LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 1 of 17
`
`

`

`STEE~BOCK
`._~Et/,ORIAL LIBRARY
`APPLIED AND ENVIRONMENTAL
`O T 1 7 2 3
`MICROBIOLOGY
`
`U.W. - MAO:SON
`
`VOLUME 69 • OCTOBER 2003 • NUMBER 10
`
`L. Nicholas Ornston, Editor in Chief (2006)
`Yale University
`Daniel J. Arp, Minireview Editor (2004)
`Oregon State University
`Mark J. Bailey, Editor (2007)
`National Environment Research Council
`Harold L. Drake, Editor (2006)
`Universitiit Bayreuth
`Katharine J. Gibson, Editor (2006)
`DuPont Experiment Station
`
`Caroline S. Harwood, Editor (2006)
`Univedity of Iowa
`Eric A. Johnson, Editor (2005)
`University of Wisconsin
`Sophia Kathariou, Editor (2007)
`North Carolina State University
`Christian P. Kubicek, Editor (2007)
`Technische Universitiit Wien
`Laura G. Leff, Editor (2006)
`Kent State University
`John F. Leslie, Editor (2006)
`Kansas State University
`
`EDITORIAL BOARD
`
`Kenneth H. Nealson, Editor (2008)
`University of Southern California
`Douglas H. Ohlendorf, Editor (2006)
`University of Minnesota
`Linda D. Stetzenbach, Editor (2006)
`University of Nevada
`Peter A. Williams, Editor (2004)
`University of Wales
`Jizhong Zhou, Editor (2008)
`Oak Ridge National Laboratory
`
`Yin-Won Lee (2004)
`John A. Leigh (2003)
`Michael Lemke (2005)
`Hiltrud Lenke (2003)
`Gregory J. Leyer (2004)
`James C. Liao (2004)
`Gunnar Liden (2004)
`Markus Linder (2005)
`H. D. Alan Lindquist (2004)
`Jon Lloyd (2005)
`John Londesborough (2004)
`G. T. Macfarlane (2005)
`Roderick I. Mackie (2004)
`Eugene L. Madsen (2005)
`Walter F. 0. Marasas
`(2004)
`Robert E. Marquis (2005)
`Terence L. Marsh (2005)
`Phyllis Martin (2004)
`Angel T. Martinez (2004)
`Esperanza Martinez-Ro-
`mero (2004)
`Juergen Marxsen (2005)
`Luke Masson (2005)
`Ann G. Matthysse (2004)
`Harold D. May (2004)
`J. Vaun McArthur (2005)
`Timothy R. McDermott
`(2005)
`Michael J. Mcinerney
`(2004)
`Mhairi McIntyre (2005)
`Robert J. C. McLean (2004)
`Phillip G. Meaden (2005)
`Aubrey F. Mendonca (2003)
`Eric S. Miller (2005)
`Robert V. Miller (2005)
`Terry L. Miller (2005)
`Kiwamu Minamisawa
`(2005)
`William W. Mohn (2005)
`Soeren Molin (2005)
`Istvan Molnar (2005)
`Mark Morrison (2005)
`Jim Mueller (2004)
`Volker Miieller (2004)
`Peter Muriana (2005)
`
`Max Haggblom (2003)
`Dittmar Hahn (2004)
`Martin W. Hahn (2004)
`Jo Handelsman (2005)
`Theo A. Hansen (2004)
`Ian M. Head (2005)
`Hermann Heipieper
`(2005)
`Alfredo Herrera-Estrella
`(2005)
`Russell T. Hill (2005)
`Mark E. Hines (2005)
`Ayala Hochman (2003)
`William E. Holben (2004)
`Christof Holliger (2004)
`Laszlo Hornok (2005)
`Richard C. Hutchinson
`(2005)
`Michael R. Hyman (2005)
`Steven Ingham (2005)
`Karl-Erich Jaeger
`005)
`Dick Janssen (2004
`Edward L. Jarroll 2004)
`Tom Jeffries (2005)
`Peter Jensen (2005)
`Carlos A. Jerez (2005)
`Mike Jetten (2004)
`D. Barrie Johnson (2005)
`David Kaplan (2004)
`Charles W. Kaspar (2004)
`Jay D. Keasling (2005)
`Lee Kerkhof (2003)
`Ronald P. Kiene (2005)
`David L. Kirchman (2005)
`Staffan Kjelleber (2005)
`Keith K. Klein (2003)
`Martin G. Klotz (2005)
`Tyler Kokjohn (2004)
`Allan Konopka (2005)
`Joel E. Kostka (2004)
`Jerome J. Kukor (2005)
`Charles F. Kulpa, Jr. (2004)
`Anna M. Lammerding
`(2004)
`Gisele LaPointe (2004)
`Michael J. Larkin (2004)
`Peter C. K. Lau (2005)
`
`82
`
`Lori L. Daane (2005)
`Steven L. Daniel
`005)
`David D'Argenio 2004)
`Atin R. Datta (20 4)
`Seana K. Davidson (2005)
`Jacqueline E. Dawson
`(2004)
`Frank B. Dazzo (2004)
`Jeffrey F. D. Dean (2005)
`Edward F. DeLong (2005)
`Edward DeMoll (2004)
`Anne Desjardins (2005)
`Thomas J. DiChristina
`(2005)
`Alan DiSpirito (2005)
`Jan Doffing (2004~
`Wouter A. Duetz 2004)
`Peter Diirre (2005
`.
`ancy DuTeau ~2 05)
`Richard Eaton 2004)
`Leo Eberl (2005
`Henry L. Ehrlich (2004)
`Lindsay D. Eltis (2004)
`David Emerson ~2003)
`Scott A. Ensign 2005)
`David Faguy (20 5)
`Joseph 0. Falkinham ill
`(2004)
`Brian Federici (2003)
`Juan Ferre (2003)
`Katharine G. Field (2004)
`Turlough Finan (2004)
`Robert Findlay (2003)
`Madilyn Fletcher (2005)
`Miroslav Flieger (2005)
`G. Shay Fout (2004)
`Clay Fuqua (2005)
`Karen A. Garrett (2004)
`Gill G. Geesey (2005)
`Michael Givskov (2005)
`L. Anne Glover (2003)
`Amanda E. Goodman
`(2003)
`Friedrich Gotz (2005)
`Lone Gram (2005)
`Irene R. Grant (2004)
`Jean Guard-Petter (2004)
`
`82
`
`Yousef Abu Kwaik (2005)
`Michael J. Adang (2003)
`Craig Altier (2005)
`Jose A. Amador (2003)
`David Archer (2004)
`Arthur Aronson (2004)
`Steven D. Aust (2004)
`Brian Austin (2005)
`Charles W. Bacon (2004)
`Alan Bakalinsky (2003)
`Katherine H. Baker (2003)
`J6zsef Baranyi (2004)
`Douglas H. Bartlett (2005)
`Gwyn A. Beattie (2005)
`J. Thomas Beatty (2003)
`Tahia Benitez (2005)
`Colin Berry (2005)
`K. Johanna Bjorkroth
`(2004)
`Paul L. E. Bodelier (2005)
`Katherine J. Boettcher
`(2005)
`Kathryn J. Boor (2004)
`Dulal Borthakur (2003)
`Thomas L. Bott (2003)
`Peter J. Bottomley (2003)
`Botho Bowien (2004)
`Axel Brakhage (2004)
`Michael G. Bramucci (2005)
`Frederick Breidt, Jr. (2004)
`Michael Breitenbach (2005)
`Neil C. Bruce (2004)
`Douglas G. Capone (2005)
`Russell W. Carlson (2004)
`Jeffrey W. Cary (2004)
`Enrique Cerda-Olmedo
`(2003)
`Darrell P. Chandler (2003)
`Dean 0. Cliver (2004)
`John D. Coates (2005)
`Yehuda Cohen (2003)
`Mary Lynne Perille Collins
`(2005)
`Michael T. Collins [004)
`Tim Coolbear (2003
`Daniel Cullen (2004
`Catherine N. Cutter (2004)
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 2 of 17
`
`

