Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 1 of 137 PageID #: 30793
`
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 1 of 137 PageID #: 30793Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 1 of 137 PageID #: 30793
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`(cid:40)(cid:59)(cid:43)(cid:44)(cid:37)(cid:44)(cid:55)(cid:3)(cid:26)(cid:3)
`
`
`
`EXHIBIT 7EXHIBIT 7
`
`(cid:3)
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 2 of 137 PageID #: 30794
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 2 of 137 PageID #: 30794
`
`\
`
`w
`E McGIRAw-HEM.
`\ Ducwnowmw or-
`
`sonlzmmrc mm
`
`
`won-mama,
`
`mmms ._
`
`_
`
`
`
`McGrow-Hill
`
`San Francisco
`New York Chicago
`Lisbon
`London Madrid Mexico City
`Milan- New Delhi
`San Juan
`Seoul
`Singapore
`Sydney
`
`Toronto
`
`
`
`=~_____——_._
`APPX 0373
`APPX 0373
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 3 of 137 PageID #: 30795
`Case 1:18—cv-00924-CFC Document 399-8 Filed 10/07/19 Page 3 of 137 PageID #: 30795
`
`On the cover: Representation of a fullerene molecule with a noble gas atom trapped
`inside. At the Permian-Triassic sedimentary boundary the noble go see helium and argon
`have been found trapped inside fullerenes. They exhibit isotope ratios quite similar to
`those found in meteroritos, suggesting that a fireball meteorite or asteroid exploded
`when it hit the Earth, causing major changes in the environment. (Image copyright ©
`Dr. luann Becker. Reproduced with permission.)
`
`-Paw-n
`
`Over the six editions of the Dictionary. material has been drawn from the‘fullowing-references: G. M. Ganjty
`et 11]., Taxonomic Outline ofrhe Procnryores, Release 2, SpfinfiersVerlag, January 2002; D. W. Linzey, Vertebrate
`Biology, McGraw—Hill, 2001; J. A. Pechenik, Biology of the Invertebrates. 4th ed, McGraw-Hill, 2000; U.S.
`Air Force Glossary ofStandardized Terms, AF Manual 11-1, vol. 1, 1972; F. Casey, ed., Compilation of Terim
`in [rgfonnation Sciences fichnology, Federal Council for Science and TochnoIogy, 1970; Commit-odom-
`Electmm‘cs Temtirtoiogy, AF Manual 11-1, vol. 3, 1970; P. W. Thrush, comp. and ed., A Dictionary ofMirtt‘ng,
`Mineral, and Related Tet-rm, Bureau of Mines, 1968; A DOD Glossary of Mapping, Charting and Geodetic
`Toms, Department of Defense. 1967; J. M. Gilliland. Solar-Terrestrial Physics: A Glossary of Terms and
`Abbreviations, Royal Aircraft Establishment Technical Report 67158, 1967; W. H. Allen, ed., Dictionont of
`Technical Terms for Aerospace Use, National Aeronautics and Space.Administration, 1965; Glossary of Stin'fo
`Terminology, Office of Aerospace Research, U.S. Air Force, 1963; Naval'th'ctt‘onar-y ofElectronic, Technicil,
`
`andImperative firms, Bureau ofNaval Personnel, 1962;11. E. Huschke,.Glossary ofMeteorology, Amenfi'én
`
`Meteorological Society, 1959; ADP Glossary, Department of the Navy, NAVSO P6097; Glossary ofAir Tr
`Control Terms, Federal Aviation Agency; A Glossary ofRange, Terminology, -'While Sands Missile Range, N
`Mexico, National Bureau of Standards, AD 467-424; Nuclear Terms: A claim 2d ed, Atomic Energrr
`Cannnission.
`|
`
`1:
`
`McGRAW-HIIJ. DICHONAKYOF SCIENTIFIC AND TECHNICALTERMS,
`
`Sixth Edition
`.
`McGraw—l-Iill Companies, Inc. All nghti
`Copyright © 2003, 1994, 1989, 1984, 1978, 1976, 1974 by The
`‘
`reserved. Printed in the United States of America. Except as pennitted under the United States Copynght Act
`of 1976, no part of this publication may be reproduced or distributed in any form or by any means. or stored
`in a database or retrieval system. without the prior written permission of the publisher.
`
`\
`
`1234567890
`
`DOWIDOW
`
`08765432
`
`ISBN 0—07-0423]3—X
`
`library of Congress Cataloging-in-Publication Data
`
`McGraw-Hill dictionary of scicnlific and technical tenns--6th ed.
`p.
`cm.
`‘
`‘
`ISBN 007-042313-X (alk. paper)
`l. Science--Diclionaries.
`2. Technology--DiclionarleS.
`and technical terms.
`
`.
`I. Title: Dictionary of scientific
`.
`
`0121MB
`503—ch
`
`2002
`
`APPX 0374
`APPX 0374
`
`
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 4 of 137 PageID #: 30796
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 4 of 137 PageID #: 30796
`
`no
`
`fenestrated membrane
`
`
`
`Fermi hole
`
`ismstoliveandgrowtnthe
`ruin
`'
`‘
`'
`'
`ond’mnlmfgdoten varifffindusuial processes for the
`absentcemo 01" products such as alcohols. acids..and Cheese
`We action ofyeasts. molds. and bacteria; alcoholic fer-incurs.
