Ex Parte Huisman et al

Patent Trial and Appeal Board

Nov 5, 2013

11924350 (P.T.A.B. Nov. 5, 2013)

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.
11/924,350 10/25/2007 Gjalt W. Huisman MBX 025 DIV CON (2) 1099
116248 7590 11/05/2013
Pabst Patent Group
1545 Peachtree Street
Atlanta, GA 30309-2492
EXAMINER
HUTSON, RICHARD G
ART UNIT PAPER NUMBER
1652
MAIL DATE DELIVERY MODE
11/05/2013 PAPER
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.
PTOL-90A (Rev. 04/07)

UNITED STATES PATENT AND TRADEMARK OFFICE
__________

BEFORE THE PATENT TRIAL AND APPEAL BOARD
__________

Ex parte GJALT W. HUISMAN, LAURA Z. LUO, and
OLIVER P. PEOPLES
__________

Appeal 2012-004554
Application 11/924,350
Technology Center 1600
__________

Before DEMETRA J. MILLS, ERIC GRIMES, and JEFFREY N.
FREDMAN, Administrative Patent Judges.

MILLS, Administrative Patent Judge.

DECISION ON APPEAL

This is an appeal under 35 U.S.C. § 134. The Examiner has rejected
the claims for obviousness. We have jurisdiction under 35 U.S.C. § 6(b).
Appeal 2012-004554
Application 11/924,350

2
STATEMENT OF CASE
Claims 11, 19, 22 and 23 are representative and read as follows:

11. A fermentation process comprising
adding to a growth medium a bacterial strain having a periplasmic space,
wherein the bacteria produce polyhydroxyalkanoates and wherein the
bacteria express a heterologous nuclease gene or a genetically modified
homologous nuclease gene, the product of which is secreted into the
periplasmic space in an amount effective to degrade at least 95% of all of the
nucleic acid released following lysis of the bacterial cells in less than 24
hours.

19. The process of claim 11 wherein the nuclease gene is
integrated into a host strain selected from the group consisting of Ralstonia
eutropha, Methylobacterium organophilum, Methylobacterium extorquens,
Aeromonas caviae, Azotobacter vinelandii, Alcaligenes latus, Pseudomonas
oleovorans, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas
aeruginosa, Pseudomonas acidophila, Pseudomonas resinovorans,
Escherichia coli, and Klebsiella.

22. The process of claim 11 wherein the bacterial strain expresses a
homologous nuclease, further comprising mutating the bacterial strain and
screening for bacteria expressing enhanced nuclease activity.

23. The process of claim 11 wherein the bacteria expresses a
homologous nuclease, further comprising genetically engineering the
nuclease to enhance nuclease activity.

Cited References

Greer, WO 94/10289, published May 11, 1994.

Miller et al., Secretion and Processing of Staphylococcal Nuclease by
Bacillus subtilis, 169 J. Bacteriology 3508-3514 (1987).

Appeal 2012-004554
Application 11/924,350

3
Bernard Atkinson & Ferda Mavituna, Biochemical Engineering and
Biotechnology Handbook (Stockton Press, 2nd. ed., 1991).1

Liebl et al., Expression, Secretion, and Processing of Staphylococcal
Nuclease by Corynebacterium glutamicum, 174 J. Bacteriology 1854-1861
(1992).

Sang Yup Lee & Ho Nam Chang, Production of Poly(hydroxyalkanoic
acid), 52 Advances in Biochemical Engineering/Biotechnology 28-58
(1995).

Boynton et al., Reduction of Cell Lysate Viscosity during Processing of
Poly(3-Hydroxyalkanoates) by Chromosomal Integration of the
Staphylococcal Nuclease Gene in Pseudomonas putida, 65 Applied and
Environmental Microbiology 1524-1529 (1999).

Leung et al., US 6,258,560 B1, Jul. 10, 2001.

Grounds of Rejection
Claim 11, 12, 14-16, 19, 22, and 23 are rejected under 35 U.S.C. §
103(a) as being unpatentable over Greer, Leung, Atkinson, and Lee in view
of Liebl or Miller.
FINDINGS OF FACT
The Examiner’s findings of fact are set forth in the Answer at pages 4-10.

