DETAILED ACTION
Claims 5, 7, 9, 11, 67 and 71-72 remain withdrawn.
All objections and rejections raised in prior Office Actions are withdrawn unless restated below.
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 .
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.
Claim(s) 1, 16, 17 and 34 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Aux (U.S. 2009/0221041 A1).
Aux, abstract, states:
The presently disclosed subject matter provides a process for starch liquefaction using at least two classes of α-amylase enzymes, wherein the starch hydrolysis pattern from at least two of these classes is different. At least one class of enzyme is provided to the liquefaction process in the form of transgenic plant material expressing at least one class of a-amylase enzyme or is provided in the form of a purified or partially-purified α-amylase enzyme preparation. The second or subsequent class(es) of α-amylase enzymes may be provided in the form of additional transgenic plant material expressing the second or subsequent class(es), or may be provided in the form of a second or subsequent purified or partially-purified α-amylase enzyme preparation.
Aux, in the claims, states:
21. A method for producing a biofuel comprising:
a) liquefying an aqueous slurry of starch-containing plant material in the presence of at least a first and a second class of α-amylase enzymes, wherein the first class of α-amylase enzymes exhibits a starch hydrolysis pattern that is different from the starch hydrolysis pattern of at least the second class of α-amylase enzymes to obtain a liquefact; and,
b) fermenting the liquefact to produce said biofuel.
26. The method of claim 21, wherein the fermentation is a yeast fermentation.
“In various embodiments of the present invention, either the first or the second class of α-amylase enzymes, or both, is provided as a crude, purified or partially-purified preparation of the α-amylase enzyme. The exogenously-added α-amylase enzyme may be de novo synthesized, or may be isolated from an organism expressing the α-amylase enzyme prior to addition of the enzyme to the starch-containing plant material, or may be through the addition of a crude extract containing at least one enzyme useful in starch conversion.” Aux, para. [0028].
“Alpha-amylase enzymes can be expressed in and isolated from any number of eukaryotic and prokaryotic organisms. Appropriate expression cassettes, vectors, transformation, and transfection techniques for a particular organism of interest will be evident to one of skill in the art.” Aux, para. [0030].
“In another embodiment, fungal hosts, such as fungal host cells belonging to the genera Aspergillus, Rhizopus, Trichoderma, Nurerospora, Mucor, Penicillium, etc., such as yeast belonging to the genera Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Schwanniomyces, etc. may be used.” Aux, para. [0032].
“Additional methods for generating an enzyme extract are described in [several references cited].” Aux, para. [0034].
“[L]iquefying an aqueous slurry [i.e. a liquefaction medium] of starch-containing plant material in the presence of at least a first and a second class of α-amylase enzymes, wherein the first class of α-amylase enzymes exhibits a starch hydrolysis pattern that is different from the starch hydrolysis pattern of at least the second class of α-amylase enzymes to obtain a liquefact [i.e. a fermentation medium],” is considered to be a disclosure of liquefying a liquefaction medium to obtain a fermentation medium as recited in claim 1. Aux, para. [0005], claim 21. “[F]ermenting the liquefact [i.e. fermentation medium] to produce said biofuel,” is considered to be a disclosure fermenting the fermentation medium with a fermenting to yeast (as recited in claim 26 of Aux) to obtain ethanol as a fermentation product.
Regarding recitation that the liquefaction medium contains a first inactivated yeast product made from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme, Aux as discussed above provides that “In various embodiments of the present invention, either the first or the second class of α-amylase enzymes, or both, is provided as a crude, and that further such alpha-amylase can be expressed in and isolated from any number of eukaryotic and prokaryotic organisms including yeasts.” Aux, para. [0028].
