`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`_____________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`_____________________
`
`
`BUTAMAX™ ADVANCED BIOFUELS LLC
`Petitioner
`v.
`GEVO, INC.
`Patent Owner
`
`_____________________
`
`CASE IPR: Unassigned
`_____________________
`
`DECLARATION OF DENNIS J. THIELE, Ph.D.
`
`
`
`
`
`
`
`BUTAMAX 1002
`
`

`

`
`
`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`TABLE OF CONTENTS
`
`B. 
`
`C. 
`
`I. 
`Overview .......................................................................................................... 1 
`II.  My background and qualifications .................................................................. 4 
`III.  List of documents considered in formulating my opinion .............................. 7 
`IV.  Person of ordinary skill in the art .................................................................... 9 
`V. 
`The '565 patent ............................................................................................... 10 
`VI.  The full scope of claims 1-19 of the '565 patent is not disclosed in the '952
`and '209 provisional applications .................................................................. 11 
`A. 
`The provisional applications do not describe the full scope of
`inactivated GRX3 and/or GRX4 as encompassed by claims 1-19 of
`the '565 patent ...................................................................................... 12 
`The provisional applications do not describe all of the claimed
`nucleotide deletions, insertions, or combinations of deletions and
`insertions into endogenous GRX3 and/or GRX4 genes as
`encompassed by claims 1-19 of the '565 patent .................................. 17 
`The provisional applications do not fully describe the full genus of
`yeast genera as encompassed by claims 1-19 of the '565 patent ......... 23 
`VII.  Basis of my analysis with respect to anticipation .......................................... 24 
`VIII.  Flint anticipates claim 5 of the '565 patent .................................................... 25 
`A. 
`Claim 5 in independent form ............................................................... 27 
`IX.  Basis of my analysis with respect to obviousness ......................................... 32 
`B. 
`Overview of Fe regulation and the roles of Fe-S cluster proteins in
`yeast ..................................................................................................... 32 
`Overview of yeast fermentation products made from glucose and the
`GPD proteins ....................................................................................... 37 
`Comparison of claim 10 to Flint in view of Valadi ............................ 41 
`Comparison of claim 10 to Anthony in view of Puig and Ojeda and
`further in view of Valadi ..................................................................... 45 
`1. 
`Claim 1 ...................................................................................... 46 
`2. 
`Claim 2 ...................................................................................... 51 
`3. 
`Claim 10 .................................................................................... 53 
`
`C. 
`
`D. 
`E. 
`
`
`
`i
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`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`F. 
`G. 
`
`H. 
`
`Comparison of claim 10 to Flint in view of Dundon .......................... 55 
`Comparison of claim 10 to Anthony in view of Puig and Ojeda and
`further in view of Dundon ................................................................... 59 
`The prior art does not teach away from modifying a yeast containing
`recombinant overexpressed DHAD .................................................... 61 
`Gevo's alleged unexpected results would have been entirely expected
`for recombinantly expressed DHAD ................................................... 65 
`Conclusion ..................................................................................................... 68 
`
`
`I. 
`
`
`
`X. 
`
`
`
`ii
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`I, Dennis J. Thiele, hereby declare as follows:
`
`I.
`
`Overview
`1.
`
`I am over the age of eighteen (18) and otherwise competent to make
`
`this Declaration.
`
`2.
`
`I have been retained as an expert witness on behalf of Butamax™
`
`Advanced Biofuels, LLC ("Butamax") for the above-captioned inter partes review
`
`("IPR"). I am being compensated for my time in connection with this IPR at my
`
`standard consulting rate. I understand that the Petition for IPR involves U.S. Patent
`
`No. 8,273,565 ("the
`
`'565 patent"), BMX1001, which resulted from U.S.
`
`Application No. 13/246,693 ("the '693 application"), filed on September 27, 2011.
`
`The '565 patent issued on September 25, 2012, from the '693 application, which is
`
`a division of U.S. Patent Appl. No. 13/228,342, filed September 8, 2011, now U.S.
