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
Applicant’s amendment filed 06 July 2026 is entered. Claims 1, 9, 16, 22, 26, 28, and 35 are amended. Claims 1-2, 4-9, 16, 19-20, 22-28, 35, 38-41, and 48-51 are pending. Claims 38-41 and 48-51 remain withdrawn. Claims 1-2, 4-9, 16, 19-20, 22-28, and 35 are under examination.
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.
Claims 1-2, 4-8, 16, 19, 22-27, and 35 remain rejected under 35 U.S.C. 103 as being unpatentable over Hu et al. (CN 104513840 A published 15 April 2015), Horbal et al. (Dual control system – A novel scaffolding architecture of an inducible regulatory device for the precise regulation of gene expression, Metabolic Engineering 37 (2016) 11–23), and Banchio et al. (A Stationary-Phase Acyl-Coenzyme A Synthetase of Streptomyces coelicolor A3(2) Is Necessary for the Normal Onset of Antibiotic Production, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2002, p. 4240–4246, Vol. 68, No. 9), and as evidenced by LibreTexts (Oxidation of Fatty Acids, https://chem.libretexts.org/@go/page/234043, accessed on 01 April 2025), Yang et al. (Enhancement of acyl-CoA precursor supply for increased avermectin B1a production by engineering meilingmycin polyketide synthase and key primary metabolic pathway genes, Microbial Biotechnology. 2024;17:e14470.), and Gibson et al. (Enzymatic Assembly of Overlapping DNA Fragments, Methods in Enzymology, Volume 498, 2011).
The limitation “…for performing a triacylglycerol decomposition pathway in a Streptomyces during a stationary growth phase” recited in claim 1 is an intended result or effect of practicing the method, but does not recite an active step to execute the method for improving the production of a polyketide compound in a Streptomyces. As such, where the prior art teaches all of the steps of claim 1, the limitation is considered to be necessarily met.
The limitation “…to obtain a plasmid triacylglycerol degradation regulation module having a sequence as set forth in SEQ ID NO: 35…” recited in claim 1 is an intended result of practicing the method steps prior to the limitation, but does not recite an active step to execute the claimed method. Practicing the amplifying steps for the cumate inducible promoter and Streptomyces gene, and the Gibson assembly step results in the plasmid triacylglycerol degradation regulation module having a sequence as set forth in SEQ ID NO: 35. Thus, where the art teaches the amplifying steps for the cumate inducible promoter and Streptomyces gene, and the Gibson assembly step of claim 1, the limitation is considered to be necessarily met.
Regarding claim 1, Hu teaches a method for improving yield of polyketide compounds in engineered Streptomyces hygroscopius (Hu Abstract and pg. 3 para. 6) comprising the steps of amplifying a promoter ERM*E using primers P3 and P4 to obtain a fragment containing the ERM*E promoter and amplifying acyl coenzyme A DPTE using primers P1 and P2 to obtain a fragment containing the complete DPTE gene (Hu pg. 4 Embodiment 2 steps 1 and 2). The ERM*E promoter and DPTE fragments were assembled into plasmid pSET152 using the Gibson assembly method of digesting the ERM*E fragment with restriction enzymes BamHI and NdeI, digesting the DPTE fragment with NdeI and XbaI, digesting the pSET152 plasmid with BamHI and XbaI, and the ligating all of the fragments together using T4 DNA ligase to obtain a plasmid pSET152erm*E-DPTEF (Hu fig. 1 and pg. 4 last para. through pg. 5 para. 1). The pSET152erm*E-DPTEF plasmid was then integrated into the genome of actinomycete Streptomyces hygroscopicus by intergenic conjugation through E. coli (Hu pg. 3 para. 6 and pg. 5 Example 3).
Hu does not teach amplifying a cumate inducible promoter using primers CuF and CuR or amplifying a gene of Streptomyces using primers 6196F and 6196R.
Horbal teaches a library of cumate inducible promoters which can be used to modulate gene expression of secondary metabolite gene clusters (Horbal pg. 14 Results section 3.1). Horbal also teaches inducing the promoters with different concentrations of cumate (denoted Cu) in order to induce the expression of the genes following the promoters (Horbal Fig. 1).
Banchio teaches Streptomyces coelicolor acyl coenzyme A synthetase encoded by the fadD1 gene, also called the sco6196 gene, as evidenced by Yang (Yang pg. 8 right col. para. 1), modulates the activation of polyketide actinorhodin synthesis (Banchio discussion).
Hu, Horbal, and Banchio do not teach the primers CuF, CuR, 6196F, and 6196R.
