DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Amendments
2. The amendment filed August 24, 2026 has been amended. Claim 8 was canceled. Claim 16 was newly added. Claims 1 and 9-10 were currently amended. Claims 3a, 3c, 3d, 4, 6-7 and 11-15 are withdrawn from consideration. Claims 1-2, 3b, 5, 9-10 and 16 are under consideration in this Office Action.
Maintained Grounds of Rejection
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 nonobviousness.
3. Claims 1-2, 3b, 5, 9-10 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Cherry et al., (US 20160115490 published 2016-04-28; priority to 2014-06-18) and Bongiorni (WO2010144283 published Dec 2010; priority to June 2009) in view of Ogura et al, (J Bacteriol. 2014 Feb;196(4):873–881).
The claims are drawn to a modified Bacillus host cell comprising: i) a mutation which reduces the amount of exopolymeric substances (EPS) and/or a mutation which reduces the amount of the biofilm extracellular matrix component TasA as compared to a control cell, and ii) a genetic modification which increases the amount of phosphorylated DegU as compared to a control cell wherein said genetic modification is a) a degU32 mutation and/or b) the overexpression of the degQ gene.
Cherry et al., teach Bacillus mutants having improved transformation efficiency, comprising a disruption of an endogenous epsA-O operon and methods for producing a polypeptide or fermentation product using the mutants [abstract]. Cherry et al., teach the mutant comprises a disruption of an endogenous epsA-O operon [para 14]. The Bacillus mutant strains may be constructed by gene deletion techniques to eliminate or reduce expression of the epsA-O operon coding sequences. Gene deletion techniques enable the partial or complete removal of the operon thereby eliminating expression. In such methods, deletion of the operon is accomplished by homologous recombination using one or more plasmids that have been constructed to contiguously contain the 5′ and 3′ regions flanking the genes [para 186]. The term “disruption” means that a coding region and/or control sequence of a referenced gene is partially or entirely modified (such as by deletion, insertion, and/or substitution of one or more nucleotides) resulting in the absence (inactivation) or decrease in expression, and/or the absence or decrease of enzyme activity of the encoded polypeptide [para 23]. Thus teaching claim 1-2.
The term “epsA-O operon” means a fifteen-gene operon known in Bacillus cells to be involved in exopolymeric substance (EPS) biosynthesis, modification, and export [para 24]. The term “disruption of an endogenous epsA-O operon” means a disruption resulting in the absence or decrease in expression of at least one coding sequence of the epsA-O operon, and/or the absence or decrease of enzyme activity of at least one encoded polypeptide of the epsA-O operon. Non-limiting examples of a disruption of an endogenous epsA-O operon include disruption of an operon promoter, and/or disruption of one or more (e.g., two, three, four, five, six, etc.) of the epsA-O operon coding sequences [para 25]. The disruption of the endogenous epsA-O operon occurs in an endogenous epsA coding sequence. In one aspect, the endogenous epsA coding sequence is inactivated. [para 63]. Thus Cherry et al., teach a mutation that inactivates the epsA-O operon just as instantly claimed by 3b. Therefore, Cherry teach methods for obtaining a Bacillus transformant, comprising transforming a heterologous polynucleotide into a Bacillus mutant, wherein the mutant comprises a disruption of an endogenous epsA-O operon [para 14]. Also described are methods of producing a polypeptide, comprising: (a) cultivating a Bacillus transformant described herein comprising a heterologous polynucleotide encoding the polypeptide; and (b) recovering the polypeptide [para 15]. Therefore, Cherry et al., teach Bacillus host cell comprising: i) a mutation which reduces the amount of exopolymeric substances (EPS) and/or a mutation which reduces the amount of the biofilm extracellular matrix component TasA, but does not teach a mutation which increases the amount of phosphorylated DegU as compared to a control cell wherein said genetic modification is a) a degU32 mutation and/or b) the overexpression of the degQ gene.
Bongiorni teach Bacillus host cells that have been genetically manipulated to have an enhanced capacity to produce proteins of interest. The enhanced production of proteins of interest by the modified Bacillus sp. Host cells is further increased in modified Bacillus sp that have at least one inactivated phr gene [abstract]. The Bacillus host cells that have been genetically manipulated to have an enhanced capacity to produce proteins of interest [para 5]. The at least one inactivated phr gene is chosen from phrA, phrE, phrC, phrF, phrG, phrI and phrK. Preferably, the inactivated phr gene is the inactivated phrA or phrE gene [para 13]. Bongiorni provided a modified Bacillus sp. host cell that comprises a recombinant nucleic acid for producing a protein of interest at a level that is greater than that produced by the unmodified precursor host cell [para 14].