`
`Patent No. 8,071,358; and of U.S. Patent Appl. No. 12/953,884, filed November
`
`24, 2010, now U.S. Patent No. 8,017,376, which claimed the benefit of U.S.
`
`Provisional Application No. 61/263,952 ("the '952 application"), BMX1010, filed
`
`on November 24, 2009, and U.S. Provisional Application No. 61/350,209,
`
`BMX1011, filed on June 1, 2010. It is my understanding that the earliest claimed
`
`priority date of the '565 patent is November 24, 2009, the filing date of the '952
`
`application. I further understand that, according to the United States Patent and
`
`
`
`1
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`Trademark Office ("USPTO") records, the '565 patent is currently assigned to
`
`Gevo, Inc.
`
`3.
`
`In preparing this Declaration, I have reviewed the '565 patent and
`
`considered each of the documents cited herein, in light of general knowledge in the
`
`art as of the earliest effective filing date of the '565 patent. In formulating my
`
`opinions, I have relied upon my experience in the relevant art and the viewpoint of
`
`a person of ordinary skill in the art ("POSA"), as described below at ¶17.
`
`4.
`
`Claim 1 of the '565 patent, from which claims 5 and 10 ultimately
`
`depend, generally encompasses a recombinant microorganism that recombinantly
`
`overexpresses a polynucleotide encoding a dihydroxy acid dehydratase (DHAD).
`
`Claim 1 further specifies that the recombinant microorganism is engineered to
`
`comprise at least one inactivated monothiol glutaredoxin selected from the group
`
`consisting of monothiol glutaredoxin-3 (GRX3), monothiol glutaredoxin-4
`
`(GRX4), and GRX3 and GRX4, wherein the inactivated GRX3 and/or GRX4
`
`results from the deletion of one or more nucleotides in the coding region of an
`
`endogenous GRX3 and/or GRX4 gene, the insertion of one or more nucleotides
`
`into the coding region of an endogenous GRX3 and/or GRX4 gene, or a
`
`combination of such a deletion and insertion. My conclusions regarding claims 5
`
`and 10 are summarized in the following paragraphs 5-7.
`
`
`
`2
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`5.
`
`Claims 5 and 10 of the '565 patent are not entitled to the benefit of the
`
`
`
`filing dates of the '952 and '209 provisional applications.
`
`6.
`
`Flint (BMX1003) discloses each and every limitation of claim 5 of the
`
`'565 patent, arranged as claimed.
`
`7.
`
`A POSA would have had a reason to arrive at claim 10 of the '565
`
`patent with a reasonable expectation of success.
`
`a. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 10 of the '565 patent by combining the disclosures
`
`of Flint (BMX1003), and Valadi (BMX1027).
`
`b. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 10 of the '565 patent by combining the disclosures
`
`of Anthony (BMX1005), Puig (BMX1006), Ojeda (BMX1007), and
`
`Valadi (BMX1027).
`
`c. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 10 of the '565 patent by combining the disclosures
`
`of Flint (BMX1003), and Dundon (BMX1028).
`
`
`
`3
`
`

`

`
`
`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`d. A POSA would have had a reason to, and would have had a
`
`reasonable expectation of success to, arrive at an embodiment within
`
`the scope of claim 10 of the '565 patent by combining the disclosures
`
`of Anthony (BMX1005), Puig (BMX1006), Ojeda (BMX1007), and
`
`Dundon (BMX1028).
`
`e. Gevo's arguments that (i) the art taught away from the claimed yeast,
`
`and (ii) the claimed yeast had some alleged unexpected property were
`
`directed to a native yeast. These arguments are therefore inapplicable
`
`to the claims in the '565 patent, which are directed to a recombinant
`
`yeast.
