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 Status
2. Claims 1-15 are under consideration in this Office Action.
Election/Restrictions
3. Applicant's election with traverse of Group B in the reply filed on March 30, 2026 is acknowledged. The traversal is on the ground(s) that the cited reference does not teach the technical feature linking the claims. This is not found persuasive because Ogura et al, teach the mutation of degU-lacZ, the introduction of a sinR mutant and the inactivation of 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. Therefore, Ogura et al., the mutated Bacillus with TasA and DegU mutations just as instantly claimed. Thus requirement is still deemed proper and is therefore made FINAL.
Information Disclosure Statement
4. The information disclosure statement (IDS) submitted on June 20, 2024 was filed. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
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.
5. Claims 1-2, 3b, 5 and 8-10 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, and ii) a mutation which increases the amount of phosphorylated DegU as compared to a control cell.
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.
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 genome comprising a rap operon that has an inactivated phrA gene and an inactivated phrE gene, and 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]. Thus teaching claim 8.
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]. 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.
Therefore, it would have been prima facie obvious at the time of applicants’ invention to apply Bongiorni teach 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/or 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 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.
Pertinent Art
6. 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
7. No claims allowed.
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
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