`

`APPLIED AND ENVIRONMENTAL MICROBIOLOGY
`STf~~·~30CK
`- ~ "L 1 ·'L'lr"'c")I\ RY
`· M c, v, U 1-1 i , • ..,
`L..1 w t 1J-\
`t 2D03
`UI • t
`'-' I
`
`No. 10
`
`Volume 69
`
`October 2003
`
`GENETICS AND MOLECULAR BIOLOGY
`
`Diversity of Nitrite Hydratase and Amidase Enzyme Genes
`in Rhodococcus erythropolis Recovered from Geographically
`Distinct Habitats
`A Potential Food-Grade Cloning Vector for Streptococcus
`thermophilus That Uses Cadmium Resistance as the
`Selectable Marker
`Identification and G'enetic Characterization of a Novel
`Proteinase, PrtR, from the Human Isolate Lactobacillus
`rhamnosus BGTlO
`
`Functional Analysis of the Gene Cluster Involved in
`Production of the Bacteriocin Circularin A by Clostridium
`beijerinckii ATCC 25752
`lSLpll Is a Functional IS30-Related Insertion Element in
`Lactobacillus plantarum That Is Also Found in Other Lactic
`Acid Bacteria
`Nuclease Activity of Saccharomyces cerevisiae Mrell
`Functions in Targeted Nucleotide Alteration
`Development of a mariner-Based Transposon for Use in
`Sorangium cellulosum
`
`ENZYMOLOGY AND PROTEIN ENGINEERING
`
`Purification and Characterization of a Novel Bacteriocin
`Produced by Enterococcus f aecalis Strain RJ-11
`
`Optimized Expression of a Thermostable Xylanase from
`Thermomyces lanuginosus in Pichia pastoris
`
`Identification of Peptides That Mimic the Pertussis Toxin
`Binding Site on Bovine Fetuin
`
`PHYSIOLOGY AND BIOTECHNOLOGY
`
`Glutathione Protects Lactococcus lactis against Oxidative
`Stress
`Engineering Escherichia coli for Increased Productivity of
`Serine-Rich Proteins Based on Proteome Profiling
`Peptide Surface Display and Secretion Using Two LPXTG(cid:173)
`Containing Surface Proteins from Lactobacillus fermentum
`BRll
`Engineering Redox Cofactor Regeneration for Improved
`Pentose Fermentation in Saccharomyces cerevisiae
`Role of Ectoine in Vibrio cholerae Osmoadaptation
`
`U W
`
`r..nAr1::o N
`• -
`•
`lVi
`L., ,~
`5754-5766
`Pedro F. B. Brandiio, Justin P.
`Clapp, and Alan T. Bull
`
`Wing Yee Wong, Ping Su, Gwen E.
`Allison, Chun-Qiang Liu, and Noel
`W. Dunn
`Irena Pastar, Ivana Tonic, Natasa
`Golie, Milan Kojic, Richard van
`Kranenburg, Michiel Kleerebezem,
`Ljubisa Topisirovic, and Goran
`Jovanovic
`Rober Kemperman, Marnix Jonker,
`Arjen Nauta, Oscar P. Kuipers, and
`Jan Kok
`Herve Nicoloff and Franc;oise
`Bringel
`
`Li Liu, Michael Usher, Yiling Hu,
`and Eric B. Kmiec
`Bryan Julien and Ruby Fehd
`
`Yukio Yamamoto, Yoshikazu
`Togawa, Makoto Shimosaka, and
`Mitsuo Okazaki
`Monica C. Triches Damaso, Marcius
`S. Almeida, Eleonora Kurtenbach,
`Orlando B. Martins, Nei Pereira,
`Jr., Carolina M . M. C. Andrade,
`and Rodolpho M. Albano
`John A. Bogdan, Wei Yuan, Karen
`0. Long-Rowe, Jawad Sarwar, Eric
`Allen Brucker, and M. S. Blake
`
`Yin Li, Jeroen Hugenholtz, Tjakko
`Abee, and Douwe Molenaar
`Mee-Jung Han, Ki Jun Jeong, Jong(cid:173)
`Shin Yoo, and Sang Yup Lee
`Mark S. Turner, Louise M. Hafner,
`Terry Walsh, and Philip M. Giffard
`
`Ritva Verho, John Londesborough,
`Merja Penttila, and Peter Richard
`Kathryn J. Pflughoeft, Katharine
`Kierek, and Paula I. Watnick
`
`5767-5771
`
`5802-5811
`
`5839-5848
`
`6032-6040
`
`6216-6224
`
`6299-6301
`
`5746-5753
`
`6064-6072
`
`6272-6279
`
`5739-5745
`
`5772-5781
`
`5855-5863
`
`5892-5897
`
`5919-5927
`
`Continued on following page
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 3 of 17
`
`

