I 1111111111111111 1111111111 1111111111 1111111111 1111111111111111 IIII IIII IIII
`US011781148B2
`
`c12) United States Patent
`Aeling et al.
`
`US 11,781,148 B2
`(IO) Patent No.:
`(45) Date of Patent:
`Oct. 10, 2023
`
`(54) BIOLOGICAL METHODS FOR PREPARING
`TERPENES
`
`(71) Applicant: Radici Chimica S.p.A., Bergamo (IT)
`
`(72)
`
`Inventors: Kimberly Ann Aeling, San Clemente,
`CA (US); Pierre-Yves De Wais,
`Encinitas, CA (US); Eric Scott,
`Encinitas, CA (US); Michael
`Waldbridge, Saugus, MA (US);
`Thomas Beardslee, Carlsbad, CA (US)
`
`(73) Assignee: Radici Chimica S.p.A., Bergamo (IT)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 245 days.
`
`(21) Appl. No.:
`
`16/630,567
`
`(22) PCT Filed:
`
`Jul. 11, 2018
`
`(86) PCT No.:
`
`PCT/US2018/041579
`
`§ 371 (c)(l),
`(2) Date:
`
`Jan. 13, 2020
`
`(87) PCT Pub. No.: WO2019/014310
`
`PCT Pub. Date: Jan. 17, 2019
`
`(65)
`
`Prior Publication Data
`
`US 2021/0079408 Al Mar. 18, 2021
`
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`3/2003 Longo
`2003/0044946 Al
`2004/0265980 Al* 12/2004 Zhang ....................... Cl2P 7/44
`435/134
`
`2013/0066035 Al
`2013/0302861 Al
`2014/0113343 Al
`2015/0010978 Al
`
`3/2013 Burgard et al.
`11/2013 Vainstein et al.
`4/2014 Davi et et al.
`1/2015 Heaps et al.
`
`FOREIGN PATENT DOCUMENTS
`
`JP
`WO
`WO
`WO
`
`2012254044 A
`WO 2008/073367
`2014100504 A2
`2018094110 A2
`
`12/2012
`* 6/2008
`6/2014
`5/2018
`
`OTHER PUBLICATIONS
`
`Juretzek et al., "Comparison of Promoters Suitable for Regulated
`Overexpression of 13-Galactosidase in the Alkane-Utilizing Yeast
`Yarrowia lipolytica", Biotechnol. Bioprocess Eng. 2000, 5: 320-
`326.*
`International Preliminary Report on Patentability of PCT/US2018/
`041579 dated Dec. 2, 2019.
`Search Report and Written Opinion of PCT/US18/41579 dated Sep.
`13, 2018.
`Written Opinion of the International Preliminary Examining Author(cid:173)
`ity of PCT/US2018/041579 dated Sep. 13, 2019.
`
`Related U.S. Application Data
`
`* cited by examiner
`
`(60)
`
`Provisional application No. 62/532,297, filed on Jul.
`13, 2017.
`
`(51)
`
`(52)
`
`(2006.01)
`(2006.01)
`(2006.01)
`(2006.01)
`
`Int. Cl.
`C12N 15181
`C12N 1116
`C12N 15152
`C12P 7144
`U.S. Cl.
`CPC ............. C12N 151815 (2013.01); C12N 1116
`(2013.01); C12N 15152 (2013.01); C12P 7144
`(2013.01)
`
`Primary Examiner - Suzanne M Noakes
`(74) Attorney, Agent, or Firm - SILVIA SALVADOR!,
`P.C.; Silvia Salvadori
`
`ABSTRACT
`(57)
`The technology relates in part to biological methods for
`producing terpenes and to engineered cells and microorgan(cid:173)
`isms capable of such production.
`
`3 Claims, 59 Drawing Sheets
`Specification includes a Sequence Listing.
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 1 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 1 of 59
`
`US 11,781,148 B2
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 2 of 291
`
`

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`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 2 of 59
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 3 of 291
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`

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`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 3 of 59
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`US 11,781,148 B2
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 4 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 4 of 59
`
`US 11,781,148 B2
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 5 of 291
`
`

`

`Fatty Acid (extracellular)
`
`TAG, PL
`
`FA ,~,,,,;,.,,,,no<,un,,,,ouoo»<~
`'//,.-;,;;::,ttt/4
`T~A.
`/////////
`
`7
`
`Co A
`
`! C~A - -
`lwtB~li
`DCA
`~
`(:'i:fti<.JX::) Peroxisome
`..-
`DCA -
`·,
`CoA1(~AA2)
`
`@) NADP'
`DCA-CoA •t- Ac-CoA ~
`c~~,t)r
`
`NADPH
`
`Mal-CoA
`
`Ac-CoA
`~ ~ Carn
`~ r-,.coA
`Ac-Carn
`
`Carn
`CoA
`
`(j:Ar:z)
`
`Acyl-CoA
`Co A «f-!~s_)
`Chain-Shortened
`Diacid
`! G l x~~
`i~
`!,--...
`~ .. JCLl) Mal
`
`Ac-Carn
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`Mitochondria
`
`(')
`
`0
`~ ....
`"'o
`N
`0
`N
`~
`
`~- Furn
`
`Mal
`
`G;,j
`rN/ ,(cid:141) Ac-Carn
`, .. r~
`\ .. _. ;,(€¥,f);
`f
`Ac-CoA ... , OAA
`
`I
`
`....,
`~,
`Succ
`\
`\
`
`I TCA I
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`Ul
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' --..l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`Succ-CoA
`
`" \
`
`Cit
`....... :\..¼
`'·· - · · ·
`1/KOl)
`
`/
`a KG
`
`i-Cit-~
`
`Mal
`
`1,,..,.--NADP'
`r,.,...NADPH
`'o/
`OAA
`
`~luconeogenesis
`
`FIG. 5
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 6 of 291
`
`

