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
The instant application is a CON of PCT/US20/19079, filed February 20, 2020, with provisional applications 62/807969 filed 2/20/2019 and 62/961351 filed 1/15/2020. Applicant’s Amendment filed January 21, 2026 is acknowledged.
Claims 1-5, and 9 are canceled, and claims 30-32 are newly added. Claims 6, 10, 12, and 15 are amended. Currently claims 6-8 and 10-32 are pending, wherein claims 16-24 are withdrawn.
Claim Objections
Claims 6 and 31 are objected to because of the following informalities:
Claim 6, line 7, change ‘yeast host cell’ to ‘fungal host cell’ to maintain consistency in claim language in line 1.
Claim 31, delete ‘from’ in line 2 due to convoluted claim language.
Appropriate correction is required.
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 6-8, 10-11, 25-26, and 30 are rejected under 35 U.S.C. 103 as being unpatentable over by Wehrs et al. (Microb Cell Fact (2018) 17:193, pgs. 1-11, cited in IDS filed 1/18/2023) in view of Xue et al. (CRITICAL REVIEWS IN BIOTECHNOLOGY 2018, VOL. 38, NO. 7, 1049–1060, cited in PTO-892 mailed 7/17/2024, hereinafter “Xue”).
Regarding claim 6, Wehrs teaches a genetically modified Saccharomyces cerevisiae host cell capable of producing indigoidine, wherein the host cell comprises a non-ribosomal peptide synthetase (NRPS) and a 4’-phosphopantetheinyl transferase (PPTase) that converts glutamine to indigoidine (abstract, pg. 3, col. 1, para 2). Wehrs teaches the engineered S. cerevisiae were grown under respiratory cell growth via carbon depletion through controlled feeding, and produced a maximum titer of 980 mg/L of indigoidine (abstract, pg. 6, col. 2, para 2). Both heterologous genes (NRPS and PPTase) were codon optimized to be expressed in the host cell and under control of the TDH3 promoter (pg. 8, col. 1, para 2).
Wehrs does not teach the host cell is oleaginous yeast.
However, Xue teaches fatty acid biosynthesis in oleaginous yeasts, which have many advantages over many other microorganisms, such as their duplication time is short (less than 1 h), they can attain high cell mass in the culture (higher than 20 g/l) and high lipid content (higher than 50% based on their dry cell weight), they can be easily modified by genetic engineering and use a wide range of substrates and a large scale fermentation using them can be easily carried out (pg. 1049, col. 2, para 1). Xue teaches in yeast, fatty acids are produced by fatty acid synthase (FAs) which comprise enzymes (such as malonyl-CoA:ACP transacylase (MAT)), that must be activated by a phosphopantetheinyl transferase (PPTase) subunit (pg. 1051, col. 2, para 2). Xue teaches yeast FAs can be autoactivated by its own PPTase in the presence of free CoA (pg. 1056, col. 1, para 2). Xue teaches similar to Saccharomyces cerevisiae, which is not an oleaginous yeast, the Fas in the oleaginous yeasts Y. lipolytica, A. melanogenum P5, C. albidus, L. starkeyi, and Trichosporon oleaginosus also have the two subunits: the b-subunit (Fas1), which contains MPT, DH, ER, and AT domains and the a-subunit (Fas2) includes PPTase, KS, KR, and acyl carrier protein (ACP) domains (pg. 1052, col. 1, para 2). Xue teaches that a conserved serine residue of an inactive form of the ACP must undergo the posttranslational modification by the covalent attachment of a 4’-phosphopantetheine (P-pant) moiety of coenzyme A to the conserved serine residue under the catalysis of the PPTase, resulting in conversion to the active form during the biosynthesis of various primary metabolites including general FAs, and lysine and some secondary metabolites such as HR-PKS associated fatty acids, melanin, and non-ribosomally synthesized peptides (NRPS), the same reaction described by Wehrs of sfp activating BpsA (pg. 1056, col. 1, para 1).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to replace the S. cerevisiae yeast host cell taught by Wehrs with an oleaginous host cell as taught by Xue. One of ordinary skill in the art would have been motivated to replace the S. cerevisiae host cell with an oleaginous yeast host cell to advantageously produce indigoidine due to oleaginous yeast having many advantages over other microorganisms, such as their short duplication time and high cell mass in culture, and can also be easily modified by genetic engineering, use a wide range of substrates, and can be easily cultivated in large-scale fermentation as taught by Xue (pg. 1049, col. 2, para 1).
