Ex Parte Fukui et al

Patent Trial and Appeal Board

Feb 19, 2013

11560937 (P.T.A.B. Feb. 19, 2013)

UNITED STATES PATENT AND TRADEMARK OFFICE
____________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
____________

Ex parte KEITA FUKUI, JUN NAKAMURA, KAYO AKIYAMA,
and HIROYUKI KOJIMA
____________

Appeal 2011-005094
Application 11/560,937
Technology Center 1600
____________

Before DONALD E. ADAMS, ERICA A. FRANKLIN, and
JACQUELINE WRIGHT BONILLA, Administrative Patent Judges.

ADAMS, Administrative Patent Judge.

DECISION ON APPEAL
This appeal under 35 U.S.C. § 134 involves claims 8, 10, 13, 14, and
16 (App. Br. 5; Ans. 2).1 We have jurisdiction under 35 U.S.C. § 6(b).

1 Pending claims 1-7 stand withdrawn from consideration (App. Br. 5).
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STATEMENT OF THE CASE
The claims are directed to a method for producing succinic acid.
Claim 8 is representative and is reproduced in the Claims Appendix of
Appellants’ Brief.
Claims 8, 10, 13, 14, and 16 stand rejected under 35 U.S.C. § 103(a)
as unpatentable over the combination of Dusch,2 Tomar,3 ‘888,4 and ‘385.5
We affirm.

ISSUE
Does the preponderance of evidence on this record support a
conclusion of obviousness?

2 Dusch et al., JP 2001-161386, published June 19, 2001 (as translated in ZA
20006039, lodged October 26, 2000; see generally Office Action entered
October 14, 2008).
3 A. Tomar et al., The effect of acetate pathway mutations on the production
of pyruvate in Escherichia coli, 62 APPL. MICROBIOL. BIOTECHNOL. 76-82
(2003).
4 Kobayashi et al., JP 11-196888, published July 27, 1999 (as translated at
http://dossier1.ipdl.inpit.go.jp/cgi-bin/tran_web_cgi...edical_NG2V13
&Ntt9=gakujutsuV13&Ntt10=&Ntt11=&Ntt12=, accessed March 23, 2009;
see generally Office Action entered October 14, 2008).
5 Hatakeyama et al., JP 11-206385, published August 3, 1999 (as translated
at http://dossier1.ipdl.inpit.go.jp/cgi-bin/tran_web_cgi_...ience_NG1_v5&
Ntt0=science_NG2_v5&Ntt10=science_NG3_v5, accessed July 17, 2008;
see generally Office Action entered October 14, 2008).
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FACTUAL FINDINGS (FF)
FF 1. Tomar’s Figure 1 is reproduced below:

Tomar’s Figure 1 illustrates “[b]iochemical pathways involved in pyruvate
accumulation in Escherichia coli. 1 Phosphotransferase system, 2 pyruvate
kinase, 3 pyruvate dehydrogenase, 4 phosphoenol pyruvate carboxylase, 5
pyruvate oxidase, 6 lactate dehydrogenase, 7 acetyl CoA synthase, 8
phosphotransacetylase, 9 acetate kinase, 10 isocitrate lyase, 11 malate
synthase” (Tomar 77: col. 1, Fig. 1, legend).
FF 2. ‘888 suggests a
method for production of succinic acid in [] coryneform
bacteria having succinic acid producing ability…, said method
comprising allowing said coryneform bacterium … to act on an
organic raw material in a reaction liquid containing carbonate
ion, bicarbonate ion or carbon dioxide to produce and
accumulate succinic acid in the reaction liquid under anaerobic
conditions … polymerizing the obtained succinic acid

(Ans. 4-5 (citing ‘888 claims 1-6 and entire document).)
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FF 3. Tomar’s Figure 1 illustrates, inter alia, that “pyruvate oxidase and
lactate dehydrogenase … divert … pyruvate (a key intermediate precursor
for succinate/succinic acid production) to form acetate or lactate (unwanted
by-products) respectively” (Ans. 6; see Tomar 77: col. 2, ll. 17-18; see also
Tomar 77, Fig. 1).
FF 4. ‘385 suggests a “method for production of succinic acid in a
coryneform bacteria having succinic acid producing ability” that involves
disruption of [the] lactate dehydrogenase gene responsible for
catalyzing the formation of lactate, an unwanted by-product of
fermentation…, said method comprising allowing said
genetically modified coryneform bacterium … to act on an
organic raw material in a reaction liquid containing carbonate
ion, bicarbonate ion or carbon dioxide to produce and
accumulate succinic acid in the reaction liquid under anaerobic
conditions.

(Ans. 5 (citing ‘385 claims 1-6 and entire document).)