`

`FA-CoA
`
`TAG, PL
`
`~ NADP+
`
`@t:NADPH,W
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+(cid:173)....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJ'1 =(cid:173)
`.....
`O'I
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' -....l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`Mal-CoA
`
`@¢01
`
`Ac-CoA ~r
`
`Ac
`
`/Vo•,,
`1
`
`,;~>(cid:141) Ac- a
`c rn
`'""'"·1
`r@N)1
`
`~,
`
`Ac-CoA" /
`\
`'{ TC,~·
`
`Cit
`
`'·, .__,
`
`11e
`
`i
`Ac-Cami --
`
`NADP+
`
`'C
`
`Mal
`~
`!
`w
`OAP:"' x.ilvGluconeogenesis
`
`NADPH
`
`FIG. 6
`
`Fatty acid (extracellular)
`
`l C~A - -
`mi CoA
`~ r
`
`,GW/>G//>6.,,'il::¢;,;(>(;/)/6/WWG/W/>GWW6/Wt;<!W'.>a0~
`
`(mN,9
`
`FA
`
`DCA
`
`CP.1)1.1,:,,,}; Peroxisome
`DCA -
`·,
`CoA1(~AA2)
`
`CoA
`
`Ac-CoA~ Ac
`~
`
`DCA-CoA
`
`•l-
`
`Acyl-CoA
`Co A «f-!~s_)
`
`Chain-Shortened
`Diacid
`
`Succ
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 7 of 291
`
`

`

`Fatty acid (extracellular)
`
`FA •~'~ •• ~:•,,,~,,,,,,.
`
`.
`
`,,u"'"""'""'•'?F TAG, PL
`
`DCA
`
`@t:NADPH,W
`
`! CoA ~
`mi C[A
`\ '~
`, (;;;~;;~,m~,~
`FA-CoA ~ NADP+
`CP.f.>f,+i:> Peroxisome
`DCA -
`·,
`CoA1(~AA2)
`
`Mal-CoA
`
`@¢01
`
`Ac-CoA -4'
`
`OAA
`
`Cit
`
`Mitochondria
`
`-
`(cAr~)}::~Ym:;. I'
`
`DCA-CoA
`
`1111- Ac-CoA
`
`Acyl-CoA
`Co A «f-!~s_)
`Chain-Shortened
`Diacid
`
`Succ
`
`,
`!,--...
`%1
`J,,JCLl) Mal
`;
`l
`I so-Cit
`
`f
`
`. . . . . . . . . . . .
`
`Glx ll GlyOx I
`
`=i-Carn
`(~?l:r:~1 CoA
`
`,r~·· ..
`:"'-1:
`F~--f:1..J:
`1 0: ,
`!
`i I..J:
`\-.._.,...J
`
`Ac-Carn
`
`Ac-CoA
`
`\
`
`Cit TC~
`)
`'·, ~ .__,
`
`Mal
`jc:NADP'
`,t
`NADPH
`OAA-
`
`Gluconeogenesis
`
`FIG. 7
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`~ ....
`~
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`-....J
`0 ....
`
`Ul
`1,0
`
`d
`rJl.
`"'""'
`.,.
`"'""'
`--..l
`00
`.,.
`"'""'
`"'""' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 8 of 291
`
`

`

`Fatty acid (extracellular)
`
`PL, TAG
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`FA-CoA
`
`.. l<r-~-T
`
`l
`TARGET
`NADP'
`~CNADPH MOLECULE
`
`DCA
`
`DCA
`
`r·············, ...
`l PEX11 J--' - - - -
`• ... ________ ,/ Peroxisome----..,
`
`Ac-CoA :t= Carn
`
`I to 13-0x 1,,i,.,•-
`
`(~~!_~
`
`CoA
`
`CoA1(!.~~~)
`
`DCA-CoA
`
`L -
`
`Succ
`
`Glx
`V1CLi')
`1· ... _ ___...,,.
`
`r1
`Ac-CoA ~l:~::
`
`Mal-CoA
`¢4«1,)t
`
`Ac-Carn
`
`Ac-Carn
`
`Mitochondria
`
`(?,,
`
`"t~ a,/ Ac-
`i,:q ~\~CoA
`
`~-1 Carn
`~7t~:t.~_ / ~,
`
`'{ TC~J
`Cit
`'·, ......_,
`
`Mal fr NADP'
`
`t~NADPH
`
`Gluconeogenesis
`
`(')
`
`0
`:-+(cid:173)....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`QO
`
`rJJ =(cid:173)
`.....
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' --..l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 9 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 9 of 59
`
`US 11,781,148 B2
`
`Ill
`·;::
`"C
`C
`0
`.c
`
`u .s
`~
`
`<(
`
`0 u
`~
`
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`LL
`
`X
`(.!J • .
`
`ra1
`_;;;.~._
`
`u
`u
`::::l
`V)
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 10 of 291
`
`

`

`Fatty acid (extracellular)
`
`! ... ~t--~ .. ~
`mi cl
`
`(rffs1®
`
`PL, TAG
`
`FA-CoA/4!
`
`DCA
`
`r·············, ...
`l PEX11 ' f - - - - - -
`• ... _______ ,J Peroxisome
`
`/"""'NADP'
`( FAS ) 1-...,_ NADPH
`I,
`6
`
`TAL
`
`{BD
`
`Mal-CoA
`¢4«1,)t
`
`Ac-CoA ~l:~::
`
`!$ Ac-Carn
`
`Mal 'r NADP'
`
`,.~NADPH
`
`OAA-
`
`Gluconeogenesis
`
`C,:.)
`~...u .....
`~AV.
`""·N/,
`~,./ Ac-Carn
`
`Carn«
`
`~~M~ j
`•.
`----
`
`CoA.1/f'Ji+:t,r:~ ..... ..._'-.,
`
`Mitochondria
`
`\
`
`\
`
`Ac-CoA
`
`, TC~·
`'\
`Cit
`
`'
`
`'·, ......_,
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+(cid:173)....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`....
`0
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' -....l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`DCA ----,
`CoA1(!.~~~)
`
`I to 13-0x 1-<l--•-
`
`DCA-CoA
`
`L
`
`-
`
`Ac-CoA :t Carn
`
`(~~~~
`
`CoA
`
`j
`
`Glx ,,,,,❖~1 GlyOx
`V,CLi)
`!'--~·
`Mal
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 11 of 291
`
`

