DSM IP ASSETS B.V.

Patent Trials and Appeals Board

Jul 29, 2020

14045683 - (D) (P.T.A.B. Jul. 29, 2020)

UNITED STATES PATENT AND TRADEMARK OFFICE
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
Address: COMMISSIONER FOR PATENTS
P.O. Box 1450
Alexandria, Virginia 22313-1450
www.uspto.gov
APPLICATION NO. FILING DATE FIRST NAMED INVENTOR ATTORNEY DOCKET NO. CONFIRMATION NO.
14/045,683 10/03/2013 Ulrike Maria MUELLER 2919208-088001 4824
84331 7590 07/29/2020
McBee Moore & Vanik, IP, LLC
7900 Westpark Drive, Suite A100
McLean, VA 22102
EXAMINER
LIU, SAMUEL W
ART UNIT PAPER NUMBER
1656
NOTIFICATION DATE DELIVERY MODE
07/29/2020 ELECTRONIC
Please find below and/or attached an Office communication concerning this application or proceeding.
The time period for reply, if any, is set in the attached communication.
Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the
following e-mail address(es):
cgmoore@mmviplaw.com
docketing@mmviplaw.com
smcbee@mmviplaw.com
PTOL-90A (Rev. 04/07)
UNITED STATES PATENT AND TRADEMARK OFFICE
____________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
____________

Ex parte ULRIKE MARIA MUELLER, LIANG WU,
LOURINA MADELEINE RAAMSDONK, and
AARON ADRIAAN WINKLER
____________

Appeal 2019-005900
Application 14/045,683
Technology Center 1600
____________

Before FRANCISCO C. PRATS, TAWEN CHANG, and
MICHAEL A. VALEK, Administrative Patent Judges.

VALEK, Administrative Patent Judge.

DECISION ON APPEAL
Appellant1 submits this appeal under 35 U.S.C. § 134(a) involving
claims to a recombinant yeast cell encoding a polypeptide having enzymatic
activity for the direct conversion of acetaldehyde into acetyl-CoA in the
cytosol. We have jurisdiction under 35 U.S.C. § 6(b). We REVERSE.

1 We use the word “Appellant” to refer to “applicant” as defined in
37 C.F.R. § 1.42(a). Appellant identifies DSM IP ASSETS B.V. as the real
party in interest. Appeal Br. 1. Herein, we refer to the Final Action mailed
November 17, 2017 (“Final Act.”); Appellant’s Appeal Brief filed January 9,
2019 (“Appeal Br.”); Examiner’s Answer mailed May 31, 2019 (“Ans.”);
and Appellant’s Reply Brief filed July 30, 2019 (“Reply Br.”).
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STATEMENT OF THE CASE
“Acetyl-coenzyme A (CoA) is an essential intermediate in numerous
metabolic pathways, and is a key precursor in the synthesis of many
industrial relevant compoun[d]s.” Spec. 1. “In cytosol, acetyl-CoA is
synthesized via the pyruvate dehydrogenase (PDH) by-pass” pathway. Id. at
2. However, according to the Specification, “the PDH bypass in yeast is not
optimal with respect to the energy” because it requires additional ATP,
which “can present a problem for synthesiz[ing] the product of interest from
cytosolic acetyl-CoA precursor, as more carbon source needs to be diverted
for ATP generation . . . thereby lowering the overall yield of the product.”
Id.
The present application proposes an “alternative metabolic route[]” to
“overcome the problems of the PDH by-pass” through the “direct conversion
of acetaldehyde to acetyl-CoA without ATP consumption, by the use of an
acetylating acetaldehyde dehydrogenase (E.C. 1.2.1.10).” Spec. 3. The
Specification describes experiments to identify particular acetylating
acetaldehyde dehydrogenases (ACDH) that are capable of driving this direct
conversion. See id. at 21–28. Based these experiments, the Specification
identifies peptide sequences for several ACDH, including SEQ ID NOs: 19,
22, 25, and 28, that were shown to complement a “delta acs2 S. cerevisiae
strain,” i.e., a yeast strain where normal cytosolic acetyl-CoA production has
been disrupted by deletion of the acetyl-CoA synthetase2 (acs2) gene.2 Id.
at 26–27 (Tables 1 and 2).