The above is considered to be an anticipatory disclosure of forming a crude enzyme extract from a recombinant yeast expressing a vector encoding an appropriate alpha-amylase and adding the same to the “aqueous slurry of starch-containing plant material” (i.e. liquefaction medium) as set forth in claim 21 of Aux wherein such a crude enzyme extract prepared from a yeast host cell is a first inactivated yeast product from a first recombinant yeast host cell, wherein the first recombinant yeast host cell comprises a first heterologous nucleic acid molecule for expressing a first heterologous enzyme and the first inactivated yeast product comprises the first heterologous enzyme being an alpha-amylase. More specifically, a crude enzyme extract from a recombinant yeast cell is within the broadest reasonable interpretation of a yeast extract.
Regarding recitation of the claims of “improve the yield of the fermentation product,” “In one embodiment, the liquefact is further processed to produce ethanol. In one embodiment, the use of at least two different classes of α-amylase enzymes in the liquefaction process results in a substrate that leads to higher ethanol yields compared to the ethanol yield from starch-containing plant material that is exposed to only one class of α-amylase enzymes.” Aux, para. [0043]. That is, Aux indicates that performing liquefaction increases product yield as compared to not performing any step of liquefaction.
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.
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.
Claim(s) 1, 2, 6, 14, 16, 17, 18 and 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Aux (U.S. 2009/0221041 A1) as applied to claims 1, 16, 17 and 34 above, and further in view of Callen et al. (U.S. 2003/0125534 A1), Balasundaram et al. (Advances in product release strategies and impact on bioprocess design, Trends Biotechnol. 27, 2009, 477-85) and Schuster et al. (Protein expression in yeast, J. Biotechnol. 84, 2000, 237-48).
Regarding specific identity of alpha-amylase as recited in claim 18, Aux states: “Other amylases such as BD1 2870, Thermococcales derived amylases, unimodal or bimodal amylases may be combined essentially as described in Example 3 to show a synergistic dual mode of action benefit.” Aux, para. [0131]. “Alpha-amylases for example, such as those described in US Patent Publication US2003/0125534 [i.e. Callen] . . . may be further characterized in regards to their respective starch hydrolysis pattern and be further employed in starch hydrolysis as described herein.”
Callen, abstract, states:
The invention relates to alpha amylases and to polynucleotides encoding the alpha amylases. In addition methods of designing new alpha amylases and methods of use thereof are also provided. The alpha amylases have increased activity and stability at acidic, neutral and alkaline pH and increased temperature.
The alpha-amylases of Callen are expressly taught to be suitable for “particularly useful in corn-wet milling processes, detergents, baking processes, beverages and in oilfields (fuel ethanol), and also for liquefaction of starch. Callen, paras. [0003] and [0051].
“In yet another aspect, the invention provides an isolated nucleic acid encoding a polypeptide having a sequence as set forth in SEQ ID Nos.: 2, 4, 6, 10, 12, 14 .. . . 74. . . 80” Callen, para. [0018]. “Another aspect of the invention is an isolated nucleic acid encoding a polypeptide or a functional fragment thereof having a sequence as set forth in SEQ ID Nos.: 2, 4, 6, 10, 12, 14 . . . 74 . . . 80 (hereinafter referred to as “Group B amino acid sequences”), and sequences substantially identical thereto.” Callen, para. [0019].
“The polypeptides of Group B amino acid sequences, and sequences substantially identical thereto or fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof, may also be used in the liquefaction and saccharification of starch. . . . In a preferred embodiment, the polypeptides or fragments thereof of this invention are thermostable at 90-95° C.” Callen, para. [0244].
SEQ ID NO: 74 of Callen has about 99% identity to recited SEQ ID NO: 63 as to be a variant of the same.
“Polynucleotides selected and isolated as hereinabove described are introduced into a suitable host cell. A suitable host cell is any cell which is capable of promoting recombination and/or reductive reassortment. The selected polynucleotides are preferably already in a vector which includes appropriate control sequences. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast.” Callen, para. [0127].
