DETAILED ACTION
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 .
The Preliminary Amendment of 5 Aug. 2024 has been entered.
Claims 1-20 are currently pending and are examined herein.
Claim Rejections - 35 USC § 112(a) (scope of enablement)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 13 and 14 are rejected under 35 U.S.C. 112(a) because the specification, while being enabling for a method of producing (S)-3HB with a productivity/concentration within the ranges of clams 13 and 14 by culturing a Clostridium sp. expressing an integrated ctfAB/atoB and further comprising a heterologous thl2 and C. ljungdahlii hbd2, does not reasonably provide enablement for such a method using any Clostridium sp. expressing a heterologous ctfAB and a hbd2. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Whether a disclosure satisfies the enablement requirement is assessed with respect to the factors set forth in In re Wands, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988); MPEP 2164.01 (a). These factors include: breadth of the claims, nature of the invention, state of the prior art, level of one of ordinary skill, level of predictability in the art, amount of direction provided by the inventor, existence of working examples and quantity of experimentation needed to make or use the invention.
All of the Wands factors have been considered with respect to the instant claims. The most relevant factors are discussed in detail below.
Breadth of the claims
Claims 13 and 14 recite a method of making (S)-3HB using a Clostridium sp. comprising a heterologous ctfAB gene and a hbd2 gene, wherein the (S)-3HB is made at a rate up to 0.083 g/L/hr for 24 hours (claim 13) and up to a concentration of 88 mM (claim 14). The claims do not require tha the hbd2 is heterologous and the instant specification evidences that hbd2 of SEQ ID 2 is endogenous to C. ljungdahlii 13528 (Published Spec. US20250101472, [0031], referring to hbd2 as CLJU_c37300; see also, GenBank: ADK16756.1, stating that locus CLJU_c37300 is a C. ljungdahlii 13528 enzyme having 100% identity to SEQ ID 2). Thus, the claims encompass methods of making (S)-3HB using any Clostridium strain comprising a heterologous ctfAB.
State of the prior art/Predictability in the art
The instant specification evidences that little was known about hbd2 of SEQ ID 2, including its function(s), activity, cofactors, etc., prior to the filing date (Spec., [0017]; [0034]; [0040]-[0044]). Thus, one of ordinary skill in the art would not have possessed knowledge or predictability for optimizing productivity of (S)-3HB in Clostridium via the hbd2 pathway.
Direction provided by the inventor/existence of working examples
The instant specification states that the rate of 0.083 g/L/hr for 24 hours and concentration of 88 mM were produced using an integrated ctfAB/AtoB construct which further expresses a heterologous thl/hbd2 construct (Spec., [0038]). The integrated ctfAB/AtoB without heterologous thl2 and C. ljungdahlii hbd2 showed significantly lower (approx.. half) (S)-3HB productivity (Spec., [0032]; Fig. 4; [0037]-[0038]; Fig. 6), and the specification concludes that the higher productivity recited in claims 13-14 is due to targeted overexpression of hbd2 ([0039]-[0040]). The specification does not contain any other working examples or guidance that would allow one of ordinary skill in the art to achieve the productivity/concentration levels cited in claims 13 and 14 via pathways/strategies other than with the exemplified strain comprising heterologous thl2 and C. ljungdahlii hbd2.
Quantity of experimentation needed to make or use the invention
In view of the lack of knowledge/unpredictability in the prior art regarding (S)-3HB production via the hbd2 pathway and the absence of examples guidance in the specification of how to achieve the productivity/concentration levels cited in claims 13 and 14 via pathways/strategies other than with the exemplified strain comprising heterologous thl2 and C. ljungdahlii hbd2, one of ordinary skill would have needed to undertake extensive and undue experimentation in order to practice the full scope of the claimed invention.
Claim Rejections - 35 USC § 102
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.
Claims 1-3, 6 and 7 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Jones et al., Nature communications 7.1 (2016): 12800 (cited in IDS of 21 Oct. 2024), as evidenced by GenBank: ADK16756.1 (2014).