`
`II. My background and qualifications
`8. My qualifications and credentials are fully set forth in my curriculum
`
`vitae, attached as BMX1023. I am an expert in the fields of molecular biology and
`
`microbial genetics, and one emphasis of my work focuses on understanding the
`
`regulation of iron and copper homeostasis in yeast. I am knowledgeable about the
`
`experimental techniques used in the fields of molecular biology and microbial
`
`genetics. I have been an expert in these fields since 1984. For the past 34 years, I
`
`have accumulated significant training and experience in the fields of molecular
`
`biology, microbial genetics, and microbial metabolism, as well as related fields.
`
`
`
`4
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`9.
`
`I received a Bachelor's Degree in Biology from the State University of
`
`
`
`New York, College at Fredonia, Fredonia, New York in 1978. I received a
`
`Master's Degree in Microbiology in 1981 and a Ph.D. in Microbiology in 1983
`
`from Rutgers University, New Brunswick, New Jersey.
`
`10. From 1984 to 1987, I was a Staff Fellow at the National Cancer
`
`Institute in Bethesda, Maryland. From 1987 to 1992, I was an Assistant Professor
`
`of Biological Chemistry at the University of Michigan Medical School, Ann Arbor,
`
`Michigan. From 1992 to 1996, I was an Associate Professor at the University of
`
`Michigan Medical School. From 1996 to 2003, I was a Professor at the University
`
`of Michigan Medical School.
`
`11. Since 2003, I have been a Professor in the Department of
`
`Pharmacology and Cancer Biology at the Duke University School of Medicine in
`
`Durham, North Carolina. In addition, since 2006, I have been the George Barth
`
`Geller Distinguished Professor of Pharmacology and Cancer Biology at the Duke
`
`University School of Medicine, and from 2006 to 2012 I served as the Vice Chair
`
`of the Department of Pharmacology and Cancer Biology at the Duke University
`
`School of Medicine.
`
`12.
`
`I have published more than 100 papers in the areas of microbial
`
`genetics and molecular biology including metal homeostasis in yeast. I serve as a
`
`
`
`5
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`reviewer for professional journals in my field and have been a member of the
`
`Editorial Boards of The Journal of Biological Chemistry, Genes and Nutrition,
`
`Eukaryotic Cell and Virulence. I am a member of multiple professional societies
`
`including
`
`the Genetics Society of America,
`
`the American Society for
`
`Microbiology, the American Association for the Advancement of Science and the
`
`American Society for Biochemistry and Molecular Biology. In addition, I have
`
`been elected a Fellow of the American Academy of Microbiology and of the
`
`American Association for the Advancement of Science. I collaborate with several
`
`prominent researchers in the fields of molecular biology and microbial genetics.
`
`13.
`
`I am the corresponding author of Puig, S., et al. "Coordinated
`
`Remodeling of Cellular Metabolism during Iron Deficiency through Targeted
`
`mRNA Degradation," Cell 120:99-110 (2005) ("Puig") which is cited in the
`
`Petition for Inter Partes Review (attached to the Petition as BMX1006). The work
`
`reported in this article was conducted under my supervision in my laboratory at the
`
`Duke University School of Medicine.
`
`14.
`
`In addition to my educational training, professional experiences, and
`
`research experiences described above, I attend multiple scientific conferences each
`
`year, and I am invited to speak at scientific seminars several times a year. I was
`
`the Keynote Lecturer at the Gordon Research Conference on the Cell Biology of
`
`
`
`6
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`Metals held in 2009, the Gordon Research Conference on Stress Proteins also held
`
`in 2009, and the Federation of American Societies for Experimental Biology
`
`(FASEB) Trace Elements Summer Conference held in 2010.
`
`15. Accordingly, I am an expert in the fields of molecular biology,
`
`microbial genetics, and microbial metabolism.
`
`III. List of documents considered in formulating my opinion
`16.
`
`In formulating my opinion, I have considered
`
`the following
`
`documents:
`
`Exhibit No.