The host cell comprises a mutation in at least one gene chosen from degU, degQ, degS, sco4, spollE, degQ and degR. Preferably, the host cell comprises a deg(Hy)32 mutation [para 30]. The method for producing a protein of interest in a host cell that comprises introducing into a precursor Bacillus sp. host cell an inactivating DNA construct comprising an inactivating polynucleotide that results in the inactivation [para 32]. A preferred Bacillus sp. host is a Bacillus sp. that includes a mutation or deletion in at least one of the following genes, degU, degS, degR and degQ. Preferably the mutation is in a degU gene, and more preferably the mutation is degU(Hy)32. In one embodiment, the host cell is a Bacillus subtilis host cell that carries a degU32(Hy) mutation [para 151]. The degU(Hy) mutation, enhances the production of the by several fold [para 294, See also Figures 10A and B]. Thus teaching claim 1. Example 6 teach Bacillus subtilis host cells that carry the degU(Hy)32 mutation enables the overexpression of the protein of interest [para 290]. Thus teaching claims 9-10.
Ogura et al., describe Bacillus subtilis DegU is a response regulator of the DegS-DegU two-component regulatory system. Phosphorylated DegU (DegU-P) controls many genes and biological processes, such as exoprotease and γ-polyglutamic acid production, in addition to the degU gene, by binding to target gene promoters [Abstract]. Ogura et al., found that SinR repressed degU [Abstract]. The prior art observed decreased expression of several genes in the DegU regulon in the slrR mutant, for example, aprE. Considering the known interaction between SlrR and SinR (Fig. 1). SinR inhibits degU expression and then SlrR titrates SinR, resulting in a positive role for SlrR in degU gene expression [Results]. The extracellular matrices are composed of EPSs synthesized by the gene products of the 15-gene epsA-O operon, TasA protein fibers, and BslA surface layer protein. The SinR repressor is one of the major regulators of the genes required for biofilm formation. SinR binds to the promoters of the epsA-O and tapA-sipW-tasA operons to repress their expression [Introduction]. Therefore, Ogura et al., teach a single Bacillus host cell comprising multiple mutation which reduces the amount of exopolymeric substances (EPS) and a mutation which increases the amount of phosphorylated DegU as compared to a control cell wherein said genetic modification is a) a degU32 mutation and/or b) the overexpression of the degQ gene.
Therefore, it would have been prima facie obvious at the time of applicants’ invention to apply Bongiorni teach DegU(Hy)32 mutation which increases the amount of phosphorylated DegU to Cherry et al’s Bacillus mutants in order to have an enhanced capacity to produce proteins of interest. Furthermore, Ogura et al., teach Bacillus host cells comprising both mutations to reduce the amount of exopolymeric substances (EPS) or reduce the amount of the biofilm extracellular matrix component TasA, and a mutation which increases the amount of phosphorylated DegU. Therefore, one of ordinary skill in the art would have a reasonable expectation of success by incorporating Cherry et al., Bongiorni and Ogura et al., by teaching epsA-O operon, and mutations to DegU and TasA to have improved transformation efficiency, and enhanced production of proteins of interest by the modified Bacillus sp.
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses combining prior art elements according to known methods to yield predictable results, thus the combination is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that "The combination of familiar element according to known methods is likely to be obvious when it does no more than yield predictable results". It is well known to take a Bacillus host cells including mutations which reduces the amount of exopolymeric substances (EPS) and/or mutation which reduces the amount of the biofilm extracellular matrix component TasA, and a mutation which increases the amount of phosphorylated DegU as compared to a control cell; however there is no change in the respective function of the mutated DegU32 and/or DegQ genes or Bacillus host cells, thus the combination would have yielded a reasonable expectation of success along with predictable results to one of ordinary skill in the art at the time of the invention. Thus, it would have been obvious to a person of ordinary skill in the art to combine prior art elements according to known methods that is ready for improvement to yield predictable results. The claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
Response to Arguments
4. Applicant's arguments filed August 24, 2026 have been fully considered but they are not persuasive. The rejection of claims 1-2, 3b, 5, 9-10 and 16 under 35 U.S.C. 103 as being unpatentable over Cherry et al., and Bongiorni in view of Ogura et al., is maintained for reasons of record.