Regarding claims 1, 2 and 7, Jones teaches a recombinant (non-naturally occurring) C. ljungdahlii strain DSM-13528 transformed with a heterologous ctfAB (CoA transferase) gene from C. acetobutylicum (p. 7-8, under Construction of CLJ ΔSADH (pTCtA)). The ctfAB gene of Jones is the same gene as used in strains of the instant specification (Published Spec. US20250101472, [0027]).
Regarding the recitation in claim 1 that the strain comprises “a hbd2 gene”, the instant specification evidences that hbd2 of SEQ ID 2 is endogenous to C. ljungdahlii 13528 (Spec., [0031], referring to hbd2 as CLJU_c37300; see also, GenBank: ADK16756.1, stating that locus CLJU_c37300 is a C. ljungdahlii 13528 enzyme having 100% identity to SEQ ID 2).
Regarding claims 3 and 6, the instant specification evidences the use of NADH as co-factor in claim 3 and the specific activity in claim 6 are inherent properties of the enzyme (Spec., [0034]) (see also, MPEP 2112.01 - Where the claimed and prior art products are identical in structure, claimed properties of the product are presumed to be inherent).
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 4 is rejected under 35 U.S.C. 103 as being unpatentable over the combination Jones as evidenced by GenBank: ADK16756.1 (2014), as applied above to claims 1-3, 6 and 7, in view of Lee et al., Bioresource technology 218 (2016): 909-917 (cited in IDS of 21 Oct. 2024).
The teachings of Jone are set forth above. Regarding claim 4, Jones further teaches that the heterologous ctfAB gene is operably linked with a heterologous thiolase gene (p. 7-8, under Construction of CLJ ΔSADH (pTCtA)). Jones teaches that the thiolase catalyzes a key step in 3-hydroxybutyrate (3-HB) synthesis upstream from ctfAB (Fig. 2, THL=thiolase, CoAT=ctfAB).
Claim 4 differs from Jones as evidenced by GenBank: ADK16756.1 (2014), as applied above to claims 1-3, 6 and 7, in that: the strain further comprises a heterologous atoB gene operably linked to the heterologous ctfAB gene.
Lee teaches a recombinant Clostridium strain for producing acetate and related solvent chemicals utilizing the same pathway as in Jones (cf. Jones, Fig. 2 vs. Lee, Fig. S1), wherein the strain comprises a heterologous ctfAB gene from C. acetobutylicum (the same ctfAB in Jones) operable linked with a heterologous thiolase (atoB from E. coli) (under 2.3. Plasmid construction). Lee teaches that AtoB (thiolase from E. coli) was selected instead of another Clostridium thiolase “because AtoB has higher activity, with no competitive inhibition by free CoA” (under 3.2. Effect of overexpressing thl (atoB) on ABE fermentation in the BEKW strain).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to make a recombinant C. ljungdahlii strain useful for producing 3-HB (and acetone) comprising a heterologous ctfAB gene operably linked to a heterologous thiolase gene as taught by Jones wherein the thiolase is AtoB as taught by Lee because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use AtoB as the thiolase in the strain of Jones because Lee teaches that AtoB has the benefits of high activity with no competitive inhibition by free CoA, and shows good productivity when operably linked to ctfAB (Lee, under 3.2. Effect of overexpressing thl (atoB) on ABE fermentation in the BEKW strain). Using AtoB as the thiolase in the strain of Jones would have led to predictable results with a reasonable expectation of success because Lee teaches that AtoB shows good productivity in a substantially similar context as Jone (operably linked to ctfAB for production of acetone via the same pathway utilized in Jones).
Claims 5 and 8-20 are rejected under 35 U.S.C. 103 as being unpatentable over the combination Jones as evidenced by GenBank: ADK16756.1 (2014) in view of Lee, as applied above to claim 4, further in view of Annan et al., Applied microbiology and biotechnology 103.11 (2019): 4633-4648.