However, it would have been a matter of routine optimization by one of ordinary skill in the art to determine adequate primer sequences in order to amplify Horbal’s cumate inducible promoter and Banchio’s fadD1 (sco6196) acyl coenzyme A synthetase gene. In depth instructions on how to design effective primers for amplifying DNA fragments for use in Gibson assembly are readily available and well known in the art, as evidenced by Gibson (Gibson pgs. 352-353 sec. 2 and Fig. 15.1). By referencing the DNA sequences of the cumate inducible promoter as disclosed by Horbal and fadD1 (sco6196) acyl coenzyme A synthetase gene as disclosed by Bachio, one of ordinary skill in the art would use the readily available guidance of Gibson Assembly method as disclosed by Hu to construct PCR primers that would predictably amplify the cumate inducible promoter and fadD1 (sco6196) acyl coenzyme A synthetase gene fragments, and prepare those fragments for Gibson assembly.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to modify Hu’s method of improving polyketide production in a Streptomyces by substituting the ERM*E promoter used in Hu’s method with Horbal’s cumate inducible promoter, and Hu’s Streptomyces gene DPTE with Banchio’s Streptomyces gene fadD1 (sco6196), thereby obtaining a triacylglycerol degradation regulation module.
One of ordinary skill in the art would have been motivated to substitute Hu’s ERM*E promoter with Horbal’s cumate inducible promoter in order to selectively control the transcription of the acyl coenzyme A synthetase gene using cumate. One of ordinary skill in the art would have a reasonable expectation of success because Horbal taught the effectiveness of a cumate inducible promoter controlling expression of downstream genes upon addition of cumate (Horbal fig. 2b).
One of ordinary skill in the art would have been motivated to substitute Hu’s acyl coenzyme A synthetase gene DPTE with Banchio’s acyl coenzyme A synthetase gene fadD1 (sco6196) in order to overexpress the transcription levels of FadD1 (SCO6196) in the Streptomyces to improve its polyketide production. One of ordinary skill in the art would have a reasonable expectation of success because Hu’s method is taught to improve the production of polyketides in a Streptomyces species by overproducing the expression of an acyl coenzyme A synthetase, and Hu’s DPTE and Banchio’s FadD1 (SCO6196) are both acyl coenzyme A synthetases produced by Streptomyces species.
Regarding claim 22, Hu teaches that polyketides are secondary metabolites (Hu pg. 2 Background), and Banchio teaches that the polyketides are synthesized during the stationary phase of the Streptomyces (Banchio Fig. 6). The obvious method of Hu in view of Horbal and Banchio improves the production of polyketides by inducing selective control over the expression of acyl coenzyme A synthetase SCO6196, which is normally expressed in the stationary phase when the secondary metabolism naturally begins and is correlated with SCO6196 levels (Banchio Figs. 3A and 6). Inducing the expression of acyl coenzyme A synthetase SCO6196 leads to increased secondary metabolites like polyketides, thereby switching the primary metabolism of Streptomyces to its secondary metabolism where the polyketides are synthesized. Therefore, performing the obvious method of Hu in view of Horbal and Banchio switches the primary metabolism of a Streptomyces to its secondary metabolism.
Regarding claims 2 and 23, acyl-coenzyme A synthetase is an enzymatic component that is part of the fatty acid (such as triacylglycerol) catabolism through β-oxidation, as evidenced by LibreTexts (Pg. 1 para. 2 and chemical equation).
Regarding claim 4, Hu teaches their method can improve the yield of polyketide avermectin (Hu pg. 2 Background).
Regarding claims 6 and 25, Hu teaches a method for improving yield of polyketide compounds in engineered Streptomyces hygroscopius (Hu Absract and pg. 3 para. 6).
Regarding claims 5, 7-8, 16, 19, 24, 26-27, and 35, these claims further limit the phrase “for performing a triacylglycerol decomposition pathway in the Streptomyces” recited in claim 1. This phrase is an intended result or effect of practicing the method, and not an active step to be performed in order to execute the method. Since Hu teaches a method for improving yield of polyketide compounds in engineered Streptomyces hygroscopius comprising the steps of claims 1 and 22, the limitations of these claims are considered necessarily met.
Claims 9 and 28 remain rejected under 35 U.S.C. 103 as being unpatentable over Hu in view of Horbal and Banchio as applied to claims 1-2, 4-8, 16, 19, 22-27, and 35 above, and further in view of Risdian et al. (Biosynthesis of Polyketides in Streptomyces, Microorganisms 2019, 7, 124; doi:10.3390/microorganisms70501 24).
The obvious method of Hu in view of Horbal and Banchio enhances the expression level of the acyl coenzyme A synthetase enzyme SCO6196. Hu and Horbal do not teach the SCO6196 has a sequence of SEQ ID NO: 31.
Banchio teaches Streptomyces coelicolor strain A3(2) (Banchio discussion). NCBI BLAST alignment shows that SEQ ID NO: 31 is 100% identical to the genome positions 221769 to 223394 of Genbank accession number AL939126.1, which corresponds to gene SCO6196 of the Streptomyces coelicolor A3(2) strain's genome (See below alignment). Thus, since Banchio's strain is Streptomyces coelicolor A3(2), the SCO6196 gene described therein is 100% identical to SEQ ID NO: 31 of the instant claims.
