Ex Parte Stoynova et al

Board of Patent Appeals and Interferences

Aug 21, 2008

11218787 (B.P.A.I. Aug. 21, 2008)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte NATALIA VIKTOROVNA STOYNOVA,
ELENA VIKTOROVNA SYCHEVA,
EKATERINA SERGEEVNA PREOBRAZHENSKAYA,
ANNA EVGENIEVNA NOVIKOVA, and
NIKOLAY GEORGIEVICH MATROSOV
__________

Appeal 2008-2024
Application 11/218,787
Technology Center 1600
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Decided: August 21, 2008
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Before DONALD E. ADAMS, RICHARD M. LEBOVITZ, and
MELANIE L. McCOLLUM, Administrative Patent Judges.

McCOLLUM, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to an
Escherichia coli producing an abnormal amino acid. The Examiner has
rejected the claims as obvious. We have jurisdiction under 35 U.S.C. § 6(b).
We affirm.
Appeal 2008-2024
Application 11/218,787

STATEMENT OF THE CASE
Claims 16 and 17 are on appeal. Claims 18 and 19 are also pending
but have been withdrawn from consideration by the Examiner. The claims
on appeal have not been argued separately and therefore stand or fall
together. 37 C.F.R. § 41.37(c)(1)(vii). We will focus on claim 16, the
broadest claim on appeal, which reads as follows:
16. An abnormal amino acid-producing Escherichia coli, wherein
the Escherichia coli has been modified to inactivate AHAS I encoded by the
ilvBN genes, AHAS II encoded by the ilvGM genes, and AHAS III encoded
by the ilvIH genes on its chromosome by disrupting the ilvBN genes, the
ilvGM genes, and the ilvIH genes, respectively, and wherein the Escherichia
coli has been further modified to have enhanced expression of the leuABCD
operon of Escherichia coli by increasing its copy number or by replacing its
native promoter with a stronger promoter, as compared to a non-modified
Escherichia coli.
Claims 16 and 17 stand rejected under 35 U.S.C. § 103(a) as obvious
in view of Guardiola,1 Herring,2 Barak,3 Bogosian,4 Kisumi,5 Vartak,6 and
Datsenko7 (Ans. 3-4).

1 John Guardiola et al., Mutants of Escherichia coli K-12 Missing
Acetolactate Synthase Activity, 120 JOURNAL OF BACTERIOLOGY 536-538
(1974).
2 Patricia A. Herring et al., Channeling Behavior and Activity Models for
Escherichia coli K-12 Acetohydroxy Acid Synthases at Physiological
Substrate Levels, 207 BIOCHEMICAL AND BIOPHYSICAL RESEARCH
COMMUNICATIONS 48-54 (1995).
3 Ze’ev Barak et al., Physiological Implications of the Specificity of
Acetohydroxy Acid Synthase Isozymes of Enteric Bacteria, 169 JOURNAL OF
BACTERIOLOGY 3750-3756 (1987).
4 Gregg Bogosian et al., Biosynthesis and Incorporation into Protein of
Norleucine by Escherichia coli, 264 JOURNAL OF BIOLOGICAL CHEMISTRY
531-539 (1989).
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Application 11/218,787

