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
Claims 1-2 and 5-22 are pending and under examination on their merits. Claims 3-4 are cancelled.
Response to Arguments
Applicant's arguments filed 5/26/2026 have been fully considered but they are not persuasive.
Applicant argues against the written description rejection of claims 1-2 and 5-22 under 35 U.S.C. 112(a) on the grounds that a BLAST alignment of SEQ ID NO: 1 indicates there are other known acetohydroxy acid synthase small subunit variants comprising sequences having at least 80% identity to SEQ ID NO: 1. Thus, Applicant argues, there is a representative number of known acetohydroxy acid synthase small subunit variants (Arguments, bottom paragraph on page 4).
In response, this argument is unpersuasive. First, it is not possible to read the alignment results presented in the arguments due to poor legibility. Regardless, sequence similarity alone is insufficient to demonstrate the function of a protein. NCBI BLAST search results often include proteins with putative functions. Therefore, the BLAST results do not serve as evidence of a representative number of species of acetohydroxy acid synthase small subunit variants. Thus, the written description rejection under 35 U.S.C. 112(a) is maintained. In addition, claims 13-15 and 22 are rejected under 35 U.S.C. 112(a) because Applicant does not have possession of the claimed genus of method conditions, as discussed in the rejection below.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
(New Rejection Necessitated by the Amendment) Claim 2 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 2 is indefinite for “the amino acid corresponding to position 42 in the amino acid sequence of SEQ ID NO: 1 is further substituted with valine.” Claim 1 does not recite any amino acid substitution at position 42, so it is unclear how the amino acid corresponding to position 42 is “further substituted.”
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
(New Rejection Necessitated by the Amendment) Claims 1-2 and 7-22 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claims contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventors, at the time the application was filed, had possession of the claimed invention.
Claim 1 recites “An acetohydroxy acid synthase small subunit (ilvN) variant having at least 80% identity to the amino acid sequence of SEQ ID NO: 3 or 5, in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with alanine.” Claim 1 is interpreted as requiring that the variant has up to 35 amino acid substitutions (0.20 ×172 amino acids) but retains the ability to regulate acetohydroxy acid synthase (the function of the acetohydroxy acid synthase small subunit). Claims 7-9 require a polynucleotide encoding the variant and claims 9-12 are drawn to a microorganism comprising the variant or a polynucleotide encoding the variant.
Claim 13 recites a method for producing L-valine, the method comprising culturing a microorganism comprising the variant in a medium. Claim 14 recites the method further comprises recovering L-valine from the medium.
Claims 13-14 are generic to any medium and any microorganism.
Claim 15 recites that the medium includes a carbon source.
Claims 17-21 further limit the sequence identity of the variant to SEQ ID NO: 3 or SEQ ID NO: 5.
Claim 22 is drawn to a method for increasing L-valine production comprising transfecting a microorganism with a polynucleotide encoding the variant of claim 1 and culturing the microorganism in a medium, wherein the microorganism comprises the acetohydroxy acid synthase small subunit variant or a polynucleotide encoding the variant, and an acetohydroxy acid synthase large subunit or a polynucleotide encoding the acetohydroxy acid synthase large subunit. Claim 22 is generic to any medium and any microorganism.
The person of ordinary skill in the art would not have recognized that the inventors were in possession of a genus of acetohydroxy acid synthase small subunit (ilvN) variants with at least 80%, at least 90%, or at least 95% identity to SEQ ID NO: 3 or 5. The person of ordinary skill in the art would also not have recognized that the inventors were in possession of the claimed genus of method conditions for producing L-valine (claims 13-15) or increasing L-valine production (claim 22).
The specification discloses producing L-valine from unmodified Corynebacterium glutamicum CJ7V as well as a T44A variant of C. glutamicum CJ7V and a A42V+T44A variant (paragraph 1 and Table 7 on page 34). The strains are cultured using the method described in Example 3-1. The specification also discloses L-valine production from unmodified, T44A, and A42V+T44A variants of C. glutamicum KCCM11201P (Table 5 on page 32). The specification discloses the culture conditions as follows: Each strain was subcultured in a nutrient medium, inoculated into a 250 ml corner-baffle flask containing 25 ml of a production medium, and cultured with shaking at 200 rpm for 72 hours at 30°C (last full paragraph on page 92). Applicant discloses a single nutrient medium and production medium (paragraphs 2-3 on page 26). Both nutrient and production medium are aqueous, contain glucose (carbon source), as well as phosphate salts and a nitrogen source. The specification discloses that branched-chain amino acids are biosynthesized by Corynebacterium from 2-ketoisocaproate as a precursor after undergoing several steps from pyruvic acid (specification, page 1, Background Art, paragraph 2).
Although Applicant generates several different mutants of Corynebacterium glutamicum KCCM11201P that are capable of producing L-valine (see Table 1 on page 26-27), only the C14 strain sequence is analyzed and only the mutations at amino acid positions 42 and 44 are disclosed (bottom two paragraphs on page 27). No further variants of the C14 variant are generated (i.e. acetohydroxy acid synthase ilvN containing amino acid substitutions at positions 42 and 44 as well as other amino acid substitutions). Furthermore, only the mutations A42V and T44A are disclosed. No other variants with different amino acid substitutions at positions 42 and 44 that retain acetohydroxy acid synthase regulatory activity are disclosed.