`tion is the best-known example. Also known as Zymosts.
`l Jermain-short ]
`r s
`S
`reorientation loo-um“(a; féMm) :iuthbgufiltfpflumn
`Whiiiiflil‘hw changes: can be a“ my“ °' “W
`catalytic aaent.
`l .{ar-man’lfi‘ShW' Bk'sel'mm'mj
`‘_
`A culture tube with a vet-u
`torment-lion tube
`[macaw]
`‘ mm m b
`cal closed lfi'fl to collect E” “fwd“??? ‘
`c“
`'3 y
`'
`anisrus.
`[ .ffl'manlu'5 on '
`.
`m [FOOD ENG] A vessel used for irrntennniiirsluch
`as a vat for fermenting mash ut brewing.
`l' M mef
`ferment oll
`[mm] Avolatile oil formed by the criteria-
`tion of plant material in which the oil was not presenl originally.
`llfarlmnhbil)
`‘H m“
`term! See femtometer.
`er'
`_
`MW [mm] -“" ""’“° “iw‘a‘fifhmifiillffii
`down area in the Fermi age model; It has
`e m
`bol'
`area. not time. Also known}: age: neulmfl 886‘ 33'1“
`in
`age of neutrons.
`{ ‘fer-me .a]
`_
`.
`_
`....mm W. s actuate
`age model which states that the_Laplacran o
`e_ «to:
`down density equals the patrol derrvauve of the lelfllB. k
`R
`density with respect to the Fermi age.
`i ferme .5]
`l. “'5
`zhan }
`,
`'
`Fermi one model
`[NUCLEO] A model used in'studytngthe
`slowing down of neutrons by elastic collisions; rt tsassumed
`that the slowing down takes place by a very large number of
`very small enefgy charges.
`1 ‘l'er-me .aj .mad-at }
`'
`Fermi beta-decay theory [Nuc PHYS] Theory in which a
`nucleon source current interacts with art electron-neutnno field
`to produce beta decay. in a manner analogous to the interaction
`of an electric current with an electromagnetic field during the
`emission of a photon of electromagnetic radiation.
`{ 'fer-rrr!
`thud-a dillel .tbe-srre }
`.
`,
`.
`Farml constant
`[NUC PHYS] A universal constant.
`intro-
`duced in beta-disintegration theory. that expresses the strength
`of the interaction between the ounsforruin'g nucleon and the
`electron-neutrino field.
`{ 'fer'rne .ithnvstcnt ]
`Fermi derivative
`[mar]. A generahgaflion of covariant dif-
`ferentiation along a curve that reduca‘to cow-lath difierenua.
`tion when the curve is geodesic; an, orthonormal
`tetrad
`constructed at each point along a timelike curve such that the
`Fermi MW“: Of the “m 810115 thcicurve is zero has (1)
`“5 drum“ basis “Wu." equal 19 "E who‘s unit tangent vector
`and (2) i“- mafia! basis vectors “Omling 310.08 the curve.
`{ {fer-rue daaiv-ad-iv }
`.'
`‘
`,
`Fermi-Dirac distribution iteration [Storm] , A function
`§P¢Crfyrns the probability mat a member of an assembly of
`Wmfmm “Elias ciecn'ons in a senficonduetor
`°r m' “n.““l’l’ 5 Wu energy state.when thumal
`eaulhbnum exists.
`“tar-rue dunk ..dis-na'hrh-shon .faulo
`shan )
`-
`
`['lterarne dim-gas }
`an 5:: Fermi gas.
`Fonnt-DlmcFphi
`
`‘
`arm rlc Itatiulca [STAT_.l\-[ECI-I] The ur'
`'
`.assemblyof identical half-into
`3 mm of m
`have wave functions
`5
`$15531“ Pin-ides; sucli particles
`.
`-.
`an Symmetry With I'CSpect m
`'cle
`mum-e “d “if! “145 Pauli excl
`‘
`-
`-
`.‘
`I
`ma di:rak sru'ris-tjk; }
`_
`Fm Principle.-
`'
`.
`Distribution of
`Fermi dletrlbutlon [soup STATE}
`metal Ira-given by the Fermi.
`of electrons to a semiconductor or
`energies
`
`-
`f
`I
`.
`.
`1 The averageenergy
`Fermi energy [5TH truer-il-
`Inns 1]] a_ metal. equal to three-fiflhs of the Fermi Insole“:
`See Fermi level:
`[ 'fer-rne-.en'ar-je ]
`'-
`
`I
`
`.
`.
`Pauli exclusion princ'iple; am on
`electron thepry ofmgmsmifl mm '5 “59d In the_free.
`the nucleons in a nucleus. Also [mum
`[
`'ffir'mfi .3” i
`.
`'
`.
`Fermi hole
`[souo m] A
`-
`in a solid in which lhe “21:31:31? smiling an electron
`that“? Pradicta that the
`
`'fpn-
`
`l
`
`Fennel (Foerriculum vulgare).
