US008367395 B2

(12) United States Patent (10) Patent No.: US 8,367,395 B2 Bailey et al. (45) Date of Patent: Feb. 5, 2013

(54) PRODUCTION OFSTEROLS IN (58) Field of Classification Search ...... None OLEAGNOUSYEAST AND FUNG See application file for complete search history. (75) Inventors: Richard B. Bailey, South Natick, MA (56) References Cited (US); Joshua Trueheart, Concord, MA (US); Kevin T. Madden, Arlington, MA U.S. PATENT DOCUMENTS (US) 2006, OO88903 A1 4/2006 Lang et al. 2009/0324800 Al 12/2009 Bailey et al. (73) Assignee: DSM IP Assets B.V., Heerlen (NL) FOREIGN PATENT DOCUMENTS (*) Notice: Subject to any disclaimer, the term of this EP O 486 290 B1 5, 1992 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 123 days. Merkulov et al. Yeast. Feb. 2000:16(3):197-206.* Chica et al. Curr Opin Biotechnol. Aug. 2005:16(4):378-84.* (21) Appl. No.: 12/443,350 Sen et al. Appl Biochem Biotechnol. Dec. 2007: 143(3):212-23.* 1-1. Veen et al. FEMSYeast Res. Oct. 2003;4(1):87-95.* (22) PCT Filed: Sep. 28, 2007 Madzak et al. J. Biotechnol. Apr. 8, 2004:109(1-2): 63-81.* Athenstaedt et al., “Lipid particle composition of the yeast Yarrowia (86). PCT No.: PCT/US2007/021091 lipolytica depends on the carbon source'. Proteomica, 6:1450-1459 (2006). S371 (c)(1), Merkulov et al., “Cloning and characterization of the Yarrowia (2), (4) Date: May 28, 2009 lipolytica squalene synthase (SQS1) gene and functional complementation of the Saccharomyces cerevisiae erg9 mutation'. (87) PCT Pub. No.: WO2008/130372 Yeast, 16:197-206 (2000). International Search Report for PCT/US2007/021091, mailed Jan. PCT Pub. Date: Oct. 30, 2008 13, 2009. International Preliminary Report on Patentability for PCT/US2007. (65) Prior Publication Data 021091, mailed Mar. 31, 2009. US 2010/0305341 A1 Dec. 2, 2010 * cited by examiner

O O Primary Examiner — Christian Fronda Related U.S. Application Data (74) Attorney, Agent, or Firm — Choate, Hall & Stewart, (60) Provisional application No. 60/848,582, filed on Sep. LLP 28, 2006. (57) ABSTRACT (51) Int. Cl. The present invention provides methods for production of one CI2N 9/00 (2006.01) or more sterol compounds. Further provided are methods and CI2N 9/10 (2006.01) systems for producing engineered oleaginous yeast or fungi CI2N I/00 (2006.01) that are capable of production of one or more sterol com CI2N IS/00 (2006.01) pounds, and compositions which utilize the produced Sterol C7H 2L/04 (2006.01) compound(s). (52) U.S. Cl...... 435/254.11: 435/183; 435/193; 435/320.1; 536/23.2 9 Claims, 37 Drawing Sheets

U.S. Patent Feb. 5, 2013 Sheet 2 of 37 US 8,367,395 B2

U.S. Patent Feb. 5, 2013 Sheet 4 of 37 US 8,367,395 B2

Non-mevalonate Mevalonate Pathway O O Pathway os-coo". Crop He3 g sco 1 2 OH Acetyl-CoA dxS CO2 FIG.3 . COA-SH OH O' O O s OP H3C 1-sca OH 10 3. Acetyl-CoA 2 NADPH +2H COASH dXr (ispC) 2 NADP 9 'oho SOH or 11Š-Ns. S-CoA OP 2 NADPH + 2H

4 CTP O OH COA-SH ispD (ygbp) PP NH2 S 12 OH OH O O N O S o-P-O-P-o N-so ATP OH OH O ADP O O- O OH - ATP Š- OPP ispE (ychB) S. ADP O 13 ATP O W-O NH2 ADP 9 y Q O O "OH r-i-o-, -o N-so S OH OH p O le. O 1 14- SOPPATP ADP ispF (ygbB) -CMP OP O ul-s-s W-O 15 OPP or \o sai-O1 V CO2 OH OH O P 7 1- OPP ispG (gCpE) 16 idi