`
`Document
`
`BMX1001
`
`BMX1003
`
`BMX1004
`
`BMX1005
`
`BMX1006
`
`BMX1007
`
`Dundon et al., U.S. Patent No. 8,273,565
`
`Flint et al., Int. Appl. Publ. No. WO 2011/1033000 A2
`
`Flint et al., U.S. Prov. Appl. No. 61/305,333
`
`Anthony et al., U.S. Appl. Publ. No. 2010/0081179
`
`Puig, S., et al., "Coordinated Remodeling of Cellular
`Metabolism during Iron Deficiency through Targeted
`mRNA Degradation," Cell 120:99-110
`(2005)
`("Puig")
`
`Ojeda, L., et al., "Role of Glutaredoxin-3 and
`Glutaredoxin-4 in the Iron Regulation of the Aft1
`Transcriptional
`Activators
`in
`Saccharomyces
`cerevisiae," J. Biol. Chem. 281(26): 17661-17669
`(2006)
`
`BMX1010
`
`Urano et al., U.S. Prov. Appl. No. 61/263,952
`
`
`
`7
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`Urano et al., U.S. Prov. Appl. No. 61/350,209
`
`File Wrapper for U.S. Appl. No. 13/246,693
`
`"Cytosolic Monothiol
`Mühlenhoff, U., et al.,
`Glutaredoxins Function in Intracellular Iron Sensing
`and Trafficking via Their Bound Iron-Sulfur Cluster,"
`Cell Metabolism, 12:373-385 (2010) ("Mühlenhoff")
`
`Pujol-Carrion, N., et al., "Glutaredoxins Grx3 and
`Grx4 regulate nuclear localisation of Aft1 and the
`oxidative
`stress
`response
`in
`Saccharomyces
`cerevisiae," J. Cell Sci. 119(2):4554-4564 (2006)
`("Pujol-Carrion")
`
`Li et al., U.S. Appl. Publ. No. 2009/0163376 (filed
`Dec. 18, 2008; published June 25, 2009)
`
`Mercier, A. and Labbé, S., "Both Php4 Function and
`Subcellular Localization are Regulated by Iron via a
`Multistep Mechanism Involving the Glutaredoxin Grx4
`and
`the Exportin Crm1," Journal of Biological
`Chemistry 284: 20249-202261 (2009) ("Mercier")
`
`Donaldson et al., U.S. Appl. Publ. No. 2007/0092957
`
`Rutherford , J.C., et al., "Aft1p and Aft2p Mediate
`Iron-responsive Gene Expression in Yeast through
`Related Promoter Elements," Journal of Biological
`Chemistry 278(30): 27636-27643 (2003) ("Rutherford
`2003")
`
`Li, H., et al., "The Yeast Iron Regulatory Proteins
`Grx3/4 and Fra2 Form Heterodimeric Complexes
`Containing a [2FE-2S] Cluster with Cysteinyl and
`Histidyl Ligation," Biochemistry 48: 9569-9581 (2009)
`
`Rutherford, J.C., et al., "A second iron-regulatory
`system in yeast independent of Aft1p," PNAS 98(25):
`
`8
`
`BMX1011
`
`BMX1012
`
`BMX1013
`
`BMX1014
`
`BMX1015
`
`BMX1017
`
`BMX1018
`
`BMX1019
`
`BMX1020
`
`BMX1021
`
`
`
`
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`14322-14327 (2001) ("Rutherford 2001")
`
`Li et al., U.S. Appl. Publ. No. 2010/0197519
`
`Curriculum vitae of Dennis J. Thiele, Ph.D.
`
`Michnick, et al., "Modulation of Glycerol and Ethanol
`Yields During
`Alcoholic
`Fermentation
`in
`Saccharomyces cerevisiae Strains Overexpressed or
`Disrupted for GPD1 Encoding Glycerol 3-Phosphate
`Dehydrogenase," Yeast 13:783-93 (1997)
`
`U.S. Ser. No. 12/637,905
`
`David L. Nelson and Michael M. Cox, Lehninger
`Principles of Biochemistry, 527-44 (3rd ed. 2000)
`
`Valadi et al., "Improved ethanol production by
`glycerol-3-phosphate dehydrogenase mutants of
`Saccharomyces cerevisiae," AMB 50:434-39 (1998)
`
`BMX1022
`
`BMX1023
`
`BMX1024
`
`BMX1025
`
`BMX1026
`
`BMX1027
`
`BMX1028
`
`Dundon et al., Int. Appl. Publ. No. WO 2007/106534
`
`
`
`
`
`IV. Person of ordinary skill in the art
`17.