2 The Specification explains that “Saccharomyces cerevisiae mutants
carrying an inactivation of the acs2 gene are not able to grow on glucose as
sole carbon source.” Spec. 14. Thus, “a delta acs2 yeast mutant . . . is
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Claims 9–12, 14–18, and 23–26 are on appeal and can be found in the
Claims Appendix of the Appeal Brief. Claim 11 is illustrative of the claims
on appeal. It reads as follows:
11. A recombinant yeast cell comprising a heterologous
nucleotide sequence encoding a polypeptide having enzymatic
activity for the direct conversion of acetaldehyde into acetyl-
CoA in the cytosol of said yeast cell wherein the polypeptide is
an acetylating acetaldehyde dehydrogenase (EC 1.2.1.10),
wherein said polypeptide has at least 70% sequence identity
with at least one amino acid sequence selected from the group
consisting of SEQ ID NOS: 19, 22, 25, and 28.
Appeal Br. 49–50. Claims 12 and 24 depend from claim 11 and additionally
require the inactivation of a gene encoding for at least one enzyme involved
in the PDH by-pass, such as acetyl-CoA synthetase, (claim 12) or acetyl-
CoA synthetase specifically (claim 24). Id. at 50–51.
The following rejections are before us for review:3
I. Claims 9–11, 15, 18, and 23–26 under 35 U.S.C. § 103 as
unpatentable over Wahlbom4, Boxma,5 and NCBI Q99X67;6

unable to grow on glucose as sole carbon source unless cytosolic acetyl-CoA
formation is complemented” in this case by expression of an ACDH capable
of direct conversion of acetaldehyde to acetyl-CoA. Id. at 21–22.
3 The written description rejection was withdrawn in Examiner’s Answer
and is not before us. Ans. 3.
4 US 2005/0153411 A1, published July 14, 2005 (“Wahlbom”).
5 Brigitte Boxma et al., The Anaerobic Chytiridiomycete Fungus Piromyces
sp. E2 Produces Ethanol Via Pyruvate:formate Lyase and an Alcohol
Dehydrogenase E, 51(5) Molecular Microbiology 1389–1399 (2004)
(“Boxma”).
6 NCBI Q99X67, “Alcohol-acetaldehyde dehydrogenase,
http//www.ncbi.nlm.nih.gove/protein/Q99X67 (2006) (“NCBI Q99X67”).
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Application 14/045,683

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II. Claims 12 and 24 under 35 U.S.C. § 103 as unpatentable over
Wahlbom, Boxma, NCBI Q99X67, and Remize;7
III. Claim 14 under 35 U.S.C. § 103 as unpatentable over Wahlbom,
Boxma, NCBI Q99X67, and van Maris;8 and
IV. Claims 16 and 17 under 35 U.S.C. § 103 as unpatentable over
Wahlbom, Boxma, NCBI Q99X67, and Donaldson.9
Appeal Br. 5.
Analysis
All of the rejections turn on common issues relating to Examiner’s
combination of Wahlbom and Boxma, i.e., the ACDH-expressing,
recombinant yeast of Wahlbom modified to express the Staphylococcus
aureus peptide sequence in Figure 3 of Boxma, and Examiner’s conclusion
that such a combination would have been obvious to one or ordinary skill in
the art in light of these references. See Final Act. 9–11 (relying on the
combination of these references “as applied to claim 11” for Rejections II–
IV). Accordingly, we consider the rejections together, focusing on claim 11
as illustrative. The same analysis, discussed below within the context of
claim 11, also applies to the other rejected claims. The issue before us is
whether a preponderance of the evidence supports Examiner’s conclusion

7 Fabienne Remize et al., Engineering of the Pyruvate Dehydrogenase
Bypass in Saccharomyces cerevisiae: Role of the Cytosolic Mg2+ and
Mitochondrial K+ Acetaldehyde Dehydrogenases Ald6p and Ald4p in
Acetate Formation during Alcoholic Fermentation, 66 Applied and
Environmental Microbiology 3151–3159 (2000) (“Remize”).
8 US 2005/0059136 A1, published March 17, 2005 (“van Maris”).
9 US 2008/0182308 A1, published July 31, 2008 (“Donaldson”).