's- OPP ispH (lytB) OH -s-s OPP 1 7 U.S. Patent Feb. 5, 2013 Sheet 5 Of 37 US 8,367,395 B2

FIG.4 FPP YEAST ERG ERG 9 trans, trans-farensyl diphosphate = presqualene - PP C-14 demethylase ERG 9 squalene squalene monooxygenase ERG 1 2,3-epoxysqualene 2, 3-oxidosqualene lanosterol synthase ERG 7 lanosterol C-14 demethylase t ERG 11 14-demethyllanosterol (4, 4-dimethyl-cholesta-8, 14,24-trienol) C-14 reductase ERG 24 4, 4-dimethylzymosterol (4, 4-dimethyl-cholesta-8,24-dienol) C-4 methyloxidase ERG 25 -a- Vitamin D3 C-4 decarboxylase ERG 26 Zymosterone 3-ketOreductase ERG 27 zymosterol (cholesta-8,24-dienol) C-24 methyltransferase ERG6 fecOStero (ergosta-8,24-dienol) A8-7 isomerase t ERG 2 episterol (ergosta, 7,24-dienol) C-5 desaturase ERG 3 ergosta-5, 7,24-treinol C-22 desaturase ERG 5 ergosta-5, 7,22,24-tetraenol C-24 reductase ERG 4 ergosterol -- vitamin D2 (ergosta-5, 7,22-trienol) U.S. Patent Feb. 5, 2013 Sheet 6 of 37 US 8,367,395 B2

FIG.5 mevatonate pathway Non-mevalonate pathway (Operates in humans) (Operates in some human pathogens/not in humans) isopentenyl diphosphate - I - Dimethylally diphosphate (IPPC) Somerase (DMAPP; C5)

Head-to-tail Condensation GPP synthase Phylosierols Geranyl diphosphate -e- Monoterpenes (GPP; C10) FPP synthase (+ IPP) Squalene Synthase Squalene -s- Farnasyl diphosphate -- Sesquiterpenes, (FPP; C15) Triterpenes

GGPP synthase Cholesterol, Bile acids (+ IPP) Steroids hormones (in Human) Geranylgeranyl diphosphate -ie- Diterpenes, Carotenoids, (GGPP; C20) Ubiquinones, Menaquinones, Plastoquinones Prenyl diphosphate synthase

(+IPP) Polyprenyl diphosphate V Polyprenyl-phosphate (Pol-P) (Pol-PP) P (e.g. C5-C5 in Mycobacteria) U.S. Patent Feb. 5, 2013 Sheet 7 Of 37 US 8,367,395 B2

FIG.6 Hydroxybenzoate soprenyl Biosynthesis Biosynthesis Acetyl-CoA

Acetoacetyl-CoA

HMG-COA -- HMG-CoA reducase Mevatonate

Tyrosine (or phenylalanine) Mevalonate-P

4OH-Phenylpyruvate Mevatonate-PP

4OH-Phenylaciate isopentenyl-PP oil- cle Protein isoprenylation t 4OH-Benzoate Decaprenyl-PP - Farnesyl-PP Z-r Galley | | - -- cis-Prenyltransferase Decaprenoyl trans Squalene Squalene

40H-benzoatetransferase Prenvlrenyltransferase transf SVnthetasey Polyprenyl-PP Decaprenyl-4OH-benzoate Cholesterol / \ Dolichol Dolichy-PP Ubiquinone Carotenoid Cholesterol Biosynthesis Ubiquinone Biosynthesis Biosynthesis U.S. Patent US 8,367,395 B2

U.S. Patent Feb. 5, 2013 Sheet 9 of 37 US 8,367,395 B2

- - - - -nm - r real are C - N - u mi " in to Wu- - -- Y- a-- Y--> -- war U.S. Patent Feb. 5, 2013 Sheet 10 Of 37 US 8,367,395 B2

**** U.S. Patent Feb. 5, 2013 Sheet 11 of 37 US 8,367,395 B2

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U.S. Patent Feb. 5, 2013 Sheet 17 Of 37 US 8,367,395 B2