`(USDA)
`.5
`
`[rusroL] One of the layers ofelaa-
`tcnutreted membrane
`tic tissue in the tunica media and tunica intima of large arteries.
`{ 'l'en-a.sn'advad 'mem.brln }
`foundation [ARCH] The arrangement of openings. espe-
`cially windows. in the wall of a building.
`[am]
`1. A trans-
`parent or windowlike break or opening in the surface.
`2. The
`presence of windowlike openings.
`[ .feu-a'stra-shon I
`tenllrothlon [0R0 maul CoflerOsPS A yellow—brown
`liquid. insoluble in water; used as a miticide and insecticide
`[or rioe. orchards. vegetables. cereals. and cotton. and for fly
`and mosquito control.
`{
`.fen-a-tro'tlrr.an ]
`Mull
`[BOT] Foeniculum Mitigate. A tall perennial herb of
`the family Umbelliferae; a spice is derived from the fruiL
`[
`'t’en-al ]
`[MATER] The essential oil obtained from fennel;
`tennel oll
`a colorless liquid with aromatic scent and bitter taste. insoluble
`in water and boiling at lbOwZZO‘C; used in medicine. perfumes.
`and liquems. Also known as oil of fennel.
`(
`'fen-cl .oil ]
`ten out See low-moor pear.
`[ ‘ten .pet ]
`Fenalre equation See Fenske-Underwood equation.
`.ske Lima-rhea }
`Poncho-Underwood equation [CHEM ENG] Equation in
`plate-ro—plare distillationucolurnn calculations relating the num-
`ber of theoretical plates needed at total reflux to overall relative
`volatility and the liquid-vapor composition ratios on upper and
`lower plates. Also known as Fenslre equation.
`“fen-sire
`'an'dar.wud Lima-rhea }
`tenant See window.
`[
`'fen-star ]
`mutation [one CHEM] C,,HITSIO,P A brown liquid
`with a boiling point of ills-141°C; used as an insecticide a'nd
`nematicide in soils.
`{
`.fen.scl-fb‘thl.ltn ]
`tentlnaeetate
`[one CHEM]
`cmH.go.Sn A yellow to
`brown. crystallinesoiid that melts at uni—125°C: used as a
`fungicide, molluscicide. and algicide for early and late blight
`on potatoes. sugarheets. peanuts. and coffee. Also‘lrnown as
`triphenyltinacetate.
`{ .fent-an'as-aMt]
`letu'on [Doctrinal] CgfluNzo A white. crystalline corn-
`pound with a melting point of 133—l34“C; soluble in water;
`used as a herbicide‘t'ci kill‘weeds and bushes.
`{ .fen'yuitni
`fonuron-TGA [Ofld'Cl-IEM] Clll-IuClJNIO; A white. crys-
`talline compomtd with a melting point of 65—68°C; nmderately
`soluble'in water; used as a herbicide for'noncrop areas.
`{ .‘fe'rt-
`'Wn'lln lwlsell l
`'
`FEP rosin See'fluotinated ethylene propylene resin.
`[ :eflelpe
`'rezvan }
`‘
`"'
`‘
`turban! 10mm] Cg'l-Il,FeNss.,[iron('fl1)dirnethytdithio~
`cashmere] A hingicide for protecting fnrits. vegetables. mel-
`ons. and ornamental plants.
`{ 'fer-bom l
`7
`ferberlld’ [MEAL] FeNO. A black mineral of the wol-
`framite solid-solution series occurring as monoclinic. prismatic
`crystals and having a-submetallic luster: hardness is 4;.5 on
`Mons scale. and specific gravity is 7.5.'
`I 'far-bom ]
`feral-tantra
`[MINERAL]
`U3(V04)_2°6HIQ
`Sulfur-yellow
`mineral cornposed'of hydrated uranium vauadate. occurring in
`scales.
`{ fur'gltmlt }
`'
`tergueonlte
`[MINERAL] YiOythJ‘ahos
`Brownish-
`black rare-earth mineral with a ten-agonal crystal form; his
`isomorphous with formanite'.
`[ Tango-saint }
`Format numbera
`[MATH] The numbers of the form F. =
`(2m) + l fora =0. 1.2.....
`['l'er-miimom-borz]
`Fern-rat’s Int theorem [MATH] The proposition. proven in
`1995. that there arena positive integer solution: of the equation
`if + y' = t’forn 2 3.
`[fer'mazllast 'thir-am]
`Format’s'prlnclple
`[names] The principle that an electro-
`magnetic wave will take a path that involves‘the least travel
`time when propagating hetuieen two points. Also known as
`least-timeprinciple; stationary timept'inciple.
`{ fer'miiz’prin-
`scipol I
`[MATH] A plane curve whose equation in
`Fermat's aph'al
`polar coordinates (all) is r‘ =a10. where a is a constant.
`I let'-
`mite .rrpI-ral-l
`Fermat's theorem [MATH] The proposition that. if p is a
`prime number and a is a positive integer which is notdivisible
`by 1:. than a’" .. l is divisible by p.