`

`Continued from preceding page
`
`Cloning, Characterization, and Functional Expression of the
`Klebsiella oxytoca Xylodextrin Utilization Operon (xynTB) in
`Escherichia coli
`Identification and Characterization of the CYP52 Family of
`Candida tropicalis ATCC 20336, Important for the
`Conversion of Fatty Acids and Alkanes to a,w-Dicarboxylic
`Acids
`Transformation of Fatty Acids Catalyzed by Cytochrome
`P450 Monooxygenase Enzymes of Candida tropicalis
`
`Lysine-2,3-Aminomutase and 13-Lysine Acetyltransferase
`Genes of Methanogenic Archaea Are Salt Induced and Are
`Essential for the Biosynthesis of ~-Acetyl-13-Lysine and
`Growth at High Salinity
`Influence of Nutritional Factors on the Nature, Yield, and
`Composition of Exopolysaccharides Produced by
`Gluconacetobacter xylinus 1-2281
`Escherichia coli Ghost Production by Expression of Lysis
`Gene E and Staphylococcal Nuclease
`Formaldehyde Fixation Contributes to Detoxification for
`Growth of a Nonmethylotroph, Burkholderia cepacia TMl, on
`Vanillic Acid
`Cloning and Characterization of the xyll Gene, Encoding an
`NADH-Preferring Xylose Reductase from Candida
`parapsilosis, and Its Functional Expression in Candida
`tropicalis
`Prospecti_ng for Novel Biocatalysts in a Soil Metagenome
`
`Effects of Minerals on Resistance of Bacillus subtilis Spores
`to Heat and Hydrostatic Pressure
`
`Formate-Dependent Growth and Homoacetogenic
`Fermentation by a Bacterium from Human Feces:
`Description of Bryantella formatexigens gen. nov., sp. nov.
`
`MYCOLOGY
`
`Mycotoxin Fumonisin B1 Increases Intestinal Colonization
`by Pathogenic Escherichia coli in Pigs
`
`Tri16 ls Required for Esterification of Position C-8 during
`Trichothecene Mycotoxin Production by Fusarium
`sporotrichioides
`Effect of pH and Oxalate on Hydroquinone-Derived
`Hydroxyl Radical Formation during Brown Rot Wood
`Degradation
`Invasion of Spores of the Arbuscular Mycorrhizal Fungus
`Gigaspora decipiens by Burkholderia spp.
`
`A Novel Extracellular Multicopper Oxidase from
`Phanerochaete chrysosporium with Ferroxidase Activity
`
`Yilei Qian, L. P. Yomano, J. F.
`Preston, H . C. Aldrich, and L. 0.
`Ingram
`David L. Craft, Krishna M .
`Madduri, Mark Eshoo, and C. Ron
`Wilson
`
`William H. Eschenfeldt, Yeyan
`Zhang, Hend Samaha, Lucy Stols,
`L. Dudley Eirich, C. Ronald Wilson,
`and Mark I. Donnelly
`K. Pfluger, S. Baumann, G.
`Gottschalk, W. Lin, H. Santos, and
`V. Muller
`
`Henri Kornmann, Philippe Duboc,
`Ian Marison, and Urs von Stockar
`
`W. Haidinger, U. B. Mayr, M. P.
`Szostak, S. Resch, and W. Lubitz
`Ryoji Mitsui, Yoko Kusano, Hiroya
`Yurimoto, Yasuyoshi Sakai, Nobuo
`Kato, and Mitsuo Tanaka
`Jung-Ku! Lee, Bong-Seong Koo,
`and Sang-Yong Kim
`
`S. Voget, C. Leggewie, A. Uesbeck,
`C. Raasch, K.-E. Jaeger, and W. R.
`Streit
`Noriyuki Igura, Yuya Kamimura, M.
`Shahidul Islam, Mitsuya Shimoda,
`and Isao Hayakawa
`Meyer J. Wolin, Terry L. Miller,
`Matthew D. Collins, and Paul A.
`Lawson
`
`Isabelle P. Oswald, Clarisse
`Desautels, Joelle Laffitte, Sylvie
`Fournout, Sylvie Y. Peres, Marielle ·
`Odin, Pierrette Le Bars, Joseph Le
`Bars, and John M. Fairbrother
`Andrew W. Peplow, Isaac R Meek,
`Melinda C. Wiles, Timothy D.
`Phillips, and Marian N. Beremand
`Elisa Varela and Ming Tien
`
`Avram Levy, Barbara J. Chang,
`Lynette K. Abbott, John Kuo, Gerry
`Hamett, and Timothy J. J. Inglis
`Luis F. Larrondo, Loreto Salas,
`Francisco Melo, Rafael Vicuna, and
`Daniel Cullen
`
`5957-5967
`
`5983-5991
`
`5992-5999
`
`6047-6055
`
`6091-6098
`
`6106-6113
`
`6128-6132
`
`6179-6188
`
`6235-6242
`
`6307-6310
`
`6321-6326
`
`5870-5874
`
`5935-5940
`
`6025-6031
`
`6250-6256
`
`6257-6263
`
`Continued on following page
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 4 of 17
`
`

