Ex Parte HO et al

Board of Patent Appeals and Interferences

Aug 28, 2008

09180340 (B.P.A.I. Aug. 28, 2008)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE BOARD OF PATENT APPEALS
AND INTERFERENCES
__________

Ex parte NANCY W. Y. HO and ZHENG-DAO CHEN
__________

Appeal 2008-3282
Application 09/180,340
Technology Center 1600
__________

Decided: August 28, 2008
__________

Before LORA M. GREEN, RICHARD M. LEBOVITZ, and
FRANCISCO C. PRATS, Administrative Patent Judges.

PRATS, Administrative Patent Judge.

DECISION ON APPEAL
This is an appeal under 35 U.S.C. § 134 involving claims to methods
and vectors for integrating multiple copies of exogenous DNA into reiterated
chromosomal DNA of cells. The Examiner has rejected the claims as
obvious. We have jurisdiction under 35 U.S.C. § 6(b). We affirm-in-part.

Appeal 2008-3282
Application 09/180,340

STATEMENT OF THE CASE
Claims 14-18, 28-30, and 32-34 are pending and on appeal (App. Br.
2). Claims 14, 29, and 34 are representative of the appealed subject matter
and read as follows:
14. A method of integrating multiple copies of
exogenous DNA into reiterated chromosomal DNA of cells,
comprising:
(a) transforming the cells with a replicative and
integrative plasmid comprising an autonomous replicating
sequence, exogenous DNA, and a first selection marker; and
(b) repeatedly replicating the cells from step (a) to
produce a number of generations of progeny cells while
selecting for cells which include the selection marker,
promoting the retention of the replicative and integrative
plasmid in subsequent generations of the progeny cells and
produce progeny cells having multiple integrated copies of the
exogenous DNA.

29. A plasmid vector comprising a functional yeast
autonomous replicating sequence and exogenous DNA
including genes encoding xylose reductase, xylitol
dehydrogenase, and xylulokinase flanked on each end by a
DNA flanking sequence which is homologous to a reiterated
DNA sequence of a target yeast cell, the plasmid vector for use
in integrating the exogenous DNA sequence into chromosomal
DNA of the target yeast to form stable integrants which ferment
xylose to ethanol.

34. A plasmid vector comprising a functional yeast
autonomous replicating sequence and exogenous DNA flanked
on each end by a DNA flanking sequence which is homologous
to a reiterated ribosomal DNA sequence of a target yeast cell,
the plasmid further comprising a selection marker in a position
other than between the DNA flanking sequences, the plasmid
vector for use in integrating an exogenous DNA sequence into
chromosomal DNA of the target yeast cell.

2
Appeal 2008-3282
Application 09/180,340

The Examiner applies the following documents in rejecting the
claims:
Hallborn1 WO 91/15588 A1 Oct. 17, 1991
Ho WO 95/13362 A1 May 18, 1995

S. Lopes et al., Factors Affecting the Mitotic Stability of High-copy-
number Integration into the Ribosomal DNA of Saccharomyces cerevisiae
YEAST VOL. 12, PP 467-477 (1996).

The following rejections are before us for review:
Claims 14-18, 28-30, and 32-34 stand rejected under 35 U.S.C.
§ 103(a) as obvious over Ho and Lopes (Ans. 3-7).2
Claims 28, 29, and 34 stand rejected under 35 U.S.C. § 103(a) as
obvious over Ho and Hallborn (Ans. 7-13).
OBVIOUSNESS -- HO AND LOPES
ISSUE
The Examiner cites Ho as teaching “recombinant yeasts containing
genes encoding xylose reductase, xylitol dehydrogenase and xylulokinase,
DNA molecules, plasmid vectors and methods useful for producing said
yeasts which are capable of fermenting xylose to ethanol and glucose to
ethanol” (Ans. 3). The Examiner further cites Ho as teaching “selection
markers (pages 15-16) and specific DNA fragments that serve as replicons
and selection markers that enable the plasmid to be replicated autonomously
in S. cerevisiae” (Ans. 4). Regarding the claim limitations requiring the
plasmid to be “integrative,” the Examiner states:

1 The Examiner’s Answer mailed September 15, 2005, lists “CA 2090122”
as the document number for the Hallborn reference (Ans. 2). However, the
document on file having the asserted publication date is WO 91/15588 A1.
2 Examiner’s Answer mailed September 15, 2005.
3
Appeal 2008-3282
Application 09/180,340

In-so-far-as Ho et al. does not explicitly teach the integration at
multiple sites, Lopes et al. teach numerous plasmids containing
various genes integrated into a RNA gene of S. cerevisiae.
Multiple copies of the plasmid were successfully integrated into
the genome (over 140 copies), which are stably maintained in
non-selective medium for generations over long periods of time
(see abstract and pages 467-475). Further, the plasmids
contained a Leu2d selection marker and various cloned genes
for stability and expression studies.