`

`Fatty acid (extracellular)
`
`! C~A - -
`I wtB~ U cl
`
`FA · ~,,,,, ,•~~,,,,,,,,,,,,,,,,,,,, •• , %f>
`(rffs1®
`
`PL, TAG
`
`FA-CoA/4!
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`r·············, ...
`l PEX11 ' , - - - - - - - -
`• ... _____ ,/ Peroxisome
`
`/"""'NADP'
`( FAS ) !-...,_ NADPH
`I 8
`
`TERPENES
`
`r-1-,
`t
`
`IPP <• DMAP
`
`Mev
`
`Mal-CoA J C,:.)
`,,/•/·+;,\r:~--... ..__"-,
`foA.1
`
`DCA
`
`DCA
`
`Ac-CoA :t
`
`2
`
`r
`_
`!CAT
`'-..........
`
`CoA
`Cam
`
`CoA1(!.~~~)
`
`DCA-CoA
`
`L -
`
`Glx
`VCLi"')
`i./~.
`!
`
`~...u .....
`~AZ/
`!'"'"'\!;'a Ac-Cam
`,
`:,;
`(
`Ac-CoA
`<CA.,,
`,
`:
`:
`•. -···
`~,.,-; l:Carn
`Carn""
`\
`'
`;
`,
`C:OA
`,
`\ TC~
`~,i.,,,,,-..• Ac-Carn mm ,m "
`Ac-CoA
`
`t
`
`Cit
`
`'·, ....._,
`
`Mal Ir NAoP·
`
`i~NADPH
`
`OAA-
`
`Gluconeogenesis
`
`Mitochondria
`
`(')
`
`0
`:-+(cid:173)....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`....
`....
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' --..l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 12 of 291
`
`

`

`ORI
`i
`I
`i
`i
`i
`
`I i
`f
`i
`i
`i
`~
`~
`~
`~
`~
`
`pAA061 re
`377l bp
`
`APr
`
`/~
`
`P(BLA)
`
`Kpn l (155q)
`
`>Fin dDI(r;14)
`
`URA3 Term
`
`.:i;xd. (7:31)
`
`(JnT(1c6:,)
`
`URA3
`
`fl?;t]
`
`EcoRf
`
`XmaI
`
`)• ..
`
`··-----.. ; ___________ _
`
`;:,,.-9~4;;,4"~.,
`
`lfapQI
`
`)(ma I
`
`I
`
`BspQI
`PGK terminator
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`0
`(') z-;-....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`....
`N
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' --..l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`FIG.12
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 13 of 291
`
`

`

`ORI
`
`) I i ii
`ii '
`
`···· i
`
`APr
`
`pAA105
`4p4lp
`
`~ ',
`
`,,./
`
`/
`
`P(BLA)
`
`URA3 Term
`
`/
`
`P-;t I
`
`&x, RI
`
`. .
`
`'~,_:<;.• 1;···
`
`·-·
`
`. ·-··· ··-··
`
`PGK promoter
`
`..,'(i-na I
`
`;,,,","
`
`BspCJ,
`
`XmaI
`
`XbaI
`
`Bsp<J
`
`•••--.h,
`
`PGK terminator
`
`EcoR[
`
`;\?A,.~, l
`~ ... u:~ ...
`.:a_
`
`URA3 Prom
`
`Oa I(ZJ;:6)
`
`URA3
`
`FIG. 13
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`~ ....
`"'o
`N
`0
`N
`~
`
`('D
`
`rJJ =(cid:173)
`('D ....
`....
`0 ....
`
`~
`
`Ul
`1,0
`
`d
`rJl.
`""'"'
`""'"'
`"' --..l
`00
`""'"'
`"'
`""'"' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 14 of 291
`
`

`

`A;t I(~w9)
`
`K"'o RI(616)
`
`PGK promoter
`
`I (77z5)
`
`ORI
`i I
`/ i
`if
`"' "' -~
`"' "' "'
`
`pAA219
`69.1.2 hp
`
`~
`
`Fro RI (2384)
`.... ·• ... •• ....... p t ·r / r, -,.,,, . . )
`,. s. t-"'t)/ 2. .
`
`·1· (,~(j(.Y/l
`
`~ •• ~ /
`
`.?\,)/
`
`APr
`
`P(BLA) ... µ----•t_
`
`.. µ•\
`
`J,,.,, ..... ,-.,._.-
`,_.,. .. -.v-"'"'" • .,,,,-..
`. .
`
`URA3 Term
`
`URA3
`
`Cla I (,1.po6)
`
`URA3 Prom
`
`PGK terminator
`
`1·1 I (. ,. -c '')
`r
`fl]() . "\... ..35 9,;:,,
`
`FIG. 14
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+(cid:173)....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`.i;...
`
`rJJ =(cid:173)
`.....
`....
`0 ....
`
`Ul
`1,0
`
`d r,r;_
`"'""'
`"'""'
`"' -....l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 15 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 15 of 59
`
`US 11,781,148 B2
`
`(9
`LL
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 16 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 16 of 59
`
`US 11,781,148 B2
`
`E
`
`... G) -0
`0 ... Q.
`-..:I' ><
`0 a..
`
`'\
`
`o:::'.
`0
`
`E
`0 ...
`
`D..
`(")
`
`~ ::,
`
`//.
`
`~
`
`~ E
`
`r-...
`Q
`
`.'\
`
`I
`
`j '\_
`
`M !
`i
`.. "
`i
`E ... G)
`~~ ; ; \
`
`(9
`LL
`
`I-
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 17 of 291
`
`

`

`ORI
`j
`
`/
`
`{ ___
`jf Jq
`~
`
`pAA153
`5&x)bp
`
`}
`
`1st I ( 2C~)
`- POX4 promoter
`
`CYP52A14
`
`APr __ "
`
`P(BLA) .. ----~
`
`/-'\
`
`.,,,,..,,,--~ .. :.
`
`,..,,,,,,,,..--
`
`URA3 Term
`
`URA3
`r-1,, I f ,,, ,,,., -J.
`,_,;, l_ •./,:)_
`
`uf.U
`
`;
`/
`,
`/
`
`\
`\
`',
`',
`\,
`\
`
`POX4terminator
`-;,, 1>J-, r (,, -1<')6 -J.
`
`..,,.._.,_~) ;• '-'.
`
`f .. /:-;. ~./ J •
`
`\URA3 Prom
`
`e •
`
`r:J)_
`•
`~
`~
`~
`
`~ = ~
`
`0
`(') z-;--....
`
`"'o
`N
`0
`N
`~
`
`('D
`('D
`
`rJJ =(cid:173)
`....
`""""
`--.J
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`"' --..l
`00
`"""'
`"' """' ~
`00 = N
`
`FIG. 17
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 18 of 291
`
`

