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(12) Patent Application Publication (10) Pub. No.: US 2015/0094483 A1 Ness Et Al US 2015 0094.483A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0094483 A1 Ness et al. (43) Pub. Date: Apr. 2, 2015 (54) BIOTRANSFORMATION USING Publication Classification GENETICALLY MODIFIED CANDDA (51) Int. Cl. (71) Applicants: Jon E. Ness, Redwood City, CA (US); CI2P 7/64 (2006.01) Jeremy Minshull, Palo Alto, CA (US) CIIC3/00 (2006.01) CI2N 5/8 (2006.01) (72) Inventors: Jon E. Ness, Redwood City, CA (US); (52) U.S. Cl. Jeremy Minshull, Palo Alto, CA (US) CPC ............. CI2P 7/6409 (2013.01); CI2N 15/815 (2013.01): CI IC3/006 (2013.01) (21) Appl. No.: 14/084.230 USPC ....................... 554/219; 435/254.22:554/213 (22) Filed: Nov. 19, 2013 Related U.S. Application Data (57) ABSTRACT (60) Division of application No. 12,775,306, filed O May A substantially pure Candida host cell is provided for the 6, 2010, now Pat. No. 8,597,923, which is a continu biotransformation of a substrate to a product wherein the host ation-in-part of application No. 12/436,729, filed on cell is characterized by a first genetic modification class that May 6, 2009, now Pat. No. 8,158,391. comprises one or more genetic modifications that collectively (60) Provisional application No. 61/176,064, filed on May or individually disrupt at least one alcohol dehydrogenase 6, 2009. gene in the Substantially pure Candida host cell. Patent Application Publication Apr. 2, 2015 Sheet 1 of 28 US 201S/0094.483 A1 Fatty Acid rt-NS Fatty Acid Fatty Acid CYP52A Type P450 s COAOxidase (HO-Fatty Acid CB-unsaturated fatty acyl CoA Fatty Alcohol Oxidase CYP52A Type P450 Hydratase OCH-Fatty Acid 8-hydroxy fatty-acyl CoA Dehydrogenase CYP32A Type P450 Dehydrogenase HOOC-Fatty Acid BOXO fatty-acyl CoA Thiolase Fatty-acyl CoA+ Acetyl CoA Patent Application Publication Apr. 2, 2015 Sheet 2 of 28 US 201S/0094.483 A1 Fatty Acid rt- * B-Oxidationis h Fatty Acid Fatty Acid CYP52AType P450 X ApOX4 ApOX3 (strain DP1) y (HO-Fatty Acid (B-unsaturated fatty acyl CoA Fatty Alcohol Oxidase CYP52AType P450 Hydratase y OCH-Fatty Acid B-hydroxy fatty-acyl CoA Dehydrogenase CYP52A Type P450 Dehydrogenase y HOOC-Fatty Acid 8-OXO fatty-acyl CoA Thiolase w Fatty-acyl COA+ Acetyl COA Figure2 Patent Application Publication Apr. 2, 2015 Sheet 3 of 28 US 201S/0094.483 A1 albicans DH 1A MQSLFRIFRGSLTTTTAASFTATAT, GATTAKTLSGSTWLRKSYKRTYSSSWLSSPE allian8 ADH E MERFFRIFEGGSLTTTTSFTTTTTTLSSTWLRESERTESSSWLSSPE tropicalis (E A4 albicans DH 24. albicans DH 2E tropicalis ADE B4 trialis DE 4.1 tropicalis ADE B11 albicans DH 1A LFFFHFINNERYCHTTTTTNTRTIMSEQIPRTRANWFDTNGGOLWYKDYFWPTFEERIE albicans DH 1B LFFFHOFMNNERYCHTTTTTTETIMSEQIPETEAWWFDTNGGOLWYEDYFWFTFEERIE tropicalis ADE 4 ELEEDIPWPTPERIE albicans ADH 2. MSWFTTE WIFETIGGELEYEDIFWFERENE albicans ADH 2B MSWFTTOKAWIFETIGGELEYEDIFWFEFENE tropicalis ADE B4 ELEEDIPWPEPEPE ... tropicalis ADH 10 . ELEEDWPWPWPEPE tropicalis ADE B11 FLYTDIPWPWPKPNE albicans DH 1A LLIHESGCHTILHWEGDWPLTELPLGGHEGGGMGENEGWEIGDFGIEW albicans DE 1E LLINESGHTILHWEGITTPLTELPLWGGHEGGGGENEGWEIGIFIEW tropicalis ADF ki LLINKSGHTILHWKGIWFLTELPLWGGHEGGGGEWKGWEIGIFIEW albicans ADH 24. LLINESGHTILHWEGIWFLTELFLWGGHEG,GWWLGEEGWEGIWW