`
`In formulating my opinions, I have considered the viewpoint of a
`
`person of ordinary skill in the art ("POSA"). I understand that a POSA is one who
`
`is presumed to be aware of all pertinent art, thinks along conventional wisdom in
`
`the art, and is a person of ordinary creativity. With regard to the '565 patent, a
`
`POSA typically would have a Ph.D. in the life sciences or a similar related
`
`discipline, and have familiarity, training, and experience in molecular biology,
`
`microbial genetics, and microbial metabolism. Alternatively, a POSA typically
`
`
`
`9
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`would have at least a scientific background such as a Bachelor's degree in the life
`
`sciences (e.g., biology, microbiology, molecular biology or biochemistry) or a
`
`similar related discipline, and have substantial familiarity, training, and experience
`
`in molecular biology, microbial genetics and microbial metabolism. A POSA also
`
`would have had knowledge of the scientific literature concerning molecular
`
`biology, microbial (e.g., yeast) genetics and microbial metabolism, that was
`
`available at a time point of interest, including knowledge about experimental
`
`techniques available in the art.
`
`V.
`
`The '565 patent
`18.
`
`I understand that this Declaration is being submitted together with a
`
`Petition for IPR of claims 5 and 10 of the '565 patent.
`
`19.
`
`I have considered the disclosure of the '565 patent in light of the
`
`earliest claimed priority date of the '565 patent, which I understand to be
`
`November 24, 2009. It is my understanding that the Patent Trial and Appeal Board
`
`("PTAB") determined that the claims of the '565 patent, including claims 5 and 10,
`
`have an effective filing date no earlier than November 24, 2010. Case IPR2013-
`
`00539, Paper 9 at pp. 15-16.
`
`20. According to the Abstract, the '565 patent specification is directed to
`
`"recombinant microorganisms comprising one or more dihydroxyacid dehydratase
`
`
`
`10
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`(DHAD)-requiring biosynthetic pathways and methods of using said recombinant
`
`microorganisms to produce beneficial metabolites derived from said DHAD-
`
`requiring biosynthetic pathways." (BMX1001, Abstract.)
`
`21. The '565 patent claims recombinant yeast microorganisms with a
`
`recombinantly overexpressed DHAD and an inactivated GRX3 and/or GRX4, as
`
`well as a method of producing isobutanol by cultivating the claimed yeast.
`
`(BMX1001, 91:15 through 91:26 and 92:58 through 92:63.)
`
`VI. The full scope of claims 1-19 of the '565 patent is not disclosed in the
`'952 and '209 provisional applications
`22. Claims 1-19 of the '565 patent broadly encompass any recombinant
`
`yeast comprising a recombinantly overexpressed polynucleotide encoding any
`
`DHAD and engineered to comprise any inactivated GRX3 and/or GRX4 protein,
`
`and which further broadly encompass: (1) any deletion of one or more nucleotides
`
`in the coding region of an endogenous GRX3 and/or GRX4 gene, or (2) any
`
`insertion of one or more nucleotides into the coding region of an endogenous
`
`GRX3 and/or GRX4 gene, or (3) any combination of such deletions and insertions,
`
`that result in the inactivated protein, such that the protein is not expressed or is
`
`expressed in a completely non-functional form. As discussed below, in view of the
`
`disclosures of the '952 and '209 provisional applications, a POSA would not have
`
`understood the applicants to have invented the recombinant yeast microorganisms
`11
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`
`
`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`encompassed by the claims of the '565 patent as of the '952 and '209 provisional
`
`application filing dates.
`
`A.