`

`P(BLA)
`
`I,
`
`URAJ Term
`
`URAJ 3' partial
`
`i
`\
`\
`i,
`\
`).
`
`I,
`:
`:
`
`I
`\
`
`1, APr
`~ \
`
`- ~:1/
`
`. . !!
`V I
`
`1
`~
`
`?
`
`POX4 terminator
`I '
`J 1\de1
`I 1
`1 ! URA3 Prom
`• !
`Fstt POX4 promoter CYP52A14 I / / URA3 5' partial
`
`,
`
`,, .•
`
`i
`
`I
`
`ORI
`
`I ! r
`
`'l ''
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`~
`
`('D
`('D
`
`QO
`
`rJJ =(cid:173)
`.....
`....
`0 ....
`
`Ul
`1,0
`
`pAA 153 linearized at C la I
`r:8ool1p·,
`
`,j~ 'J ~..;
`
`_,"
`
`d r.,;_
`"""'
`"""'
`"' --..l
`00
`"""'
`"' """' ~
`00 = N
`
`FIG. 18
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 19 of 291
`
`

`

`Zeo(R)
`····••. ~ ....
`
`pUC origin
`
`/
`/ '
`
`b~::;:,:oter :;---- ~ ,,,,- lac promoter
`*,'~ .. --....
`••..
`410·
`·.A])f'
`/?Y
`i~[JV
`&::::/
`Kan promoter
`.......................... #
`
`SP6 promoter
`...... ~::--·'.·.·::
`.. .
`.· ... / ....
`dV'2'Z..x) t1(X) (Pf i
`//t
`\jfi}~
`~~~A .. ~~
`" . " ,OJ
`\;J[\
`URA3 31 partial (F23 to stoi
`\!lillllfrt;·················"
`
`,,,,,,=,==:
`
`AA36 7
`
`cc~~~::: ::~i.::: '
`
`f.'J;,;,tff.1()(')'.)'1
`, .J...
`.{ •/,. , t
`oNQ2fJJ (1CO ()¾-;'
`
`.
`
`;J
`
`'
`
`'.' ( )
`
`}~
`i·i•··· ...... !"' .
`..................... ..,..-,\,
`--_ ·~
`,. ·.:··/
`···•• ................... -✓,((:~.
`/:::\~/
`.. :";,49....
`/(/
`. .... ..
`..
`,.,,.,,✓" A{)>,.
`\<,J:/ ······...
`,.ff:;
`·· ...
`,,)i::{>y ~
`URA3 51 partial (M1 to L22)
`URA:v:rl:.:~:· ///;~/ ~ ·
`
`P ~"' hp
`
`URA3 Term
`
`~fd>.,""f';fi{' (1{'<''>((;~~}
`,v,.,;~v;~
`JJrY.-?,£A,.J,
`oN(!XB ("(Dar
`• 70)
`I
`
`P.9. t I (2165)
`
`POX4 promoter
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`~
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`....
`0 ....
`
`1,0
`
`Ul
`1,0
`
`PO X4 terminator
`
`/
`
`\
`\
`'\.
`\\
`CYP52A14
`
`d r.,;_
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`FIG.19
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 20 of 291
`
`

`

`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`..,.o
`N
`0
`N
`~
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`N
`0
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`..,.
`--..l
`00
`"""'
`..,.
`"""' ~
`
`00 = N
`
`FIG.20
`
`,,_
`
`\,J;,,
`
`NdeI r 1.}2n)
`URA3Prom
`U RA'l 5' partiru ( M 1 to L22)
`
`10
`
`0.0r
`
`:::·; oAfaaE.(
`
`POX4terminator
`
`P;f r
`10
`< ,,A \._
`
`90'1
`;:_~)/
`
`URA3Term
`
`CYP52A14
`
`OX4 promoter
`
`1P
`
`;
`
`URA3 ~ partial (F23 to stop)
`
`aANm (im rn~
`
`1,
`
`i, •
`
`pAA367 antibiotic~free cassette
`3804 bp
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 21 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 21 of 59
`
`US 11,781,148 B2
`
`>
`b.O
`0
`0
`E
`0
`..r:
`m
`a I,!)
`
`M
`<(
`a:
`:::J c..
`
`M
`<(
`a:
`:::J
`f-
`
`cYl
`<(
`0:::
`::J
`
`>
`b.O
`0
`0
`E
`0
`..r:
`
`Ln a I,!)
`
`M
`<(
`a:
`:::J c..
`
`M
`<(
`a:
`:::J
`f-
`
`cYl
`<(
`0:::
`::J
`
`M
`<(
`a:
`:::J c..
`
`m
`a I,!)
`
`M
`<(
`a:
`:::J c..
`
`Lt) a I,!)
`
`<(
`'"" N
`(9
`LL
`
`en
`'"" N
`(9
`LL
`
`u
`'"" N
`(9
`LL
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 22 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 22 of 59
`
`US 11,781,148 B2
`
`>
`b.O
`0
`0
`E
`0
`..r:
`
`N) a I,!)
`
`M
`<(
`a:
`:::J c..
`
`M
`<(
`a:
`:::J
`f-
`
`cYl
`<(
`0:::
`::J
`
`M
`<(
`a:
`:::J c..
`
`>
`b.O
`0
`0
`E
`0
`..r:
`
`Lt) a I,!)
`
`N
`N
`(9
`LL
`
`>
`b.O
`0
`0
`E
`0
`..r:
`m
`a I,!)
`
`M
`<(
`a:
`:::J c..
`
`>
`b.O
`0
`0
`E
`0
`..r:
`
`Ln a I,!)
`
`n
`Q)
`
`Q)
`
`<(
`
`N
`Q)
`
`Q)
`
`<(
`
`N
`C
`0
`+-'
`Q)
`
`Q)
`0
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 23 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 23 of 59
`
`US 11,781,148 B2
`
`(9
`LL
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 24 of 291
`
`