Regarding claims 7-8, Xue teaches many oleaginous yeasts accumulate lipids to levels corresponding to 50% of their cell dry weight; furthermore, under conditions of nitrogen limitation, some oleaginous yeasts such as Rhodosporidium sphaerocarpum, Rhodosporidium toruloides, and Rhodosporidium fluvialis may accumulate lipids to levels exceeding 70% of their cell dry weight (pg. 1050, col. 1, para 2). Xue teaches the yeast strain 2F5 of R. toruloides could produce higher amount of lipid from inulin and larger lipid particles in its cells than any other yeast strain tested in this study (pg. 1050, col. 2, para 3). Xue teaches R. toruloides and R. glutinis both have two ACPs in the α-subunit of Fas2 (Figure 3), which ACP is a key player in interactive substrate shuttling, and the authors suggest the presence of tandem ACPs may improve the efficacy of fatty acid biosynthesis by providing a higher intermediate concentration in the reaction chamber of the Fas, leading to enhanced lipid biosynthesis (pg. 1052, col. 1, para 1).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to replace the S. cerevisiae yeast host cell taught by Wehrs with R. toruloides yeast as taught by Xue. One of ordinary skill in the art would have been motivated to substitute R. toruloides as a host cell, due to their unique Fas2 subunits having two ACPs which would increase fatty acid synthesis, thereby improving cell biosynthesis as taught by Xue.
Regarding claims 6 and 25-26, although Wehrs does not teach the genetically modified fungal host cell is capable of producing indigoidine with a yield of 2900-19,500 mg/L (claim 6) or 3700-19,500 mg/L (claim 25) or 16,500-19,500 mg/L (claim 26), “where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977).”. Thus, an engineered R. toluroides comprising a bacterial NRPS (BpsA) and a Bacillus subtilis PPTase sfp, as taught by Wehrs and Xue, would inherently be capable of producing the recited indigoidine yields as claimed.
Regarding claims 10-11 and 30, Wehrs teaches the genetically modified fungal host cell comprising the NRPS is heterologous to the host cell, specifically, using the bacterial blue pigment synthetase (BpsA) from Streptomyces lavendulae (claims 10-11) and integrate a 4’-phosphopantetheinyl transferase (PPTase), needed to activate the apo-NRPS into its holo-form via the addition of a coenzyme A-derived phosphopantetheine moiety, that is a sfp PPTase from Bacillus subtilis (claim 30) (pg. 2, col. 2, para 2; pg. 3, col. 1, para 2).
Claims 12-14 and 27-29 are rejected under 35 U.S.C. 103 as being unpatentable over Wehrs in view of Xue as applied to claims 6-8, 10-11, 25-26, and 30 above, and further in view of Tao (US 20160168619 A1, cited in PTO-892 mailed 7/17/2024).
Wehrs does not explicitly teach the NRPS/BpsA has an amino acid sequence at least 90%, 95%, 99%, or 100% identical to SEQ ID NO: 1. Wehrs also does not teach the NRPS comprises SEQ ID NO:2 at positions 937-1012 of SEQ ID NO:1, nor that the NRPS comprises SEQ ID NO: 3 at position 1005-1012 or SEQ ID NO: 4 at position 1005-1025 of SEQ ID NO:1 (a coiled coil structure).