The Examiner relies on Guardiola for disclosing “the role of AHAS I-
III in the biosynthesis of L-amino acid Valine and generation of mutants of
E.coli K-12 missing acetolactate synthase activity (acteohydroxy synthase)”;
on Herring for disclosing “the role of AHAS I and AHAS III in the
conversion of α-ketobutyrate to branched L-amino acids such as Isoleucine
and Valine in E.coli K-12”; and on Barak for disclosing “the physiological
implications of the specificity of acetohydroxy acid synthase isozymes
(AHAS I-III) of enteric bacteria . . . and the details regarding the gene (map
location) and structure of ilvBN, ilvGM, and ilvIH of E.coli K12” (Ans. 4).
The Examiner relies on Kisumi, Bogosian, and Vartak for disclosing “the
structure and the role of leuABCD operon in the biosynthesis of abnormal
amino acids such as Norvaline and Norleucine” (id.). The Examiner relies
on Datsenko for disclosing “methods for one-step inactivation of any
chromosomal gene of interest in E.coli K12 using PCR products” (id. at 6).
The Examiner concludes that it would have been obvious
to modify an E.coli bacterial strain wherein the genes encoding
AHAS I-III are disrupted to relieve the microorganism from the
biosynthetic pathway proceeding towards L-amino acid
biosynthesis of Valine, Leucine and Isoleucine to have
enhanced expression of leuABCD operon . . . to increase the
production of intermediates like α-KV and α-KC shown to be
5 Masahiko Kisumi et al., Biosynthesis of Norvaline, Norleucine, and
Homoisoleucine in Serratia marcescens, 80 J. BIOCHEM. 333-339 (1976).
6 Narendra B. Vartak et al., A Functional leuABCD Operon is Required by
Leucine Synthesis by the Tyrosine-Repressible Transaminase in Escherichia
coli K-12, 173 JOURNAL OF BACTERIOLOGY 3864-3871 (1991).
7 Kirill A. Datsenko and Barry L. Wanner, One-step Inactivation of
Chromosomal Genes in Escherichia coli K-12 using PCR Products,
97 PNAS 6640-6645 (2000).
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Application 11/218,787

required for producing abnormal amino acids such as
Norleucine and Norvaline.
(Id. at 7.)
Appellants contend that the Examiner erred in concluding that
claim 16 would have been obvious over the applied references.
FINDINGS OF FACT
1. Acetohydroxy acid synthase (AHAS) is also known as
acetolactate synthase (Barak 3750).
2. “At least three different AHAS isozymes [AHAS I-III] . . . have
been recognized in the enteric bacteria,” including Escherichia coli (id. at
3750-3751).
3. The AHAS enzymes are involved in the formation of valine and
isoleucine from pyruvate and 2-ketobutyrate (2-KB) in Escherichia coli.
Specifically, the AHAS enzymes catalyze the condensation of pyruvate with
a second pyruvate to form 2-acetolactate (AL), a precursor of valine, and
catalyze the condensation of pyruvate with 2-KB to form 2-aceto-
2-hydroxybutyrate (AHB), a precursor of isoleucine. (Herring 48-49;
Barak 3750.)
4. Guardiola discloses the isolation of an Escherichia coli K-12
mutant missing AHAS activity and therefore requiring isoleucine and valine
for growth (Guardiola 536).
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Application 11/218,787

5. Kisumi Figure 1 is reproduced below:

This Figure sets forth the “[r]egulatory mechanisms of isoleucine, valine,
and leucine biosynthesis in Serratia marcescens” (Kisumi 334). As depicted
in this Figure, the Ile-Val biosynthetic enzymes, including AHAS (“Active
acetaldehyde”), are involved in the formation of isoleucine and valine. In
addition, this Figure demonstrates that the Leu biosynthetic enzymes are
involved in the formation of leucine.
6. Kisumi discloses that norvaline is formed from α-ketobutyrate
by the leucine biosynthetic enzymes in Serratia marcescens (Kisumi 337).
7. Kisumi also discloses that “[n]orleucine may be formed from
α-ketovalerate, the α-ketoacid corresponding to norvaline, by the leucine
biosynthetic enzymes” (id.).
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8. Kisumi Figure 3 is reproduced below:

Kisumi Figure 3 sets forth “[p]ostulated biosynthetic pathways of branched-
chain amino acid analogs” (Kisumi 338). As depicted in this Figure, the
formation of norvaline (Nva) and norleucine (Nle) appears to be catalyzed
by the Leu enzymes but does not appear to be catalyzed by the AHAS
(“Active acetaldehyde”) enzymes.
9. Vartak discloses that, in “wild-type strains of Escherichia coli,
the first three steps in leucine biosynthesis are catalyzed by enzymes
encoded by the leuABCD operon” (Vartak 3864).
10. Bogosian discloses that norleucine formation in Escherichia
coli is “dependent upon the leucine biosynthetic enzymes, as deletion of the
leucine operon from an otherwise wild-type strain prevented the biosynthesis
of norleucine” (Bogosian 538).
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Application 11/218,787