`(
`'fer.nutz .thir-arn ]
`let-morn [Broom] An agent that can initiate femientarlon
`and other metabolic processes.
`{ li‘or’lment ]
`termentatttm [manor-o] An enzymatic transformation of
`organicsubsuates. especially carbohydrates. generally accom-
`panied by the evolution of gas: a physiological counterpart of
`
`APPX 0375
`APPX 0375
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 5 of 137 PageID #: 30797
`
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 5 of 137 PageID #: 30797Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 5 of 137 PageID #: 30797
`
`(cid:40)(cid:59)(cid:43)(cid:44)(cid:37)(cid:44)(cid:55)(cid:3)(cid:27)(cid:3)
`
`
`
`EXHIBIT 8EXHIBIT 8
`
`(cid:3)
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 6 of 137 PageID #: 30798
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 6 of 137 PageID #: 30798
`
`||||||l|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
`US 20070141687A1
`
`(19}
`
`United States
`
`(12) Patent Application Publication (10) Pub. No.: US 2007/0141687 A1
`
`Porro et al.
`(43) Pub. Date:
`Jun. 21, 2007
`
`INCREASE IN STRESS 'I‘()I.ER.-\N(_'IC WITH
`ASCORBIC ACID DURING FERMl-IN'EVI‘IOX
`
`Publication Classification
`
`(51)
`
`(52)
`
`(51)
`
`1111.0.
`(2006.01)
`2/56
`(“121’
`(2000.01)
`('er ms
`(2006.01)
`CIZN V21
`(2006.01)
`(UN 15/?!
`11.19.01.
`................... 4351139;435(252.3:4351254.2:
`4351433
`
`.-\BS'!‘RA(."I‘
`
`A ntelhod oi‘ increasing stress tolerance in reeoinbimnn
`organisms that have been engineered for industrial produc—
`tion is described. Stress tolerance is increased by making
`|.-ascorbic acid available to the recombinant organism.
`either by exogenous addition to the culture medium or by
`endogenous production from l)—glucose by the rteombiimnt
`organism. To enable endogenous production. the recombi—
`nant organism is transformed with a coding region encoding
`11 1111111110513 cpiniemse (ME). a coding region encoding an
`L-gaiaetose dehydrogenase (LGDl I). and a D-arubinnno-l.
`4-Iactone oxidasc (ALO). The recombinant organism may
`he further lranslbrmed with a ll'lytlifll‘lsillji phospiulmst‘
`(Mll‘).
`
`(54)
`
`(76)
`
`(21.3
`
`(22}
`
`(63]
`
`(30)
`
`Inventors: Danilo l’orro. lirba (Como) (IT): Paola
`Branduardi. Milano {1T}: Diethard
`Mattanm’ich. Wien (AT): Michael
`Sauer. Wien (All)
`
`Correspondence Address:
`RA‘I‘N I‘IRPRES’I‘IA
`HO. BOX l596
`WILMINGTON. DF. 19899018)
`
`Appl. No;
`
`ll!546,95l
`
`Filed:
`
`()et. 12. 2006
`
`Related 0.3. Application Data
`
`Conlinualion-in-part of application No.
`liled on Apr. 13. 2005.
`
`111r 105.162.
`
`Foreign Application Priority Data
`
`Apr.
`
`'1. 2005
`
`(US) _____________________________ I’fvl‘IUSflt'v‘IZRSéi
`
`APPX 0376
`
`APPX 0376
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 7 of 137 PageID #: 30799
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 7 of 137 PageID #: 30799
`
`Patent Application Publication Jun. 2], 2007 Sheet 1 of 15
`
`US 2007;0141687 A]
`
`Figure 1
`
`D'Mannose
`
`D—Glucose
`
`ATP
`
`ADP
`
`J
`
`D-Glc-G-P
`
`1 I
`D-Fructose-S-P
`in
`D-Mannose-B-P
`is
`D-Mannose-1 -P
`
`GTPPF’i :DI F
`
`GDP-D-Mannose
`3E
`GDP-L-Galactose
`
`GMP (-2“; D
`L-Galactose-1-P
`
`H44 C
`
`L-Galactose
`
`NAD aNADH
`
`B
`
`L-Galactono-1,4Jactone
`
`2H
`
`A
`
`L-Ascorbate
`
`APPX 0377
`
`APPX 0377
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 8 of 137 PageID #: 30800
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 8 of 137 PageID #: 30800
`
`Patent Application Publication Jun. 2], 2007 Sheet 2 of 15
`
`US 2007;0141687 A]
`
`0 "M H202 -AA
`
`Figure 2A
`
`
`
`010 20
`
`3O
`
`40
`
`50
`
`60
`
`70
`
`80 90100
`
`Tirmfl'l)
`
`APPX 0378
`
`APPX 0378
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 9 of 137 PageID #: 30801
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 9 of 137 PageID #: 30801
`
`Patent Application Publication Jun. 2], 2007 Sheet 3 of 15
`
`US 2007;0141687 A]
`
`0,3 M H302 -AA
`
`Figure ZB
`
`
`
`0
`
`10
`
`20
`
`3G
`
`40
`
`50
`
`60
`
`3’0
`
`30
`
`90
`
`100
`
`T'srmfl'l)
`
`APPX 0379
`
`APPX 0379
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 10 of 137 PageID #: 30802
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 10 of 137 PageID #: 30802
`
`Patent Application Publication Jun. 2], 2007 Sheet 4 of 15
`
`US 2007;0141687 A]
`
