(12) (10) patent n0.: us 7,009,045 b2 united states patent · 2018-07-10 · us 7,009,045 b2 page 2...

United States Patent

US007009045B2

(12) (10) Patent N0.: US 7,009,045 B2 Abbas et al. (45) Date of Patent: Mar. 7, 2006

(54) TRANSFORMATION SYSTEMS FOR 5,118,625 A 6/1992 Aiba et al. FLAVINOGENIC YEAST 5,120,655 A 6/1992 Foster et al.

5,126,248 A 6/1992 Matsuyama et al.

(75) Inventors: Charles Abbas, Champaign, IL (US); 2 gregfg t 1 n - _ 7 7 ee ner e a .

ill?“ ‘1% ligmnovsl?y’. LVS'AGJA)’ 5,210,023 A 5/1993 Grimmer et al. 1“ 0V ' ayura’ “V ( ,)> _ 5,212,087 A 5/1993 Fournier et al.

Barbara V- Kshanovska, LYN (UA)> 5,231,007 A 7/1993 Heefner et al. Kostlalltyll V- _Dl_11ytFuk,_LV1V(UA); 5,268,273 A 12/1993 Buckholz Kateryna A- slblrna, Lv1v(UA); 5,334,510 A 8/1994 Usui et al. AIldI‘ii A. Sibirny, LViV (UA) 5,460,949 A 10/1995 Saunders et al.

5,589,355 A 12/1996 KoiZumi et al. (73) Assignee: Archer-Daniels-Midland Company, 5,665,600 A 9/1997 Hagenson etal.

Decatur, IL (Us) 5,700,643 A 12/1997 Kawasaki 5,716,808 A 2/1998 Raymond

( * ) Notice: Subject to any disclaimer, the term of this 2 1g; g‘lilymgnd t 1 ~ ~ , , am on e a .

gusenct liszxgengedzgg adjusted under 35 5,821,090 A 10/1998 Revuelta Doval et al. ' ' ' ( ) y ays' 5,837,528 A 11/1998 Perkins et al.

_ 5,854,039 A * 12/1998 Raymond et al. ......... .. 435/490 (21) APPl- N°~~ 09/903,508 5,866,404 A 2/1999 Bradshaw et al.

_ 5,871,957 A 2/1999 Kawasaki et al. (22) P116011 Jul- 13, 2001 5,891,672 A 4/1999 Wang et al.

_ _ _ 5,925,538 A 7/1999 Perkins et al. (65) Prior Publlcatlon Data 5,932,701 A 8/1999 Black et al.

6,001,597 A 12/1999 Raymond et al. Us 2003/0082815 A1 May 1’ 2003 6,017,728 A 1/2000 Black et al.

Related US‘ Application Data 6,146,866 A 11/2000 Vntanen et al.

(60) Provisional application No. 60/218,244, ?led on Jul. (Continued) 14, 2000, provisional application No. 60/290,667, ?led on May 15, 2001, provisional application No. FOREIGN PATENT DOCUMENTS 60/288,491, ?led on May 4, 2001. EP 0 967 287 A2 12/1999

(51) Int- Cl- (Continued) C07H 21/04 (2006.01) CIZN 1/16 (200601) OTHER PUBLICATIONS

C12N 15/81 (200601) Clyne et al. Identi?cation of autonomously replicating (52) US Cl- ~~~~~~~~~~~~~~~~ -- 536/241; 435/3201; 435/440; sequence (ARS) elements in eukaryotic cells. Methods 13:

435/471; 435/476; 435/483; 435/243; 435/255.1; 221-233, 1997* 435/255.4; 435/255.5; 536/231

(58) Field of Classi?cation Search ............. .. 536/231, (Continued) 536/241; 435/320.1, 440, 471, 476, 483,

435/243, 2551, 2554, 2555 Primary Examiner—James Ketter See application ?le for complete search history. Assistant Examiner—DaVid A~ Lambertson

(74)Att0rney, Agent, or Firm—Craig G. Cochenour; Duane 56 References Cited A. SteWart, III; Buchanan In ersoll PC ( ) 8

