Ex Parte Kuwabara et al

Board of Patent Appeals and Interferences

Apr 29, 2010

10351306 (B.P.A.I. Apr. 29, 2010)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte YOKO KUWABARA, OSAMU KURAHASHI, and
TSUYOSHI NAKAMATSU
__________

Appeal 2009-007889
Application 10/351,306
Technology Center 1600
__________

Decided: April 29, 2010
__________

Before ERIC GRIMES, JEFFREY N. FREDMAN, and STEPHEN WALSH,
Administrative Patent Judges.

GRIMES, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to a method
of making arginine, which the Examiner has rejected as anticipated and
obvious. We have jurisdiction under 35 U.S.C. § 6(b). We affirm the
rejection for obviousness.

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STATEMENT OF THE CASE
Claims 5 and 7 are on appeal. The claims have not been argued
separately and therefore stand or fall together. 37 C.F.R. § 41.37(c)(1)(vii).
Claim 5 is representative and reads as follows:
5. A method for producing L-arginine, comprising the steps of:
culturing a coryneform bacterium which has been modified to
enhance an activity of intracellular glutamate dehydrogenase as
compared to an unmodified bacterium by increasing copy number of a
gene which encodes the glutamate dehydrogenase and whereby L-
arginine producing ability of the bacterium is increased in a medium
to produce and accumulate L-arginine in the medium; and
collecting the L-arginine from the medium.

Claims 5 and 7 stand rejected under 35 U.S.C. § 102(b) as anticipated
by Börmann-El Kholy1 as evidenced by Hoischen2 (Ans. 3), and under 35
U.S.C. § 103(a) as obvious in view of Katsumata,3 Umbarger,4 Börmann,5
and “site directed mutagenesis techniques known in the art” (Ans. 4).