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U.S. Patent US 8,367,395 B2

U.S. Patent Feb. 5, 2013 Sheet 20 Of 37 US 8,367,395 B2

FIG.9A Schematic diagram of certain plasmids used herein

pMB4591 6680 bp

kan R

hmg1-trunc

Ban HI

Acc65. Kpni Mll I Spel ACC65 Kpni Sna BI U.S. Patent Feb. 5, 2013 Sheet 21 Of 37 US 8,367,395 B2

FIG.9B Schematic diagram of certain plasmids used herein Accó51 Kpni

ampr carb(i-)

pMB4660 : 8046 bp exon3 ill Mlul kan R &\ RN / Spe

Xho

Aw Ni (8261) HindIII (360) -

ERG9Orf pMB4789 pcrmut (TioA)(1890)s KAN erg9::URA3 -- BstEIII (2231.) Spe I (2290) Alw NI (5935) F1 Ori --- AfLII (3267)

URA3Orf Apa I (5220) Sma I (3695) Aw NI (5134) N. Nsi I (4204) ERG9 3'flank U.S. Patent Feb. 5, 2013 Sheet 22 Of 37 US 8,367,395 B2

FIG.9C Schematic diagram of certain plasmids used herein Acc65 (250) KpnI (254) w Spel (453) NheI (658) NotI (665) Mlul (672) Spel (810) AMP Sal I (1166)

if ura3 (1549)

KANr

Xbal (2734) F1 Or.

Spel (8353) Mlul (1) / Spel (139) carrP(-)

Spel (6349) Kpnl (6150) i. XbalNot I (2303)(232 I) AccG5I (6146) U.S. Patent Feb. 5, 2013 Sheet 23 Of 37 US 8,367,395 B2

FIG.9D pUC ori ampR

Kan R

pMB4628 8470 bp exOn2

pMB4638 9596 bp exOn3 U.S. Patent Feb. 5, 2013 Sheet 24 of 37 US 8,367,395 B2

FIG.9F

Kan R

ampR pMB4692

Kan R U.S. Patent Feb. 5, 2013 Sheet 25 Of 37 US 8,367,395 B2

FIG.9H pUC ori

pMB4698 8653 bp

ACC65 (250 FIG.91 / ci. Ea Spel (453)

HMG1 trune AMPrs BamHI (1783) AccG5 (1883) Kpni (1887) MIul (2178) KANr Spel (2316)

Xba I (4240) URA3Of U.S. Patent Feb. 5, 2013 Sheet 26 of 37 US 8,367,395 B2

FIG.9/ pUC ori AMP Acc65 (1008) Kpnl (1012) Spel (1211) KANr. Ay 4- Nhe I (1453) CrtW (Parva.)

Mlu I (2220) F1 Ori A Spel (2358) N. AccG5 (2588) Xba I (5832) - Kpnl (2592) Not I (5814) S-Accesi (4030) ADE 1 of Kpnl (4034)

FIG.9K pUC ori AMPr AccG5I (1008) Kpnl (1012) Spel (1211) KANr

crtW (Aurant.)

i-Miul (2377) fis Spel (2515)

- Acc65 (2745) Xbal (5989) Kpnl (2749) Nott (5971)

AccG5 (4187) ADE 1 of Kpnl (41.91) U.S. Patent Feb. 5, 2013 Sheet 27 Of 37 US 8,367,395 B2

FIG.9L AccG5I (241), Kpn I (245) Spel (444) Nhe I (650) Not I (657) Miu I (664)

AMP

Bam HI (2028) Spel (2034)

Sal I (2398)

URA3

Acc 65I (250) Kpn I (254) Spel (453)

Mlul (882) CrtZ (Xanth.) Mlul (1178) AMP Spel (1334) Sal I (1690)

KANr URA3Orf U.S. Patent Feb. 5, 2013 Sheet 28 of 37 US 8,367,395 B2

FIG 10A Vitamin D Vitamin D is a steroid hormone that functions to regulate specific gene expression following interaction with its intracellular receptor. The biologically active form of the hormone is 1,25-dihydroxy vitamin D3 (1,25 (OH)2 D3, also termed calcitriol). Calcitriol functions primarily to regulate calcium and phosphorous homeostasis.