1 We note that page 7 of the Answer includes a typographical error
indicating that, “Atkinson et al. teach that Alcaligenes eutrophus (Ralstonia
eutrophus) has been studied in detail due to its ability to accumulate large
amounts of P(3HB) (i.e. ability to grow to cell densities of approximately 85
g/l and produce P(3HB) at 61.5 g/l, or 80% wt/wt of dry cell mass, page 30
through 32).” This reference citation, however, is from Lee page 32, section
3.2, not Atkinson pages 30-32.
Appeal 2012-004554
Application 11/924,350

4
Discussion
ISSUE
The Examiner concludes that
One of ordinary skill in the art would have been motivated to
genetically engineer a Alcaligenes eutrophus (Ralstonia eutrophus)
bacterial strain to express the Staphylococcal aureus nuclease as taught
by Liebl et al. or Miller et al. and Leung and Swartz, or a homologous
nuclease gene that has been modified to enhance nuclease activity, so
that this bacterial strain would produce and excrete the nuclease into
the bacterial periplasmic space as part of a fermentation process for the
synthesis of industrially important molecules, such as polyhydroxy-
alkanoates. A nuclease excreted into the periplasmic space as a result of
such a genetically engineered bacterial strain would inherently result in
the degradation of at least 95% of all the nucleic acid released
following lysis of the cells in less than 24 hours. The basis of this is
that as shown by Leung and Swartz, in the data shown in Table 3 of US
6,258,560, those harvested broths in which nuclease was expressed in
the periplasmic space showed significantly less viscosity relative to
those harvested broths in which no nuclease was expressed even
without 37°C incubation. The motivation for producing a nuclease by a
genetically engineered bacterial strain used in the fermentation process
is to reduce the amount of nucleic acids in the medium which result in
an increase in the viscosity of the medium, causing problems in the
downstream processing steps, as taught by Greer et al. and Leung and
Swartz. Greer et al. give further motivation for genetically engineering
a bacterial strain to express a nuclease, because they teach that purified
preparations of nucleases are expensive and a bacterial strain that was
genetically engineered to express a nuclease activity would not require
an external nuclease or hydrogen peroxide to be added to the
fermentation. One would have had a reasonable expectation of success
because both Liebl et al. and Miller et al. were able to express
functional Staphylococcal aureus nuclease in different bacterial
species, specifically Corynebacterium glutamicum and Bacillus subtilis
and Liebl et al. teach that the Staphylococcal aureus nuclease is a heat-
stable biochemically well characterized enzyme. One would have been
further motivated to engineer the bacterial strain to secrete the nuclease
into the growth medium in an effective amount to enhance the recovery
Appeal 2012-004554
Application 11/924,350

5
of product from the growth medium. Alternatively one would have
been motivated to engineer a homologous nuclease to increase its
nuclease activity for the same reasons as stated above for the
introduction of the heterologous Staphylococcal nuclease.

(Ans. 8-9.)

Appellants argue that, “[n]one of the prior art recognizes that the use
of the gram negative organism in combination with expression of high levels
of nuclease, which then accumulates within the periplasmic space, provides
a significant economic advantage.” (App. Br. 9.) Appellants rely on the
Declaration of Peoples as evidence of the economic advantage associated
with the claimed method. (App. Br. 32.)
Appellants further argue that, to the extent that the Examiner relies on
Leung for providing a motivation to express nuclease which is secreted into
the periplasmic space, such reliance is misplaced because Leung is not prior
art to this application. (App. Br. 11.) Appellants argue that, “[n]one of
Miller of Liebl would lead one to express nuclease into the periplasmic
space.” (App. Br. 24.)
Appellants provide separate arguments for claim 19-23 on page 28-30
of the Brief.
The issue is: Does the cited prior art support the Examiner’s
obviousness rejection?

PRINCIPLES OF LAW
“In rejecting claims under 35 U.S.C. § 103, the examiner bears the
initial burden of presenting a prima facie case of obviousness. Only if that
burden is met, does the burden of coming forward with evidence or
Appeal 2012-004554
Application 11/924,350

6
argument shift to the applicant.” In re Rijckaert, 9 F.3d 1531, 1532 (Fed.
Cir. 1993) (citations omitted). In order to determine whether a prima facie
case of obviousness has been established, we consider the factors set forth in
Graham v. John Deere Co., 383 U.S. 1, 17 (1966): (1) the scope and content
of the prior art; (2) the differences between the prior art and the claims at
issue; (3) the level of ordinary skill in the relevant art; and (4) objective
evidence of nonobviousness, if present.
“The combination of familiar elements according to known methods
is likely to be obvious when it does no more than yield predictable results.”
KSR Int’l Co. v. Teleflex Inc., 550 U.S. 398, 416 (2007).
The priority statute, 35 U.S.C. § 120, Benefit of earlier filing date in
the United States, recites that
An application for patent for an invention disclosed in the
manner provided by section 112(a) (other than the requirement
to disclose the best mode) in an application previously filed in
the United States, or as provided by section 363 of this title,
which is filed by an inventor or inventors named in the
previously filed application shall have the same effect, as to
such invention, as though filed on the date of the prior
application, if filed before the patenting or abandonment of or
termination of proceedings on the first application or on an
application similarly entitled to the benefit of the filing date of
the first application and if it contains or is amended to contain a
specific reference to the earlier filed application. [emphasis
added.]