`Figure 20
`
`013660nm
`
`0
`
`I0
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`BO
`
`90
`
`I00
`
`Tirm:(h}
`
`APPX 0380
`
`APPX 0380
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 11 of 137 PageID #: 30803
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 11 of 137 PageID #: 30803
`
`Patent Application Publication Jun. 2], 2007 Sheet 5 of 15
`
`US 2007;0141687 A]
`
`0.3 mM H302 +AA
`
`Figure 3A
`
`
`
`0
`
`IO
`
`20
`
`30
`
`40
`
`50
`
`60
`
`7G
`
`80
`
`90
`
`100
`
`Time{h)
`
`APPX 0381
`
`APPX 03 81
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 12 of 137 PageID #: 30804
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 12 of 137 PageID #: 30804
`
`Patent Application Publication Jun. 2], 2007 Sheet 6 of 15
`
`US 2007;0141687 A]
`
`1 MM H202 +AA
`
`Figure BB
`
`
`
`
`
`D
`
`ID
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`80
`
`90
`
`100
`
`Threw)
`
`APPX 0382
`
`APPX 0382
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 13 of 137 PageID #: 30805
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 13 of 137 PageID #: 30805
`
`Patent Application Publication Jun. 21, 2007 Sheet 7 of 15
`
`US 2007;0141687 A]
`
`
`
`Figure 3C
`
`‘ -MM+M©¢0M33nsfl
`
`+1410 vMED -
`
`
`
`m
`
`+LuongM
`+1.1. 4OQEI'AA so mga'l
`+Lno Iaueo rl
`
`5
`h
`.5
`4
`
`,5
`3
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`°
`2
`.5
`1
`.5
`D
`
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`
`.5
`
`O
`
`
`
`
`
`-.
`-_WW
`
`APPX 0383
`
`APPX 0383
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 14 of 137 PageID #: 30806
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 14 of 137 PageID #: 30806
`
`Patent Application Publication Jun. 2], 2007 Sheet 8 of 15
`
`US 2007;0141687 A]
`
`0.3 mM H202 -AA
`
`Figure 4A
`
` 0
`
`ID
`
`20
`
`30
`
`40
`
`50
`
`60
`
`3'0
`
`80
`
`90
`
`IDG
`
`APPX 0384
`
`APPX 0384
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 15 of 137 PageID #: 30807
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 15 of 137 PageID #: 30807
`
`Patent Application Publication Jun. 2], 2007 Sheet 9 of 15
`
`US 2007;0141687 A]
`
`1 HM H202 -AA
`
`Figure 4B
`
`ODfiwnm
`
`0102030405060703090|oo
`
`'I'ime(h)
`
`APPX 0385
`
`APPX 0385
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 16 of 137 PageID #: 30808
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 16 of 137 PageID #: 30808
`
`Patent Application Publication Jun. 2], 2007 Sheet 10 of 15
`
`US 2007;0141687 A]
`
`0 mM H202 - AA
`
`Figure 5A
`
`OD660nm
`
`Tim? U1)
`
`APPX 0386
`
`APPX 0386
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 17 of 137 PageID #: 30809
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 17 of 137 PageID #: 30809
`
`Patent Application Publication Jun. 2], 2007 Sheet 11 of 15
`
`US 200770141687 A]
`
`2 m“ H202 ‘ M
`
`Figure 5b
`
`
`
`
`
`Tim: (h)
`
`APPX 0387
`
`APPX 0387
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 18 of 137 PageID #: 30810
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 18 of 137 PageID #: 30810
`
`Patent Application Publication Jun. 21, 2007 Sheet 12 of 15
`
`US 2007f014l687 A]
`
`Figure 6
`
`
`
`full ammYMLDovw
`open areasYMLoo'nv AID, LDGH, ME, MIP
`
`1D“
`F31 He‘s”
`
`APPX 0388
`
`APPX 0388
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 19 of 137 PageID #: 30811
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 19 of 137 PageID #: 30811
`
`Patent Application Publication Jun. 21, 2007 Sheet 13 of 15
`
`US 2007i014l687 A]
`
`
`
`pH 2,2
`
`Figure 7
`
`
`
`-D'—BY4742c
`
`+ 3V4742 ALO LGDH ME MIP
`
`
`
`
`
`OD660am
`
`(a)
`
`4.0
`3.5
`
`3.0
`
`2.5
`
`5
`E 2.0
`3
`
`1.5
`
`1.0
`
`0,5
`
`0.0
`
`(b)
`
`
`
`
`
`0
`
`50
`
`100
`
`150
`
`time (h)
`
`LA 38 gfL
`
`
`
`
`
`
`
`i
`’
`
`
`
`
`
`-CI— BY4?42¢
`+ awn: ALO LGDH ME MIP
`
`
`
`0
`
`20
`
`4o
`
`60
`
`an
`
`D
`
`100
`
`120
`
`140
`
`160
`
`D
`
`time (h)
`
`APPX 0389
`
`APPX 0389
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 20 of 137 PageID #: 30812
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 20 of 137 PageID #: 30812
`
`Patent Application Publication Jun. 21, 2007 Sheet 14 0f 15
`
`US 2007;0141687 A]
`
`pH 2,2
`
`Figure 8
`
`
`
`
`
`‘D— Byrd-742:
`—I— Bwuz ALO LGDH ME MIP
`
`
`
`
`
`
`0.2 V
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`time (h)
`
`LA 38 gfL
`
`
`
`
`
`A ‘5.