(Ans. 4.)
Based on these teachings, the Examiner finds that one of ordinary skill
in the art would have been prompted to combine the references’ teachings
“because the method taught by Ho et al. introduces DNA into the same yeast
taught by Lopes” (id. at 5). Therefore, the Examiner concludes:
[I]t would have been obvious to one of ordinary skill in
the art to arrive at the claimed invention as a whole by
combining the teachings of Ho et al. and Lopes et al. because
Ho et al. teach the simultaneous fermentation of xylose and
glucose into ethanol from the yeast S. cerevisiae, as ethanol is
said to be an ideal liquid fuel for automobiles and Lopes et al.
teach a method of making transformants stably maintained in
non-selective medium for multiple generations over long
periods of time.

(Id. at 4)
Appellants separately argue three distinct claim groupings.
Specifically, Appellants argue that the Examiner erred in finding claims 14-
16, 18, 30, 32, and 33 obvious because the Examiner failed to show a
teaching or suggestion in the prior art for all of the claimed elements (App.
Br. 4-7), and because the Examiner failed to show that one of ordinary skill
in the art would have been motivated to practice the claimed invention (id. at
7-8). With respect to claims 28 and 34, Appellants argue that the Examiner
4
Appeal 2008-3282
Application 09/180,340

failed to show a teaching or suggestion for all of the elements in those
claims (id. at 8-9). Lastly, with respect to claims 17 and 29, Appellants
assert that, given the references’ teachings, one of ordinary skill in the art
would not have been motivated to practice the invention recited in those
claims (see id. at 9-11).
We select claims 14, 34, and 29, respectively, as representative of the
argued claim groupings. See 37 C.F.R. § 41.37(c)(1)(vii). The issue with
respect to this rejection, then, is whether the Examiner has established a
prima facie case that one of ordinary skill in the art would have considered
claims 14, 34, and 29 obvious in view of Ho and Lopes.
PRINCIPLES OF LAW
Recently addressing the question of obviousness, the Supreme Court
reaffirmed that under the controlling inquiry, “the scope and content of the
prior art are to be determined; differences between the prior art and the
claims at issue are to be ascertained; and the level of ordinary skill in the
pertinent art resolved.” KSR Int' l Co. v. Teleflex Inc., 127 S. Ct. 1727, 1734
(2007) (quoting Graham v. John Deere Co. of Kansas City, 383 U.S. 1, 17-
18 (1966)).
In proceedings before the Patent and Trademark Office,
the Examiner bears the burden of establishing a prima facie
case of obviousness based upon the prior art. “[The Examiner]
can satisfy this burden only by showing some objective
teaching in the prior art or that knowledge generally available to
one of ordinary skill in the art would lead that individual to
combine the relevant teachings of the references.”

In re Fritch, 972 F.2d 1260, 1265 (Fed. Cir. 1992) (citations omitted,
bracketed material in original). Thus, as the Supreme Court pointed out in
KSR, “a patent composed of several elements is not proved obvious merely
5
Appeal 2008-3282
Application 09/180,340

by demonstrating that each of its elements was, independently, known in the
prior art.” KSR, 127 S. Ct. at 1741.
While holding that some rationale must be supplied for a conclusion
of obviousness, the Court nonetheless rejected a “rigid approach” to the
obviousness question, and instead emphasized that “[t]hroughout this Court's
engagement with the question of obviousness, our cases have set forth an
expansive and flexible approach . . . .” Id. at 1739. The Court also rejected
the use of “rigid and mandatory formulas” as being “incompatible with our
precedents.” Id. at 1741; see also 1742-43 (“Rigid preventative rules that
deny factfinders recourse to common sense, however, are neither necessary
under our case law nor consistent with it.”).
The Court thus reasoned that the analysis under 35 U.S.C. § 103 “need
not seek out precise teachings directed to the specific subject matter of the
challenged claim, for a court can take account of the inferences and creative
steps that a person of ordinary skill in the art would employ.” Id. at 1741.
The Court further advised that “[a] person of ordinary skill is . . . a person of
ordinary creativity, not an automaton.” Id. at 1742.
Regarding hindsight reasoning, the Court stated that “[a] factfinder
should be aware, of course, of the distortion caused by hindsight bias and
must be cautious of arguments reliant upon ex post reasoning. Rigid
preventative rules that deny factfinders recourse to common sense, however,
are neither necessary under our case law nor consistent with it.” Id. at 1742-
1743 (citations omitted).
FINDINGS OF FACT (“FF”) PERTINENT TO CLAIMS 14 AND 34
1. Claim 14 recites a method of integrating multiple copies of exogenous
DNA into reiterated chromosomal DNA of cells. The first step of the
6
Appeal 2008-3282
Application 09/180,340