“Another aspect of the invention is a method of making a polypeptide having a sequence as set forth in Group B amino acid sequences, and sequences substantially identical thereto. The method includes introducing a nucleic acid encoding the polypeptide into a host cell, wherein the nucleic acid is operably linked to a promoter, and culturing the host cell under conditions that allow expression of the nucleic acid.” Callen, para. [0025]
Callen, in the claims, further provides:
106. A host cell as claimed in claim 47, 102, 103 or 105, wherein the host is selected from the group consisting of prokaryotes, eukaryotes, funguses, yeasts, plants and metabolically rich hosts.
As such, Aux teaches that the alpha-amylases taught by Callen should be used in the methods of Aux such that at the time of filing an ordinarily skilled artisan would have been motivated to do the same.
As set forth above, the features of at least claim 1 are considered to be anticipated by Aux. However, Aux and Callen do not directly illustrate working embodiments of an alpha-amylase expressed in a yeast host cell and such alpha-amylase applied to a liquefying step.
Balasundaram, page 477-78, teaches: “S. cerevisiae and E. coli are widely used production
hosts for a variety of recombinant proteins, although frequently the products of interest are not secreted into the culture medium, which would be ideal, but are instead retained at various locations within the cell, as shown in Figure 1.” “Mechanical methods include bead mills, high-pressure homogenizers and cavitation, which can be generated through either sonication or fluid flow.” Balasundaram, pages 478-49. Figure 1 of Balasundaram, Fig. 1, in part shows:
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As such, Balasundaram teaches that it is established in the prior art to produce alpha-amylase in S. cerevisiae (yeast cell) in a manner that the alpha-amylase is at least associated with the cell wall such that bead milling is required to recover the enzyme. If the recombinant protein, such as alpha-amylase, is associated with a cell-wall of a yeast cell, then the nucleic acid encoding the same necessarily allows for expression of encoded alpha-amylase in association with a cell membrane, which is understood as including the cell wall.
Again, neither Aux nor Callen teach specific methodology of expressing an alpha-amylase as taught therein (e.g. SEQ ID NO: 74 of Callen). However, Balasundaram teaches that it is known in the prior art to express alpha-amylases a cell-wall bound product requiring physical disruption to recover the alpha-amylase, e.g. bead mill, homogenization . Although Balasundaram does not teach expression of specific thermophilic alpha-amylases, one having ordinarily skill in the art would have recognized the methods of Balasundaram as having general applicability. Again, Aux directly states that S. cerevisiae (i.e. Saccharomyces) can be employed as a host cell for expressing alpha-amylase and that a crude enzyme extract can be employed in the methods of Aux. As such, after bead milling of a recombinant yeast cell to recover alpha-amylase, Aux instructs to the ordinarily skilled artisan that the alpha-amylase need not be further purified or processed, wherein the product of bead milling a recombinant yeast is within the broadest reasonable interpretation of a yeast extract. As such, at the time of filing an ordinarily skilled artisan would have been motivated to express an appropriate alpha-amylase, e.g. SEQ ID NO: 74 of Callen, in a S. cerevisiae host cell in a manner to be cell-wall associated and recovering such alpha-amylase for application to liquefaction methods of Aux by bead milling the same to produce a yeast extract containing the alpha-amylase, since Aux directly teaches that alpha-amylase can be recombinantly expressed in S. cerevisiae and Balasundaram teaches well-understood methods of producing alpha-amylase in S. cerevisiae.