ANALYSIS
We have decided this case in conjunction with Appeal No. 2012-
005719, Application No. 10/607,903. In addition, we have reviewed our
Appeal 2012-004554
Application 11/924,350

7
Decisions in related Appeal Nos. 2002-001357, Application No. 09/281,363,
and 2003-001996, Application No. 09/456,940. We further acknowledge
that the claims before us include modifications not present in our earlier
Decisions.
In the present case, for the most part, we agree with the Examiner’s
fact finding, statement of the rejection and responses to Appellants’
arguments as set forth in the Answer. We find that the Examiner has
provided evidence to support a prima facie case of obviousness for the
subject matter of claim 11 and its dependent claims, except claim 22. We
provide the following additional comment to argument set forth in the
Answer.
Appellants argue that, “[n]one of the prior art recognizes that the use
of the gram negative organism in combination with expression of high levels
of nuclease, which then accumulates within the periplasmic space, provides
a significant economic advantage.” (App. Br. 9.) Appellants rely on the
Declaration of Peoples as evidence of the economic advantage associated
with the claimed method. (App. Br. 32.)
We are not persuaded by Appellantsʼ argument or Declaration
evidence.
The Examiner acknowledges that
the references do not specifically disclose secretion of
nuclease into the periplasmic space of gram negative bacteria,
this limitation previously argued by appellants is considered to
be an inherent property of the bacterial cells made obvious by
Atkinson et al. [sic, Lee; see note 1 above], which express the
Staphylococcal aureus nuclease as taught by Liebl et al. or
Miller et al. The inherency of the secretion of the nuclease in
Appeal 2012-004554
Application 11/924,350

8
the obvious bacterial strain is based upon the reference of
Boynton. . .

(Ans. 19.)

We agree with the Examiner’s response to this argument set forth on
pages 19-20 of the Answer, and find acceptable the Examiner’s use of
Boynton to show that the R. eutropha cells taught by Lee inherently secrete
nuclease into the periplasmic space.
With respect to the claimed level of nuclease production, secretion
and degradation, we agree with the Examiner that, in Boynton,

PHA producing R. eutropha cells were generated using the
same nuclease encoding gene taught by Leibl [sic] et al. above
(See Boynton et al. Materials AND Methods, p 1524 and Figure
3, p 1527 and supporting text p 1526). Boynton et al. teach that
the transformant “R. eutropha secreted nuclease into the
periplasm but not into the growth medium” (p1526,
Construction of nuclease integrants of other PHA producers).
Thus the bacterial strain that is obvious over the above
references inherently produces the nuclease such that it is
secreted into the periplasmic space. Boynton et al. further
characterize the nuclease activity of the periplasmic nuclease of
the transformed R. eutropha cells in the data provided in Figure
3 and the supporting text, in which they show the result of
chromosomal DNA treatment with periplasmic fractions of R.
eutropha MBX917 (::nuckan) (lane 4) and R. eutropha
NCIMB40124HD untransformed parent strain (lane 5) at 37°C
for 1 hour. As is evident from the results of Figure 3, after only
one hour of incubation with the transformed R. eutropha
periplasmic fraction, all of the visible high molecular weight
DNA was digested to smaller molecular weight fragments.
Based upon this level of digestion of high molecular weight
chromosomal DNA for only one hour at 37°C, clearly the level
of nuclease produced in the transformed R. eutropha strain
Appeal 2012-004554
Application 11/924,350

9
would digest at least 95% of all nucleic acid released following
lysis of the bacterial cells in a 24 hour period.

(Ans. 19-20.) Therefore, consistent with our Decision in Appeal No. 2002-
1357 (page 7), we find that Appellants have not provided sufficient evidence
to show that the nuclease levels of Miller, Liebl, and Leung would not
provide the level of product recovery contemplated by claim 11 on appeal.
We are unconvinced by Dr. Peoples’ Declaration which argues that
the expense and use of nucleases is prohibitive. (Declaration 2.) The fact
that Greer and Dr. Peoples’ Declaration found that the use of nucleases is
expensive does not mean that the prior art was not aware of the viscosity
problem associated with nucleic acids and fermentation product recovery, or
unaware that nucleases could be used in place of peroxide to address the
viscosity problem. The process made obvious by the cited references
involves expressing a nuclease gene in recombinant cells, not buying
purified nucleases to add to the medium. Appellants have not explained why
the expense of commercially available nucleases would discourage a skilled
artisan from practicing the method suggested by the prior art.
Appellants further argue that, to the extent that the Examiner relies on
Leung for providing a motivation to express nuclease which is secreted into
the periplasmic space, such reliance is misplaced because Leung is not prior
art to this application. (App. Br. 11.) Appellants argue that, “[n]one of
Miller of [sic] Liebl would lead one to express nuclease into the periplasmic
space.” (App. Br. 24.)
We are not persuaded by Appellantsʼ argument that Leung is not prior
art to the present application. In particular, Appellants argue that
Appeal 2012-004554
Application 11/924,350