claimed method is transforming the cells with “a replicative and integrative
plasmid.” The plasmid must have “an autonomous replicating sequence,”
exogenous DNA, and a first selection marker.
In the second step, the cells from the first step are repeatedly
replicated to produce a number of generations of progeny cells while
selecting for cells which include the selection marker. Claim 14 provides
that the replication step promotes the retention of the replicative and
integrative plasmid in subsequent generations of the progeny cells and
produces progeny cells having multiple integrated copies of the exogenous
DNA.
2. Claim 34 recites a plasmid vector for use in integrating an exogenous
DNA sequence into chromosomal DNA of a target yeast cell. The
exogenous DNA must be flanked on each end by a DNA flanking sequence
which is homologous to a reiterated ribosomal DNA sequence of a target
yeast cell. The plasmid must also have a selection marker in a position other
than between the DNA flanking sequences.
Claim 34 also requires the plasmid to have a “functional yeast
autonomous replicating sequence.”
3. The Specification does not define “autonomous replicating sequence,”
or “functional yeast autonomous replicating sequence.” However, the
Specification states:
The yeast DNA replication origin, e.g. the ARS
[(autonomous replicating sequence)] containing DNA fragment,
can be obtained from yeast chromosomal DNA or from
chromosomal DNA of other organisms, so long as the DNA
fragment can function as an active replication origin to support
autonomous replication of plasmid in the host chosen for hcn
[(high copy number)] integration. DNA fragments which
7
Appeal 2008-3282
Application 09/180,340

function as ARSs can readily be isolated by incorporating
randomly-digested DNA fragments into an E. coli plasmid,
followed by transformation of the desired host organism, e.g. a
Saccharomyces yeast, with the resulting bank of plasmids, as
reported in the literature . . . .

(Spec. 27 (citations omitted).)
4. The Specification discloses that when testing candidate sequences for
their ability to autonomously replicate in yeast, “[a]ny properly-sized
restriction fragments that can make pUCKm6 function effectively as a yeast
plasmid must contain an effective ‘ARS’ and can be used to construct
replicative/integrative vectors such as pLNH-ST for high-copy-number
integration of exogenous gene(s) into the chromosomes of S. cerevisiae”
(Spec. 30-31).
5. Ho discloses “genetically engineered yeasts capable of simultaneously
fermenting the two major sugar constituents of cellulosic biomass, glucose
and xylose, to ethanol” (Ho 1). Ho discloses that such yeasts “can be
constructed by cloning a xylose reductase gene, a xylitol dehydrogenase
gene, and a xylulokinase gene in yeasts capable of fermenting glucose to
ethanol” (id.).
6. Ho discloses that the genes encoding the desired xylose-metabolizing
enzymes can be delivered to yeast using a plasmid designated as pLSK15
(Ho 15). Ho discloses that:
pLSK15 . . . is a low copy number plasmid with a copy number
of approximately 10 in yeast (S. cerevisiae). It contains the
yeast 2µ replicon which enables the plasmid to be replicated
autonomously in S. cerevisiae and closely related species.
pLSK15 also contains the geneticin (kanamycin) resistance
gene (KmR) and ampicillin resistance gene (ApR and also ampr)
8
Appeal 2008-3282
Application 09/180,340

which serve as selection markers in S. cerevisiae and other
yeasts.

(Id. at 15-16.)
7. Ho discloses that the genes encoding the desired xylose-metabolizing
enzymes can also be delivered to yeast using a plasmid designated as
pUCKm10, which “is a high copy-number plasmid (i.e. plasmid with a copy
number of about 50 or more) with a copy number close to 100 in S.
cerevisiae” (Ho 16). Ho states:
pUCKm10 is a pUC9 derivative containing the identical 2µ
replicon, and the KmR, and ApR genes present in pLSK15.
These specific DNA fragments serve as the replicon and
selection markers that enable the plasmid to be replicated
autonomously in S. cerevisiae (and in related yeasts) and also
enable the yeast transformants containing the plasmid to be
distinguished from the untransformed host cells.