1 Elke R. Börmann-El Kholy et al., “Glutamate Dehydrogenase Is Not
Essential for Glutamate Formation by Corynebacterium glutamicum,” 59
APPLIED AND ENVIRONMENTAL MICROBIOLOGY NO. 7, 2329-2331 (1993).
2 Christian Hoischen et al., “Membrane Alteration Is Necessary but Not
Sufficient for Effective Glutamate Secretion in Corynebacterium
glutamicum” 172 JOURNAL OF BACTERIOLOGY NO. 6, 3409-3416 (1990).
3 Ryoichi Katsumata et al., WO 0 261,627 A2, published March 3, 1988.
4 H. E. Umbarger, “Amino Acid Biosynthesis and Its Regulation,” 47 ANN.
REV. BIOCHEM. 533-606 (1978).
5 E. R. Börmann et al., “Molecular analysis of the Corynebacterium
glutamicum gdh gene encoding glutamate dehydrogenase,” 6 MOLECULAR
MICROBIOL. No. 3, 317-326 (1992).
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I.
Issue
The Examiner has rejected claims 5 and 7 as anticipated by Börmann-
El Kholy with evidence provided by Hoischen. The Examiner finds that
Börmann-El Kholy expressly discloses most of the limitations of claim 5 and
Hoischen’s disclosure shows that Börmann-El Kholy’s process inherently
included a step of collecting L-arginine from bacterial culture medium
(Ans. 3).
Appellants contend that “[t]here is nothing in Hoischen et al. which
demonstrates that collecting L-arginine is necessarily present in the process
described by Bormann-El Kholy et al.” (Appeal Br. 4).
The issue with respect to this rejection is: Does the evidence of
record support the Examiner’s finding that the process disclosed by
Börmann-El Kholy included a step of collecting L-arginine from culture
medium, as required by the claims?
Findings of Fact
1. Börmann-El Kholy discloses a bacterial strain overexpressing
glutamate dehydrogenase (gdh) that “was obtained by the transformation of
C. glutamicum WT with plasmid pEKgdh, resulting in C. glutamicum
(pEKgdh). Plasmid pEKgdh carries the C. glutamicum WT gdh gene.”
(Börmann-El Kholy 2329, right col.)
2. Börmann-El Kholy discloses that “standard glutamate
fermentations (6) were performed with . . . C. glutamicum (pEKgdh)” (id. at
2330, left col.).
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3. Börmann-El Kholy states that glutamate secretion rates were
“[d]etermined by the method described in reference 6” (id. at 2330, footnote
b to Table 3).
4. Reference “6” in FFs 2 and 3 is Hoischen (Börmann-El Kholy
2330, right col.).
5. Hoischen describes “Extraction of intracellular amino acids” as
follows:
Cells were separated from the medium by silicone oil
centrifugation, using silicone oil (PN 200; Bayer) as the
separation layer and 20% HClO4 as the acid bottom layer. . . .
The sedimented cells . . . were extracted by sonication. . . .
After cooling on ice, the extracts were centrifuged and the
supernatants were used for amino acid determination.
(Hoischen 3410, left col.)
Analysis
Hoischen describes a process that includes separating cells from
culture medium, sonicating the cells, and centrifuging the sonicated cells to
separate intracellular amino acids from insoluble cell components. Thus,
while Hoischen describes collecting amino acids (which would include
arginine) from cells that have been separated from culture medium, it does
not describe collecting any amino acids from the culture medium itself.
Interpreting claim 5’s limitation of “collecting the L-arginine from the
medium” to read on the process described by Hoischen would be an
unreasonably broad interpretation of the claim language.
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Conclusion of Law
The evidence of record does not support the Examiner’s finding that
the process disclosed by Börmann-El Kholy included a step of collecting L-
arginine from culture medium, as required by the claims.
II.
Issue
The Examiner has rejected claims 5 and 7 under 35 U.S.C. § 103(a) as
obvious in view of Katsumata, Umbarger, Börmann, and “site directed
mutagenesis techniques known in the art” (Ans. 4). The Examiner finds that
Katsumata discloses producing L-arginine using corynebacteria transformed
with a gene related to arginine synthesis, although not glutamate
dehydrogenase (GDH) (id. at 4-5). The Examiner finds that Umbarger
discloses that GDH is involved in arginine biosynthesis and that an increase
in the glutamate produced by GDH would be expected to drive an increase
in production of arginine through mass action (id. at 5). The Examiner finds
that Börmann discloses an isolated GDH gene from Corynebacterium
glutamicum (id.) and concludes that it would have been obvious to use that
gene in Katsumata’s process with a reasonable expectation that doing so
would provide increased production of arginine (id. at 5-6).
Appellants contend that the “regulation of L-amino acid biosynthesis
is very complicated. Therefore, it cannot be expected that . . . a reaction far
from the final product in a biosynthesic [sic] pathway will, in fact, affect the
eventual production of the desired amino acid.” (Appeal Br. 5.) Appellants
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cite Börmann-El Kholy, Eikmanns,6 and the Nakamura Declaration7 as
evidence supporting their position (id. at 5-6).
The issue with respect to this rejection is: Does the evidence of
record support the Examiner’s position that a skilled worker would have
reasonably expected increased expression of glutamate dehydrogenase to
increase production of arginine in recombinant bacteria?
Additional Findings of Fact
6. Katsumata discloses a process for producing L-arginine comprising
culturing corynebacteria transformed with a gene encoding an arginine-
biosynthesis enzyme and recovering L-arginine from the culture medium
(Katsumata 2: 49-54).
7. Katsumata discloses that transformation of corynebacteria with any
of the following genes results in higher L-arginine productivity:
N-acetylglutamate kinase, N-acetyl-γ-glutamyl-phosphate reductase,
N-acetylornithine-δ-aminotransferase, ornithine carbamyltransferase,
N-acetylglutamate synthetase, and N-acetylornithine deacetylase (id. at 2:
20-29).
8. Umbarger discloses that the “glutamate family of amino acids
consists of glutamate itself and those amino acids that derive all or most of
their carbon chains from glutamate: glutamine, proline, and arginine”
(Umbarger 535).