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that the recombinant yeast of claim 11 would have been obvious over the
teachings in the cited references.
Examiner finds Wahlbom teaches “multiple vectors comprising
‘acetaldehyde dehydrogenase (acylating) (E.C.1.2.1.10)” that are used to
transform “yeast host S. cerevisiae.” Final Act. 6. Examiner acknowledges
that Wahlbom does not expressly teach a sequence that has at least 70%
identity to the recited sequences. Id. at 6. However, Examiner determines
that Boxma teaches an amino acid sequence that “has 100% sequence
identity to instant SEQ ID NO:28.” Id. at 6–7.
Specifically, Examiner relies on one of the nine sequences, i.e., the
one from Staphylococcus aureus, shown in the alignment of sequences for
different alcohol dehydrogenase E (ADHE) enzymes in Boxma Figure 3. Id.
at 7. Examiner determines that a skilled artisan would have understood that
the enzyme corresponding to this sequence is “bifunctional” with acetylating
acetaldehyde dehydrogenase activity and therefore that it is “equivalent” to
the “acylating (acetylating) acetaldehyde dehydrogenase (EC 1.2.1.10)” that
Wahlbom teaches may be “introduced into yeast such as S. cerevisiae for
ethanol production.” Id. According to Examiner, it would have been
“obvious to try” the Staphylococcus aureus sequence because it is one of
“only nine species (set forth in Figure 3 of Boxma)” and therefore it is one
of a “finite number” of solutions known to a skilled artisan. Ans. 16–17.
Moreover, while neither Wahlbom, nor Boxma, teaches the addition of such
an enzyme for the recited activity of directly converting acetaldehyde into
acetyl-CoA, Examiner finds that such activity would be an “inherent
property” of the articulated combination of Wahlbom and Boxman. Ans. 16;
see also Final Act. 8.
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Appellant argues that Examiner’s rejection of claim 11 is deficient for
several reasons. In particular, Appellant disputes Examiner’s “obvious to
try” rationale because “Boxma teaches that ACDH genes are commonly
found through eukaryotes and throughout the phyla Firmicutes,
Proteobacteria, and Cyanobacteria.” Reply Br. 5. Thus, urges Appellant,
Boxma does not teach a “finite number” of solutions. Id.
Moreover, Appellant points out that Wahlbom attempted to transform
S. cerevisiae to express ACDH enzymes from “two different species of
bacteria (Lactococcus lactis and Clostridium acetobutylicium)” and “in
neither case could the corresponding enzymatic activity be detected.”
Appeal Br. 26 (citing Wahlbom ¶ 55). Appellant acknowledges that
Wahlbom was able to successfully transform yeast cells with a third ACDH,
i.e., one from Entamoeba hisolytica (Ehadh2), but argues that Wahlbom’s
results do “not teach or suggest enzymatic activity for the direct conversion
of acetaldehyde into acetyl-CoA in the cytosol of yeast cells, as claimed.”
Id. at 28.
Appellant additionally relies on the results in Example 3, which show
that, unlike the four ACDH enzymes corresponding to the sequences recited
in claim 11, expression of Ehadh2 did not complement the deletion of acs2
in S. cerevisiae. Id. at 29–30, 32–33 (citing Spec. 26–27 (Tables 1 and 2)).
According to Appellant, these results show that Ehadh2 “does not have
enzymatic activity for the direct conversion of acetaldehyde into acetyl-CoA
in the cytosol of yeast” and, therefore, “a skilled artisan could not reasonably
conclude that all [ACDH] proteins from different species are functionally
equivalent.” Id. at 30–32.
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Based on the cited art and present record, we determine that Appellant
has the better position. Examiner has not identified any rationale for
selecting the Staphylococcus aureus sequence in Figure 3 of Boxma over
any other peptide sequence known to exhibit ACDH activity. Instead, the
rejection is premised on the notion that there are a finite number of known
ACDH enzymes and, therefore, it would be obvious to try all of the
sequences in Boxma Figure 3 in Wahlbom’s system. See Ans. 17 (“In this
case, finding the [ACDH] from Staphylococcus aureus out of only nine
species (set forth in Figure 3 of Boxma) is an obvious to try; wherein said
‘nine species’ is a ‘finite number for solution. . . .’”).
The record, however, does not support such a finding. To the
contrary, Boxma evidences that enzymes with alcohol dehydrogenase and
aldehyde dehydrogenase activity10 such as those corresponding to the
sequences in Figure 3 are found across a wide variety of microorganisms,
including firmicutes, proteobacteria, cyanobacteria, and eukaryota. See
Boxma 1394 (Fig. 4) (describing phylogenetic analysis). Thus, Examiner’s
finding that a skilled artisan would be selecting from a finite set of only nine
options is directly contradicted by Boxma.
Even for just the nine sequences in Boxma Figure 3, the record does
not support Examiner’s finding that one of ordinary skill in the art would
have considered those sequences to be “equivalent” for use in Wahlbom’s
system. See Final Act. 7. In addition to the sequence from Staphylococcus
aureus, Figure 3 aligns sequences from Lactococcus lactis and Clostridium
acetobutylicium as well non-bacterial microrganisms like Entamoeba