(Id.)
8. Ho does not disclose that its plasmids are “integrative,” as required by
claim 14. Nor does Ho disclose that its plasmids contain exogenous DNA
flanked by sequences homologous to the ribosomal DNA of a target yeast
cell.
9. Lopes discloses the development of “a novel type of vector called
pMIRY2 (for Multiple Integration in the Ribosomal DNA of Yeast) that
integrates into the ribosomal DNA locus of Saccharomyces cerevisiae in up
to 140 copies which are stably maintained over long periods of growth under
non-selective conditions” (Lopes 467).
10. Lopes states that “[i]ntroduction of homologous or heterologous genes
by means of [pMIRY2] … led to protein levels similar to those observed
using a YEp vector of comparable copy number. Therefore pMIRY2 is a
9
Appeal 2008-3282
Application 09/180,340

potentially very useful vector for biotechnological application” (Lopes 467
(citation omitted)).
11. Lopes discloses that pMIRY2 “is basically composed of a 4·5 kb
BglII-B fragment of yeast rDNA (comprising part of the 26S rRNA-gene
NTS1, 5S RNA-gene and NTAS2: see Figure 1), a 450 bp chloroplast DNA
marker fragment from Spirodela oligorhiza, the LEU2d gene and pBR322
sequences, still containing the AmpR-gene” (Lopes 468). When exogenous
DNA is put into the plasmid it is flanked by the ribosomal DNA sequences
(see id. at 469-70).
12. Lopes discloses that “[y]east vectors suitable for high-level expression
of heterologous proteins should combine a high copy number with high
mitotic stability” (Lopes 467 (abstract)). Lopes discloses, however, that
“insertion of a (foreign) gene [into pMIRY2] drastically reduced [the]
mitotic stability of the resulting vector in comparison to its parent” (Lopes
467 (abstract)).
13. Lopes investigated the loss of mitotic stability of pMIRY2 by creating
a variety of related plasmids, and discovered that
Plasmid size is an important, but not the only determinant of
mitotic stability. Stable maintenance is only observed when the
complete plasmid has a size no larger than that of the rDNA
unit (9.1 kb). In addition stability depends upon the nature of
the rDNA fragment present in the plasmid, required for
targeting its integration.

(Lopes 467 (abstract)).
14. With respect to the size requirement of the heterologous DNA within
the pMIRY-type plasmids, Lopes specifically discloses:
Apparently, there is a relatively sharp transition from a
mitotically stable to an unstable vector somewhere around a
10
Appeal 2008-3282
Application 09/180,340

size of approximately 9-10 kb, i.e. about the length of an rDNA
unit. Even lengthening pMIRY2 by 0.4 kb . . . results in
reduction of stability (see above). In conclusion, pMIRY-type
vectors are only stably maintained in the rDNA cluster if their
size is smaller than or at most equal to the size of the rDNA
unit (9.1 kb).

(Lopes 473.)
ANALYSIS – CLAIM 14
Appellants’ arguments do not persuade us that the Examiner failed to
make a prima facie case that claim 14 would have been obvious to one of
ordinary skill in the art in view of Ho and Lopes. We note that Ho does not
explicitly disclose the use of an integrative plasmid as recited in claim 14
(FF 8).
However, Lopes discloses that its ribosomal DNA-containing
plasmids integrate as many as 140 copies into the yeast rDNA locus, are
stably maintained for long periods in yeast (FF 6), and, given the proper
configuration (FF 9-11), can result in protein levels that make it useful in
biotechnological applications (FF 7). We therefore agree with the Examiner
that one of ordinary skill seeking to express the xylose-metabolizing genes
taught by Ho would have been prompted to render vectors containing those
genes integrative, to achieve the advantages of chromosomally integrating
vectors taught by Lopes.
Appellants first argue that the Examiner improperly relied on a
document that was not positively recited in the statement of rejection (App.
Br. 5). Specifically, Appellants assert that Lopes’ statement at page 467,
that its pMIRY2 plasmid maintains stability over a long period (FF 6), refers
11
Appeal 2008-3282
Application 09/180,340

to a separate document which the Examiner improperly failed to cite (App.
Br. 5).
We are not persuaded by this argument. “It is well settled that a prior
art reference is relevant for all that it teaches to those of ordinary skill in the
art.” In re Fritch, 972 F.2d 1260, 1264. Appellants point to no evidence of
record showing that the statement on page 467 of Lopes is so incorrect or
unreliable that one of ordinary skill would ignore it. We therefore do not
agree that Lopes’ statement should be disregarded.
Appellants argue that the combination of Ho and Lopes fails to teach
or suggest all of the limitations of claim 14 because the 2 µm origin of
replication of Ho and a yeast autonomous replicating sequence origin “are
recognized in the art as different replication origins” (App. Br. 6 (citing
Gietz 817)).3
We are not persuaded by this argument. It is well settled that during
examination, the PTO must interpret terms in a claim using “the broadest
reasonable meaning of the words in their ordinary usage as they would be
understood by one of ordinary skill in the art, taking into account whatever
enlightenment by way of definitions or otherwise that may be afforded by
the written description contained in the applicant’s specification.” In re
Morris, 127 F.3d 1048, 1054 (Fed. Cir. 1997).
In the instant case, the Specification does not provide a specific
definition for “autonomous replicating sequence” as recited in claim 14.
However, the Specification does state that “the ARS containing DNA
fragment, can be obtained from yeast chromosomal DNA or from