Regarding claim 6, Balasundaram suggests a recombinant alpha-amylase as a cell wall-bound product. However, Fig. 1 of Balasundaram illustrates cell products (i.e. recombinant proteins) can also be expressed in the cytoplasm of a yeast cell, which also requires release for the recovery of the product such as bead milling. While Balasundaram does not specifically illustrate cytoplasmic expression of an alpha-amylase, in the absence of any particular unexpected result, an ordinarily skilled artisan at the time of filing would have been motivated to express an appropriate alpha-amylase in a yeast host cell by any appropriate means since any such means will result in production of alpha-amylase. Schuster teaches cytoplasmic yeast expression systems for S. cerevisiae (i.e. heterologous nucleic acid allows for intracellular expression, see abstract) and notes that “high mannose glycosylation typical for yeast secreted proteins may require deglycosylation in order to unmask the functionally important domains. To direct the recombinant proteins into the cytoplasm is an alternative avoiding high glycosylation at the cost of lower yields.” Schuster, page 246, left col. As such, Schuster teaches that intracellular expression may increase protein yields and reduce other difficulties associated with glycosylation. Again, Aux teaches that a crude extract can be employed such that purification of the enzyme (which may be assisted by secreted expression) is not required for the methods of Aux. Regardless, an ordinarily skilled artisan would have been motivated to employ either an intracellular or secreted expression of an appropriate alpha-amylase (for example, as taught by Callen) in yeast (e.g. S. cerevisiae) with an expectation of being able to produce the needed alpha-amylase enzyme. Again, both Aux and Callen teach the appropriateness of recombinant expression of an alpha-amylase in a yeast host cell wherein such expression must be carried out by known methodology. Balasundaram makes clear that when the alpha-amylase is cell-associated, mechanical disruption such as bead milling is required which necessarily produces a yeast extract (as recited in claims 2 and 3) wherein Aux teaches that such crude enzyme extracts can be used directly in the liquefaction step of Aux.
Claim(s) 1, 16, 17, 31 and 32 and 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Aux (U.S. 2009/0221041 A1) as applied to claims 1, 16, 17 and 34 above, and further in view of McBride et al. (WO 2014/035458 A1) (previously cited).
Regarding claims 31 and 32, McBride, abstract, further relates to ethanol fermentation using S. cerevisiae. McBride, para. [0032] states: “In some embodiments of the invention, the recombinant yeast host cell comprising at least one saccharolytic enzyme further comprises a deletion or alteration of one or more glycerol producing enzymes. In some embodiments, the recombinant yeast host cell further comprises a deletion or alteration of GPD1.” “Because glycerol is a major by-product of anaerobic production of ethanol, many efforts have been made to delete cellular production of glycerol.” McBride, para. [0351].
McBride is directed towards fermentations of lignocellulosic hydrolysates. Regardless, the discussion in McBride of “glycerol is a major by-product of anaerobic production of ethanol” is the case regardless of the specific source of fermentable sugars fermented to ethanol. Since in methods of Aux, as in McBride, ethanol and not glycerol is the desired product, an ordinarily skilled artisan at the time of filing would have been motivate to utilize a S. cerevisiae having genetic modification for reducing production of GDP1 (a native enzyme that functions to produce glycerol) as taught by McBride in order to achieve the benefit of reduced glycerol production that can be coproduced in ethanol fermentation with S. cerevisiae.
Claim(s) 1, 2, 6, 14, 16, 17, 18, 31, 32 and 34 (all non-withdrawn claims) is/are rejected under 35 U.S.C. 103 as being unpatentable over Aux (U.S. 2009/0221041 A1), Callen et al. (U.S. 2003/0125534 A1), Balasundaram et al. (Advances in product release strategies and impact on bioprocess design, Trends Biotechnol. 27, 2009, 477-85) and Schuster et al. (Protein expression in yeast, J. Biotechnol. 84, 2000, 237-48) as applied to claims 1, 2, 6, 14, 16, 17, 18 and 34 above, and further in view of McBride et al. (WO 2014/035458 A1).
Regarding claims 31 and 32, McBride, abstract, further relates to ethanol fermentation using S. cerevisiae. McBride, para. [0032] states: “In some embodiments of the invention, the recombinant yeast host cell comprising at least one saccharolytic enzyme further comprises a deletion or alteration of one or more glycerol producing enzymes. In some embodiments, the recombinant yeast host cell further comprises a deletion or alteration of GPD1.” “Because glycerol is a major by-product of anaerobic production of ethanol, many efforts have been made to delete cellular production of glycerol.” McBride, para. [0351].