`

`Kan(R).
`
`lac promoter
`
`SP6 promoter
`~ ,,,,,,,,.
`.. ,<!···· oAA2377 (100.0%)
`
`\-··-· c, CA r, 3' honwiOSJY
`
`Zeo{R)
`
`b!a promomr
`
`,
`
`pUC origin
`
`/
`
`/
`
`;·~ 6=Ttn:nf :::;:M
`
`ti
`
`Kan promoter_, ....... . .
`
`pAA1519
`(i1ilotv
`
`~ft
`,,/~,,_--::ff\
`ttdB (no A "JG(
`w',,,/'.:.l
`
`). ... /t······· Promoter URA3
`o::3°7:~:e:o .o•i.; · ,,:~~\:+:;t,.;:_;::'c:::,·',.t:=:;DJi(#f:'9::Yi
`I
`CvCA12 s· homology
`/
`f
`Promoter URAJ
`
`··Terminator U RAJ
`
`v
`
`Cv URA3
`
`Zeo(R).
`b!a promoter
`
`·······,,.,/:, ..
`
`, /
`
`'"W 1·_,- rnnu:::=:frt:u
`
`,i:1UC origin
`
`_lac promoter
`
`--;.;,_ ....... SPG promoter
`oAA2372 {1 !JC: .0%)
`CvCA12 5' homology
`__ .,_.,~
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`0
`(') z-;--....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`N
`.i;...
`
`0 ....
`
`Ul
`1,0
`
`Kan promoter·--·········,J
`
`,,:;Jiij
`,,,,,. ...... ,,
`ccdB (no A 113)
`
`,..,. ... ~
`
`pAA1520
`;o~il bp
`
`' '
`/
`Promoter URAJ
`
`0:~~l::::~w,Cy(~
`
`/
`
`CvURAJ
`
`Promoter URAJ
`
`terminator U RA3
`
`FIG. 24
`
`d
`rJl.
`""'"'
`""'"'
`"' --..l
`00
`""'"'
`"'
`""'"' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 25 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 25 of 59
`
`US 11,781,148 B2
`
`r--- 00 O"l
`<D
`O"l O"l O"l O"l
`'<Cl"
`'<Cl"
`'<Cl"
`'<Cl"
`0 0 0 0
`z z z z
`9 9 9 9
`0 0 0 0
`UJ UJ UJ UJ
`(/) (/)
`(/)
`(/)
`
`rlrl.----lrl
`
`~···~
`
`'.
`
`<(
`LO
`N
`
`(_)
`
`co
`LO
`N
`(9
`LL
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 26 of 291
`
`

`

`pU c orig i~
`
`!
`(
`i
`
`lac promoter
`r
`.
`i. M13 reverse prime
`
`~~ \ ' \
`/ _SP6 primer
`, \<_)~\A---· .:::: .. _____ SP6 promoter
`* ~,1{
`. -'>·-.. :
`·· .... _______ CvU RA3 fragment (M1 to
`ff\----~----·········t vU R A3 C v PO X4
`'l-:c,;::,c:::::·----
`. - -
`11:>
`t..-A447?2 ( 1 m tY'/oJ
`
`pAA1164
`796;:$ l)p
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`(,H
`
`L22}
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`N
`O'I
`
`0 ....
`
`Ul
`1,0
`
`Zeo(R)
`bla p remoter
`
`Kan(R]
`
`Kan promoter
`
`··'"'·
`ccd B (no ATG} .,
`4:=:::\:>.
`"•
`·,.,1llll;lri
`M13 (40) forward primer '.
`M13 (~20) forward primer ~- ···--... _>#~
`
`CvUR~ fragme: ~~:~:!:;~:;:~:,/
`
`CvURA3
`
`i
`
`/ ' /
`
`H DE Promoter
`
`POX5 (F98G}
`
`'
`
`.
`
`oAfo..47?:3 (100.Cf¾)
`
`d r.,;_
`"""'
`"""'
`"' --..l
`00
`"""'
`"' """' ~
`00 = N
`
`FIG.26
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 27 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 27 of 59
`
`US 11,781,148 B2
`
`~
`in
`ci z
`Q
`a w
`
`(/)
`
`in
`0
`l!')
`
`0 z
`Q
`a w
`
`(/)
`
`r--
`0
`l!')
`
`ci z
`g
`a w
`
`(/)
`
`©
`0
`l!')
`
`b z
`g
`a w
`
`(/)
`
`LO
`
`:::-i
`:---i
`
`t::.1
`f:.:
`(.).f
`t---f
`t---! ~---l
`D.< C:...: Li.-:
`, .. :,
`
`[:{;
`:~~.
`:S :.-··
`
`t:[~
`::!:\
`
`U)
`W..;
`l
`~::...
`
`,:t..:
`!'·••:
`
`:::--:
`.x~
`
`\:;:;
`~--~
`
`:-:r~
`
`r--..
`N
`(9
`LL
`
`((.:
`::..·.
`~---i
`~:<~ O}
`::: ... ~
`~--~ ~ en
`.:-:r:
`~--~
`~--~
`;::;_;
`;?~
`:-L
`t··i ((! ::'--,
`~~)
`(j)
`~ :~:.-:i:.
`c.:(
`E:---
`:=:J~
`(j)
`(/) :-.........
`:::r:
`CX::
`:---l
`:---·
`~=-z:
`f.··:
`!./)
`~---~
`:8 ~l~
`({;
`
`\-·-·:;
`::'.:'.::
`i:.: .. ;
`
`... ::~
`
`rl
`
`rl rl rl rl
`
`,n u;
`o, ,n
`.;.'•
`.; .. ,
`rl •. ·
`f;
`~::::
`f;::
`+J
`rd
`,··• .. : :··-:
`•o:;-
`;x;
`:>-,
`i:'-·-:
`f:···<
`~ ,,;-: H C·
`,. __ ..
`U () C)
`-~ +
`·+·
`v--:
`:··-:
`
`:··-i
`
`;::,.-:
`,_
`(~)
`
`::--:
`;~) , ___ :
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 28 of 291
`
`