10
This application was filed on October 25, 2007, as a
continuation of pending U.S. Serial No. 10/607,903 filed July
27, 2003, which is a continuation of U.S. Serial No.
09/456,940, filed December 7, 1999, now abandoned, which is
a divisional of U.S. Serial No. 09/281,363, filed March 30,
1999, now abandoned, which claims priority to U.S. Serial No.
60/079,938, filed March 30, 1998.
Thus, this application has a priority date of March 10
[sic, 30?], 1998, as evidenced by the official filing receipt (a
copy of which was attached to the Evidence Appendix of the
Appeal Brief).
The examiner has, for the first time on appeal, denied
applicants' claim to priority to U.S.S.N. 60/079,938 ("the '938
application") filed on March 30, 1998, for the aspect of the
invention drawn to "a genetically modified homologous
nuclease gene, the product of which is secreted into the
periplasmic space".

(Reply Br. 2-3.)

However, we agree with the Examiner that
Appellants initial submission that Leung (which has a
priority date of 10/28/1998) is not prior art to this application, is
not persuasive on the basis that appellants do not recieve [sic]
priority for that aspect of the invention that is drawn to "a
genetically modified homologous nuclease gene, the product of
which is secreted into the periplasmic space ... " to provisional
application 60/079,938 filed on 3/30/1998. Thus the earliest
filing date for which appellants are granted priority is
3/30/1999. Thus Leung is prior art to the claimed invention.
[emphasis added.]

(Ans. 10.) This finding of lack of priority to the March 30, 1998 filing date
of the present application for the “genetically modified homologous nuclease
gene, the product of which is secreted into the periplasmic space,” is
Appeal 2012-004554
Application 11/924,350

11
consistent with our Decision in Appeal No. 2003-19962 where we found that
there was no written descriptive support in the Specification under 35 U.S.C.
§112 for “a single bacterial strain which comprises a genetic modification of
a homologous nuclease gene.” (2003-1996 Decision 4-6.) Thus, the March
30, 1998 priority document fails to meet the requirements of 35 U.S.C. §§
120, 112, for the claimed subject matter and Appellants are not entitled to
the priority date of March 30, 1998.
We have reviewed the passages of the priority application reproduced
by Appellants in the Reply Br. Pages 6-7, however, we find no written
description under 35 U.S.C. § 112 of a single bacterial strain which
comprises a genetic modification of a homologous nuclease gene.
Appellants do not argue the specific disclosure of Leung on the merits, and,
therefore, we find that Leung is prior art to the pending claims and has not
been rebutted by Appellants.

Claims 19 - 23
Appellants provide separate argument for claim 19-23 on page 28-30
of the Brief.
With respect to claim 19, Appellants argue that “[n]o reason is
provided for why one would go to the trouble of integrating the nuclease into
the chromosome” (App. Br. 28). However, claim 19 requires only that the

2 As noted above, the application on appeal is a continuation of application
10/607,903, which is a continuation of application 09/456,940. The ‘940
application was the subject of Appeal No. 2003-1996. The application on
appeal therefore shares the same Specification with the application in
Appeal No. 2003-1996.
Appeal 2012-004554
Application 11/924,350

12
“nuclease gene is integrated into a host strain,” not that it is integrated into
the chromosome of the host strain. We interpret claim 19 as encompassing
integration of a plasmid into the host strain, and not requiring integration
into the host strain DNA. Appellants admit on page 11 of the Brief that the
combination of Atkinson with Liebl or Miller discloses bacterial strains with
an integrated plasmid in the host strain which expresses a heterologous
nuclease gene. We affirm the rejection of claim 19. For the reasons set
forth with respect to claim 11 herein and in the Examiner’s Answer we also
affirm the rejection of claim 23.

Claim 22
Claim 22 recites that, “the bacterial strain expresses a homologous
nuclease, further comprising mutating the bacterial strain and screening for
bacteria expressing enhanced nuclease activity.”
Appellants argue that,
the Examiner has not identified how the cited art renders
the limitation of claim 22 obvious. Further, the Examiner's
discussion of Leung (even if Leung were relevant, does not
address how Leung renders this limitation obvious). There is
nothing about mutation in Leung.

(Reply Br. 13-14.)
We agree with Appellants and do not find that the Examiner has
provided supporting evidence in the prior art of mutating a bacterial strain
expressing a homologous nuclease gene in order to enhance nuclease
activity. The rejection of claim 22 is reversed.

Appeal 2012-004554
Application 11/924,350

13
CONCLUSION OF LAW
The cited references support the Examiner’s obviousness rejection,
except that the rejection of claim 22 is reversed.
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-IN-PART

lp