`
`The provisional applications do not describe the full scope of
`inactivated GRX3 and/or GRX4 as encompassed by claims 1-19 of
`the '565 patent
`23. Claims 1-19 of the '565 patent are directed to recombinant yeast
`
`microorganisms and a method of using a recombinant yeast microorganism
`
`comprising an "inactivated" GRX3 and/or GRX4.
`
`24. A POSA reading the '952 and '209 provisional applications would
`
`understand the applications as being primarily directed to expression and
`
`overexpression of GRX3 and/or GRX4.1 For example, ¶¶[00155] and [00156] of
`
`1 See, e.g., BMX1010 at ¶¶[0023] and [00116] and BMX1011 at ¶¶ [0023]
`
`and [00122], disclosing GRX3 and GRX4 as a "chaperone protein" that when
`
`expressed can assist the folding of a DHAD exhibiting cytosolic activity;
`
`BMX1010 and BMX1011 at ¶[0028], disclosing overexpression of one or more
`
`genes including GRX3 and GRX4 as leading to increased iron levels in the cytosol
`
`and mitochondria for availability in producing Fe-S cluster-containing proteins in
`
`the cytosol, which BMX1011 further discloses includes cytosolic DHAD;
`
`BMX1010 at ¶¶[0030] and [00189] and BMX1011 at ¶¶[0033] and [00212],
`
`disclosing overexpression of GRX4 for increasing glutathione levels in the cytosol.
`
`
`
`12
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`the '952 application and ¶¶[00179] and [00180] of the '209 provisional application,
`
`which are under the sections describing ways to enhance DHAD activity, discuss
`
`alleged knowledge regarding GRX3 and GRX4 in yeast and list nine specific
`
`"embodiments" associated with GRX3 and/or GRX4 expression. Eight of these
`
`embodiments discuss overexpression of GRX3 and/or GRX4.2 Indeed, the header
`
`of the section including these GRX3/GRX4 embodiments in the '209 provisional
`
`application, the later-filed of the two provisional applications, is entitled
`
`"Enhancing DHAD Activity by Increased GRX3/GRX4 Activity and/or
`
`Expression." As such, even at the later priority date of the '209 provisional
`
`See also, BMX1010 at ¶¶[00154], [00155], [00156], [00158], claim 33, claim 55,
`
`and claim 66, and BMX1011 at ¶¶[00178], [00179], [00180], [00182], claim 33,
`
`claim 55, and claim 66.
`
`2 (1) "GRX3 is overexpressed," (2) "GRX4 is overexpressed," (3) "GRX3
`
`and GRX4 are overexpressed," (4) "GRX3, GRX4, or GRX3 and GRX4 are
`
`deleted or attenuated," (5) "GRX3 and Aft1 are overexpressed," (6) "GRX4 and
`
`Aft1 are overexpressed," (7) "GRX3 and Aft2 are overexpressed," (8) "GRX4 and
`
`Aft2 are overexpressed," and (9) "One or both of: Aft1, Aft2 is overexpressed
`
`either alone or in combination with: GRX3 or GRX4." BMX1010 at ¶¶ [00155]
`
`and [00156] and BMX1011 at ¶¶ [00179] and [00180].
`
`
`
`13
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`

`

`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
`
`
`
`application, it would have been apparent to a POSA reading the '209 application
`
`that the applicants considered increasing GRX3 and GRX4 expression to be the
`
`focus of any change in GRX3 and GRX4 activity, and not deletion or
`
`"inactivation."
`
`25. Furthermore, ¶[00156] of the
`
`'952 provisional application and
`
`¶[00180] of the '209 provisional application state that the embodiments disclosed
`
`in those paragraphs "can also be combined with increases in the extracellular iron
`
`concentration to provide increased iron in the cytosol or mitochondria of the cell."
`
`As such, ¶[00156] of the '952 application and ¶[00180] of the '209 application
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`confusingly describe achieving the same effect of increased iron in the cytosol or
`
`mitochondria as a result of either "overexpression" or "deletion or attenuation" of
`
`GRX3 and/or GRX4. As such, it would have been apparent to a POSA reading the
`
`provisional applications that the applicants had not yet settled on what they
`
`believed to be the invention when they filed the provisional applications.