US. PATENT DOCUMENTS

3,932,390 A 1/1976 Daigle et al. 4,102,923 A 7/1978 Pepperman, Jr. et al. 4,745,057 A 5/1988 Beckage et al. 4,794,081 A 12/1988 Kawai et al. 4,808,537 A 2/1989 Stroman et al. 4,837,148 A 6/ 1989 Cregg 4,855,231 A 8/1989 Stroman et al. 4,879,231 A 11/1989 Stroman et al. 4,882,279 A 11/ 1989 Cregg 4,885,242 A 12/ 1989 Cregg 4,925,794 A 5/1990 Isogai et al. 4,929,555 A 5/1990 Cregg et al. 4,935,350 A 6/1990 Patel et al. 4,937,193 A 6/1990 Hinchliffe et al. 4,943,529 A 7/1990 Van den Berg et al. 4,990,446 A 2/1991 Oberto et al. 4,997,767 A 3/ 1991 NoZaki et al. 5,041,384 A 8/1991 Wilson et al.

(57) ABSTRACT

This invention is directed to the transformation of the ?avinogenic yeasts, Pichia guilliermondii and Candida famata, and mutants thereof, by electroporation (elec trotransformation) and by spheroplast transformation. The invention is also directed to nucleic acid constructs such as vectors, plasmids, and ARS sequences Which transform ?avinogenic yeasts, and mutants thereof, at a high level and in a stable manner so as to result in stably transformed yeast host cells Which express/produce recombinant products. This invention also is directed to ?avinogenic yeasts, Pichia guilliermondii and Candida famata, and mutants and tem perature sensitive mutants thereof, Which produce or over produce ribo?avin.

19 Claims, 40 Drawing Sheets

US 7,009,045 B2

US. PATENT DOCUMENTS

6,376,222 B1

FOREIGN PATENT DOCUMENTS

4/2002 Babyak et a1.

W0 WO 91/18103 A1 11/1991 W0 WO 95/26406 10/1995 W0 WO 99/61623 12/1999

OTHER PUBLICATIONS

Seifert, H.S., et al., “Shuttle mutagenesis: A method of transposon mutagenesis for Saccharomyces cerevisiae,” Proc. Natl. Acad. Sci. USA 83:735-739, The National Acad emy of Sciences (1986). Tan, X., et al., “The Hansenala polymorpha PER8 Gene Encodes a Novel Peroxisomal Integral Membrane Protein Involved in Proliferation,” J. Cell Biol. 128:307-319, The Rockefeller University Press (1995). Database EMBL ’Online!, Database Entry SC35112, Database Accession No. U35112 (1995). Database EMBL ’Online!, Database Entry AF347016, Database Accession No. AF347016 (Mar. 19, 2001). International Search Report for International Patent Applica tion No. PCT/US01/22083, mailed Oct. 10, 2002. Becker, D.M., and Guarente, L., “High-Ef?ciency Transformation of Yeast by Electroporation,” Meth. Enzymol. 194:182-187, Academic Press, Inc. (1991). Birnboim, HO, and Doly, J., “A rapid alkaline extraction procedure for screening recombinant plasmid DNA,” Nucl. Acids Res. 7:1513-1523, Oxford University Press (1979). Boretsky, Y, et al., “Identi?cation of an ARS element and development of a high e?iciency transformation system for Pichia guilliermondii, ” Curr. Genet. 36:215-221, Springer Verlag (Oct. 1999). Cannon, R.D., et al., “Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae, ”Mol. Gen. Genet. 221 :210-218, Springer-Verlag (1990). Clyne, R.K., and Kelly, T.J., “Genetic analysis of an ARS element from the ?ssion yeast Schizosaccharomyces pombe, ” EMBO J. 14:6348-6357, Oxford University Press (1995). Cregg, J .M., et al., “Pichia pastoris as a Host System for Transformations,” Mol. Cell. Biol. 5 :3376-3385, American Society for Microbiology (1985). Dialog File 351, Accession No. 12891639, DerWent WPI English Language abstract for WO99/61623 (Document AN 1). Dialog File 351, Accession No. 10444341, DerWent WPI English Language abstract for WO 95/26406 (Document AL1). Faber, K.N., et al., ‘Highly-e?icient electrotransformation of the yeast Hansenala polymorpha, ’ Curr. Genet. 25 :305-310, Springer-Verlag (1994). Fayura, LR, and Kashchenko, V.E., “Thermostable ribo?avin kinase in yeast Pichia guilliermondii, ” Ukr Biokim. Zh. 69:21-25, Naukova Dumka (1997). An English language abstract is at the end of the document. Fedorovych, D., et al., “Hexavalent chromium stimulation of ribo?avin synthesis in ?avinogenic yeast,” Biometals 14:23-31, KluWer Academic Publishers (Mar. 2001). Iimura, Y, et al., “Yeast Transformation Without the Spheroplasting Process,” Agric. Biol. Chem. 47:897-901, The Agricultural Chemical Society of Japan (1983).

