DETAILED ACTION
Acknowledgment and entry of the Amendment submitted on 10/23/25 is made. Claims 1-9 are currently pending.
Applicants’ arguments are rendered moot due to the new grounds of rejection which were necessitated by the amendment to claim 1.
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.
Claim(s) 1-9 is/are rejected under 35 U.S.C. 103 as being unpatentable over Kumpfuller et al (Appl. Microbiol. Biotechnol. 2015, vol. 100, pp. 1209-1220; provided by Applicants), Tsuge et al (1) (2015. Seibutsu-kogaku kaishi. 93(9): 527-529; provided by Applicants) and Tsuge et al (2) (WO 2015/111248; corresponding to US 2017/0009243) in view of Cook et al (Medchemcomm. 2019 Apr 25;10(5):668–681).
Kumpfuller et al indicates that a plasmid including a gene cluster with a length of 30 kb or longer encoding a polyketide synthase derived from Saccharopolyspora erythraea was constructed and expressed in Bacillus subtilis. Kumpfuller also indicates that eryAI, eryAII, and eryAIII were expressed separately in three cassettes each having a promoter and a terminator, and that these have an RBS (abstract, page 1213, right column, last paragraph to page 124, right column, third paragraph, fig. 1).
However, Kumpfuller differs from the invention in claim 1 of the present application in that it does not specify that a DNA construct including a tandem repeat of DNA encoding polyketide synthase is introduced into Bacillus subtilis.
Meanwhile, the Tsuge documents each describe a multiple DNA fragment ligation method using a Bacillus subtilis plasmid transformation system, and indicate that DNA fragments serving as ligation materials are prepared so as to have specific protruding ends of 3-4 bases using a restriction enzyme such as Type IS; a tandem-repeat DNA construct is prepared by ligating the DNA fragments using the complementarity of the protruding ends; and by introducing said construct into Bacillus subtilis, DNA ligated as a circular plasmid DNA is ultimately obtained. Furthermore, it is indicated that the design is such that the length of each DNA fragment is made as equal as possible, by computer simulation, on the basis of the sequence of the target long- chain DNA, and that this facilitates equimolar consistency of the respective DNA fragments. Furthermore, it is indicated that, with said method, it is possible to integrate a large number of DNA fragments, on the order of approximately 50, making it possible to easily construct plasmids including long-chain DNA (Tsuge 2, page 528, right column, last paragraph to page 529, right column, first paragraph; document 3, claims, examples). Since the inventions described in all three cited references relate to the preparation of plasmids including long-chain DNA, in the invention described in Kumpfuller, it would have been prima facie obvious to one of ordinary skill in the art to use the methods described in documents Tsuge 1 & 2 when creating a plasmid including a polyketide synthase gene cluster having a length of 30 kb or more.
Additionally, Cook et al teach leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous host. Cook teaches that there is considerable interest in engineering these enzymes to alter substrate specificity, increasing their potential as a source of novel natural products. However, these large enzymes pose several challenges to manipulating the synthesis of their products. Genetic manipulation of the biosynthetic gene clusters (BGCs) is nontrivial because clusters often range from 20 to 100 kb in size (with some >100 kb), and contain repetitive DNA sequences due to the modular structure of their encoded megaenzymes. The reference recites that, unfortunately, the common heterologous expression workhorse, Escherichia coli, has not proven to be a widely useful host for producing complex natural products including polyketides and non-ribosomal peptides. Therefore, many groups have turned to non-model bacteria for heterologous production.
Section 3 of Cook recites that the ideal heterologous host for producing polyketides and non-ribosomal peptides should have a growth rate suitable for industrial fermentations, a characterized synthetic biology toolbox, and a sufficient supply of precursor metabolites (Fig. 2). Important genomic features include GC content and codon usage similar to the native host, unless the BGC can be codon optimized for the heterologous host. A lack of native BGCs reduces competition for limited metabolite supplies and simplifies downstream purifications. Alternatively, competing BGCs can be deleted from the chromosome using genome editing methods described in this review. Methods for integrating BGCs into the chromosome of heterologous hosts include transposition, phage integration, and homologous recombination. All three of these tools are available.