3 R. Daniel Gietz et al., Genetic Transformation of Yeast, 30 BioTechniques
816-831 (20021
12
Appeal 2008-3282
Application 09/180,340

chromosomal DNA of other organisms, so long as the DNA fragment can
function as an active replication origin to support autonomous replication of
plasmid in the host” (Spec. 27 (FF 3) (emphasis added)). Moreover, the
Specification discloses that “[a]ny properly-sized restriction fragments that
can make pUCKm6 function effectively as a yeast plasmid must contain an
effective ‘ARS’ and can be used to construct replicative/integrative vectors”
(Spec. 30-31 (FF 4) (emphasis added)).
Given the Specification’s use of the term “ARS” as encompassing
sequences that allows a vector to function as a yeast plasmid (FF 3, 4), we
do not agree with Appellants that the term as used in claim 14 is limited to
any particular term of art, nor do we agree that it would be reasonable to
exclude the yeast 2 µm origin of replication from the scope of the recitation
“autonomous replicating sequence” in claim 14. Thus, Appellants assert that
the yeast 2 µm differs from an ARS as the terms are used in the art, but they
have not met their burden in clarifying their differences in the context of
how “autonomous replicating sequence” is described in the Specification.
Appellants argue that “[t]he plasmids of Ho et al. are able to replicate,
but they do not integrate. Thus, they are not replicative and integrative
plasmids. Accordingly, Ho et al. do not teach or suggest a method including
the use of a replicative and integrative plasmid comprising an autonomous
replicating sequence” (App. Br. 6). Appellants similarly argue that, while
Lopes’ vectors can replicate in E. coli, “the plasmid vectors do not include
an autonomous replicating sequence that permits both replication and
integration in the same cell. Accordingly, Lopes et al. do not teach or
suggest a method including the use of a replicative and integrative plasmid
comprising an autonomous replicating sequence” (id.). Appellants conclude
13
Appeal 2008-3282
Application 09/180,340

that because neither Ho nor Lopes discloses the use of a vector that is both
replicative and integrative, “when combined the cited documents do not
teach or suggest all the claim limitations of independent claims 14-18 and
30. Thus, the Examiner has failed to present a prima facie case that
independent claims 14-18 and 30 are obvious in view of the cited art” (App.
Br. 7).
We are not persuaded by this argument. It is well settled that “[n]on-
obviousness cannot be established by attacking references individually
where the rejection is based upon the teachings of a combination of
references. . . . [The reference] must be read, not in isolation, but for what it
fairly teaches in combination with the prior art as a whole.” In re Merck &
Co., Inc., 800 F.2d 1091, 1097, 231 USPQ 375, 380 (Fed. Cir. 1986).
Therefore, the teachings of the individual references may not be
viewed alone, but instead must be viewed alongside the teachings of the
other reference. In the instant case, in the context of delivering exogenous
DNA to yeast, Ho teaches the desirability of using plasmids containing a
yeast autonomous replication sequence in order to maintain an adequate
intracellular copy number (see FF 6, 7), and Lopes teach that using
ribosomal DNA integrating sequences allows for mitotically stable
integration of exogenous DNA into the yeast chromosome (FF 9, 10, 13).
Given Lopes’ disclosure of the desirability of both high copy number and
mitotic stability (FF 12), we agree with the Examiner that one of ordinary
skill in the art would have been prompted to combine the two approaches so
as to meet those objectives when delivering the xylose-metabolizing genes.
We are therefore not persuaded by Appellants that the cited combination of
references fails to suggest all of the limitations in claim 14.
14
Appeal 2008-3282
Application 09/180,340