Kasiiske, A., et al., “E?icient Electropulse Transformation of Intact Candida maltosa Cells by Different Homologous Vector Plasmids,” Yeast 8:691-697, John Wiley & Sons (1992). Klinner, U., et al., “Hybridization of Pichia gailliermondii by Protoplast Fusion,” in Advances in Protoplast Research: Proceedings of the 5th International Protoplast Symposium, FerencZy, L. and G.L. Farkas, eds., Pergamon Press, pp. 113-118 (1980). KunZe, G., et al., “Transformation of the industrially important yeasts Candida maltosa and Pichia guilliermondii,” J. Basic. Microbiol. 25:141-144, Akademie-Verlag (1985). KunZe, G., et al., “Transformation of Candida maltosa and Pichia gailliermondii by a plasmid containing Sac charomyces cerevisiae ARG4 DNA,” Curr. Genet. 9:205 209, Springer-Verlag (1985). KunZe, G., et al., “Transformation of the Industrially Important Yeasts Candida maltosa and Pichia guilliermondii,” Acta Biotechnol. 6:28, Akademie-Verlag (1986). Liauta-Teglivets, O., et al., “Molecular Cloning of the GTP Cyclohydrolase Structural Gene RIB1 of Pichia guillermondii Involved in Ribo?avin Biosynthesis,” Yeast 11:945-952, John Wiley & Sons (1995). Logvinenko, E.M., et al., “Biosynthesis of 6,7-dimethyl-8 ribityllumaZine in Extracts of the Yeast Pichia guilliermondii,” Biokhimiya (Biochemistry) 47:778-783, Plenum Publishing Corp. Consultants Bureau, Plenum, NeW York (1982). Logvinenko, E.M., et al., “Cloning of the RIB7 Gene Encoding the Ribo?avin Synthase of the Yeast Pichia guilliermondii, ” Genetika 29:922-927, MAIK Nauka (1993). An English language abstract is at the end of the document. Mauersberger, S., et al., “Candida maltosa,” Chapter 12; Nonconventional Yeasts in Biotechnology: A Handbook, Wolf, K. ed., Springer-Verlag, pp. 411-580 (1996). Neilhoc, E., et al., “High Efficiency Transformation of Intact Yeast Cells by Electric Field Pulses,” Bio/Technol. 8:223 227, Nature Publishing Co. (1990). Neistat, M.A., et al., “Transformation of Hansenala polymorpha, Pichia guilliermondii, Williopsis satarnas Yeasts by a Plasmid Carrying the ADE2 Gene of Sac charomyces cerevisiae, ” Mol. Gen. Mikrobiol. Virasol. 12: 19-23, Meditsina, (1986). An English language abstract is at the end of the document. Piredda S., and Gaillardin, C., “Development of a Transformation System for the Yeast Yamadazyma (Pichia) ohmeri, ” Yeast 10:1601-1612, John Wiley & Sons (1994). Pla, J., et al., “Cloning of the Candida albicans HIS] gene by direct complementation of a C. albicans histidine auxotroph using an improved double-ARS shuttle vector,” Gene 165:115-120, Elsevier Science B.V. (1995). Prillinger, H., et al., “Phytopathogenic Filamentous (Ashbya, Eremothecium) and Dimorphic Fungi (Holleya, Nematospora) With Needle-Shaped Ascospores as NeW Members Within the Saccharomycetaceae, ” Yeast 13:945 960, John Wiley & Sons (1997). Reiser, J ., et al., “Transfer and Expression of Heterologous Genes in Yeasts Other Than Saccharomyces cerevisiae,” Adv. Biochem. Engin./Biotech 43:73-102 in Applied Molecular Genetics, J. Reiser, Springer-Verlag (1990).

