple single construct vectors, each containing a gene. Thus, one of ordinary skill in the art would be motivated to use individual vectors with same strong promoter containing each a single phaA gene, phaB gene and phaC gene and transform them into yeast host as suggested by Pi. Since Pi teaches this strategy is successful in yeast hosts, one of ordinary skill in the art would have a reasonable expectation of success.
Applicant argues the claimed invention achieves a technical effect that is unexpected in view of the teachings of Safak.
Safak, Li and Petrasovits all teach a desire to increase production of PHB, as discussed above. Thus, one of ordinary skill in the art would be motivated to combine the teachings of the prior art in order to achieve the expected result of an increase in the PHB production.
In response to applicant's argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
Applicant argues the prior art Li and Petrasovits teach 1:1:1 ratio of gene expression and do not teach there is a difference among expression levels of the first gene, the second gene, and the third gene in the at least one selected strain of the plurality of yeast transformants, and the PHB accumulation of the at least one selected strain is the highest.
In response to the argument, it is not clear in the argument where in the Li reference the expression levels of the three genes are referred to teach the ratio. Petrasovits teaches in figure 8 transformants have different levels of PhaB protein compared to PhaA protein. The limitation “wherein there is a difference among expression levels of the first gene, the second gene, and the third gene in the at least one selected strain of the plurality of yeast transformants, and the PHB accumulation of the at least one selected strain is the highest” simply expresses the intended result of a process step positively recited.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/MARY A CRUM/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657