Appellants argue that the cited prior art fails to provide motivation for
practicing the process of claim 14 (App. Br. 7-8). Specifically, Appellants
assert that “[t]he mere fact that references can be combined or modified does
not render the resultant combination obvious unless the prior art also
suggests the desirability of the combination” (id. at 8 (quoting M.P.E.P. §
2143.01)). Moreover, Appellants urge, “the use of the same yeast in each
cited document does not provide any suggestion as to the desirability of
making the combination.” (id.) Thus, Appellants conclude, “the Examiner
has not provided any motivation to combine the cited documents, and
therefore the Examiner has failed to present a prima facie case that claims
14-16, 18, 28-30, and 34 are obvious in view of the cited art” (id.).
We are not persuaded by this argument. The obviousness analysis is
not limited to the explicit statements made in the prior art, but must take into
account “the inferences and creative steps that a person of ordinary skill in
the art would employ” based on the references’ teachings. KSR Int' l Co. v.
Teleflex Inc., 127 S. Ct. at 1741.
In the instant case, for the reasons discussed above, we agree with the
Examiner that, being a person of ordinary creativity and common sense,
KSR, 127 S. Ct. at 1742-43, one of ordinary skill in the art seeking to express
the xylose-metabolizing genes taught by Ho would have been prompted to
render vectors containing those genes integrative, to achieve the advantages
of chromosomally integrating vectors taught by Lopes. We therefore do not
agree with Appellants that the Examiner failed to make a prima facie case
with respect to claim 14.
Thus, we affirm the Examiner’s obviousness rejection of claim 14
over Ho and Lopes. Because claims 15, 16, 18, 30, 32, and 33 were not
15
Appeal 2008-3282
Application 09/180,340

argued separately from claim 14, they fall with that claim. 37 C.F.R.
§ 41.37(c)(1)(vii).
ANALYSIS – CLAIM 34
Appellants present separate argument with respect to claims 28 and
34. As noted above, we select claim 34 as representative of the argued claim
grouping. 37 C.F.R. § 41.37(c)(1)(vii).
Appellants’ arguments do not persuade us that the Examiner failed to
make a prima facie case that one of ordinary skill in the art would have
considered claim 34 obvious in view of Ho and Lopes. As discussed above,
we agree with the Examiner that one of ordinary skill would have considered
it obvious to combine the approaches of Lopes and Ho to prepare a plasmid
having a yeast autonomous replicating sequence as taught by Ho (FF 6, 7),
and rDNA sequences flanking as taught by Lopes (FF 9-11) to deliver Ho’s
xylose-metabolizing genes to yeast.
Moreover, given the Specification’s apparently more general use of
the term “ARS” as encompassing sequences that allow a vector to function
as a yeast plasmid (FF 3, 4), we do not agree with Appellants that the term
“yeast autonomously replicating sequence” in claim 34 is limited to any
particular term of art, nor do we agree that it would be reasonable to
interpret claim 34 as excluding the yeast 2 µm origin of replication disclosed
by Ho.
Thus, Appellants’ arguments do not persuade us that the cited
references fail to teach or suggest all of the claimed limitations. We
therefore affirm the Examiner’s rejection of claim 34. Because claim 28 was
argued in the same group as claim 34, it falls with claim 34. See 37 C.F.R.
§ 41.37(c)(1)(vii).
16
Appeal 2008-3282
Application 09/180,340

FINDINGS OF FACT PERTINENT TO CLAIM 29
15. Claim 29 recites a plasmid vector for use in integrating the exogenous
DNA sequence into chromosomal DNA of the target yeast to form stable
integrants which ferment xylose to ethanol. The plasmid must have a
functional yeast autonomous replicating sequence and exogenous DNA
flanked on each end by a DNA flanking sequence which is homologous to a
reiterated DNA sequence of a target yeast cell.
The exogenous DNA in the plasmid must include genes encoding
xylose reductase, xylitol dehydrogenase, and xylulokinase.
16. To prepare plasmids capable of conferring xylose-metabolizing ability
to yeasts, Ho discloses that the “XR [(xylose reductase)], XD [(xylose
dehydrogenase)], and XK [(xylulokinase)] genes fused to the proper
promoters were . . . cloned on pLSK15 . . . or pUCKm10” (Ho 15).
16. Example 2 of Ho discloses the fusion of the pyruvate kinase promoter
to the xylitol dehydrogenase (XD) gene (Ho 21-22). Ho discloses that the
construct “contains -910 to -1 promoter fragments from the pyruvate kinase
gene and +1 to +1963 nucleotides from the Pichia XD gene” (id. at 22).
17. Example 3 of Ho discloses that the xylulokinase gene used by Ho to
transform yeast is about 2.4 kb, with the translated region containing 2118
nucleotides (Ho 22).
18. The combined length of the translated regions of Ho’s xylulokinase
and xylitol dehydrogenase genes is therefore 1.9 kb (XD) plus 2.1 kb (XK),
or about 4.0 kb. Including promoters the combined length of these genes is
about 2.9 (XD) plus 2.4 (XK), or about 5.3 kb (see also App. Br. 10-11).
17
Appeal 2008-3282
Application 09/180,340