10 Examiner appears to equate the latter with acetylating acetaldehyde
dehydrogenase activity. See Final Act. 7.
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histolytica. Boxma 1393 (Fig. 3). As Appellant points out, Wahlbom
attempted to transform S. cerevisiae to express ACDH from both
Lactococcus lactis and Clostridium acetobutylicium, but no activity was
detected. Wahlbom ¶¶ 55–56. In contrast, Wahlbom was able to transform
S. cerevisiae to successfully express the Entamoeba histolytica–derived
enzyme, Ehadh2. Id. ¶ 84. Given these results, Examiner has not shown
that a skilled artisan would consider the nine sequences in Boxma Figure 3
to be functionally equivalent, much less demonstrated that there would have
been a reasonable expectation of success in selecting another sequence of
bacterial origin, i.e., the sequence from Staphylococcus aureus, when
Wahlbom reports that no activity was detected when S. cerevisiae was
transformed using sequences from other bacteria.
In addition, we are persuaded by Appellant’s argument that
Wahlbom’s results with Ehadh2 do not evidence the direct conversion of
acetaldehyde into acetyl-CoA. See Appeal Br. 29–30, 32–33. Wahlbom
teaches that overexpression of Ehadh2 increased ethanol yield and xylose
uptake rate in its system. Id. ¶ 84. Those results, however, do not evidence
the direct conversion of acetaldehyde into acetyl-CoA. Indeed, Wahlbom
teaches that expression of Ehadh2 in its system was intended to drive the
reverse reaction, i.e., conversion of acetyl-CoA to acetaldehyde, which was
then converted to ethanol. Id. at Fig. 5. Examiner appears to acknowledge
that the cited references do not teach anything about the direct conversion of
acetaldehyde into acetyl-CoA, as recited in claim 11, instead finding that
such activity would have been inherent in the combination of Wahlbom’s
yeast with Boxma’s Staphylococcus aureus sequence. See Ans. 13. As
explained above, however, Examiner’s rationale for why that combination
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would have been obvious is premised on a finding that a skilled artisan
would find ADHE enzymes of different microbial origin to be “equivalent.”
Final Act. 7. The results in Example 3 of Appellant’s Specification showing
that expression of Ehadh2 did not complement acs2 deletion, while the
expression of other ADHE enzymes did, further shows that such enzymes
are not functionally equivalent and, therefore, further demonstrates that
Examiner has not articulated a sufficient rationale for combining the
references.
For all of these reasons, the rejection of claim 11 is not supported by a
preponderance of the evidence. Examiner’s rejections of Appellant’s other
claims, all of which ultimately depend from claim 11, are deficient for the
same reasons.
In addition to the reasons for reversing Examiner’s rejection of the
independent claim, we agree with Appellant that Examiner’s rejection of
dependent claims 12 and 24 should be reversed for another reason––i.e., that
Examiner’s finding that Remize “suggests that deletion of [acs2]” (Final
Act. 9) is not supported by Remize’s actual teachings. See Appeal Br. 39–
41. As Examiner found, Remize teaches that “overexpression (high
expression) of [acs2] in a S. cerevisiae strain did not result in enhanced
acetate utilization despite 4-7 fold increase in [acs2] activity . . . and that
high expression level of [acs2] triggers perturbation of host metabolic flux.”
Final Act. 9 (citing Remize 3152, 3157). However, Examiner erred in
concluding that Remize’s teachings regarding the overexpression of acs2
would have made it obvious to inactivate expression of the gene encoding
that enzyme. Id.
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Based on the record before us, we agree with Appellant that the
“absence of a specific results when an enzyme is overexpressed [as taught in
Remize] does not mean that it may be desirable to delete that enzyme.”
Appeal Br. 40. This is particularly so because Remize’s statement that “[a]
high expression level of [acs2] might trigger perturbations of metabolic
flux” refers to “perturbations” that might be triggered when acs2 is
overexpressed at the “four- to sevenfold” increase in activity achieved there.
Remize 3157, left column. Nothing in Remize, nor in the other cited
references, suggests that such problems may occur when acs2 is expressed at
normal levels, nor that inhibiting acs2 expression would successfully avoid
them. Indeed, if anything, Remize teaches that it would be disadvantageous
to inhibit acs2 because “[a] mutant with [acs2] disrupted can no longer grow
on glucose.” Remize 3152, right column. Thus, Examiner has not
sufficiently shown that the cited prior art suggests the inactivation of a gene
encoding acs2 or any other acetyl-CoA synthase. We reverse the rejection
of claims 12 and 14 for this reason in addition to those noted above for claim
11.

DECISION SUMMARY
Claims
Rejected
35 U.S.C. § Reference(s)/Basis Affirmed Reversed
9–11, 15,
18, 23–26
103 Wahlbom, Boxma,
NCBI Q99X67
9–11, 15,
18, 23–26
12, 24 103 Wahlbom, Boxma,
NCBI Q99X67,
Remize
12, 24
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Application 14/045,683

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Claims
Rejected
35 U.S.C. § Reference(s)/Basis Affirmed Reversed
14 103 Wahlbom, Boxma,
NCBI Q99X67, van
Maris
14
16, 17 103 Wahlbom, Boxma,
NCBI Q99X67,
Donaldson
16, 17
Overall
Outcome
9–12, 14–
18, 23–26

REVERSED