However, Tao teaches a colorimetric assay for L-glutamine for analyzing cell cultures that converts the L-glutamine to indigoidine (abstract). Tao teaches BpsA from S. lavendulae was expressed by transforming E. coli with a plasmid comprising the BpsA gene [0024]. Tao teaches the BpsA (SEQ ID NO: 4) used in the method has an amino acid sequence with 100% identity to instant SEQ ID NO: 1 (See sequence comparison below). Likewise, Tao teaches the conserved region of instant SEQ ID NO: 2 is also located in Tao’s SEQ ID NO: 4 (See highlighted area in sequence comparison below). Tao teaches SEQ ID NO: 4 also comprises instant SEQ ID NO: 3 at position 1005-012 and instant SEQ ID NO: 4 at position 1005-1025 (See highlighted area in sequence comparison below). Tao teaches the assay consists of an indigoidine synthetase (BpsA), adenosine triphosphate (ATP), Mg2+ and L-glutamine (para 3). Tao found the measured concentrations of L-glutamine based on the production of indigoidine were highly consistent with the actual concentrations based on the standard curve developed (para 47).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to replace the NRPS/BpsA taught by Wehrs with the NRPS/BpsA having an amino acid sequence identical to the SEQ ID NO:4, as taught by Tao, in the genetically engineered fungal host cell taught by Wehrs, as modified by Xue. One of ordinary skill in the art would have been motivated to use Tao’s SEQ ID NO:4 based on Tao’s successful and consistent results, which would advantageously convert glutamine to indigoidine effectively.
Sequence comparison of Tao’s SEQ ID NO: 4 & instant SEQ ID NO: 1 (highlighted portions of instant SEQ ID NO’s: 2 (red), 3 (green), & 4 (yellow)).
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Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over Wehrs in view of Xue and Tao as applied to claims 12-14 and 27-29 above, and further in view of Takahashi et al. (THE JOURNAL OF BIOLOGICAL CHEMISTRY, 2007, VOL. 282, NO. 12, pp. 9073–9081, cited in PTO-892 mailed 7/17/2024, hereinafter “Takahashi”), as evidenced by PubChem (O-(pantetheine-4'-phosphoryl)serine | C14H28N3O9PS | CID 57339273, [retrieved on 2025-02-10]. Retrieved from the internet: <URL: pubchem.ncbi.nlm.nih.gov/compound/O-_pantetheine-4_-phosphoryl_serine)>, cited in PTO-892 mailed 2/14/2025).
Wehrs teaches the 4’-phosphopantetheinyl transferase (PPTase) is needed to activate the apo-NRPS into its holo-form via the addition of a coenzyme A-derived phosphopantetheine moiety (pg. 2, col. 2, para 2; pg. 3, col. 1, para 2).
Neither Wehrs nor Tao teach the serine at position 972 of instant SEQ ID NO: 1 is an O-(pantetheine 4’-phosphoryl) serine.
However, Takahashi teaches cloning and characterization of blue pigment synthetase A (BPSA), which is a single module type NRPS (abstract). At position 972 in the T-domain of BPSA is a 4’-phosphopantetheinylated serine with Svp (pg. 9079, col. 2, para 1). Svp is a PPTase enzyme from S. verticilus and the 4’-phosphopantetheinyl transferase (PPTase) transfers the 4'-phosphopantetheine group (pg. 9077, col. 2, para 2). As evidenced by Pubchem, O-(pantetheine-4'-phosphoryl)serine is a synonym for 4'-phosphopanthetheine-serine (pg. 1, “Synonyms”). Takahashi teaches during MS analysis, a peak corresponding to a peptide with Ser972 containing the carbamidemethylated 4’-phosphopantetheinyl residue, was used as a precursor ion for the MS/MS analysis (pg. 9076, col. 2, para 1).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to replace the NRPS/BpsA taught by Wehrs with the NRPS/BpsA having an amino acid sequence identical to the SEQ ID NO:4, as taught by Tao, and modify the NRPS/BpsA sequence by further replacing the serine at position 972 of Tao’s sequence with an O-(pantetheine-4'-phosphoryl)serine as taught by Takahashi, which would confirm the NRPS as being 4’-phosphopantetheinylated by the PPTase as suggested by Takahashi in the genetically engineered fungal host cell taught by Wehrs, as modified by Xue. One of ordinary skill in the art would have been motivated to utilize well-known and common laboratory practices in verifying successful enzyme transformation by altering to a 4’-phosphopantetheinylated serine at position 972 in the T-domain of the NRPS as taught by Takahashi.
Claims 31-32 are rejected under 35 U.S.C. 103 as being unpatentable over Wehrs in view of Xue as applied to claims 6-8, 10-11, 25-26, and 30 above, and further in view of Castaneda et al. (Journal of Biotechnology 280 (2018) pgs. 11–18, hereinafter “Castaneda”).