albicans DH 2B LLIESGCHTILHEGIFLTELFLGGHEGGFLGEEGEGDGE ... tropicalis ADH B4 LLINESGHTILHTTEGITTPLITELPLGGHEGGIGINEGINEGILGET tropicalis. DE 10 LLWREYSGCHSDLHTTEGDTTPIPELPLGGHEGGGMGDWKGWEGILGIE tropicalis ADE B11 LLWHEYSGWCHS IIHF WKGIWFP:SELFTWGGHEGGSWWIGEMWGWENGILGIEM * * : * * * * * * * * : * : * * * * * * albicans DH 1A LNGSCMSCEFCOGAEFNCGE, DLSGTHDGSFETDAW, EIFGTDLAITWPIL albicans DH 1E LNGSCMSCEFCOGAEFMCGEADLSGTHDGSFESTADAWKIPAGTDLNWAFIL tropicalis ADE 4 LNGSCMSCEFCOGEFNCGEDLSGTHIGSFETDRIPTILEWPIL albicans ADH 24. LNGSCLNCEC3GAEFMCAEADLSGTHDGSFOQYATADAWRIFAGTDLANWAFIL albicans DH 2B LNGSCLNCEC3GEFNCEDLSGTHIGSFOOTDAWRIFTDLNWFIL ... tropicalis ADE B4 LNGSCMRCECGEPNCPDLSGTHDGSFTDAWRIPAGTDLNAPIL tropicalis ADE 10 LNGSCMRICEFCOGAEFNCSRADMSGTHDGTFY TADAWKIPEGDMASIAFIL tropicalis ADE Bll LNGSCMNCEYCOGAEPNCPHDWSGSHDGTFYIT.I.W.EFPGSDLSIPIS * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * : * : * : *, *, * * * alias DH 1. CGTELET DL, GTISGGGGLGSLFORMGLRIDGGIEEGEFES .albicans DH1B C.G.T.YKLET DLRGOT...ISGGGGLGSL SERMGLRWIDGGDEKGEF.ES tropicalis ADE 4 CGTELETILGWISGGGGLGSLWMGLRFIDGGDEEGFES albicans ADH2A C.GWTWYELETELEGOTWISGAGGLGSLWYK.MGYRLAIDGGEDKGEFE albicans ADH 2B CGTELETELEGISGGGLGSLOYEMGYRFIDGGEDEGEFE Figure 3A Patent Application Publication Apr. 2, 2015 Sheet 4 of 28 US 201S/0094.483 A1 C. tropicalis ADH E4 CGSTELET DLRGINAISGGGLSLINEAMGRWAIDGGDEGEFE C. tropicalis DH 10 CAGNTELEMADLLGAISG. GGGLSLGWEAMGRSLIDGGDERGEFE C. tropicalis DH B11 CGSTELET GLOFGTWISGGGLSLWEMGLEWIDGGDERGWFE if f * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * C. albicans ADH 1A LG.E.YWDFTEDEDIYEAWEKATIGGRHGAINSYSERIDSWEEWRFLGEWWLWGLFA C. allicans ADS 1B LG.E.YWDFTEDEDIYEAWEETIGGPEGAINSYSEEIDSWEEWRFLGEWWLWGLF). C. tropicalis ADH 4 LG.E.YIDFLEEEDISEE TIGGPHIRESSEKIISERFLELGLF C. albicans ADH2A LGETFIDFTREKDSJEANEKATNGGPHGWINSWSERIGSTEYWRTLGEWWLWGLFA C. alaicans ADH2E LGETFIDFTEEEDWWEAWEETINGGPHGNINSWSERIGSTEWRTLGEWWLWGLF). C. tropicalis DH B4 LGEWFWDFLEERDIYGAWEKATIGGPHGAWNWSISERINSYTYWRTLGRWWLWGLFA C. tropicalis DH 10 LGESYIDFLEEDIYSAIREATGGGPHGWITFSYSEEINSSEYWRTLGEWWLWSLF). C. tropicalis ADHB11 LGEWFWDFTEENYSEIIETIGGHGSINESISEEINSWEWRTLGTWWLWGLF kit. ::fff; ; ; ; kit. .; if f : * : * * * * : h it. if C. albicans ADH 1A HAEWTFWFTWWESIEIEGSYWGNREDTAETIFFSRGLIECPIKIWGLSDLPEWFELM C. allicans ADH 1E HETFFIESIEIEGSY GNREDTEIDFFSRGLIECFIEIWGLSDLFENFELM C. tropicalis ADH i4 GSEWTGWFEWWESIEIEGSYWGNREDTAEWDFFSRGLIECPIEIWGLSELPOSFELM C. albicans ADH2A GAEISTFWFDAWIRTIQIEGSYWGNREDTAEWIFFTRGLIRCPIRIWGLSELPENYELM C. allicans ADH2E GAEISTFWFDANIETIIEGSYWGNREDTAEWDFFTRGLIECFIEIWGLSELFEWYELM C. tropicalis ADH E4 GSENSAFWFISWESIQIEGSYWGNREDTAEWIFFSRGLIECFIEWGLSELFENYELM C. tropicalis DH 10 GGELTPLFESNARSIOIRTTCWGNREDTTEIDFFWRGLIDCPIEWAGLSEWPEIFDLM C. tropicalis DH B11 GELEFIFNNESIOIEGSYWGNRRDTESDFFRGLECFIEWGLSELFEIFELL f; ; ; ; ; ; ; ; ; ; ; ; * * * * * * * * * * * * * * * * * * * *; ; ; ; ; ; ; Calbicans ADH.4. EEGKILSRYWLDTS- SEID MO;172 C. albicans ADHB EEGEILGRYWLDTSE SEID MO; 173 C. tropicalis ADH4 --------------- SEC ID NO;155 C. albicans ADH2A EEGKILGRYWLDMDE SEC ID MO:174 C. albicans ADH 2B EEGEILGRYWLDRIDE SEID NO:15 C. tropicalis ADH B4 --------------- SEC ID MO: 154 C. tropicalis ADH.10 --------------- SEC ID MO:152 C. tropicalis ADE B11 --------------- SE II) NO; 151 Figure3B Patent Application Publication Apr. 2, 2015 Sheet 5 of 28 US 201S/0094.483 A1 ReCOmbinase ReCOmbinase Site Site Targeting Recombinase Targeting Sequence enCOcing gene Selective marker Sequence W ||'''''''''^ || || || W Restriction site to release targeting Restriction site to release targeting Construct from sequences required for Construct from sequences required for bacterial propagation bacterial propagation Figure 4 Patent Application Publication Apr. 2, 2015 Sheet 6 of 28 US 201S/0094.483 A1 RecombinaseSite Targeting Construct RecombinaseSite Targeting Recombinase Targeting Sequence 1 enCOding gene Selective marker SeQuence 2 e A Z. B Target Target Sequence Sequence 2 GenOmic DNA DNA region to be replaced Homologous recombination Integrant : W:8 ReCOmbinase %: NE GenOmic DNA enCOdinCggene Cene Selective marker ReCOmbinase ReCOmbinase site site EXCised DNA W KN D Figure 5 Patent Application Publication Apr. 2, 2015 Sheet 7 of 28 US 201S/0094.483 A1 ReCOmbinase ReCOmbinase Site site ReCOmbinase encoding gene Selective marker : A GenOmic DNA Induction of reCOmbinase ReCOmbinase WXNE B : GenOmic DNA ReCOmbinase Recombinase site encoding gene Selective marker Illllllllllllllllllllll ............................. C Figure 6 Patent Application Publication Apr. 2, 2015 Sheet 8 of 28 US 201S/0094.483 A1 Insertion Sequences ReCOmbinase ReCOmbinase Site Site Targeting Recombinase Targeting Sequence enCOding gene Selective marker Sequence WI W Restriction site to release targeting Restriction site to release targeting Construct from sequences required for Construct from sequences required for bacterial propagation bacterial propagation Figure 7 Patent Application Publication Apr. 2, 2015 Sheet 9 of 28 US 201S/0094.483 A1 Insertion ReCOmbinase ReCOmbinase Sequences Targeting Construct Site Site Targeting Recombinase Targeting Sequence? encoding gene Selective marker W|8E: A Z NE B Target Target Sequence Sequence 2 Genomic DNA DNA region to be replaced Homologous recombination Insertion Sequence Integrant EWI:E2%8. ReCOmbinase enCOcinq Cene Selective marker GenOmic DNA 99 ReCOmbinase ReCOmbinase Site Site Excised DNA Z: N D Patent Application Publication Apr. 2, 2015 Sheet 10 of 28 US 201S/0094.483 A1 ReCOmbinase ReCOmbinase Site site ReCOmbinase encoding gene Selective marker EW : ^ N Insertion GenOmic DNA Sequences Induction OfreCOmbinase Insertion Sequences ReCOmbinase Site WAIIII: NE B GenOmic DNA Recombinase Recombinase site encoding gene Selective marker :3''''''''y Figure9 Patent Application Publication Apr. 2, 2015 Sheet 11 of 28 US 201S/0094.483 A1 Original genomic sequence Target Sequence 2 W. NEA A ReCOMOinase ReCOmbinase Selective marker ReCombinase Site encoding gene site Sequence 1 A A A A A A B A A A C A S Patent Application Publication Apr. 2, 2015 Sheet 12 of 28 US 201S/0094.483 A1 Targeting Targeting Sequence Selective marker Sequence Figure 11 Patent Application Publication Apr. 2, 2015 Sheet 13 of 28 US 201S/0094.483 A1 Fatty Acid rt- B-Oxidation3. h Fatty Acid Fatty Acid CYP52AType P450 X X ApOx4 ApOX5 (strain DP1) y (HO-Fatty Acid (B-unsaturated
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