| HO Ergosterol

HO

7-Dehydrocholesterol Vitamin D3

Active calitriol is derived from ergosterol (produced in plants) and from 7 dehydrocholesterol (produced in the skin). Ergocalciferol (vitamin D2) is formed by uv irradiation of ergosterol. In the skin 7-dehydrocholesterol is converted to cholecalciferol (vitamin D 3) following uv irradiation. Vitamin D2 and D3 are processed to D 2-calcitriol and D3-calcitriol, respectively, by the same enzymatic pathways in the body. Cholecalciferol (or egrocalciferol) are absorbed from the intestine and transported to the liver bound to a specific vitamin D-binding protein. In the liver cholecalciferol is hydroxylated at the 25 position by a specific D3-25 U.S. Patent Feb. 5, 2013 Sheet 29 Of 37 US 8,367,395 B2

FIG 10B

hydroxylase generating 25-hydroxy-D3 25-(OH) D3 which is the major circulating form of vitamin D. Conversion of 25-(OH)D3 to its biologically active form, calcitriol, occurs through the activity of a specific D3-1- hydroxylase present in the proximal convoluted tubules of the kidneys, and in bone and placenta. 25-(OH)D3 can also be hydroxylated at the 24 position by a specific D3-24-hydroxylase in the kidneys, intestine, placenta and cartilage.

HC

HO 25-hydroxyvitamin D3 1,25-dihydroxyvitamin D3 Calcitriol functions in concert with parathyroid hormone (PTH) and calcitonin to regulate serum calcium and phosphorous levels. PTH is released in response to low serum calcium and induces the production of calcitriol. In contrast, reduced levels of PTH stimulate synthesis of the inactive 24,25-(OD) 2D 3. In the intestinal epithelium, calcitriol functions as a steroid hormone in inducing the expression of CalbindinD28K, a protein involved in intestinal calcium absorption. The increased absorption of calcium ions requires ConComitant absorption of a negatively charged counter ion to maintain electrical neutrality. The predominant counter ion is Pi. When plasma calcium levels fall the major sites of action of calcitriol and PTH are bone where they stimulate bone resorption and the kidneys where they inhibit calcium excretion by stimulating reabsorption by the distal tubules, the role of calcitonin in calcium homeostasis is to decrease elevated serum calcium levels by inhibiting bone resorption. Clinical Significance of Vitamin D Deficiency As a result of the addition of vitamin D to milk, deficiencies in this vitamin are rare in this country. The main symptom of vitamin D deficiency in children is rickets and in adults is osteomalacia. Rickets is characterized improper mineralization during the development of the bones resulting in Soft bones. Osteomalacia is characterized by demineralization of previously formed bone leading to increased softness and susceptibility to fracture. U.S. Patent Feb. 5, 2013 Sheet 30 Of 37 US 8,367,395 B2

Coenzyme Q10 OH O H3CO CH3 H3CO CH3

H3CO N H H3CO N H H CH OH CH3 1O OH 3 1O Ubiquinol (CoQH2) Semiquinone radical (CoQH)

O

H3CO nS H CH3 O 1 O Ubiquinone (CoQ) U.S. Patent Feb. 5, 2013 Sheet 31 Of 37 US 8,367,395 B2

FIG 11B

O

21 3

O PHYLOOUNONE

O

21

O MENACRUNONES

VITAMINK U.S. Patent Feb. 5, 2013 Sheet 32 Of 37 US 8,367,395 B2

FIG. 11C Vitamin E - Structures and Chemistry

(8) HO H HO O H y-tocopherol O 3 SAM S -tocopherol g g SAM HO H HO O H C-tocopherol O B-tocopherol Tocopherols

OH

TOCotrienol Structure U.S. Patent Feb. 5, 2013 Sheet 33 of 37 US 8,367,395 B2

FIG. 12

Pyruvate + Glyceraldehyde-3-phosphate DOXP sythase (dxs) CO2 1-Deoxy-D-xylulose-5-phosphate (DOXP) DOXP reductoisomerase (dxr or ispC) (+ NADPH) 2-C-Methyl-D-erythritol-4-phosphate (MEP) CDP-ME synthase (ispD) (+ CTP) 4-(Cytidine-5'-diphospho)-2-C-methyl-D-erythritol (CDP-ME) CDP-ME kinase (ispE) IV (+ ATP) 2-Phospho-4-(Cytidine-5'-diphospho)-2-C-methyl-D-erythritol (PCDP-ME) MECP synthase (ispF) CMP 2-C-Methyl-D-erythritol-2,4,cyclodiphosphate (MECP) V | HMBPP synthase (ispG)