11. Bogosian also discloses that the “proposed biosynthetic
pathway for norleucine in E. coli is the same as that proposed for
S. marcescens” by Kisumi (id.).
12. Bogosian Figure 6 is reproduced below:

Bogosian Figure 6 sets forth the “[p]roposed pathway of norleucine and
norvaline biosynthesis in E. coli” (Bogosian 536). As depicted in this
Figure, the proposed biosynthetic pathway for norvaline in E. coli is also the
same as that proposed for S. marcescens by Kisumi. In addition, this Figure
shows that pyruvate and α-ketobutyrate are used to form norvaline and
norleucine.
13. Datsenko discloses a one-step method to disrupt chromosomal
genes in E. coli K-12 (Datsenko 6640).
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Application 11/218,787

ANALYSIS
Guardiola describes an Escherichia coli K-12 mutant missing AHAS
activity (Finding of Fact (FF) 4). AHAS activity is involved in the
formation of valine and isoleucine from pyruvate and 2-ketobutyrate in
Escherichia coli (FF 3 & 5). However, AHAS activity is not thought to be
involved in the formation of norvaline and norleucine from pyruvate and
2-ketobutyrate (FF 6-8 & 11-12). The leuABCD operon encodes enzymes
involved in the formation of norleucine (FF 7-12). Thus, we agree that the
Examiner has set forth a prima facie case that it would have been obvious to
modify an Escherichia coli to inactivate AHAS (“to relieve the
microorganism from the biosynthetic pathway proceeding towards L-amino
acid biosynthesis of Valine . . . and Isoleucine”) and to enhance expression
of the leuABCD operon in order to produce abnormal amino acids norleucine
and norvaline (Ans. 7).
Appellants argue, however, that “the fact that the alleged prima facie
case of obviousness requires the combined teachings of seven references
actually suggests non-obviousness of the claimed subject matter” (Br. 6).
We are not persuaded by this argument. Instead, we agree with the
Examiner that the number of references being applied is not of itself
evidence of non-obviousness (Ans. 9). In re Gorman, 933 F.2d 982, 986
(Fed. Cir. 1991) (“[t]he criterion . . . is not the number of references, but
what they would have meant to a person of ordinary skill in the field of the
invention”).
Appellants also argue that “the little bits of knowledge culled from
each individual reference, if combined by the person of ordinary skill in the
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art, would . . . not suggest to that person the totally unexpected result which
is achieved by the present claimed invention” (Br. 6). In particular,
Appellants argue
as shown in Table 2 of the specification, [for] the strain
NS1118/pBR-leuABCD, in which all the AHASes are
inactivated and expression of the leuABCD operon is enhanced,
production of norleucine was more than double as compared to
the strain NS1118, in which only the AHASes are inactivated.
Therefore, this prominent effect of combining the inactivation
of all of the AHASes with increasing the expression of the
leuABCD operon on the production of norleucine in E. coli,
could not have been predicted from the combination of
teachings of the references, and hence represents an entirely
unexpected result.
(Id. at 9.)
We are not persuaded by this argument. As noted by the Examiner,
Bogosian discloses that the leuABCD operon encodes enzymes involved in
the formation of norleucine (FF 9-10). Thus, we agree with the Examiner
that it would not have been unexpected that increasing the expression of the
leuABCD operon would increase the production of norleucine (Ans. 12-13).
In addition, Appellants argue that the “teachings of these seven references
are NOT the combination of familiar elements according to known methods
which results in a predictable result” (Br. 6).
We are not persuaded by this argument. Instead, we agree with the
Examiner that the “basic strategy of up-regulating the expression of genes of
necessary enzymes and down-regulating the genes of competing enzymes is
well known in the art and [that] the cited references . . . provide all the
necessary pieces one would need to apply this basic strategy for the
production of [an abnormal] amino acid” (Ans. 9). In addition, as discussed
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above, Appellants have not shown that the combination provides results that
would not have been predictable.
CONCLUSION
We conclude that the Examiner has set forth a prima facie case that
claim 16 would have been obvious over the applied references, which
Appellants have not rebutted. We therefore affirm the rejection of claim 16
under 35 U.S.C. § 103(a). Claim 17 falls with claim 16.
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

clj

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