6 Bernhard J. Eikmanns et al., “Nucleotide sequence, expression and
transcriptional analysis of the Corynebacterium glutamicum gltA gene
encoding citrate synthase,” 140 MICROBIOLOGY 1817-1828 (1994).
7 Declaration under 37 C.F.R. § 1.132 of Jun Nakamura, submitted July 10,
2006.
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9. Umbarger’s Figure 1 is reproduced below:

(Id.) The figure shows the biosynthesis of the glutamate family of amino
acids (id., legend to Figure 1).8
10. Glutamate dehydrogenase catalyzes the conversion of α-
ketoglutarate to glutamate (Börmann 317); i.e., reaction 1 in the upper left
corner of Umbarger’s Figure 1.
11. The Examiner points out that glutamate is a reactant in the first
step leading to arginine production (reaction 8) (Ans. 5) and is also required
for reactions 11 and 12a (id. at 9-10).
12. The Nakamura Declaration includes Figure 1, reproduced below:

8 The portion of the figure showing biosynthesis of lysine in fungi has been
omitted because it is not relevant to amino acid biosynthesis in
corynebacteria.
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The figure shows “the arginine biosynthetic pathway of coryneform
bacterium” (Nakamura Declaration, ¶ 4). Glutamate dehydrogenase
catalyzes the reaction connecting the two sets of cyclic reactions.
13. The enzymes in the declaration’s Figure 1 that are labelled ArgB,
ArgC, ArgD, and ArgJ respectively catalyze reactions 9, 10, 11, and 12a in
Umbarger’s Figure 1; the declaration shows the N-acetylglutamate by-
product of reaction 12a being used as the substrate for reaction 9.
14. Both the reaction catalyzed by ArgD (Umbarger’s reaction 11)
and the reaction catalyzed by ArgJ (Umbarger’s reaction 12a) require
glutamate as a reactant.
15. The Nakamura Declaration describes an experiment to measure
the specific activity of the arginosuccinate synthetase (ArgG) enzyme from
Brevibacterium lactofermentum (Nakamura Declaration, ¶ 4).
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16. The Nakamura Declaration presents a comparison of the specific
activity of arginosuccinate synthetase (0.014 U/mg) with previously reported
specific activities of glutamate dehydrogenase (1.8 U/mg), ornithine
acetyltransferase (ArgJ) (0.16 U/mg), and N-acetylglutamate kinase (ArgB)
(0.10 U/mg) (id. at Table 1).
17. The Examiner finds that “specific activity alone does not
determine the rate limiting step in the arginine biosynthetic pathway”
(Ans. 11).
Principles of Law
“Obviousness does not require absolute predictability of success. . . .
For obviousness under § 103, all that is required is a reasonable expectation
of success.” In re O’Farrell, 853 F.2d 894, 903-04 (Fed. Cir. 1988).
“After evidence or argument is submitted by the applicant in response
[to the prima facie case of obviousness], patentability is determined on the
totality of the record, by a preponderance of evidence with due consideration
to persuasiveness of argument.” In re Oetiker, 977 F.2d 1443, 1445 (Fed.
Cir. 1992).
Analysis
Katsumata discloses that overexpression of any one of six enzymes
involved in arginine biosynthesis will lead to increased production of
arginine in transformed corynebacteria. One of those genes is N-
acetylglutamate kinase – ArgB in the Nakamura Declaration, reaction 9 in
Umbarger – which catalyzes a reaction six steps removed from the reaction
that forms arginine.
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Umbarger discloses that two molecules of glutamate are required (in
reactions 11 and 12a) to produce each molecule of ornithine that can be
converted to arginine. In other words, each turn of the
ArgB/ArgC/ArgD/ArgJ cycle of reactions shown in the Nakamura
Declaration’s Figure 1 requires input of two glutamate molecules (among
other reactants) and results in formation of one molecule of ornithine that
can be converted to arginine.
We agree with the Examiner that Umbarger’s disclosure of the
importance of glutamate to the reactions in the arginine biosynthetic
pathway, and Katsumata’s disclosure that any of six arginine biosynthesis
genes (including acetylglutamate kinase) can be used to increase arginine
production, would have led a skilled worker to reasonably expect that
overexpression of glutamate kinase in a transformed bacterium would also
result in increased arginine production.
Appellants argue that because of the complicated regulation of amino
acid biosynthesis, it is unpredictable that “a reaction far from the final
product in a biosynthesic [sic] pathway will, in fact, affect the eventual
production of the desired amino acid” (Appeal Br. 5). In support of their
position, Appellants cite Börmann-El Kholy as disclosing that GDH is
dispensable for the glutamate synthesis required for growth and that
glutamate secretion in C. glutamicum cannot be enhanced by increasing
GDH activity (id. at 5-6). Appellants also cite Eikmanns as disclosing that
secretion of glutamate in C. glutamicum is not enhanced by increasing the
level of citrate synthase (id. at 6). Finally, Appellants point to the data in the
Nakamura Declaration showing that the specific activity of GDH is “more
than 10 times” higher than that of some other arginine biosynthesis enzymes
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as evidence that “GDH is not involved in a rate-determining step of the
arginine biosynthetic pathway” (id.).
We have considered Appellants’ evidence but conclude that it does
not outweigh the evidence of record supporting a reasonable expectation of
success. Although Börmann-El Kholy and Eikmanns provide the
disclosures that Appellants rely on, the fact that GDH and citrate synthase
overexpression does not increase glutamate secretion does not provide
sufficient basis for concluding that increasing GDH expression would be
unlikely to lead to increased glutamate in cells9 or that increased glutamate
would be unlikely to lead to increased arginine synthesis.
With regard to the specific activity data shown in the Nakamura
Declaration, the Examiner has found that “specific activity alone does not
determine the rate limiting step in the arginine biosynthetic pathway”
(Ans. 11). Appellants have not provided evidence to the contrary, and the
Examiner’s finding makes scientific sense: the reactions shown in
Umbarger require reactants and an enzymatic catalyst. It therefore follows
that the overall rate of the reaction could be limited either by the activity of
the catalyst or by the availability of the reactants.
Katsumata provides evidence that increasing the activity of enzymes
in the arginine biosynthesis pathway would be expected to increase arginine
production even if the reactions they catalyze occur prior to a step catalyzed
by an enzyme with a low specific activity. That is, the Nakamura
Declaration provides evidence that the specific activity of arginosuccinate

