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 1-14 are pending and under examination on their merits.
Claim Objections
Claims 11-12 are objected to because of the following informalities: the Latin scientific names Corynebacterium and Corynebacterium glutamicum are not italicized. Appropriate correction is required.
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.
Claims 1-14 are 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 1 recites “An acetohydroxy acid synthase small subunit (ilvN) variant in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.” It is unclear if the claim is open or closed with respect to additional amino acid substitutions. The amino acid substitution at position 44 renders the acetohydroxy acid synthase a variant, but additional substitutions in the synthase would also constitute a variant. It is further uncertain whether claims 1-4 and 7-14 require the variant sequence to have any portion of SEQ ID NO:1.
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 another amino acid.” 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.” Applicant may consider amending claim 2 to recite “The variant of claim 1, further comprising an amino acid substitution at position 42 of SEQ ID NO: 1.”
Claims 2-14 are rejected for depending from a rejected base claim and not rectifying the sources of indefiniteness discussed above.
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.
Claims 1-4 and 7-14 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 claim(s) 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 inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 1 recites “An acetohydroxy acid synthase small subunit (ilvN) variant in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.” Claim 1 is interpreted as open and having an unlimited number of additional substitutions so long as the polypeptide has 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 a target substance from the medium.
Claims 13-14 are generic to any medium, any microorganism, and any target substance.
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 an unlimited number of amino acid substitutions. 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.
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), which do not recite any conditions for culturing the microorganism (e.g. any precursors required for the synthesis of L-valine) and are generic to recovering any target substance (claim 14). Furthermore, the claims do not recite any of the other enzymes that must be present in the microorganism for the biosynthesis of L-valine.
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.
Claims 13-14 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph. The specification is enabling for producing L-valine by culturing a microorganism comprising the acetohydroxy acid synthase small subunit and the acetohydroxy acid synthase large subunit. The specification does not reasonably provide enablement for producing L-valine by culturing a microorganism comprising the small subunit without further comprising the large subunit of acetohydroxy acid synthase.
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.
Per MPEP 2164.01(a), the following eight factors should be considered when determining whether the person of ordinary skill in the art would face undue experimentation to make and/or use the invention: (1) The nature of the invention; (2) the state of the prior art; (3) the relative skill of those in the art; (4) the predictability or unpredictability of the art; (5) the breadth of the claims; (6) the amount of direction or guidance presented; (7) the presence or absence of working examples; and (8) the quantity of experimentation necessary. While it is not essential that every factor be examined in detail, those factors deemed most relevant should be considered.
Nature of the invention. Claims 13-14 are drawn to a method of producing L-valine comprising culturing the microorganism of claim 9 in a medium. Claim 13 recites the method of claim 13, further comprising recovering a target substance from the medium. Claim 9 is drawn to a microorganism comprising the variant of claim 1. Claim 1 recites an acetohydroxy acid synthase small subunit (ilvN) variant in which the amino acid corresponding to position 44 in the amino acid sequence of SEQ ID NO: 1 is substituted with another amino acid.
Breadth of the claims. Claims 13-14 are extremely broad. The culturing step recited in claim 13 only requires a generic medium. The claim does not require a carbon substrate within the medium or any precursors for the biosynthesis of L-valine. Claim 14 recites a generic recovering step of any target substance and is not limited to L-valine.
State of the prior art and unpredictability. 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).
Guidance in the specification and working examples. The specification does not provide any working examples in which L-valine is produced by a microorganism comprising ilvN (the regulatory subunit of AHAS) but not ilvB (the catalytic subunit of AHAS). Rather, the working examples in the specification disclose mutations of ilvN in different strains of Corynebacterium but no knockouts or deletions of ilvB: see Table 5 on page 32 and Table 7 on page 34.
Amount of experimentation necessary. The person of ordinary skill in the art would have faced undue experimentation in order to practice the claimed invention without the microorganism further comprising the catalytic subunit of AHAS (ilvB) because the regulatory subunit (ilvN) cannot perform the biosynthesis of L-valine by itself.
Taking these factors into account, undue experimentation would be required by one of ordinary skill in the art to practice the full scope of the claimed invention. Thus, the claims are not fully enabled by the disclosure.
Examiner’s Comment
The prior art does not teach an amino acid sequence consisting of SEQ ID NO: 3 or 5. The prior art does not teach an amino acid sequence with 98.8% identity to SEQ ID NO: 1 and the amino acid substitutions A42V and T44A.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/CANDICE LEE SWIFT/Examiner, Art Unit 1657