As described above in the 103 rejection, Wehrs teaches indigoidine production by transforming a yeast with BpsA and sfp, and teaches the engineered S. cerevisiae was grown under respiratory cell growth phase via carbon depletion through controlled feeding, and produced a maximum titer of 980 mg/L of indigoidine (abstract, pg. 6, col. 2, para 2). Wehrs hypothesized that efficient formation of indigoidine as a product of the TCA cycle takes place predominantly during the respiratory metabolic state and not during fermentative growth, and using glucose as a carbon source caused a delay in visible pigmentation but increased growth rate of the colonies as compared to glycerol., because glycerol is a non-fermentable carbon source, cells are required to shift into respiratory metabolic state, which leads to a decrease in growth rate but an increased flux through the TCA cycle (pg. 3, col. 2, para 2). When Wehrs tested excess sugar starting sugar concentrations, there was a 48 hour delay of indigoidine production, this absence of pigment production in cultures with high initial sugar concentrations could originate from nitrogen limitations of these cultures at later stages of their growth, which is in agreement with results from observations made by Brown and Johnson when analyzing the effect of sugar concentrations on cell yield and metabolites of S. cerevisiae cultures (pg. 5, col. 1, para 1). Xue also teaches high lipid accumulation in the oleaginous yeasts is obtained under limiting nitrogen concentration conditions, when the oleaginous yeasts are grown in the nitrogen limitation medium, the activity of the AMP deaminase is high, leading to decreases in the cellular content of AMP and the activity of isocitrate dehydrogenase (a component of the TCA cycle that is dependent on the presence of AMP (pg. 1054, col. 2, para 5). As a result, citric acid (CA) accumulates in mitochondria and is finally exported into the cytosol for FA biosynthesis (Figure 2), suggesting that the mitochondria of the oleaginous yeasts can synthesize and accumulate more CA than those of nonoleaginous yeasts, leading to enhanced secretion of CA into the cytosol for FA biosynthesis (pg. 1054, col. 2, para 5).
Neither Wehrs nor Xue teach the yeast host cell is grown in a medium having a carbon to nitrogen (C/N) ratio of 4-40.
However, Castaneda teaches comprehensive analysis of a metabolic model for lipid production in R. toruloides (title). Castaneda teaches the nitrogen source incorporated from the culture medium to support cell growth leads to three key compounds: ammonium (NH4+), glutamate (glut) and glutamine (glum), and are linked by a glutamine synthetase (GLN1) that synthesizes glutamine by ammonia and glutamate (pg. 12, sec. 2.2.1). Castaneda teaches with glucose as the sole carbon and energy source, a flux distribution for optimal growth (case 1) is shown in Fig. 3A, and as expected, the fluxes through glycolysis and TCA cycle were high, showing these pathways, coupled to the respiratory chain, provide the ATP needed for cell mass synthesis; in addition, due to the incorporation of nitrogen precursors in the cell mass reaction, the central nitrogen metabolism was unblocked resulting in a net flux through ammonium transport reaction (NH4) and the anabolic reactions GDH1 and GLN1 (pg. 15). Castaneda suggests this model will be used to simulate the simultaneous cell mass and TAG production under different C/N ratios (pg. 17). Thus, it would be obvious to skew the metabolic pathway towards anabolic reactions during cell mass synthesis to increase glutamine production, and thus result in high levels of indigoidine.
Therefore, 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 change the medium and grow the genetically engineered yeast cell that produces indigoidine taught by Wehrs, with the substituted yeast host cell that is an oleaginous host cell taught by Xue, in a medium with varying ratios of C/N that would affect the R. toluroides metabolic pathways as taught by Castaneda. One of ordinary skill in the art would have been motivated to adjust the C/N ratio as low as 4, since nitrogen limitation affects growth as disclosed by Wehrs, and is important in skewing towards the cell growth respiratory state with anabolic reactions, producing higher levels of glutamine, that can then be reacted with the NRPS and PPTase to result in high indigoidine levels.
Response to Arguments
Applicant’s arguments with respect to claims 1, 3-15, and 25-29 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/JESSICA EDWARDS/
Examiner, Art Unit 1657
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