19. The smallest plasmid used by Lopes to integrate exogenous DNA into
the yeast chromosomal rDNA locus is pMIRY1, which is 6.1 kb (Lopes
469).
20. Thus, not including the xylose reductase gene, combining the XD and
XK genes with Lopes’ smallest plasmid results in a plasmid ranging in size
from 10.1 kb (promoterless XD plus XK plus pMIRY1) to 11.4 kb
(promoter-containing XD plus XK plus pMIRY1).
ANALYSIS -- CLAIM 29
We agree with Appellants that the Examiner has not established a
prima facie case that claim 29 would have been obvious to a person of
ordinary skill in the art in view of Ho and Lopes. As discussed above, Lopes
discloses that stable maintenance of its plasmid “is only observed when the
complete plasmid has a size no larger than that of the rDNA unit (9.1 kb)”
(Lopes 467 (abstract) (FF 13)), and that “[e]ven lengthening pMIRY2 by 0.4
kb . . . results in reduction of stability (Lopes 437 (FF 14)).
In contrast, even assuming the absence of promoters, and the absence
of the xylose reductase gene required by claim 29, the combination of
Lopes’ smallest plasmid with two of the three genes recited in claim 29 is
10.1 kb (FF 20), which is larger than the plasmid size stated by Lopes as
being stable (see also App. Br. 11). We therefore agree with Appellants that
one of ordinary skill in the art would not have been prompted to include all
three of Ho’s xylose-metabolizing genes in a single plasmid when using
Lopes’ rDNA integrating sequences to deliver the genes to yeast.
We therefore also agree with Appellants that the Examiner has failed
to establish a prima facie case of obviousness for claim 29. Claim 17 also
recites that “the exogenous DNA includes genes encoding xylose reductase,
18
Appeal 2008-3282
Application 09/180,340

xylitol dehydrogenase, and xylulokinase.” Therefore, for the same reasons
applied regarding claim 29, we conclude that he Examiner has not
established a prima case of obviousness with respect to claim 17.
The Examiner argues:
It is noted that claim 17 recites genes encoding XR, XD and
XK, however, note that the first selection marker could be all
three or any one of the three based on the present claim
language. Note also that Ho et al. disclose recombinant yeasts
containing genes encoding XR, XD and XK, thus the
appellant's statement that the combination of the references
might result in reduced mitot[ic] stability is not persuasive.

(Ans. 7.)
We are not persuaded by this argument. As discussed above, Lopes
discloses that even small increases in size significantly affect the stability of
Lopes’ plasmid. Thus, we do not agree with the Examiner that one of
ordinary skill in the art would have been prompted to include all three of
Ho’s xylose-metabolizing genes in a single plasmid when using Lopes’
rDNA integrating sequences to deliver the genes to yeast.
We therefore reverse the Examiner’s obviousness rejection of claims
17 and 29.
OBVIOUSNESS -- HO AND HALLBORN
ISSUE
Claims 28, 29, and 34 stand rejected under 35 U.S.C. § 103(a) as
obvious over Ho and Hallborn (Ans. 7-13). Relying on Ho for the teachings
discussed above, the Examiner states:
In-so-far-as Ho et al. does not explicitly teach the integration at
multiple sites, Hallborn et al. teach recombinant yeasts that
ferments xylose to ethanol, having genes integrated (multiple
copies) into the yeast chromosome. The genes taught by
19
Appeal 2008-3282
Application 09/180,340

Hallborn et al. encode xylose reductase and xylitol
dehydrogenase. Hallborn et al. also teach yeast from
Saccharomyces and a method of transforming cells with
integrative plasmids. Additionally, the vector taught by
Hallborn et al. autonomously replicates multiple copy plasmids
(see pages 1-9).
(Ans. 8.)
Based on the references’ teachings, the Examiner concludes that one
of ordinary skill in the art would have considered it obvious “to arrive at the
claimed invention as a whole because Ho et al. and Hallborn et al. teach the
fermentation of sugars to ethanol (i.e. xylose and glucose) using the same
yeast strain” (id. at 8-9). The Examiner finds that a person of ordinary skill
would have been “motivated to combine the teachings of the references
because Ho et al. disclose that ethanol is an ideal liquid fuel for automobiles
and Hallborn et al. disclose a method to perform stable transformations over
time. Therefore, the claimed invention was prima facie obvious” (id. at 9).
Appellants contend that the Examiner did not establish a prima facie
case of obviousness because the combined disclosures of the two references
do not teach or suggest all of the limitations in the claims (App. Br. 12-15),
and because the references failed to provide motivation and a reasonable
expectation of success (id. at 15-17).
The issue with respect to this rejection, then, is whether the Examiner
has established a prima facie case that one of ordinary skill in the art would
have considered claims 28, 29, and 34 obvious in view of Ho and Hallborn.
FINDINGS OF FACT
21. Each of claims 28, 29, and 34 recites a plasmid vector. Each of those
claims requires the plasmid to have “a functional yeast autonomous
replicating sequence” and exogenous DNA.
20
Appeal 2008-3282
Application 09/180,340