`

`_pUC origin
`
`!
`,
`
`B.spQ1 (7&))
`
`·==L. /¥~~l~c-;~::::oter
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`N
`QO
`
`0 ....
`
`Ul
`1,0
`
`Zeo(R)_
`
`bla promoter
`\
`
`~
`
`Bsp.~I (9585)
`
`f-_, ~ nn1· ('n9'7 -)
`,7,..,.,,, j
`
`J,.',J-" ·,:_-
`
`\\
`
`)
`
`\
`\
`\
`
`\,
`
`Ji0IL..
`
`········- oAA0785 (100.0%)
`
`ccdB (no ATG)
`------...
`
`T7 promoter
`
`-----.
`
`B.sp()_I (7985) ....
`
`------~
`
`----- ~t~f b~.
`{tf
`
`,,,,,.,,..,.,. .. :"~✓ ......... ,,._.....-- ✓} --~
`
`oAA.0784 (97.8%)
`
`CvACC1 5' lntron {partial)
`
`, /
`
`pAA245
`10388bp
`
`CvACC1
`
`FIG. 28
`
`d r.,;_
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 29 of 291
`
`

`

`APr
`
`, .. , ...... ,._/
`
`URA3 ~~:LA) ---~r
`(Jal (~:3 _______ , ,
`URA3 Pro~_ _llf _,
`
`~pe I (8t)73)
`
`.......................... ?:f?:?J✓✓ ..
`Hf@
`.. ~~:~---
`
`ORI
`
`I
`
`!
`i.
`
`... ~---
`~.
`~
`~
`
`pAA326
`
`11692.bp
`
`. Pst I ( 2r)<))
`
`PGK Prom
`-,J f 'r'J?i-'/)J'
`-
`Tr_
`__ _....·~ 11 U .. s. . t/ , .,_,(
`
`<~::::It\. Cv ACC1 5 • intron (pa rtia I
`
`\
`............ w.-1, .......... l fftJ ·-..
`
`\ft\
`fttI
`
`r::=:::=::l
`/::ffl
`::::=::::::r-
`/tti/
`
`7 !..~1 (' Q8Q)
`.?.1.uu
`.. 2o o
`'
`
`Y'l-.,-, I ( ''""'8.)
`
`l,)J~/
`
`,(A(;'\,{
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`~ ....
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`N
`1,0
`
`0 ....
`
`Ul
`1,0
`
`\,.
`
`C1aHA677')
`
`·-., .. ,_
`CvACC1
`
`FIG. 29
`
`d
`rJl.
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`PGK Term
`
`jJ@rr-
`
`,~
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 30 of 291
`
`

`

`I I
`
`/
`
`I
`I
`
`ORI
`
`HOE promoter
`
`CvACC1 partial {M1 to S642)
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`Hi.tl(;;;,:>:}.)
`I
`,· I
`'I .,
`:1
`'I
`1
`/
`
`>Jx? 't (21,7)
`
`-•-----~-••-•~•--••-----••••••- •~ u - • •~
`
`Cia I( 1616)
`
`(')
`
`0
`:-+(cid:173)....
`
`"'o
`N
`0
`N
`(,H
`
`AP<-~,
`
`*
`.J
`~
`~
`
`p (BLA) _________ !A
`
`s=,=====~
`
`URA3 Term
`
`''•..-::-;{
`
`_,.,-/
`
`pAA1634
`862,l bp
`
`URA3
`
`Cfol(j-,925)
`
`Spel (fif;.c;~ J
`
`,,.,.,.,,....
`
`URA3 Prom
`
`PGK
`
`' .'>¾..,_,
`
`_/,/
`
`)).Ji('
`§::~::_-:_-------51138
`S1153
`
`·,._
`
`···• ... __
`
`'•.~======~~
`
`S1158
`
`('D
`('D
`
`rJ'1 =(cid:173)
`.....
`(,H
`0
`
`0 ....
`
`Ul
`1,0
`
`CvACC1 partial (R643 to stop
`
`FIG. 30
`
`d r.,;_
`"""'
`"""'
`"' --..l
`00
`"""'
`"' """' ~
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 31 of 291
`
`

`

`Ztc(Rt
`
`bla promoter
`K.an(R
`\
`
`\
`
`pUC origin
`
`Kan prom-
`,
`eccl e ! no A TG
`
`/
`~\,, ,
`.,.,., ... ,,.,..
`,,.,.,,,,.,-.w.-/.•.
`
`1 .. pramottr
`SPI promctsr
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`1,~rr
`HDEProm_ if pAA2247
`
`i,:::,:::
`
`1401obp
`
`IGRS 5 homolog~
`T7' promotlr ·•·,..
`\.1,,,,,
`.,,,,,,,, ··
`""'",;;--<;>;;fm=====>,!r---.
`.... ~ /::::/
`IGRI 3' homo l0g~ )h% \{f:}:---,.....
`URA3 Term
`,,~~r-~RA3
`
`H-~:::=
`
`H--
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`(,H
`
`rJJ =(cid:173)
`.....
`....
`0 ....
`
`Ul
`1,0
`
`1\
`
`ACC1 S11HA
`
`S11HA
`
`FIG. 31
`
`d
`rJl.
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 32 of 291
`
`