`
`26. Based on the requirement that the yeast of the claims comprise
`
`inactivated GRX3 and/or GRX4 that do not have any activities, the claims broadly
`
`encompass: (1) a yeast in which the GRX3 and/or GRX4 proteins are not
`
`expressed, (2) a yeast in which GRX3 and/or GRX4 proteins are expressed in
`
`completely non-functional forms, and (3) a yeast in which one of GRX3 or GRX4
`
`
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`14
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`protein is not expressed, while the other is expressed in a completely non-
`
`functional form, provided such deletions, insertions, or combinations of deletions
`
`and insertions that result in complete inactivation of GRX3 and/or GRX4 protein
`
`do not occur in the regulatory regions associated with the endogenous genes.
`
`27. The '565 patent and the provisional applications do not define the term
`
`"inactivated" as recited in claim 1 of the '565 patent.
`
`28. The term "inactivated" is not defined in the provisional applications,
`
`and expression of GRX3 and/or GRX4 proteins in completely non-functional
`
`forms is not described. There are no working examples of recombinant yeast in
`
`which GRX3 and/or GRX4 are deleted. At best, the '952 provisional application
`
`sparsely discloses that yeast may be engineered to "delete" or "attenuate" GRX3
`
`and/or GRX4 genes, while the '209 provisional application sparsely discloses that
`
`yeast may be engineered to "delete," "reduce," or "attenuate" GRX3 and/or GRX4
`
`genes.3 Reduction and attenuation of GRX3 and/or GRX4 genes would be
`
`3 BMX1010 at ¶¶[0028], [00154], [00156], [00157], [00158], and claim 56,
`
`and BMX1011 at ¶¶[00178], [00180], [00181], [00182], and claim 56 generically
`
`disclose deletion and/or attenuation of GRX3 and/or GRX4 genes. BMX1011 at
`
`¶[0031] also generically discloses deletion, reduction, and/or attenuation of GRX3
`
`and/or GRX4.
`
`
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`15
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`
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`understood to accomplish less than complete inactivation of all GRX3 and/or
`
`GRX4 activities. Therefore, since "inactivated" means the absence of all GRX3
`
`and/or GRX4 activity, it is my understanding that "inactivated" GRX3 and/or
`
`GRX4 would not result from yeast that have been engineered to "reduce" or
`
`"attenuate" GRX3 and/or GRX4 genes.
`
`29.
`
`It is my understanding that the term "inactivated" with respect to
`
`GRX3 and/or GRX4 was discussed by the applicants and the Examiner during
`
`prosecution of U.S. Application No. 13/246,693 ("the '693 application"). In that
`
`discussion the applicants indicated the meaning of "inactivate" is "to render
`
`inactive so that GRX3 and GRX4 protein have no activity thereof" in contrast to
`
`partially functional GRX3 and GRX4 proteins. See File Wrapper for the '693
`
`application, Examiner-Initiated Interview Summary dated May 17, 2012
`
`(BMX1012). As such, it is my understanding that "inactivated" means that the
`
`GRX3 and/or GRX4 protein lacks all activity and excludes GRX3 and/or GRX4
`
`proteins having reduced, attenuated or partial activities.
`
`30. A POSA would understand the generic disclosure of yeast engineered
`
`to "delete" GRX3 and/or GRX4 genes to only encompass, at best, a complete
`
`deletion of the GRX3 and/or GRX4 genes, such that the corresponding proteins are
`
`not expressed.
`
`
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`16
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`31. For example, the '952 provisional application at ¶[00268] and the '209
`
`
`
`provisional application at ¶[00299] disclose that "when expression is to be
`
`repressed or eliminated, the gene for the relevant enzyme, protein or RNA can be
`
`eliminated by known deletion techniques." Therefore, the '952 and '209 provisional
`
`applications do not disclose expression of GRX3 and/or GRX4 proteins that are
`
`non-functional as a result of partial deletions of the endogenous genes.