`
`_. 'N
`
`_. '0
`
`E 0.8
`g
`
`8 0.6
`
`0.4
`
`0.0
`
`(a)
`
`4.0
`
`3.5
`
`3.0
`
`2.5
`
`§ 2 0
`
`1.0
`
`0.5
`
`0.0
`
`(b)
`
`
`
`
`E_
`
`3 '43— Emma:
`1.5 '3an AU) LGDH ME MIP
`
`_.
`
`
`
`
`
`
`
`0
`
`20
`
`40
`
`60
`
`80
`
`100
`
`120
`
`time (h)
`
`APPX 0390
`
`APPX 0390
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 21 of 137 PageID #: 30813
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 21 of 137 PageID #: 30813
`
`Patent Application Publication Jun. 21, 2007 Sheet 15 of 15
`
`US 2007!014168? Al
`
`Figure 9
`
`Effect of adding Ascorbic acid on Lactic accumulation
`
`*——«
`
`ll
`
`5'
`
` onat660nm
`
`Nw-b-MGNHJOOVO
`
`+ lactfcontrol
`
`+ lactf+0.3 A5
`
`+ law-#06 A5
`
`+
`
`lactf+0.16 As
`
`- - -|:I- - - ODr'control
`
`—fl— ODf+0.3 As
`
`—O— 00:40.6 As
`
`0Dl+0. 16 As
`
`
`
`36
`
`48
`
`60
`
`7'2
`
`Fermentation time (Hr)
`
`APPX 0391
`
`APPX 0391
`
`33 L
`
`3=
`
`aa E9f
`
`l03 3J
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 22 of 137 PageID #: 30814
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 22 of 137 PageID #: 30814
`
`US 200700141687 Al
`
`Jun. 21, 200?
`
`INCREASE IN STRESS TOLERANCE WI'I'I-l
`ASCORBIC ACID DURING FERMENTA'I'ION
`
`RELA'I‘l-El) APPIJCKI‘IONS
`
`[0001] Tltis application is a continuation-in-part of U.S.
`Ser. No.
`lll|05.162.
`filed on Apr. 13. 2005. which is
`incorporated lterein by reference. This application claims
`priority frotn U.S. Scr. No. 1 13105.162 and front PC‘I‘t’USOG;f
`012854. filed on Apr. 7. 2006. also ittcorporatcd herein by
`reference.
`
`FIELD OF THE INVENTION
`
`[0002] The present invention relates generally to tlte [ield
`of increasing stress tolerance in organisms used for indus-
`trial production. More particularly. it relates to a process for
`making [_-ascorbic acid available to organisms during indus‘
`trial production.
`
`BACKGROUND
`
`[0003] Microorganisms and cells can be easily grown on
`an industrial scale and are Irequently employed in the
`contmercial production of compounds such as organic acids.
`amino acids. vitamins. polyols. solvents. biofuels. therapeu—
`tics. vaccines. proteins. and peptides. Both prokaryotic and
`eukaryotic microorganisms are today easily and sttccessfully
`used for the production of hcterologous proteins as well as
`for the production of natural or engineered metabolites.
`Among prokaryotes. Escherichia coli and Bacillus sttbtt'lt's
`are often used. Among etlkaryotes. the yeasts. Snceharonre
`res c-erevt'sfae and Kittyt'temmyces hurls. are otlen used.
`l-Iowever.
`in an industrial process. wherein the organism is
`used as a means for production. stress on the organism
`typically leads to lower or zero production of the product.
`lower or zero productivity.
`:1
`lower or zero yield of the
`product. or two or more thereof. Bacteria. yeast. other fungi.
`cultured animal cells. and cultured plant cells sltow similar
`responses to stress. (Close. D. C. et al.. Oxidative Stress.
`Exercise, and .rl‘gfrrg. H. M. Alessio. A. F.. Hagert'rtarl. Eds.
`(2006). pp. 9—23; Sugiyanta. K.. et al._. (2000). J Biol. Chem.
`275. 553545540; Mongkotsuk. S. and Helmann. J.
`l).
`(2002). Molecular Microbiology 45. 9-15). Techniques for
`minimizing. stress would therefore be useful for improving
`industrial production by these organisms.
`
`[0004] Stresses may have cellular (internal or intracellu-
`lar) origins. environmental (extemal or extracellular) ori-
`gins. or both. Classical examples of the internally-originat-
`ing stresses include protein and metabolite overproduction
`(in terms of weightlvolume) and protein and metabolite
`overprodttctivity (in terms of weightlvolumc per unit tithe).