`

`Zeo(Rl
`
`bla promoter
`
`. ···*,,:>····-:---;;
`
`yuc origin
`':.. .... ·.·.: ... · .. /tt, ... ,·
`
`/
`
`Kon(R)·--/
`
`Kan promoter_,., .. ,.,.
`
`pAA 1613 re
`:59mbp
`
`lac promoter
`t,-,;:;' ___ SP6 promoter
`~~f\ ........ .,··oM5512('100.0%)
`id
`CRC1 3' homology
`,·:::=:~
`
`U RA3 Prom
`
`:·••,•.:'.<"r7r,. __ rr...
`
`--r
`\
`
`\,
`
`URA3
`
`/
`
`\ ·,
`URA3Term
`
`\V ,,,/::i
`,.,.,,,..,. ... .,.,,,,,.,. .......
`T7 promoter
`
`,/~ii~\,.
`
`ccctB (no ATGf'
`
`__ .. ----··
`o,4',55·I1 (100.0%)
`
`/
`CRC1 !i' homolog-y
`
`URA3 Prom
`
`FIG. 32
`
`e •
`
`r:J).
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`..,.o
`N
`0
`N
`~
`
`Zeo(R) __
`
`pUCorigin
`
`/
`
`/
`
`, .. /~'+. :::<
`
`·.
`
`blapromoter
`
`··«>~:.'x,·>
`Kan(RL. .... -. J1J)Fi
`
`''" pcomot,, _ ... 1
`
`pAl'.\17 a 1temp re
`
`6os4 hp
`
`cc-:lB (no ATG)
`
`/
`
`T7 promoter
`
`ofv\5511 (100.0%)
`
`/
`C RC i 6' homology
`
`URA3 Prom
`
`lac promoter
`
`•·~_::.:;::. ___ SF'5 promoter
`,. ···
`0M½·12O00.o•i::,)
`
`RC1 3' homology
`
`) .. .,, __
`
`RA3 Prom
`
`URA3Term
`
`URA3
`
`('D
`('D
`
`rJ'1 =(cid:173)
`.....
`~
`N
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`..,.
`-....l
`00
`"""'
`..,.
`"""' ~
`
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 33 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 33 of 59
`
`US 11,781,148 B2
`
`E
`2
`a.
`II") ~
`~ f!,
`:::, ~ E
`
`./ § ~
`~~
`
`··•:•::::::·
`
`(9
`LL
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 34 of 291
`
`

`

`.,,,,.,./
`Elt@::::gj[G:tt;;ff;
`
`pUC origin
`
`lac promoter
`
`_M13 reverse primer
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`Zeo(R}
`
`-~- ;,...,d:::,.,
`bla promote~
`------------lWtu:;t:;::;;,
`Kan(RI_ ___ ✓
`
`> ,,.~;: ____ SP6 primer
`,At/:f/·"!:q ···-
`SP6 promoter
`... 1/ ,4 ,,;;: · •.
`···:cr-,i>-.
`i-/\ ..
`oAA2209 ,100.0%)
`\\·. __ ,. URA3 frag~ent (M1 to L22;
`
`' URA3 Prom
`
`POX4 Term
`
`~-~?
`Ai,,--
`/ff
`
`,✓ ·cRC1
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`(,H
`
`rJJ =(cid:173)
`.....
`
`('D
`('D
`
`(,H
`.i;...
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`Kan promoter__ __ . I
`
`... ::::::::,
`,ff
`,:-:-:-:,(cid:173)
`•:-:-:-:,-
`
`pAA2311
`68:e6bp
`
`.-{fr
`ccdB (no ATG)
`\j:[\
`M13 (-40) forward prime·~--..
`M13 (-20)forward prim~r .. __ ::?;'~:::.i
`T7T:,::::; ' /
`
`··;--·,<
`
`oAA2206 (100.0%)
`
`/
`
`"
`
`/
`
`URA3 fragment (F23 to stop)
`
`/
`
`·-- ...
`G6PI promoter
`
`URA3 Term
`
`FIG. 34
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 35 of 291
`
`

`

`lac promoter
`./· SP6 promoter
`
`/'
`
`/
`
`.
`/ /
`
`bla promoter
`
`Zeo(R)
`'\
`'
`\\
`
`Kan(R)" ,~~ !
`
`f "'---·Mn'"~-, L., .. ,,.,:.y
`
`,;,
`
`t
`K
`.-:::;v7Jt)='•:--·
`an promo er .
`,:-=··············
`.=<:::>==··
`---------.,
`<Lu
`---~
`
`ccd 8 ( n O AT G)
`
`,,.,.,.,.----
`
`______ 5:@L_
`,:.:-:-:-:-:,
`,:::::::::::;:-
`[ff/
`
`T7 prom oter---~-~----:::,;:;t:i:::/'l,,
`
`pAA879
`9795 hp
`
`Cv F ,:~A3~~:~1:o:r---\,,,\
`'---/
`
`... ,,,.,,,...,,--,,.-"'
`
`, /
`
`POX4 Term
`
`/
`
`-·
`CvCPRB
`
`pUCorigin
`/'
`/
`/
`/
`
`/
`--/~--- ______ oA.A.3557 (100.0%)
`FAA1 51 homology
`,/41:flrt._
`
`"W?),----
`
`U RA3 Prom
`
`..:.-.~:1
`\-;.•,,:•i
`
`\\________
`+};2"
`--------
`J jf f ~
`
`/-,/----- U RA3
`
`-, ................. ...
`
`URA3Term
`
`URA3 Prom
`
`POX4 Promoter
`
`FIG. 35
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`~
`
`rJJ =(cid:173)
`('D ....
`
`('D
`
`~
`Ul
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 36 of 291
`
`

`

`P(LAC)
`
`oAA3356 (66.7%)\\.
`·. \
`
`POX4 3' homology(8162--9078)
`
`oAA'.3159 (692%) ..
`
`ORI
`
`/
`{
`I
`I
`I
`l
`
`)>
`
`pAA208
`9522 hp
`
`",AP•
`
`i;:f:~:,,,.----·P(BLA)
`
`oAA3354 (57A%)
`
`POX4 6' homology(2222-3046)
`
`URA3 Prom (3067-3470)
`
`URA3 (3471-4274)
`
`~ !
`
`•
`
`\
`URA3 Term (4276-4641)
`
`,·
`oAA3156 (74.0%)
`
`URA3 Prom (46484961)
`
`FIG. 36
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`(,H
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`(,H
`O'I
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 37 of 291
`
`

`

`Zeo{R)
`
`bla promoter
`
`_pUC origin
`
`lac promoter
`
`Kan(R) ,,/2:r ,,,.,.·.·'.·:_,.,,;.,,·, __ -;>'
`e'<c--···~~3 reverse primer
`4/✓,' ', ~:::,~;::oter
`
`Kan promoter_
`
`ccdB {no ATG)
`
`M13 (-40) forward primer
`
`M13 (•20) forward primer
`\\
`T7 primer . <:\
`··.·:::>.-.::W~-
`_ _:.,. ~
`T7 promoter
`
`pAA850
`8715 bp
`
`oAA:335ti (100.0%)
`
`POX4 5' homology
`
`U RA3 Prom
`
`of ... A?,,357 (100.0%)
`
`POX4 3' homology
`
`j>i
`,ii}-···- · POX4 Prom
`,/.;>;
`
`/< 1·. "PEX11
`
`POX4 term
`
`RA3 Prom
`
`URA3
`
`URA3 Term
`
`FIG. 37
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`~
`
`('D
`('D
`
`rJJ =(cid:173)
`.....
`~
`-....J
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"""'
`"""'
`"' -....l
`00
`"""'
`"' """' ~
`00 = N
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 38 of 291
`
`