FF 5. “Pyruvate oxidase is encoded by the poxB gene and … would be an
obvious next candidate for mutation as a means to generate additional
pyruvate” (Tomar 81: col. 1, l. 7 - col. 2, l. 2; Ans. 6).
FF 6. Dusch suggests the amino acid sequence of pyruvate oxidase, which
corresponds to Appellants’ SEQ ID NO: 49 (Ans. 4).
FF 7. Dusch suggests “coryneform bacteria … wherein the activity of [the]
poxB gene encoding the pyruvate oxidase enzyme has been
attenuated/decreased as a result of integration mutagenesis” (id.; see Dusch
translation 5: 18-27 (Dusch’s “invention … relates to a process for the
fermentative production of amino acids … using coryneform bacteria …
[wherein] the nucleotide sequences which code for the poxB gene are
attenuated…. [T]he term ‘attenuation’ means reducing or suppressing the
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intracellular activity of one or more enzymes … in a microorganism”); see
also id. at 19: 10 (“Integration mutagenesis of the poxB gene”)).
FF 8. Examiner finds that the “pyruvate carboxylase gene (pyc) [is] one of
the key genes involved in the production of organic acids such as succinic
acid” (Ans. 5).
FF 9. Dusch and ‘888 both suggest “the overexpression/increased activity
of [the] pyc gene” in coryneform bacteria (id. at 4-5)
FF 10. Vemuri’s6 Figure 4 is reproduced below:

Vemuri’s Figure 4 illustrates “[b]iochemical pathways for the synthesis of
succinate from glucose in E. coli” (Vemuri 1722: FIG. 4, legend).

6 G. N. Vemuri, et al., Effects of Growth Mode and Pyruvate Carboxylase on
Succinic Acid Production by Metabolically Engineered Strains of
Escherichia coli, 68(4) APPLIED AND ENVIRONMENTAL MICROBIOLOGY
1715-1727 (2002).
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ANALYSIS
Appellants provide separate arguments for the following groups of
claims: (I) claims 8, 13, 14, and 16; and (II) claim 10.7 Claims 8 and 10 are
representative.
Based on the combination of Dusch, Tomar, ‘888, and ‘385, Examiner
concludes that, at the time Appellants’ invention was made, it would have
been prima facie obvious to produce succinic acid by a method that allows a
coryneform bacterium, comprising: (1) modifications that disrupt the genes
encoding, and thereby decrease the activity of, pyruvate oxidase and lactate
dehydrogenase and (2) having a succinic acid-producing ability, to act on an
organic raw material in a reaction liquid containing carbonate ion,
bicarbonate ion or carbon dioxide under anaerobic conditions to produce and
accumulate succinic acid in the reaction liquid (see Ans. 6-8; FF 1-9).

Claim 8:
Appellants contend “that the cited documents, either alone or in
combination, fail to suggest that a decrease in pyruvate oxidase activity,
much less a decrease in pyruvate oxidase activity as a result of modification
of a gene encoding SEQ ID NO: 49 or a variant thereof, plays a role in
succinic acid biosynthesis” (App. Br. 17). We are not persuaded.
Methods of producing succinic acid, wherein coryneform bacteria are
allowed to act on an organic raw material in a reaction liquid containing