US 7,009,045 B2

Rose, M.D., and Roach, J.R., “Cloning Genes by Complementation in Yeast,” Meth. Enzymal. 194:195-230, Academic Press, Inc. (1991). Scorer, C.A., et al., “Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastaris for High level Foreign Gene Expression,” Bia/Technalagy 12:181 184, Nature Publishing Co. (1994). Shavlovskii, G.M., et al., “Flavinogenous Mutant of the Yeast Pichia gailliermandii With Damaged Transport of Iron,” Mikrabialagiia 45:313-318, Nauka (1976). An English language abstract is at the end of the document. Shavlovskii, G.M., et al., “Activity of the second step ?avinogenesis enZyme, 2,5-diamino-6-hydroxy-4 ribosylaminopyrimidine-5‘-phosphate reductase, in the yeast Pichia gailliermandii,” Mikrobiologiya/Microbiology 50: 752-755, Consultants Bureau, NeW York, Plenum Publish ing Corp. (1981). Sherman, E, et al., “Laboratory Course Manual for Methods in Yeast Genetics,” Cold Spring Harbor Laboratory Press, pp. 91-103,117-122(1986). Sibirnyi, A.A., et al., “Active Transport of Ribo?avin in the Yeast Pichia gailliermandii. Detection and Some Properties of the Cryptic Ribo?avin Permease,” Biakhimiia 4211841 1851, Nauka (1977). An English language abstract is at the end of the document. Sibirnyi, A.A., et al., “Effect of Glucose and its Derivates on Systems of Ribo?avin Uptake and Excretion in Yeast Pichia guilliermandii, ” Biakhimiia 43:1414-1422, Nauka (1978). An English language abstract is at the end of the document. Sibirnyi, AA, and Shavlovskii, G.M., “On Inhibition of Alkaline Phosphatase I of Pichia gailliermandii Yeast in vitro and in vivo,” Ukr. Biakhim. Zh. 50:212-217, Naukova dumka (1978). An English language abstract is at the end of the document. Sibirny, A.A., “Pichia gailliermandii,” in: Nonconventional Yeasts in Biotechnology: A Handbook, Wolf, K., ed., Springer-Verlag, pp. 255-275 (1996). Sreekrishna, K., and Kropp, K.E., “Pichia pastaris,” in Nonconventional Yeasts in Biotechnology, K. Wolf, ed., Springer-Verlag, pp. 215-226 (1996). Stahmann, K.-P., et al., “Three biotechnical processes using Ashbya gassypii, Candida famata, or Bacillus sabtilis complete With chemical ribo?avin production,” Appl. Micrabial. Biatechnal. 53:509-516, Springer-Verlag (May 2000). Steiner, S., and Philippsen, P., “Sequence and promoter analysis of the highly expressed TEF gene of the ?lamentous