In other words, Applicant’s disclosure is limited to two species of the claimed invention: a variant with the amino acid substitution T44A and a variant with both the amino acid substitutions A42V and T44A.
The prior art does not teach the amino acid substitution T44A or any other amino acid substitution at position 44 of SEQ ID NO: 1, so there are no species of the claimed invention taught by the prior art.
Liu et al. (Enzyme and Microbial Technology 129 (2019): 109357), published just two years before the effective filing date of the claimed invention, teaches that as of the year 2019, the mechanism for the substrate specificity of C. glutamicum AHAS remains unknown (Abstract). Liu teaches that AHAS comprises the subunits IlvB and ilvN (Abstract). IlvB is the catalytic subunit and ilvN is the regulatory subunit (page 2, left column, paragraph 1). AHAS catalyzes the first reaction in the biosynthetic pathway to branched chain amino acids L-valine, L-leucine, and L-isoleucine (Abstract). Liu teaches variants of AHAS with mutations at positions 138 and 404 in the large subunit (IlvB) confer substrate selectivity to L-valine (Abstract).
Yin et al. (Metabolic engineering 14.5 (2012): 542-550) teaches that the amino acid substitutions P176S, D426E, and L575W in the large subunit (ilvB) of AHAS may be important for enhanced L-isoleucine production in a strain of C. glutamicum (page 546, left column, top paragraph).
Although crystal structures of complexes of E. coli ilvN (small subunit of acetohydroxy acid synthase) with valine or isoleucine have been solved (see Abstract of Bansal et al., Biochemistry 58.15 (2019): 1992-2008), the crystal structure of Corynebacterium glutamicum acetohydroxy acid synthase (AHAS) has not been solved.
Blombach et al. (Applied and Environmental Microbiology, p. 419–427) teaches that the AHAS of C. glutamicum differs from E. coli: in contrast to C. glutamicum, E. coli possesses three AHAS isozymes (I, II, and III), differing in their regulation and biochemical properties (page 420, left column, paragraph 1). The regulatory subunit of E. coli shares only 39% identity with ilvN of C. glutamicum (page 420, left column, paragraph 1). Blombach teaches C-terminal truncations of the ilvN gene render the enzyme insensitive to feedback inhibition by L-valine (Abstract and page 420, left column, paragraph 1).
Elisakova (Applied and Environmental Microbiology, Jan. 2005, p. 207–213) teaches introducing mutations at positions 20-22 into the small regulatory AHAS subunit encoded by ilvN of C. glutamicum (Abstract and Table 4). The M13 mutant, corresponding to G20D, I21D, and I22F was feedback resistant to all three branched amino acids and produced more L-valine than the wild-type (Abstract). Elisakova cultures C. glutamicum in CGXII medium for 48 h (60 ml in 500-ml flasks) at 30 °C and 120 rpm and subsequently recovers valine from the supernatant of the cultures (page 209, left column, bottom paragraph, “Determination of valine production”).
Wada et al. (Bioscience, biotechnology, and biochemistry 72.11 (2008): 2959-2965) teaches a C-terminal truncation of ilvN of C. glutamicum lacking 53 amino acid residues from the C-terminus (paragraph bridging left and right columns on page 2962) with increased L-valine production compared to wild-type (Abstract). Wada cultures C. glutamicum in medium VI, which is a fermentation medium with 100 g/L of glucose as well as mineral salts such as ammonium sulfate (page 2960, Materials and Methods, left column, bottom paragraph). Wada cultures C. glutamicum for 72 h at 30 °C and determines valine concentration in the supernatant (page 2960, Materials and Methods, right column, Valine fermentation and Analytical method paragraphs). Wada teaches the biosynthetic pathway for branched amino acids in Corynebacterium (Fig. 1), which includes ilvBN as well as several other enzymes.
In summary, the prior art teaches species of acetohydroxy synthase variants primarily with mutations in the large subunit, which is responsible for the catalytic activity of the enzyme. A smaller number of species with mutations in the small subunit (regulatory subunit) is taught by the prior art. These mutations are primarily C-terminal truncations or mutations distant from the presently claimed mutations at positions 42 and 44. The person of ordinary skill in the art would have been unable to predict the combined effect of the amino acid substitutions on the regulatory activity of the small subunit without understanding the three-dimensional structure of the enzyme (i.e. the structure-function correlation of the enzyme).
Therefore, the person of ordinary skill in the art would not have recognized that the inventors had possession of the claimed genus of acetohydroxy acid synthase small subunit (ilvN) variants. The person of ordinary skill in the art would also not have recognized that the inventors had possession of the claimed genus of method conditions (claims 13-14 and newly added claim 22), which do not recite any conditions for culturing the microorganism (e.g. any precursors required for the synthesis of L-valine). Furthermore, claims 13-14 do not recite any of the other enzymes that must be present in the microorganism for the biosynthesis of L-valine.
Allowable Subject Matter
Claims 5-6 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter: the prior art does not teach the amino acid sequence consisting of SEQ ID NO: 3 or 5.
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CANDICE LEE SWIFT whose telephone number is (571)272-0177. The examiner can normally be reached M-F 8:00 AM-4:30 PM (Eastern).
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657
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