`

`Continued from preceding page
`
`PUBLIC HEALTH MICROBIOLOGY
`
`Detection, Quantitation, and Phylogenetic Analysis of
`Noroviruses in Japanese Oysters
`
`Identification of Naegleria fowleri in Domestic Water Sources
`by Nested PCR
`
`PCR Detection and Analysis of the Free-Living Amoeba
`Naegleria in Hot Springs in Yellowstone and Grand Teton
`National Parks
`Assessment of the Elfects of Holding Time and Temperature
`on Escherichia coli Densities in Surface Water Samples
`
`Single-Cell Enumeration of an Uncultivated TM7 Subgroup
`in the Human Subgingival Crevice
`Teschoviruses as Indicators of Porcine Fecal Contamination
`of Surface Water
`
`Comparison of Campylobacter Isolates from Poultry and
`Humans: Association between In Vitro Virulence Properties,
`Biotypes, and Pulsed-Field Gel Electrophoresis Clusters
`
`ENVIRONMENTAL MICROBIOLOGY AND
`BIODEGRADATION
`
`Substrate Preferences in Biodesulfurization of Diesel Range
`Fuels by Rhodococcus sp. Strain ECRD-1
`Anaerobic Degradation of Flavonoids by Clostridium
`orbiscindens
`
`Stimulating the In Situ Activity of Geobacter Species To
`Remove Uranium from the Groundwater of a Uranium(cid:173)
`Contaminated Aquifer
`
`Utilization of Acyl-Homoserine Lactone Quorum Signals for
`Growth by a Soil Pseudomonad and Pseudomonas aeruginosa
`PAOI
`Bacterial Chemotaxis to Naphthalene Desorbing from a
`Nonaqueous Liquid
`Distribution of the Coenzyme M Pathway of Epoxide
`Metabolism among Ethene- and Vinyl Chloride-Degrading
`Mycobacterium Strains
`
`Tomoko Nishida, Hirokazu Kimura,
`Mika Saitoh, Michiyo Shinohara,
`Masahiko Kato, Shinji Fukuda,
`Tetsuya Munemura, Toshiyuki
`Mikami, Ayumi Kawamoto, Miho
`Akiyama, Yumiko Kato, Kanako
`Nishi, Kunihisa Kozawa, and Osamu
`Nishio
`Francine Marciano-Cabral, Rebecca
`MacLean, Alex Mensah, and Laurie
`LaPat-Polasko
`Kathy B. Sheehan, Jennifer A. Fagg,
`Michael J. Ferris, and Joan M.
`Henson
`Misty L. Pope, Michelle Bussen,
`Mary Ann Feige, Lois Shadix,
`Sharon Gonder, Crystal Rodgers,
`Yildiz Chambers, Jessica Pulz, Ken
`Miller, Kevin Connell, and Jon
`Standridge
`Cleber C. Ouvemey, Gary C.
`Armitage, and David A. Reiman
`Miguel Angel Jimenez-Clavero,
`Carlos Fernandez, Jose Antonio
`Ortiz, Javier Pro, Gregoria
`Carbonell, Jose Vicente Tarazona,
`Neftalf Roblas, and Victoria Ley
`Eric Nadeau, Serge Messier, and
`Sylvain Quessy
`
`Roger C. Prince and Matthew J.
`Grossman
`Lilian Schoefer, Ruchika Mohan,
`Andreas Schwiertz, Annett Braune,
`and Michael Blaut
`Robert T. Anderson, Helen A.
`Vrionis, Irene Ortiz-Bemad, Charles
`T. Resch, Philip E. Long, Richard
`Dayvault, Ken Karp, Sam Marutzky,
`Donald R. Metzler, Aaron Peacock,
`David C. White, Mary Lowe, and
`Derek R. Lovley
`Jean J. Huang, Jong-In Han, Lian(cid:173)
`Hui Zhang, and Jared R.
`Leadbetter
`Aaron M. J. Law and Michael D .
`Aitken
`Nicholas V. Coleman and Jim C.
`Spain
`
`5782-5786
`
`5864-5869
`
`5914-5918
`
`6201-6207
`
`6294-6298
`
`6311-6315
`
`6316-6320
`
`5833-5838
`
`5849-5854
`
`5884-5891
`
`5941-5949
`
`5968-5973
`
`6041-6046
`
`Continued on f allowing page
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 5 of 17
`
`