fungus Ashbya gassypii,” Mal. Gen. Genet. 242:263-271, Springer-Verlag (1994). Takagi, M., et al., “Construction of a Host-Vector System in Candida maltasa by Using an ARS Site Isolated from Its Genome,” J. Bacterial. 167:551-555, American Society for Microbiology (1986). Thompson, J.R., et al., “An Improved Protocol for the Preparation of Yeast Cells for Transformation by Electroporation,” Yeast 14:565-571, John Wiley & Sons (1998). Voronovsky, A., and Sybirny, A.A., “Development of clon ing and expression transformation systems for nonconventional yeasts,” Biapalymers and Cell 151122-132, Naukova Dumka (Mar.-Apr. 1999). An English language summary is at the end of the document. Voronovsky, A.Y., “Development of Genetic Transformation Systems for the Flavinogenic Yeasts Pichia gailliermandii and Candida famata,” Dissertation, pp. 1-138, Academy of Sciences of the Ukraine (Jun. 2001) (translation attached as document AS15). Voronovsky, A.Y., “Development of Genetic Transformation Systems for the Flavinogenic Yeasts Pichia gailliermandii and Candida famata,” pp. 1-97, Ralph McElroy Translation Company, Austin, Texas (Jun. 2001). Yang, C., et al., “Conservation of ARS Elements and Chromosomal DNA Replication Origins on Chromosomes III of Saccharamyces cerevisiae and S. carlsbergensis,” Genetics 152:933-941, Genetics Society of America (Jul. 1999). Yang, V.W., et al., “High-E?iciency Transformation of Pichia stipitis Based on Its URA3 Gene and a Homologous Autonomous Replication Sequence, ARS2,” Appl. Env. Micrabial. 60:4245-4254, American Society for Microbiol ogy (1994). Zakalskii, A.E., et al., “Cloning of the RIB1 gene coding for the enZyme of the ?rst stage of ?avinogenesis in the yeast Pichia gailliermandi, GTP cyclohydrolase, in Escherichia cali cells,” Genetika 26:614-620, MAIK Nauka (1990). An English language abstract is at the end of the document. Zvjagilskaia, RA, et al., “The Respiration System of the Yeast Pichia gailliermandii at Different Levels of Flavinogenesis,” Mikrabialagiia 47: 975-984, Nauka (1978). An English language abstract is at the end of the document.

* cited by examiner

U.S. Patent Mar. 7, 2006 Sheet 1 0f 40 US 7,009,045 B2

14

EW SiNVWHOASNVHl ,, 01

U.S. Patent Mar. 7, 2006

(Hc SI

I

Sheet 4 0f 40

IocZ

LEU2

prom. lucZ

US 7,009,045 B2


prom. lucZ


3.? Nazi and? as 5% E0 M2

Illa-Ill

2.? 2%? ll-IIII

~02 aumxsmm b2

“6: ED 0%

N2:

3 llul iumxEmmnx aumxsmm


mo: N02

Sm:

2 um V. E m a m g

=. 1 . 1 . .

.1‘. s. .12....0 1.1%... ..v


Candida famata C8818 TOTAL DNA

Bum HI; Sou 3Al dephosphorylotion PARTIAL RESTRICTION

I? B

I \\7< //

0 O 9 O

Candida famata CBS18 GENOME LIBRARY ON THE VECTOR pIQLZ

FiG.9


11497

5077 4749 4507

2838 2560 2459 2140 1986 1790

1159 1093

805

Id:

US 7,009,045 B2 A0.

f we

0 Ill u 2.? 292% 2.2,. g 3%? we 3

M m. 7, u M U.S. Patent


FIG.12

U.S. Patent US 7,009,045 B2

Q _ gm 3 02

£2.52 1' = a a _m Q

m _ Q“ E .2

f Nugéoa II. m. a; 8.1 egg 2 z x d

m I m m 01 OZ om E

m a

I mam _m Q m _ ma ~02 b2 8.52 l'

29 um E I n_ OI OI 0m E

7 u

M I a n. a an

_ E E E

Nusépa II.

2x 8.1 mwmoa _ _

m % £3 E IQMEZZ


3.01

. _ II III III

. m a Nossoa A 1 1

III a l 5% I .I I. _

I II “III II

I II I a II

. P. O Q

AMHIIMMIM V 2w: I _ III N E III

_ _ _\ I _ f \ _ _

I a I I III III“ II

I II I a II

EEEI \ _ II III III r

20. 3,1 EMIIIIII _ II .I x \ \

II II III “III II

I Im I a II

EEEI _ II N 02 III

5 mi :WIIIII _ _ z I.