McBride is directed towards fermentations of lignocellulosic hydrolysates. Regardless, the discussion in McBride of “glycerol is a major by-product of anaerobic production of ethanol” is the case regardless of the specific source of fermentable sugars fermented to ethanol. Since in methods of Aux, as in McBride, ethanol and not glycerol is the desired product, an ordinarily skilled artisan at the time of filing would have been motivate to utilize a S. cerevisiae having genetic modification for reducing production of GDP1 (a native enzyme that functions to produce glycerol) as taught by McBride in order to achieve the benefit of reduced glycerol production that can be coproduced in ethanol fermentation with S. cerevisiae.
Response to arguments
Headman and Eksteen are no longer relied upon. Regardless, that addition of an appropriate alpha-amylase enzyme to a liquefaction would be expected to result in some degree of liquefying starch, which is all that is required by claim 1. There is no technical reason why such an alpha-amylase present in a crude extract prepared from a yeast or purified would prevent activity of the alpha-amylase to accomplish liquefying in either scenario. Aux as cited expressly teaches that a crude enzyme can be used for liquefaction.
Applicant argues:
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The claims directly recite use of an enzyme in “secreted form” (see end of claim 6).
Applicant argues:
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The burden is on applicant to establish that results are unexpected and significant. MPEP 716.02(b). "[A]ppellants have the burden of explaining the data in any declaration they proffer as evidence of non-obviousness." Ex parte Ishizaka, 24 USPQ2d 1621, 1624 (Bd. Pat. App. & Inter. 1992); MPEP 716.02(b). “An affidavit or declaration under 37 CFR 1.132 [or data from the specification] must compare the claimed subject matter with the closest prior art to be effective to rebut a prima facie case of obviousness.” MPEP 716.02(e). "Expected beneficial results are evidence of obviousness of a claimed invention, just as unexpected results are evidence of unobviousness thereof." In re Gershon, 372 F.2d 535, 538, 152 USPQ 602, 604 (CCPA 1967); MPEP 716.02(c)(II). “Whether the unexpected results are the result of unexpectedly improved results or a property not taught by the prior art, the ‘objective evidence of nonobviousness must be commensurate in scope with the claims which the evidence is offered to support.’" MPEP 716.02(d).
None of the claims are limited to adding an alpha amylase. For example, addition of a protease would not be expected to have any particular unexpected results based on the present record. Further, Example III, and Figure 11 describes all examples including a commercial enzyme, which is not recited in any of the claims. For this reason, the examples are not commensurate in scope with the claims for any unexpected results. Further, in is noted that in Figure 11 the lowest Torque was seen with “Commercial Enzyme #1” such that the specification demonstrates that embodiments of the claims are inferior to use of “Commercial Enzyme #1.” It is noted that self-manufacture of an alpha-amylase enzyme being less expensive that buying a commercial product is not in itself an unexpected result.
The non-statutory double patenting rejections in the prior Office Action have been reevaluated. U.S. 9,206,444; 11,332,728; 16/493,245; 11,198,881; and 11,814,629 relate to yeast cells expressing a glucoamylase or protease. While the claims encompass a glucoamylase or protease, it is generally understood in the prior art that an alpha-amylase is required for liquefaction and a glucoamylase is employed for a subsequence saccharification. See Fig. 2 of Zyl et al. (Engineering yeasts for raw starch conversion, Appl. Microbiol. Biotechnol. 95, 2012, 1377-88), reproduced in part below:
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As such, there is not considered sufficient motivation to modify the yeast cells of the reference claims to further express an alpha-amylase and apply the same to liquefaction (for example, as taught by Aux) wherein at least an alpha-amylase is understood in the prior art as needed for liquefaction and glucoamylase is not critically necessary. In 16/493,245, claims have been amended to be limited to a method of making a food product.
Conclusion
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/TODD M EPSTEIN/Primary Examiner, Art Unit 1652