`

`Continued from preceding page
`
`Coaggregation among Nonflocculating Bacteria Isolated
`from Activated Sludge
`
`Characterization of Multiple-Substrate Utilization by
`Anthracene-Degrading Mycobacterium frederiksbergense
`LBS0lT
`The High Salt Requirement of the Moderate Halophile
`Chromohalobacter salexigens DSM3043 Can Be Met Not Only
`by NaCl but by Other Ions
`Petroleum Pollution Bioremediation Using Water-Insoluble
`Uric Acid as the Nitrogen Source
`
`MICROBIAL ECOLOGY
`
`Elfects of Iron Limitation on Adherence and Cell Surface
`Carbohydrates of Corynebacterium diphtheriae Strains
`
`Comparing Denitrification Estimates for a Texas Estuary by
`Using Acetylene Inhibition and Membrane Inlet Mass
`Spectrometry
`
`Community Composition and Functioning of Denitrifying
`Bacteria from Adjacent Meadow and Forest Soils
`
`Compound-Specific Isotopic Fractionation Patterns Suggest
`Dilferent Carbon Metabolisms among Ch/oroftexus-Like
`Bacteria in Hot-Spring Microbial Mats
`
`Molecular Diversity of Sulfate-Reducing Bacteria from Two
`Dilferent Continental Margin Habitats
`
`RpoS-Dependent Stress Response and Exoenzyme
`Production in Vibrio vulnificus
`
`Marker Rescue Studies of the Transfer of Recombinant
`DNA to Streptococcus gordonii In Vitro, in Foods and
`Gnotobiotic Rats
`Radioisotopic, Culture-Based, and Oligonucleotide
`Microchip Analyses of Thermophilic Microbial Communities
`in a Continental High-Temperature Petroleum Reservoir
`
`Patterns of Community Change among Ammonia Oxidizers
`in Meadow Soils upon Long-Term Incubation at Dilferent
`Temperatures
`
`Anushree Malik, Masashi Sakamoto,
`Shohei Hanazaki, Masamitsu
`Osawa, Takanori Suzuki, Masaki
`Tochigi, and Kazuo Kakii
`Lukas Y. Wick, Natacha Pasche,
`Stefano M. Bernasconi, Oliver Pelz,
`and Hauke Harms
`Kathleen O'Connor and Laszlo N.
`Csonka
`
`Omry Koren, Vishnia Knezevic,
`Eliora Z. Ron, and Eugene
`Rosenberg
`
`Lilian de Oliveira Moreira, Arnaldo
`Feitosa Braga Andrade, Marcio
`Damasceno Vale, Sonia Maria Silva
`Souza, Raphael Hirata, Jr., Lidia
`Maria Oliveira Buarque Asad,
`Nasser Ribeiro Asad, Luiz Henrique
`Monteiro-Leal, Jose Osvaldo
`Previato, and Ana Luiza Mattos(cid:173)
`Guaraldi
`Melody J. Bernot, Walter K. Dodds,
`Wayne S. Gardner, Mark J.
`McCarthy, Dmitri Sobolev, and
`Jennifer L. Tank
`J. J. Rich, R. S. Heichen, P. J.
`Bottomley, K. Cromack, Jr., and D.
`D . Myrold
`Marcel T. J. van der Meer, Stefan
`Schouten, Jaap S. Sinninghe
`Damste, Jan W. de Leeuw, and
`David M . Ward
`Xueduan Liu, Christopher E.
`Bagwell, Liyou Wu, Allan H. Devol,
`and Jizhong Zhou
`A Hiilsmann, T. M. Rosche, 1.-S.
`Kong, H. M. Hassan, D. M. Beam,
`and J. D . Oliver
`Mitra Kharazmi, Silke Sczesny,
`Michael Blaut, Walter P. Hammes,
`and Christian Hertel
`Elizaveta A Bonch-Osmolovskaya,
`Margarita L. Miroshnichenko,
`Alexander V. Lebedinsky, Nikolai
`A Chernyh, Tamara N. Nazina,
`Valery S. Ivoilov, Sergey S. Belyaev,
`Eugenia S. Boulygina, Yury P.
`Lysov, Alexander N. Perov, Andrei
`D. Mirzabekov, Hans Hippe, Erko
`Stackebrandt, Stephane L'Haridon,
`and Christian Jeanthon
`Sharon Avrahami and Ralf Conrad
`
`6056-6063
`
`6133-6142
`
`6334-6336
`
`6337-6339
`
`5907-5913
`
`5950-5956
`
`5974-5982
`
`6000-6006
`
`6073-6081
`
`6114-6120
`
`6121-6127
`
`6143-6151
`
`6152-6164
`
`Continued on fol/owing page
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 6 of 17
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`