`

`i~; _L:;:;;1:;;:~E_;;':;Y ::::: ;;./;: i:;!; ;·~tfa:? s?.! ;;:~:::; i:::~ r $N~,rvr::,~i1;,1f~K ? ;:::J;l
`:::1 F<I1
`})ft
`, .•. ,.19 .... Hi
`B.rn L < ... Lx.,l,·d.,;1,..,,,, .. : .,.g, • .. , .. ,If .. , .• ,'-'·' ,.
`~fF~
`:m~tr~;·tJfl@x~ FJJi::; (;\/fil\::; t,J (?tt}.i:i err F·~:/ttJ·T 'i -t:iit~:\tli.':I:/~ i~)-~i.1 ·i c·;(: F· ::::;:t~1i~ E:j~-:!:;ji(it:~ 1~1
`liiO
`:-v'Q(; I :E:J·~~r !~\_'T :F rrE f/r I'YE. F'\/ I }J ;~p P· H J ::}t~ ~r'K\· .. :J~=t I C;(;J~ .. r.d~:{ S l\ ~r l{ c: ftJ'v.· ;.\J~t IJ< \t}{ 2 0 0
`(; K. [::-y{_;\lff"T f-.V.,J~/F' L:l~t I);-;; N .H [) .L-I:~ l?(i\l~f \l{3I) :r r;t\ K.l:1(;J.:.~ I.Jc; I rn~I c~·v~ I (2 E~ E)}f~)T{ 1 2 .5 0
`30·0
`3~7)0
`400
`450
`SOG
`.SSG
`·600
`
`50
`
`r)
`V
`
`SEQ ID NO: 508
`
`e •
`
`00
`•
`~
`~
`~
`
`~ = ~
`
`(')
`
`0
`:-+-....
`
`"'o
`N
`0
`N
`~
`
`rJ'1 =(cid:173)
`.....
`
`('D
`('D
`
`~
`QO
`
`0 ....
`
`Ul
`1,0
`
`d r.,;_
`"'""'
`"'""'
`"' -....l
`00
`"'""'
`"'
`"'""' ~
`
`00 = N
`
`ii:l!!i~~~~~~iiii!mti~~i;!;f:?!!~~!!l~
`1~~,~•~:~!;~-~:,~~,,~~~~~li~~:~~.~~1-~,:;!;;~!;;
`
`J\E.A:" I I)E;f~.J? Q·i-\C.~r;<; Fi c_;. Y s ::3 Y t~ (_; "E't;f:tf\ Y s r/f.{\l\l Qc::•:r1~1E1 c_; 1)r1 t,J I :tr~t-1t;J\l i\.KJfr:--1
`
`I?(;]\ :r }tI.lJ :{ (; I I ~r 'f? [J\l ;?'P;~: I s ;:-; E~-.H x:-: p i:-.!i:~ t\J<\/R .P·JfV\/(/L ~r [;(; ftt}f~/r rjt/(f?:l:~i
`;\1~\I c~r~.Y f)(9t~\TY:E;tt YF~E~l\lJ<.l-\jr\~·1? PI::t~~.r.K1~\:r=y r.9.K . .i\1.:E; 6 3 6
`
`~ '
`J
`K8A1~le
`highlight
`
`?~IC) t. r~ .P (} t.
`:S, (:· (J .r (~
`
`:::,.;·
`
`8
`,.,
`
`{
`
`6
`
`FIG. 38
`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 39 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 39 of 59
`
`US 11,781,148 B2
`
`0
`'SI"
`lJ')
`<(
`<(
`0
`
`....
`
`'SI"
`lJ')
`<(
`<(
`0
`
`""C ;;: -(.9
`
`00
`CJ)
`LL
`
`> (!J
`....
`(.9
`00
`CJ)
`LL
`
`""C
`;;:
`
`-LL
`
`CJ)
`N
`'SI"
`$
`
`>
`~
`LL
`CJ)
`N
`'SI"
`$
`
`<(
`0
`.2!1
`0
`
`0
`0
`.2!1
`0
`
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 40 of 291
`
`

`

`Zeo(R)
`
`pUC origin
`
`/
`
`/
`
`(::nw:::;:;f G;fon
`
`_;,;.:,•;
`
`bla promoter
`,,
`..... , ..... ·-.((:;N?D
`
`•~orom::[R} f
`
`pAA 1117
`&417 l,p
`
`Jae promoter
`
`PXA1 S' homology
`
`/ / SP6promoler
`... ·-'\J/ oAA?J.ml (1llJf.J<Yo}
`<?[
`::111jrc ····-
`}0
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`
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`
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`
`/ /
`
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`~=:~,, .. /"'.:::l
`
`ccdB (no ATG)
`
`T7prnmoter
`oAA2fl64 (100 o<>;f · ·
`PXA1 :r homology
`
`URAJ Prom
`
`/
`
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`
`U RAJ
`
`Zeo(Rl
`
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`
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`
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`
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`
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`
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`tl
`PXA1 3' homolorn
`·-:=:\,,...-·.,.~ .. --
`
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`
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`
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`
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`
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`
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`
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`
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`
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`
`FIG.40
`
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`
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`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 41 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 41 of 59
`
`US 11,781,148 B2
`
`I.D ii~~~~ ,:j" CJ') i~l~ili
`
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 42 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 42 of 59
`
`US 11,781,148 B2
`
`~'««
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`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 43 of 291
`
`

`

`U.S. Patent
`
`Oct. 10, 2023
`
`Sheet 43 of 59
`
`US 11,781,148 B2
`
`(9
`LL
`
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`
`LCY Biotechnology Holding, Inc.
`Ex. 1001
`Page 44 of 291
`
`

`

`Genomic
`CAT2
`
`CAT21lmts
`