Applicants argue that there is no reason to combine the references and there is no 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, Ogura et al., teach Bacillus host cells comprising both mutations to reduce the amount of exopolymeric substances (EPS) or reduce the amount of the biofilm extracellular matrix component TasA, and a mutation which increases the amount of phosphorylated DegU. Moreover, it would have been prima facie obvious at the time of applicants’ invention to apply Bongiorni teach DegU(Hy)32 mutation which increases the amount of phosphorylated DegU and overexpression of degQ to Cherry et al’s Bacillus mutants in order to have an enhanced capacity to produce proteins of interest.
Accordingly, one of ordinary skill in the art would have a reasonable expectation of success by incorporating Cherry et al., Bongiorni and Ogura et al., by teaching epsA-O operon, and mutations to DegU and TasA to have improved transformation efficiency, and enhanced production of proteins of interest by the modified Bacillus sp. Therefore, Applicants arguments are not found persuasive.
Additionally, Applicants argue that would not have been a reasonable expectation of success regarding the degU32 strain improved expression. In this case, Bongiorni teach Bacillus subtilis host cells that carry the degU(Hy)32 mutation and the SigH construct that enables the overexpression of YmaH protein. Bacillus subtilis host cells that do not carry the degU(Hy)32 mutation and do not carry a construct that enables overexpression of ymaH [para 290]. Bacillus subtilis host cells that do not carry the degU(Hy)32 mutation and do carry a construct that enables overexpression of ymaH [para 290]. Therefore, Bongiorni teach it is reasonable to employ the degU32 mutation and improved expression because the prior art already taught the expectation.
Finally, it would have been obvious to a person of ordinary skill in the art to combine prior art elements according to known methods that is ready for improvement to yield predictable results. The claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary. Therefore, the rejection is maintained contrary to Applicants arguments.
Pertinent Art
5. The prior art made of record and not relied upon is considered pertinent to applicant’s disclosure.
Argianas, Alexander, "Characterization of Exopolysaccharide (EPS) Produced by Bacillus subtilis Mutants" (2015). Master's Theses. 3124.
Cairns et al., (Mol. MIcrobio. 2013 Aug 14;90(1):6-21) teach inhibition of flagellar rotation acts as a mechanical trigger to activate the DegS–DegU two-component signal transduction system. We postulate that inhibition of flagellar rotation could function as a mechanical trigger to activate bacterial signal transduction cascades in many motile bacteria upon contact with a surface.
Daahl et al., ( J of Biol. Chem. Vol.267, No. 20, Issue of July 15, pp. 14509-14514, 1992) teach Two classes of mutations were identified in the degS and degU regulatory genes of Bacillus subtilis, leading either to deficiency of degradative enzyme synthesis (degS or degU mutations) or to a pleiotropic phenotype which includes overproduction of degradative enzymes and the loss of genetic competence (degB(Hy) or degU(Hy) mutations).
Ma et al., (Protein Expression and Purification. 142. Sept 2017) teach the optimization and modification is a useful strategy to improve the homologous overproduction of other extracellular proteins in B. subtilis
Olmos et al., (MoI. Gen. Genet., 253:562-567 [1997]) teach expression is regulated by a complex network of activators and repressors that includes the products of hpr, degU and sinR. In order to understand the effect of these gene products on subtilisin expression, strains carrying combinations of the degU32(Hy), hpr2 and sinR null mutations, were constructed. We found that in all the genetic backgrounds tested, the sinR null mutation decreased aprE expression. Also, by measuring alkaline phosphatase synthesis and the formation of heat-resistant spores, as indicators of sporulation, we found that some of the mutant strains showed alterations in the sporulation process.
Conclusion
6. No claims allowed.
7. 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.
8. Any inquiry concerning this communication or earlier communications from the examiner should be directed to JA-NA A HINES whose telephone number is (571)272-0859. The examiner can normally be reached Monday thru Thursday.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor Peter Paras, can be reached on 571-272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/JANA A HINES/Primary Examiner, Art Unit 1645