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Hu, Horbal, and Banchio do not teach an improvement in yield of polyketide compounds in the species Streptomyces coelicolor.
Risdian teaches that Streptomyces coelicolor produces polyketides actinorhodin (Risdian pg. 2-3 bridging para.) and germicidin A (Risdian pg. 11 sec. 4.1 para. 1). Risdian teaches that germicidin A is known to inhibit spore germination and be antibacterial against various Gram-positive bacteria (Risdian pg. 11 sec. 4.1 para. 1).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to apply the obvious method of Hu in view of Horbal and Banchio to the Risdian’s Streptomycin coelicolor in order to advantageously enhance production of antibacterial and anti-spore polyketide compound germicidin A produced by Risdian’s Streptomyces coelicolor. One of ordinary skill in the art would have a reasonable expectation of success because Hu taught their method of improving polyketide yield to be useful in engineered Streptomyces cells, and so one of ordinary skill in the art would reasonably expect the method of Hu in view of Horbal and Banchio to successfully work in Streptomyces coelicolor.
Claim 20 remains rejected under 35 U.S.C. 103 as being unpatentable over Hu in view of Horbal and Banchio as applied to claims 1-2, 4-8, 16, 19, 22-27, and 35 above, and further in view of Zhang et al. (Suitable extracellular oxidoreduction potential inhibit rex regulation and effect central carbon and energy metabolism in Saccharopolyspora spinosa, Microbial Cell Factories 2014, 13:98).
Hu, Horbal, and Banchio do not teach a step of increasing a NADH/NAD+ ratio in the Streptomyces.
Zhang teaches that polyketides are synthesized in the stationary growth phase of fermentation, and that the ratio of NADH/NAD+ has significant influence on polyketide production (Zhang Abstract). Zhang also teaches that the NADH/NAD+ ratio is significantly higher in stationary growth phase than in either lag or exponential growth phases under normal conditions (Zhang Fig. 2 control group). Therefore, Zhang teaches that a high NADH/NAD+ ratio is present in stationary growth phase, which allows for the production of polyketides secondary metabolites.
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date of the present invention to increase the ratio of NADH/NAD+ of the Streptomyces of Hu in view of Horbal and Banchio. One of ordinary skill in the art would have been motivated to do so in order to advantageously promote the secondary metabolism conditions which positively impact therapeutic polyketide production under normal growth conditions, such as a high NADH/NAD+ ratio. One of ordinary skill in the art would have had reasonable expectations of success in this endeavor because Zhang teaches that polyketides are synthesized in the stationary growth phase, the ratio of NADH/NAD+ has significant influence on polyketide production, and the NADH/NAD+ ratio is significantly higher in stationary growth phase than in either lag or exponential growth phases under normal conditions. Thus, a high NADH/NAD+ ratio is present in stationary growth phase, which enhances the production of therapeutic polyketides secondary metabolites.
Response to Arguments
Applicant's arguments filed 06 July 2026 have been fully considered but they are not persuasive.
Regarding Applicant’s arguments that amended claims 1 and 22 recite that by adding cumate, the triacylglycerol decomposition pathway can be performed without addition of external carbon sources during the stationary growth phase of the Streptomyces, but reference Hu required the addition of oil (another carbon source) in order to improve the yield of polyketides (Remarks pg. 8-9), Hu teaches that the medium used to grow the Streptomyces bacteria is supplemented with natural oils (Hu abstract and claim 9). However, Hu never discloses that this oil is introduced during the stationary growth phase of the Streptomyces. Rather, the Streptomyces is initially cultivated in a medium supplemented with natural oil that acts as a cheap natural raw material to provide synthetic precursors for the polyketide compounds (Hu abstract). Hu teaches the addition of the oil external carbon source at the very beginning of culturing, not during the stationary growth phase. Hu does not teach that any additional carbon source is added once the Streptomyces bacteria reach stationary growth phase. Thus, Hu still teaches the invention, and Applicant's new limitation fails to obviate the 103.
Regarding Applicant’s arguments that Horbal and Banchio do not teach that expression of a specific gene of a specific strain can be controlled at a specific time point to improve the yield of polyketides (Remarks pg. 9), Horbal teaches a library of cumate inducible promoters used to modulate gene expression in secondary metabolite clusters (Horbal pg. 14 Results section 3.1). Banchio teaches Streptomyces coelicolor strain A2(3) acyl coenzyme A synthetase encoded by the fadD1 gene (sco6196), which modulates the activation of polyketide actinorhodin synthesis (Banchio discussion). Horbal does not teach or suggest that these cumate inducible promoters are unable to modulate gene expression at specific time points. Rather, Horbal teaches that they can be induced at any time that cumate is added. One of ordinary skill in the art would reasonably conclude that Horbal's promoters would function at any time point, regardless of specific strains and specific genes.
Conclusion
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.
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/Alexander M Duryee/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
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