`among others. Examples of extentally-originating stresses
`include high osmolarity. high salinity. oxidative stress. ltigh
`or low temperature. non-optimal pll. presence of organic
`acids. presence oftoxic compounds. and macro- and micro-
`nutriettt starvation.
`
`[0005] Stress is typically caused by stressors (or stimuli).
`Stressors are negative influences on a cell that require the
`cell to dedicate more etl‘on to maintain equilibrium than is
`required in tlte absence of the stressor. This greater effort can
`lead to a higher or lower metabolic activity. lower growth
`rate.
`lower viability. or lower productivity. among other
`cll'ects. Stressors are agents of a physical. chemical or
`biological nature that represent a change in the usual intra~
`
`cellular or extracellular conditiotts for any given life form.
`It follows that while a specific condition (tag. a temperature
`of 65° C.) may be stressful {or even lethal) to a certain
`species that normally lives at 3?” (I. it ma . be optimal for
`a thermophilic organism.
`
`the cellular level. stress can damage DNA.
`[0006] At
`lipids. proteins. membranes. and other molecules and mac-
`rotnolccttlcs. induce apoptosis (prograuunod cell death). cell
`necrosis and cell Iysis. and intpair chl integrity and cell
`viability. These eficcts are often mediated by the generation
`ol'reactive oxygen species (R05).
`
`[0007] R05 can be generated through both intracellular
`and extracellular stimuli. The majority of endogenous R05
`are produced through leakage of these species from the
`mitochondrial electron transport chain. In addition. cytosolic
`enzyme systems. including NADPH oxidases and lay-prod-
`ucts of peroxisomrt]
`tnctaholism. are also endogenous
`sources of ROS. Generation of ROS also can occur through
`exposure to numerous exogenous agents and events includ-
`ing ionizing radiation. UV light. chemotherapeutic drugs.
`environmental toxins, and hyperthemiia. Oxidative damage
`caused by intracellular ROS can result in DNA base modi-
`fications. single- and double-strand DNA brmks. and the
`fomiution ofapuriniclapyrintidinic lesions. tnany of which
`are toxic nnd’or mutagenic. Therefore. the resulting DNA
`damage may also be a direct contributor to deleterious
`biological consequences (liltany. B. et al.. (2004) Nucleic
`Acids Research 32. 3712—3723).
`
`[0008] One example of an industrial process known to be
`hampered by stress responses is the production of lactic acid
`by bacteria or yeast. During a typical lactic acid fermenta-
`tion. the accumulation of lactic acid in the medium also
`causes a drop in pH of the medium. The stress of low pH is
`amplified by the ability of the organic free acid to diliuse
`through the membrane and dissociate in the higher 1311 of the
`cytoplasm. The accumulation of lactic acid inhibits cell
`growth and metabolic activity. The toxicity of these stresses
`is mediated at least in part by reactive oxygen species. As a
`result. the extent of lactic acid production is greatly reduced
`by the accumulation of lactic acid in the medium.
`
`[0009] The addition of Ca(0ll)3. CaC03. NaOll. or
`Nt—QOH to the fermentation mediunt to neutralize the lactic
`acid and to thereby prevent the pH drop is a conventional
`operation in industrial processes to counteract the negative
`effects of free lactic acid accumulation. These processes
`allow the production of lactate(s) by maintaining the pH at
`a constant value in the range of about 5 to ‘l. which is well
`above the pKa of lactic acid (3.86).
`
`I-lowever. this netttmtiration procedure has major
`[0010]
`disadvantages. Additional operations are required to regen—
`erate free lactic acid from its salt and to dispose of or recycle
`the neutralizing cation. which adds expense to the process.
`The added operations and expense could be lessened if free
`lactic acid could be accumulated by organisms growing at
`low pH values. To this end. the use of recombinant yeast that
`are engineered for industrial production of free lactic acid.
`and. in particular. recombinant yeast from strains showing
`greater tolerance for extrente environmental conditions have
`been described. Engineered strains of recombinant yeast
`functionally transformed with a gene for lactate dehydroge-
`nase (LDl l} in the genera Saecharompes. Ztgosaccbamttut-
`ces. Tortilaspora. and Kluveroutt-ws have been produced as
`
`APPX 0392
`
`APPX 0392
`
`

`

`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 23 of 137 PageID #: 30815
`Case 1:18-cv-00924-CFC Document 399-8 Filed 10/07/19 Page 23 of 137 PageID #: 30815
`
`US 200700141687 Al
`
`Jun. 21, 200?
`
`oxidative stress. Yapl activates genes required for the
`response to oxidative stress: deletion oi'this gene leads to the
`observed phenotype.
`
`FIG. 3 shows the impact of two stressors on yeast
`[0019]
`growth. FIGS. 3H-3b show the optical density at 660 nm of
`B‘MMZ wt (A) and YMLt'lt'l'i'w (- .-) yeast in the presence of
`H201 in medium +I—ascorbic acid. Flti. 3:” shows the optical
`density at 660 1m1 of wild type yeast GRFc. CENPK
`113-5D. and BY4741 in the presence of-10 gtl lactic acid and
`zero. or increasing levels of ascorbic acid.