7 Appellants separately list claims 13, 14, and 16 and contend that “the
obviousness rejection based upon [these] claim[s] … is without appropriate
basis for at least the reasons set forth by Appellant with respect to claim 8”
(App. Br. 31-32). Therefore, claims 13, 14, and 16 stand or fall with claim
8.
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carbonate ion, bicarbonate ion or carbon dioxide to produce and accumulate
succinic acid in the reaction liquid under anaerobic conditions, were known
at the time of Appellants’ claimed invention (FF 2 and 4).
Tomar’s Figure 1 illustrates that pyruvate is an intermediate, i.e., a
precursor, for succinic acid production (FF 1 and 3). Therefore, at the time
of Appellants’ claimed invention, a person of ordinary skill in this art would
have recognized that pathways diverting pyruvate away from the production
of succinic acid, or otherwise reducing the intracellular concentration of
pyruvate, would have been detrimental to the production of succinic acid
(see, e.g., FF 4). Tomar’s Figure 1 illustrates that pyruvate oxidase and
lactate dehydrogenase divert pyruvate away from the production of succinic
acid (FF 1).
Disruption of lactate dehydrogenase inhibits the conversion of
pyruvate to lactate (see FF 1 and 3). ‘385 suggests a method of producing
succinic acid, wherein a coryneform bacteria comprising a disruption of the
lactate dehydrogenase gene is allowed to act on an organic raw material in a
reaction liquid containing carbonate ion, bicarbonate ion or carbon dioxide
to produce and accumulate succinic acid in the reaction liquid under
anaerobic conditions (FF 4).
Tomar suggests that pyruvate oxidase would have been “an obvious
next candidate for mutation as a means to generate additional pyruvate” (FF
5). Stated differently, Tomar suggests the disruption of pyruvate oxidase to
inhibit the conversion of pyruvate to acetate (id.; see FF 3). Dusch suggests
the modification of the pyruvate oxidase gene, which encodes a protein
having the amino acid sequence of Appellants’ SEQ ID NO: 49, in
coryneform bacteria (FF 6-7).
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Therefore, notwithstanding Appellants’ contentions to the contrary,
the combination of prior art relied upon by Examiner suggests that decreases
in pyruvate oxidase and lactate dehydrogenase activities play a role in
succinic acid biosynthesis by inhibiting pyruvate from entering pathways
that result in unwanted products (Cf. App. Br. 17).
For the foregoing reasons we are not persuaded by Appellants’
contention that “it was not known that … the enhancement or attenuation of
any gene involved in L-amino acid production [(e.g., pyruvate oxidase)] was
also effective in succinic acid production” (App. Br. 17; see also id. at 23;
Cf. FF 5-7).
For the same reasons we are not persuaded by Appellants’ contention
that Examiner “fail[ed] to establish or provide a reason as to why one of
ordinary skill in the art would have combined the art cited to arrive at the
invention as claimed” (id. at 18; see also id. at 21 “the cited art fails to
specifically motivate the skilled artisan to practice … [the claimed]
method”).
Appellants failed to establish that succinate/succinic acid production
in coryneform bacteria follows a different pathway than that set forth in
Tomar’s figure 1 (see FF 1; Cf. Ans. 11 (the “TCA cycle is conserved across
various organisms including prokaryotes and eukaryotes”)). Therefore, we
are not persuaded by Appellants’ contentions that “Tomar is not germane to
the claimed subject matter” or that “[u]nder anaerobic conditions the TCA
cycle does not proceed well” (App. Br. 18 and Reply Br. 2).
We recognize, but are not persuaded by, Appellants’ reference to “the
Official Action mailed September 3, 2008” in support of their contention
that the obviousness rejection before this panel “can only be characterized as
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a 180 degree shift in [Examiner’s] position” (id. at 18-19; see also Reply Br.
3-4). We recognize that in response to Examiner’s September 3, 2008
Office Action, Appellants amended the claims and contended that “any (and
every) coryneform bacteria can be used, as the succinic acid-producing
pathway including pyruvate oxidase and lactate dehydrogenase is conserved
in coryneform bacteria” (December 10, 2008, Amendment Under 37 C.F.R.
§ 1.11: 8-9; Cf. Ans. 19-21). As discussed above, Appellants failed to
establish that succinate/succinic acid production in coryneform bacteria
follows a different pathway than that set forth in Tomar’s figure 1.
Therefore, we are not persuaded by Appellants’ contention that “the
documents in combination fail to establish any nexus between pyruvate
oxidase function in E. coli and the role of poxB disruption in coryneform
bacteria in succinic acid production” (App. Br. 20; Reply Br. 4; Cf. Ans. 12-
13 (“The key points … of entry or diversion of the desired precursors, the
metabolic pathway, the specific enzymes, their structure correlated to
function are also known in the art for many of the industrially important
microorganisms such as E.[ ]coli and Corynebacteria”)).
Notwithstanding, Appellants’ unsupported contentions regarding the
differences between E. coli and coryneform bacteria, discussed above, we
recognize Appellants’ reliance on Vemuri, which addresses the “Effects of
Growth Mode and Pyruvate Carboxylase on Succinic Acid Production by
Metabolically Engineered Strains of Escherichia coli” (Vemuri, Title; App.
Br. 24). To be clear, Vermuri illustrates part, but not all, of the pathway set
forth in Tomar (see FF 10; Cf. FF 1; see also App. Br. 27). Nevertheless,
Appellants contend that “Vermuri fails to disclose pyruvate oxidase[’s]
involvement in succinate production at all, much less as a ‘key enzyme’ in
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succinic acid production” (App. Br. 24; Reply Br. 4-5). We are not
persuaded. The question is not whether pyruvate oxidase is involved in
succinic acid production, but is instead whether pyruvate concentration in a
cell is reduced or otherwise diverted away from succinic acid production, by
entering a pathway, involving pyruvate oxidase, that leads to an unwanted
product, e.g., acetate. As discussed above, the combination of prior art
relied upon by Examiner suggests that succinic acid production will benefit
from inhibiting pyruvate from entering into pathways leading to the
production of unwanted products, e.g., lactate and acetate (FF 3; Cf. App.
Br. 27 (“the Office has failed to acknowledge Appellant’s arguments and
evidence … that one of ordinary skill in the art would not have been
motivated by the teachings of Tomar to attenuate pyruvate oxidase activity
in a method of succinic acid production”)).
For the foregoing reasons and those set forth by Examiner, we are not
persuaded by Appellants’ contentions relating to Bott8 (see Ans. 27-29; Cf.
App. Br. 25-26 and 27-28; Reply Br. 5-6).

Claim 10:
We are not persuaded by Appellants’ contention that the combination
of prior art relied upon by Examiner fails to suggest “’polymerizing the
obtained succinic acid’” (App. Br. 30; Cf. FF 2).

8 Michael Bott and Axel Niebisch, The respiratory chain of
Corynebacterium glutamicum, 104 J. BIOTECH. 129-153 (2003).
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CONCLUSION OF LAW
The preponderance of evidence on this record supports a conclusion
of obviousness. The rejection of claims 8 and 10 under 35 U.S.C. § 103(a)
as unpatentable over the combination of Dusch, Tomar, ‘888, and ‘385 is
affirmed. Claims 13, 14, and 16 are not separately argued and fall with
claim 8.
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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