I I III III

I II I a II

U.S. Patent

cttgactgat ctgaatattt ggttttccaa aaagaacaac gataaagctg tcgactacgt tatgggaaat tgttttttat aatcatagtt tcatgatttt ctgttacacc aattcttttt ccttatcacg tttctccttc ttgataaatg tatgtagatt tgtcgnttct ttatgaattt caanggattt cttaacttnt ggcgacagca ttccaaanct tttactgctc anacnaaaaa ggcgatnggg

Mar. 7, 2006

tacgaattcg ttttaacgat aatttaaatt ttaaagtcac caaagtcaca cgttaaggcc gg’ctcaagaa ttgttgtatt tcatgatttt ctgttacacc taactttttg

agctcggtac tttttaataa gattttttag tatgacaaat agcaaggcta gtttctgaca ggtattgact tttttttttt tagagaaaat

Sheet 15

ccggggatca ttttgcaatc aaagtcctaa tcagaaatac actaagatcc gagtaaaatt taaactccat tagagaaaat cctccaatat tgtggngccc

taactttttg tgtggngccc tcctccttgt ccttatcacg ttgagccatt agtatcaatt ttgagccatt agtatcaatt tgcttacctg ttgataaatg tgcttacctg tattccttta tatgtagatt gcgtatatag gcgtatatag attatgaatt catttataaa cttaactcaa tcgqggacgc tcccgacttc tttactggtn ttcaagggct nggcttttca tgacctntnt

tttcgtctac tcatttataa gtttatgtcc atatcataaa atccccgctt gqngg?actg ttcatgggcc taccttntca gcaggtcaan ttggcaacga

tttcgtctac cctatgaaca aggttatgtn aatatcataa aaaagagaat 899099569 tggaacccct taccccagtt ggcaggtcaa gcaatttcac tngggtgacc

FIG.15

0f 40

tgaatgctag atttaagaaa aaaatagaat aatcgtatga tctaggggga cttgagggaa 'caaatggtca cctccaatat ataaattagg tcctccttgt caatattaat gttaaagtgc tattccttta catcctcctt cctatgaaca tattccattt tgtaatttcg aaaaagagaa ctttttaagc gtggacccct aaatcccagt ctgaacctgc tgacaattca atcntgcnca tatntttgca

US 7,009,045 B2

tctttttaca aaccacaaat cataccaaat tccgttcatg tcctctagag ctttcaccat ggtcattgag ataaattagg aatcatagtt caatattaat gttaaagtgc aattcttttt catcctcctt tttc’cccttc tattccattt tgtaatttcg ggcgtttcta tctttttaag aagggatttt aaatctnttc ntgatcctgc atccaaanct catcntgcnc aaaananagt aac

60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1493


tatgcacatt cgcacgccga ggtgcagcgt ttaggcgcgg ctcaacggaa gccaacggcc 60 gccacaaatt gtccggaaag tcgccgaaac tgatccactg gtaccacagc cccataagaa 120 ccccctttaa tattaaaaac cgttcttcag ccacttttga tcacattgtt tgcagccgcc 180 cgttgctgcc atccaaacac cacgcgtccc ccgcaccctt ttacggtgcc cactgcattg 240 gaatttgcat aaaacagcct cacgaagtgg attaatt’ctt agagcactca agtcatcatg 300 ctgcaatctc tgcatcatga aatgactccc gttgatacag ggaactcaga ccgcaagcgg 360 cgaagagtca caagagcgtg tgatgtgtgt cgactctaga ggatccccgg gtac 414


4: x '30. (D

Z

‘T' 2 O:

E Q o

2.

|\ F

N g 0

CD Ll.

LEU2

prom. locZ

(12) (10) patent n0.: us 7,009,045 b2 united states patent · 2018-07-10 · us 7,009,045 b2 page 2...

Documents