`
`FIG. 4 shows the optical density at 650 run of
`[0020]
`BY4742 wt (A): YML007w expressing ALO. LDGI'I and
`ME (El): and YML007‘w expressing ALO. LDGI']. ME and
`MIP (I) yeasts in the presence of oxidative stress (FIGS.
`40-40).
`
`FIG. 5 shows the optical density at 660 nm ofwild
`[0021]
`
`type GRFc (A): GRF] 8U expressing AID. IDGI-I and MI?
`
`
`
`(
`); and GRFISU expressing ALO, I..I)GH_. MF. and Mll’
`(I) yeast strains in the absence (FIG. 5a) and presence (2
`mM of “202) of oxidative stress. (FIG. 515).
`
`FIG. 6 shows ROS (upper panels) and viability
`[0022]
`(bottom panels) determination by flow cytometric analyses
`of S. ccrevisiae cells producing (YML007w ALO. LDGII.
`ME. MIP. open area) or not producing (YMLOOTW. full area)
`ascorbic acid when grown in minimal glucose medium in the
`presence (tight) or absence (left) of hydrogen peroxide.
`
`FIG. 7 shows growth curves of strains BY4T-‘42c
`[0023]
`
`
`
`(
`) and BY4742 ALO. LDGI'I. ME. MIP (I) inoculated in
`minimal glucose medium at pH 2.2 (a). or in minimal
`glucose medium pH 3.0 containing 38 gfl of lactic acid (b).
`
`FIG. 8 shows growth curves of strains BY4742c
`[0024]
`(___) and BY4?42 ALO. LDGII. ME. MIFI (I) that were lirst
`grown for 24 h ill minimal glucose medium under nonlim-
`iling conditions. and then transferred to minimal glucose
`medium at pH 2.2 (a). or to minimal glucose medium pH 3
`containing 38 g/l of lactic acid (b).
`
`
`
`
`
`described in U.S. Pat. Nos. 6.429.006 and 7.049.108. While
`these recombinant strains show improved eiliciency of lactic
`acid production at low pil. they are still adverser affected
`by stresses. In addition, it may be necessary to use organisms
`or strains that are less tolerant of extreme environmental
`conditions for the industrial production of specific corn-
`pounds.
`
`[0011] Ascorbic acid is a known antioxidant that is pro-
`duced in all higher plants and many higher animals. Ascor-
`bic acid has been shown to modulate the heat shock response
`in yeast through an ell'ect on ROS(C. Moraitis and B. P. G.
`Curran. (2004). Year! 2i. 313—323). and to improve cell
`viability and redtlce proteolysis of the end product of high
`cell-density fennentation (Xiao. A. et al.
`(2006). Appl.
`Microbiol. Bioteclutol. 72. 837-844). These effects suggest
`that ascorbic acid could improve stress tolerance in general
`in organisms utilized for industrial production.
`
`[0012] We have shown that recombinant yeast that are
`functionally transformed to produce L-ascorbic acid.
`the
`biologically active enantiomer.
`from D—glthosc produce
`lower levels of ROS and exhibit
`improved growth and
`viability under conditions of low pH. oxidative stress. and in
`the presence of high concentrations of lac1ic acid. (Brando-
`ardi. R. et al.. International Specialised Symposium on
`Yeast. ISSYZS. Systems Biology oneast - Front Models to
`Applications. “L-ascorbic acid production from D-glucose
`in metaboloic engineered Shocker-(moves cerevist'ac and its
`ellcct on strain robustness." Hanasaari. Bspoo. Finland. Jun.
`21. 2006).
`
`[0013] Accordingly. it would be advantageous to indus-
`trial fermentation processes if the organisms and cells used
`for
`industrial production could endogenously produce L
`ascorbic acid from l)~glucose.
`
`SUMMARY OF 'l‘l-ll-i INV‘EN'I'ION
`
`invention relates to a method of
`[0014] The present
`increasing stress tolerance in a recombinant organism that is
`engineered tor industrial production ol'at least one product.
`The method comprises making [.—ascorbic acid available to
`the reCombinant organism.
`
`In one embodiment. ascorbic acid is Inade avail-
`[0015]
`able by functionally transforming the recombinant organism
`with a coding region encoding a mannose epimerase (ME),
`a coding region encoding an I.-galactose dehydrogenase
`(LGDII). and a coding region encoding a D-arabinono-l.4-
`lact'one oxidasc (ALO). In a further embodiment. the func-
`tionally transformed. recombinant organism is further func—
`tionally transformed with a coding region encoding a
`myoinositol phosphatase (MW).
`
`the L-ascorbic acid is
`In another embodiment.
`[0016]
`made available by culturing the recombinant organism in
`culmre medium containing an effective amount of I.-ascorm
`bic acid.
`
`DESCRIPTION OF TIIE DRAWINGS
`
`1 shows the main plant pathway for the
`FIG.
`[0017]
`synthesis of [.—ascorbic a