`

`Continued from preceding page
`
`Detection of Prochwrothrix in Brackish Waters by Specific
`Amplification of pcb Genes
`
`Detection of the Free-Living Forms of Sulfide-Oxid'izing Gill
`Endosymbionts in the Lucinid Habitat (Thalassia te;studinum
`Environment)
`
`GEOMICROBIOLOGY
`
`Immobilization of Arsenite and Ferric Iron by
`Acidithiobacillus ferrooxidans and Its Relevance to Acid Mine
`Drainage
`
`'
`FOOD MICROBIOLOGY
`
`Membrane Fluidity Adjustments in Ethanol-Stressed
`Oenococcus oeni Cells
`
`Application of Host-Specific Bacteriophages to the Surface
`of Chicken Skin Leads to a Reduction in Recovery of
`Campylobacter jejuni
`
`PLANT MICROBIOLOGY
`
`Conditional Survival as a Selection Strategy To Identify
`Plant-Inducible Genes of Pseudomonas syringae
`Use of Bromodeoxyuridine Immunocapture To Identify
`Active Bacteria Associated with Arbuscular Mycorrhizal
`Hyphae
`
`INVERTEBRATE MICROBIOLOGY
`
`Dual Resistance to Bacillus thuringiensis CrylAc and Cry2Aa
`Toxins in Heliothis virescens Suggests Multiple Mechanisms
`of Resistance
`Population Dynamics of Vibrio fischeri during Infection of
`Euprymna scolopes
`
`Phylogenetic Diversity, Abundance, and Axial Distribution of
`Bacteria in the Intestinal Tract of Two Soil-Feeding
`Termites (Cubitermes spp.)
`Axial Dynamics, Stability, and Interspecies Similarity of
`Bacterial Community Structure in the Highly
`Compartmentalized Gut of Soil-Feeding Termites
`(Cubitermes spp.)
`Diversity of Wolbachia Endosymbionts in Heteropteran Bugs
`
`Spiralin Is Not Essential for Helicity, Motility, or
`Pathogenicity but Is Required for Efficient Transmission of
`Spiroplasma citri by Its Leafhopper Vector Circulifer
`haematoceps
`
`Ulrike GeiB, Ingo Bergmann,
`Miriam Blank, Rhena Schumann,
`Martin Hagemann, and Arne
`Schoor
`Olivier Gros, Martine Liberge,
`Abdelaziz Heddi, Chaque
`Khatchadourian, and Horst Felbeck
`
`K. Duquesne, S. Lebrun, C. Casiot,
`0. Bruneel, J.-C. Personne, M .
`Leblanc, F. Elbaz-Poulichet, G.
`Morin, and V. Bonnefoy
`
`M. Gra<;a Da Silveira, Elena A.
`Golovina, Folkert A. Hoekstra,
`Frank M. Rombouts, and Tjakko
`Abee
`Robert J. Atterbury, Phillippa L.
`Connerton, Christine E. R. Dodd,
`Catherine E. D. Rees, and Ian F.
`Connerton
`
`6243-6249
`
`6264-6267
`
`6165-6173
`
`5826--5832
`
`6302-6306
`
`Maria L. Marco, Jennifer Legac,
`and Steven E. Lindow
`Veronica Artursson and Janet K.
`Jansson
`
`5793-5801
`
`6208-6215
`
`Juan Luis Jurat-Fuentes, Fred L.
`Gould, and Michael J. Adang
`
`Jessica McCann, Eric V. Stabb,
`Deborah S. Millikan, and Edward
`G. Ruby
`Dirk Schmitt-Wagner, Michael W.
`Friedrich, Bianca Wagner, and
`Andreas Brune
`Dirk Schmitt-Wagner, Michael W.
`Friedrich, Bianca Wagner, and
`Andreas Brune
`
`Yoshitomo Kikuchi and Takema
`Fukatsu
`Sybille Duret, Nathalie Berho, Jean(cid:173)
`Luc Danet, Monique Garnier, and
`Joel Renaudin
`
`5898-5906
`
`5928-5934
`
`6007-6017
`
`6018-6024
`
`6082-6090
`
`6225-6234
`
`Continued on following page
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 7 of 17
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`