Despite the advantageous growth characteristics of B. subtilis, it is limited as a heterologous host for polyketides and non-ribosomal peptides because of the low GC content of its chromosome. For example, a refactored pathway for the synthesis of 6-deoxyerythronolide B, the precursor to erythromycin, in B. subtilis resulted in a titer of 2.6 μg L–1, (Krumpfuller) much lower than what is possible in E. coli. Several natural products have been discovered from low-GC bacteria, including other Bacilli and some cyanobacteria. One example of these compounds is a class of antibiotics called polymyxins. These compounds are bactericidal towards Gram-negative bacteria, and their primary mode of action is increasing cell wall permeability. Cook describes a study by Kim that demonstrated the utility of B. subtilis for heterologously producing non-ribosomal peptides from other low-GC Bacilli by engineering a strain of B. subtilis to produce polymyxins, antibiotics natively produced by Paenibacillus polymyxa. Integrating the polymyxin BGC into the chromosome of B. subtilis yielded a strain capable of producing up to 200 mg L–1 of polymyxins. The authors also succeeded in engineering strains towards the selective production of polymyxins B and E by replacing domains in the polymyxin NRPS with homologous domains with the desired substrate specificity.
Section 4 of Cook recites that actinobacteria are the most abundant source of sequenced BGCs encoding PKSs and NRPSs, and Streptomyces species have been the most common hosts for the heterologous expression of these BGCs (Fig. 3b and c). They are also the most common heterologous hosts for the discovery of novel polyketides and non-ribosomal peptides through the heterologous expression of BGCs from metagenomic libraries and cryptic BGCs (Fig. 3d).
Cook concludes (section 5) that researchers have put considerable effort towards identifying alternative heterologous hosts for the discovery and production of polyketides and non-ribosomal peptides. Streptomyces species remain the dominant candidates, and this trend will hold in the foreseeable future as actinobacteria continue to be rich sources for bioactive natural products.
Since the inventions described in the cited references relate to the preparation of plasmids including long-chain DNA, PKS, in the invention described in Kumpfuller, it would have been prima facie obvious to one of ordinary skill in the art to use the methods described in documents Tsuge 1 & 2 when creating a plasmid including a polyketide synthase gene cluster having a length of 30 kb or more. Further, Cook et al specifically teaches competing BGCs can be deleted from the chromosome using genome editing methods which include integrating BGCs into the chromosome of heterologous hosts include transposition, phage integration, and homologous recombination. Cook et al teach leveraging synthetic biology for producing bioactive polyketides and non-ribosomal peptides in bacterial heterologous host. They recite that Streptomyces species have been the most common hosts for the heterologous expression of these BGCs (Fig. 3b and c). They are also the most common heterologous hosts for the discovery of novel polyketides and non-ribosomal peptides through the heterologous expression of GCs from metagenomic libraries and cryptic BGCs. The instant claims broadly recite GC contents of 65% or less which would be in the range recited in the references cited above as Bacillus subtilis was well known to have a low GC content (see Cook above). Additionally, the instant claims do not provide the DNA or plasmid which encodes a PKS which is 70 mol % or less and it appears it would inherently be that which is disclosed in the prior art references. The Tsuge documents teach a multiple DNA fragment ligation method using a Bacillus subtilis plasmid transformation system, and indicate that DNA fragments serving as ligation materials are prepared so as to have specific protruding ends of 3-4 bases using a restriction enzyme such as Type IS; a tandem-repeat DNA construct is prepared by ligating the DNA fragments using the complementarity of the protruding ends; and by introducing said construct into Bacillus subtilis, DNA ligated as a circular plasmid DNA is ultimately obtained. The molar concentration ratio is within range of the method taught by Tsuge. The very broadly claimed methods are described in these prior art reference, and Cook et al specifically teaches the use of Streptomyces as a host cell.
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.
Correspondence regarding this application should be directed to Group Art Unit 1645. Papers related to this application may be submitted to Group 1600 by facsimile transmission. Papers should be faxed to Group 1600 via the PTO Fax Center located in Remsen. The faxing of such papers must conform with the notice published in the Official Gazette, 1096 OG 30 (November 15,1989). The Group 1645 Fax number is 571-273-8300 which is able to receive transmissions 24 hours/day, 7 days/week.
Information regarding the status of an application may be obtained from the Patent Application Information Retrieval (PAIR) system. Status information for published applications may be obtained from either Private PAIR or Public PAIR. Status information for unpublished applications is available through Private PAIR only. For more information about the PAIR system, see http://pair-direct.uspto.gov. Should you have questions on access to the Private PAIR system, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free).
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jennifer E. Graser whose telephone number is (571) 272-0858. The examiner can normally be reached on Monday-Thursday from 8:00 AM-6:30 PM.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Gary Nickol, can be reached on (571) 272-0835.
Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (571) 272-0500.
/JENNIFER E GRASER/Primary Examiner, Art Unit 1645 11/13/25