`
`32.
`
`In view of the disclosures of the '952 and '209 provisional applications
`
`as discussed above, a POSA would not have understood the applicants to have
`
`invented recombinant yeast microorganisms comprising, among other things, (1) a
`
`yeast in which the GRX3 and/or GRX4 proteins are not expressed, (2) a yeast in
`
`which GRX3 and/or GRX4 proteins are expressed in completely non-functional
`
`forms, and (3) a yeast in which one of GRX3 or GRX4 proteins is not expressed,
`
`while the other is expressed in a completely non-functional form at the filing dates
`
`of the '952 and '209 provisional applications.
`
`B.
`
`The provisional applications do not describe all of the claimed
`nucleotide deletions, insertions, or combinations of deletions and
`insertions
`into endogenous GRX3 and/or GRX4 genes as
`encompassed by claims 1-19 of the '565 patent
`33. Claims 1-19 of the '565 patent encompass any deletion of one or more
`
`nucleotides, any insertion of one or more nucleotides, or any combination of any
`
`deletion and any insertion of one or more nucleotides in an endogenous yeast
`
`
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`17
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`
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`GRX3 and/or GRX4 gene that results in any "inactivated" GRX3 and/or GRX4
`
`protein, in which, as discussed above, the protein is not expressed or is expressed
`
`in a completely non-functional form.
`
`34. The deletions, insertions, or combinations of deletions and insertions
`
`that result in inactivation of GRX3 and/or GRX4 protein are not limited by the
`
`claims to any portion of the coding regions of the endogenous GRX3 and/or GRX4
`
`genes. As such, it is my understanding that the claims encompass any deletion, any
`
`insertion, or any combination thereof in any portion of the coding region that
`
`results in an inactivated GRX3 and/or GRX4 protein.
`
`35.
`
`It is my understanding that the Examiner rejected claims reciting
`
`deletions, insertions and combinations thereof in regulatory regions as lacking
`
`written description and enablement during prosecution of the '693 application. See
`
`File Wrapper for the '693 application, Office Action dated May 1, 2012 at pp. 3-7
`
`(BMX1012). It is also my understanding that the applicants deleted recitation of
`
`regulatory regions from the claims in response to the Examiner's rejection. See File
`
`Wrapper for the '693 application, Amendment under 37 CFR § 1.116 dated May 8,
`
`2012 at p. 2 (BMX1012). Therefore, it is my understanding that the claims of the
`
`'565 patent do not encompass deletions, insertions, and combinations of deletions
`
`
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`18
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`Inter Partes Review of USPN 8,273,565
`Declaration of Dennis J. Thiele, Ph.D. (Exhibit BMX1002)
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`
`
`and insertions that occur in the regulatory regions of the endogenous GRX3 and/or
`
`GRX4 genes.
`
`36. As discussed above, the '952 and '209 provisional applications at best
`
`only disclose complete deletion of GRX3 and/or GRX4 and do not provide any
`
`disclosure regarding partial deletions that would result in a GRX3 and/or GRX4
`
`protein lacking all functional activities.
`
`37. Additionally, the '952 and '209 provisional applications do not
`
`describe structural features common to GRX3 and/or GRX4 endogenous genes
`
`from all of the yeast genera encompassed by the claims that would allow for
`
`inactivation of GRX3 and/or GRX4 protein function with less than complete
`
`deletion of the genes.
`
`38. The '952 and '209 provisional applications make only a passing,
`
`generic reference to any type of engineering besides complete deletion, and no
`
`description for any of these is provided that would be understood by a POSA
`
`reading the applications as describing inactivated GRX3 and/or GRX4 proteins.
`
`For example, the '952 and '209 provisional applications at ¶¶[0067] and [0073],
`
`respectively, disclose that:
`
`[t]he term "engineer" refers to any manipulation of a
`microorganism that results in a detectable change in the
`microorganism, wherein the manipulati