The teachings of Jones and Lee are set forth above.
Regarding claim 8, Jones further teaches a method of making 3-HB, comprising adding a carbon source to a solution comprising the strain (p. 2-4, under Engineered CLJ strain for acetone production; Fig. 2).
Regarding claims 18-19, Jones teaches producing 3-HB under mixotrophic conditions with sugar and syngas as carbon sources (p. 2-4, under Engineered CLJ strain for acetone production; Fig. 2).
Regarding claim 20, Jones teaches carrying out the fermentation under anaerobic conditions (p. 8, under Batch fermentations of CLJ ΔSADH (pTCtA) and High density cell recycle fermentations).
Claims 5 and 8-20 differ from the combination Jones as evidenced by GenBank: ADK16756.1 (2014) in view of Lee, as applied above to claim 4, in that: the heterologous atoB gene and the heterologous ctfAB gene are both integrated into the chromosome of the Clostridium sp. (claims 5, 12); the 3-HB is the (S)-isomer (claim 8); (S)-3- hydroxybutyrate is made at a rate up to 0.083 g/L/hr for 24 hours (claim 13); (S)-3- hydroxybutyrate is made at up to a concentration of 88 mM (claim 14); and (S)-3-hydroxybutyrate is made at a rate that is up to 2.5-fold greater than a naturally occurring Clostridium sp. (claim 15).
Annan teaches that the PyrE locus is commonly used to stably integrate heterologous genes into acetogenic Clostridia strains, and further demonstrates that multi-gene heterologous biosynthetic pathways can be stably integrated into the PyrE locus in C. ljungdahlii 13528 (the C. ljungdahlii strain taught by Jones) (under Introduction, Strains and Results).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to make a recombinant C. ljungdahlii strain useful for producing 3-HB comprising a heterologous ctfAB gene operably linked to a heterologous AtoB gene as taught by Jones in view of Lee wherein the ctfAB/AtoB construct is stably integrated into the PyrE locus of the strain as taught by Annan because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use integrate the ctfAB/AtoB construct into the chromosome to create a stable strain comprising the heterologous genes without the need for transformation, etc. One would have been motivated to select the PyrE locus as the site for integration because Annan teaches that the site is well known/characterized for integration in acetogenic Clostridia. Integrating the ctfAB/AtoB construct of Jones in view of Lee into the PyrE locus of the host C. ljungdahlii strain as taught by Annan would have led to predictable results with a reasonable expectation of success because Annan teaches that the PyrE site can be used to integrate several different heterologous multigene biosynthetic pathways into the same C. ljungdahlii 15328 strain used by Jones.
Regarding the recitation that the 3-HB is the (S)-isomer in claim 8, the instant specification evidences that the same strain as taught by the cited combination comprising ctfAB/AtoB integrated into the PyrE locus of C. ljungdahlii 15328 (referred to as the “integrated 3HB strain”) produces only the (S)-isomer (Spec., [0036]; Fig. 5C).
Regarding claims 13 and 14, it is noted that the claims require only a productivity/concentration “up to” 0.083 g/L/hr/88 mM. Thus, any amount of 3-HB production would meet the limitations of claims 13-14. Jones teaches a 3-HB productivity of 0.29 g/L over 168 hours (~ 2.8 mM) (p. 2-4, under Engineered CLJ strain for acetone production).
Regarding claim 15, the instant specification evidences that 3-HB production in C. ljungdahlii requires the heterologous ctfAB (coA tranferase) (Spec., [0033] – “we saw no 3HB production in this strain [wild-type], suggesting that ctfAB is a critical gene”). Thus, the ctfAB/AtoB integrated strain of the cited combination would make 3-HB at a rate that is up to 2.5-fold greater than a naturally occurring Clostridium sp.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ROBERT J YAMASAKI whose telephone number is (571)270-5467. The examiner can normally be reached M-F 930-6 PST.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Louise Humphrey can be reached at 571-272-5543. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657
Prosecution Insights