`

`Continued from preceding page
`
`METHODS
`
`Development of an Improved Selective Agar Medium for
`Isolation of Yersinia pestis
`
`Establishment of an Arbitrary PCR for Rapid Identification
`of Tn917 Insertion Sites in Staphylococcus epidermidis:
`Characterization of Biofilm-Negative and Nonmucoid
`Mutants
`
`Development of a PCR-Enzyme Immunoassay Oligoprobe
`Detection Method for Toxoplasma gondii Oocysts,
`Incorporating PCR Controls
`Rapid Screening for Freshwater Bacterial Groups by Using
`Reverse Line Blot Hybridization
`
`Molecular Analysis of the rjb O Antigen Gene Cluster of
`Salmonella enterica Serogroup 0:6,14 and Development of a
`Serogroup-Specific PCR Assay
`
`A Model To Estimate the Optimal Sample Size for
`Microbiological Surveys
`Statistical Approaches for Estimating Actinobacterial
`Diversity in Marine Sediments
`
`An Alternative Efficient Procedure for Purification of the
`Obligate Intracellular Fish Bacterial Pathogen Piscirickettsia
`salmonis
`Comparison of Velocity Profiles for Different Flow Chamber
`Designs Used in Studies of Microbial Adhesion to Surfaces
`
`Species-Specific Peptide Ligands for the Detection of
`Bacillus anthracis Spores
`
`Multiplex Real-Time PCR Method To Identify Shiga Toxin
`Genes stxl and stx2 and Escherichia coli 0157:H7/H(cid:173)
`Serotype
`
`LETTERS TO THE EDITOR
`
`Bacterial Activity in South Pole Snow Is Questionable
`
`Terminal Restriction Fragment Length Polymorphism Data
`Analysis
`
`Raphael Ber, Emanuelle Mamroud,
`Moshe Aftalion, Avita! Tidhar,
`David Our, Yehuda Flashner, and
`Sara Cohen
`Johannes K.-M. Knobloch, Max
`Nedelmann, Kathrin Kiel, Katrin
`Bartscht, Matthias A. Horstkotte,
`Sabine Dobinsky, Holger Rohde,
`and Dietrich Mack
`Kellogg J. Schwab and James J.
`McDevitt
`
`Gabriel Zwart, Erik J. van Hannen,
`Miranda P. Kamst-van Agterveld,
`Katleen Van der Gucht, Eva S.
`Lindstrom, Jeroen Van Wichelen,
`Torben Lauridsen, Byron C. Crump,
`Suk-Kyun Han, and Steven Declerck
`Collette Fitzgerald, Rachel
`Sherwood, Linda L. Gheesling,
`• Frances W. Brenner, and Patricia I.
`Fields
`S. F. Altekruse, F. Elvinger, Y.
`Wang, and K. Ye
`James E. M. Stach, Luis A.
`Maldonado, Douglas G. Masson,
`Alan C. Ward, Michael Goodfellow,
`and Alan T. Bull
`Vitalia Henriquez, Marfa Veronica
`Rojas, and Sergio H. Marshall
`
`D. P. Bakker, A. van der Plaats, G.
`J. Verkerke, H . J. Busscher, and H .
`C. van der Mei
`David D. Williams, Orsolya
`Benedek, and Charles L.
`Turnbough, Jr.
`Karen C. Jinneman, Ken J.
`Yoshitomi, and Stephen D.
`Weagant
`
`Letter: Stephen G. Warren, aQd
`Stephen R. Hudson. Reply: Edward
`J. Carpenter, and Douglas G.
`Capone
`Letter: Alastair Grant, and Lesley A.
`Ogilvie. Reply: Christopher B.
`Blackwood, Terry Marsh, Sang(cid:173)
`Hoon Kim, and Eldor A. Paul
`
`5787-5792
`
`5812-5818
`
`5819-5825
`
`5875-5883
`
`6099-6105
`
`6174-6178
`
`6189-6200
`
`6268-6271
`
`6280-6287
`
`6288-6293
`
`6327-6333
`
`6340-634]
`
`6342-6343
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 8 of 17
`
`