22. Claims 28 and 34 require the exogenous DNA to be “flanked on each
end by a DNA flanking sequence which is homologous to a reiterated
ribosomal DNA sequence of a target yeast cell.” Claim 29 requires the
exogenous DNA to be “flanked on each end by a DNA flanking sequence
which is homologous to a reiterated DNA sequence of a target yeast cell.”
23. As discussed above, Ho discloses a plasmid having a functional yeast
autonomous replicating sequence and exogenous DNA (FF 5-7).
24. As also discussed above, Ho does not disclose that its plasmids
contain exogenous DNA flanked by sequences homologous to the ribosomal
DNA of a target yeast cell.
25. Hallborn discloses “recombinant yeast strains transformed with xylose
reductase and/or xylitol dehydrogenase enzyme genes” (Hallborn 1).
Hallborn states that “[a] yeast strain transformed with the xylose reductase
gene is capable of reducing xylose to xylitol and consequently of producing
xylitol in vivo. If both of these genes are transformed into a yeast strain, the
resultant strain is capable of producing ethanol on xylose containing medium
during fermentation” (id.).
26. Hallborn discloses that the gene encoding xylose reductase (XR) can
be introduced into yeast using a plasmid “capable of replicating
autonomously when transformed into the recipient yeast strain” (Hallborn
7).
27. Hallborn discloses:
Alternatively, the gene coding for XR can also be integrated
into the yeast chromosome, into the ribosomal RNA locus, for
instance. For this purpose the ribosomal sequences of a suitable
plasmid, eg. plasmid pIRL9 are released, and cloned
appropriately to BS+ vector. The gene coding for XR, coupled
in between suitable yeast promoter and terminator regions, is
21
Appeal 2008-3282
Application 09/180,340

released from the hybrid vector comprising the gene and cloned
into the plasmid obtained at the previous stage. From this
resulting plasmid the expression cassette, flanked by ribosomal
sequences can be released. This fragment is cotransformed into
a yeast with an autonomously replicating plasmid carrying a
suitable marker for transformation. The plasmid can be later on
removed from the cells containing the xrd gene integrated in the
chromosome by cultivating the cells in non-selective
conditions. This way, recombinant strains can be obtained
which carry no extra foreign DNA such as bacterial vector
sequences. If a polyploid yeast strain, such as VTT-A-63015, is
used the gene can be integrated also to an essential locus such
as the PGKI or the ADH1 locus.

(Hallborn 7.)
ANALYSIS
We agree with Appellants that the Examiner has not made a prima
facie case of obviousness for claims 28, 29, and 34. Each of claims 28, 29,
and 34 recites a plasmid having both a yeast autonomous replicating
sequence and exogenous DNA flanked by DNA sequences homologous to
reiterated sequences in a target yeast cell (FF 21, 22).
While Hallborn discloses a DNA vector having the XR gene “flanked
by ribosomal sequences” (Hallborn 7 (FF 27)), that construct is not
circularized or placed into a plasmid, but is instead is used as a “fragment . .
. cotransformed into a yeast with an autonomously replicating plasmid
carrying a suitable marker for transformation” (id.). Thus, rather than
suggesting the desirability of placing the rDNA sequences within an
autonomously replicating plasmid, as required by claims 28, 29, and 34,
Hallborn teaches that the reiterated sequences homologous to the yeast host
should be kept separate from the autonomously replicating plasmid used to
cotransform the yeast.
22
Appeal 2008-3282
Application 09/180,340

Because Hallborn teaches that its integrating sequences should be kept
separate from the autonomously replicating plasmid, we do not agree with
the Examiner that one of ordinary skill would have been prompted to add
those sequences to the plasmids of Ho. We therefore agree with Appellants
that the Examiner has not made a prima facie case that the plasmids recited
in claims 28, 29, and 34 would have been obvious to one of ordinary skill in
the art in view of Ho and Hallborn.
We therefore reverse the Examiner’s obviousness rejection of claims
28, 29, and 34 as obvious over Ho and Halborn.
SUMMARY
We affirm the Examiner’s rejection of claims 14-16, 18, 28, 30, and
32-34 as obvious over Ho and Lopes.
We reverse the Examiner’s rejection of claims 17 and 29 as obvious
over Ho and Hopes.
We reverse the Examiner’s rejection of claims 28, 29, and 34 as
obvious over Ho and Hallborn.
No time period for taking any subsequent action in connection with
this appeal may be extended under 37 C.F.R. § 1.136(a)(1)(iv).

AFFIRMED-IN-PART

dm
Mueting, Raasch & Gebhardt, P.A.
P.O. Box 581336
Minneapolis, MN 55458
23