9 Börmann-El Kholy in fact found increased intracellular glutamate in cells
that overexpressed GDH (Börmann-El Kholy, abstract), even though the
glutamate was not secreted.
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synthase (ArgG) is about 10-fold lower than that of N-acetylglutamate
kinase (ArgB) and ornithine acetyltransferase (ArgJ). Yet Katsumata
discloses that overexpression of any of several enzymes – including N-
acetylglutamate kinase – results in increased arginine production, and most
if not all of Katsumata’s enzymes precede arginosuccinate synthase (ArgG)
in the arginine biosynthesis pathway. Thus, it is apparent from Katsumata
that the low specific activity of arginosuccinate synthase does not prevent
overexpression of enzymes farther back in the biosynthesis pathway from
increasing the production of arginine.
Conclusion of Law
The evidence of record supports the Examiner’s position that a skilled
worker would have reasonably expected increased expression of glutamate
dehydrogenase to increase production of arginine in recombinant bacteria.
SUMMARY
We reverse the rejection of claims 5 and 7 as anticipated by Bormann-
El Kholy but affirm the rejection of claims 5 and 7 as obvious in view of
Katsumata, Umbarger, Bormann, and known site-directed mutagenesis
techniques known in the art.
TIME PERIOD FOR RESPONSE
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a).

AFFIRMED

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