`

`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2003, p. 5983-5991
`0099-2240/03/$08.00+0 DOI : I0.1128/AEM.69.10.5983-5991.2003
`Copyright © 2003, American Society for Microbiology. All Rights Reserved.
`
`Vol. 69, No. 10
`
`Identification and Characterization of the CYP52 Family of Candida
`tropicalis ATCC 20336, Important for the Conversion of Fatty Acids
`and Alkanes to a,w-Dicarboxylic Acids
`David L. Craft,* Krishna M. Madduri, t Mark Eshoo,:j: and C. Ron Wilson
`Biotechnology Group, Cognis Corporation, Cincinnati, Ohio 45232
`
`Received 20 March 2003/Accepted 25 July 2003
`
`Candida tropicalis ATCC 20336 excretes a,w-dicarboxylic acids as a by-product when cultured on n-alkanes
`or fatty acids as the carbon source. Previously, a P-oxidation-blocked derivative of ATCC 20336 was con(cid:173)
`structed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the
`next steps in strain improvement, which were directed toward the isolation and characterization of genes
`encoding the w-hydroxylase enzymes catalyzing the first step in the w-oxidation pathway. Cytochrome P450
`monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the
`hydroxylase complex responsible for the first and rate-limiting step of w-oxidation of n-alkanes and fatty acids.
`10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the
`accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their
`putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52Al 3 and
`CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse
`transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold
`increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52Al 7 and CYP52A18
`was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was
`observed. These results id~ntify the CYP52 and NCP genes as being involved in a,w-dicarboxylic acid produc(cid:173)
`tion by C. tropicalis and provide the foundation for biocatalyst improvement.
`
`a ,w-Dicarboxylic acids (a,w-diacids) are versatile chemical
`intermediates useful as raw materials for the preparation of
`perfumes, polymers, adhesives, and macrolide antibiotics. Cur(cid:173)
`rently, only three diacids, nonanedioic (azelaic), decanedioic
`(sebacic), and dodecanedioic, are available at a quantity and
`cost acceptable for commercial applications. Long-chain diac(cid:173)
`ids with more than 12 carbons offer potential advantages over
`shorter-chain diacids, but their limited commercial availability
`and high price have prevented widespread growth in many of
`these applications.
`Several species of yeasts belonging to the genus Candida
`excrete a ,w-diacids as a by-product when cultured on n-alkanes
`or fatty acids as the carbon source (22). One such yeast, Can(cid:173)
`dida tropicalis ATCC 20336, is the subject of this paper.
`In Candida spp., n-alkanes and fatty acids are metabolized
`by enzymes present in the [3-oxidation and w-oxidation path(cid:173)
`ways. For alkanes, the first reaction occurs in the w-oxidation
`pathway with the formation of the corresponding alcohol. This
`reaction is catalyzed by a cytochrome P450 hydroxylase com(cid:173)
`plex, which consists of a cytochrome P450 monooxygenase (the
`CYP protein) and the accompanying NADPH cytochrome
`P450 reductase (NCP). A terminal carboxy function is ulti(cid:173)
`mately formed from two additional oxidation steps, catalyzed
`by a fatty alcohol oxidase and a fatty aldehyde dehydrogenase.
`Fatty acid formed via the w-oxidation pathway or introduced as
`
`• Corresponding author. Mailing address: Biotechnology Group,
`Bldg. 53, 4900 Este Ave., Cincinnati, OH 45232. Phone: (513) 482-
`2368. Fax: (513) 482-2862. E-mail : craftdl@one.net.
`t Present address: Dow AgroSciences LLC, Indianapolis, IN 46268.
`:j: Present address: Buck Institute, Novato, CA 94945 .
`
`a substrate can be oxidized through the same w-oxidation path(cid:173)
`way to the corresponding aliphatic a ,w-dicarboxylic acid. Fi(cid:173)
`nally, the diacids as well as the fatty acid substrates can be
`activated to the corresponding acyl-coenzyme A ester and then
`metabolized through [3-oxidation to yield energy, CO2 , and
`water. In Candida spp., the [3-oxidation pathway is localized in
`the peroxisomes. A necessary step to produce high yields of
`aliphatic diacids from C. tropicalis ATCC 20336 was to elimi(cid:173)
`nate [3-oxidation so as not to consume the desired product.
`When [3-oxidation was blocked by disrupting the POX4 and
`POX5 genes, encoding acyl-coenzyme A oxidase, the enzyme
`catalyzing the first step in the pathway, the result was a dra(cid:173)
`matic increase in the production of diacids. The resultant [3-ox(cid:173)
`idation-blocked strain was designated C.
`tropicalis ATCC
`20962 (13).
`Once the [3-oxidation-blocked strain was available, the next
`step toward increasing diacid production was directed toward
`improving the flow through the w-oxidation pathway by over(cid:173)
`expressing the enzymes constituting the cytochrome P450 hy(cid:173)
`droxylase complex. Cytochromes P450 (P450s) are terminal
`monooxygenases of a multicomponent enzyme system. They
`constitute a superfamily of proteins which exist widely in na(cid:173)
`ture having been isolated from a variety of organisms (9). An
`alkane-inducible family of P450s, CYP52, has been described
`in several different Candida species (11, 20). These P450s and
`their corresponding reductase, NADPH cytochrome P450 re(cid:173)
`ductase (NCP), constitute the hydroxylase complex responsible
`for the first and rate-limiting step of w-oxidation of n-alkanes
`and fatty acids (3, 4). In order to alleviate this rate-limiting step
`in the conversion of n-alkanes and fatty acids to their corre(cid:173)
`sponding aliphatic diacids, it is necessary to increase the
`
`5983
`
`LCY Biotechnology Holding, Inc.
`Ex. 1070
`Page 9 of 17
`
`

`

`5984
`
`CRAFf ET AL.
`
`APPL. ENVIRON. MICROBIOL.
`
`CYP52 protein responsible for the conversion. One method to
`effect the increase in CYP52 protein would be overexpression
`of a specific CYP52 gene.
`Prior to overexpression, it is imperative to identify the
`CYP52 enzymes responsible for the w-oxidation of the target
`n-alkanes and fatty acids. Quantitative competitive reverse
`transcription-PCR (QC RT-PCR) was used to identify CYP52
`genes induced by selected fatty acid feedstreams. The identity
`of the CYP52 gene(s) involved in w-oxidation and the level of
`mRNA transcription for each CYP52 was determined with this
`highly specific and sensitive procedure.
`This paper describes the isolation and characterization of 10
`members of the CYP52 gene family from Candida tropicalis
`ATCC 20336. A sensitive and specific QC RT-PCR was used to
`characterize the specific CYP52 mRNA transcription levels in
`C. tropicalis ATCC 20962 in response to oleic acid, octadecane,
`and a commercially available fatty acid feedstock. Transcrip(cid:173)
`tional induction patterns identified specific CYP52 genes in(cid:173)
`volved in the conversion of long-chain fatty acids and n-alkanes
`(> 12 carbons) into their corresponding dicarboxylic acid.
`The isolation, characterization, and identification of the spe(cid:173)
`cific CYP52s from C. tropicalis A TCC 20336 will allow the
`development of commercially useful strains for the production
`of aliphatic dicarboxylic acids from n-alkane and fatty acid
`feeds tocks.
`
`MATERIALS AND METHODS
`
`Strains. Candida tropicalis ATCC 20336, a natural isolate from an oil field, was
`the wild-type strain used to generate genomic DNA and genomic libraries and