1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP) HMBPP reductase (isph) W (+ NADH)

isopentenyl diphosphate Dimethylally diphosphate (IPP) (DMAPP) U.S. Patent Feb. 5, 2013 Sheet 34 of 37 US 8,367,395 B2

FIG. 13 UBOUNONE BIOSYNTHESIS Phenylalanine, tyrosine and Chorismate MenF - - - - -CO 54.42 tryptophan biosynthesis ISOchorismate UbiC 4-Hydroxybenzoate O 2-SUCCinyl-6-hydroxy Q 24-cyclohexadiene all-trans-Hexapreny Polyisopentenyl- all-trans-Octaprenyl 1-carboxylate diphosphate C Odiphosphate O diphosphate 251. Coq2 25,139 UbiA MenG 4-Hydroxy-3- 3-Octaprenyl-4- 2-SUCCinyl 3-Hexaprenyl-4- O polyprenyl- hydroxyoenzoate benzoate hydroxybenzoate benz0ate Ubi) . . . 4.1.1 - UbiX Spiroclactone () 62.126 MenE 3-Hexaprenyl-4,5- , t dihydroxybenzoate 2-0ctaprenylphenol 2-SUCCInyl 2-Polyprenyl benzoyl-CoA 2.1.1.114 COc3 phenol Ubi shape-cy 2-Octaprenyl-6- 4.1.3.36 MenB 5-methoxybenzoate C hydroxyphenol () 4-polyprenyl 1,4-Dihydroxy phenol 2,164 UbiG 2-naphthoyl-CCA 2-Hexaprenyl-6- 2-0 6 methoxyphenol SE 3.12. 1413. Coq6 1,4-Dihydroxy 1.14.13. UbiH O 2-naphthoate 2-Hexaprenyl- 2-0ctaprenyl 6 methoxy-14- O 6-methoxy-1,4- benzoquinone benz0Cuinone 211. Cogs 2.1.1 - Ubi 2-Damethyl 2-Phylyl-1,4- menaquinone naphthoovinone

2-Hexaprenyl- 2-Octaprenyl 3-methyl-6-methoxy- O l 3.nety-6-methoy UbiE 1,4-benzoquinone 1,4-benzoquinone 1413. Coq7 Ubiquinol 1.14.1d-Ubif 2-0ctaprenyl-3-methy Menaquinone Phylloquinone 2-Hexaprenyl-3-methyl- 5-hydroxy-3-methoxy 5-hydroxy-6-methoxy 1,4-benzoquinone 1,4-benzoquinone 2.1.1.114 CO(3 2.1.164 UbiG O Ubiquinone

U.S. Patent US 8,367,395 B2

U.S. Patent Feb. 5, 2013 Sheet 37 Of 37 US 8,367,395 B2

US 8,367,395 B2 1. 2 PRODUCTION OF STEROLS IN sterol-producing capabilities as compared with otherwise OLEAGNOUSYEAST AND FUNG identical organisms that lack the modification(s). In some embodiments, the present invention provides a CROSS REFERENCE TO RELATED recombinant fungus. In certain embodiments, the recombi APPLICATIONS nant fungus is oleaginous in that it can accumulate lipid to at least about 20% of its dry cell weight; and the recombinant This application is a national phase application under 35 fungus produces at least one sterol compound, and can accu U.S.C. S371 of PCT International Application No. PCT/ mulate the produced sterol compound to at least about 1% of US2007/021091, filed Sep. 28, 2007, which is copending its dry cell weight; wherein the recombinant fungus com with, shares at least one common inventor with, and claims 10 prises at least one modification as compared with a parental priority to U.S. provisional patent application No. 60/848, fungus, which parental fungus both is not oleaginous and 582, filed Sep. 28, 2006, the contents of which are hereby does not accumulate the sterol compound to at least about 1% incorporated by reference in their entirety. of its dry cell weight, the at least one modification being selected from the group consisting of sterologenic modifica BACKGROUND OF THE INVENTION 15 tions, oleaginic modifications, and combinations thereof, and wherein the at least one modification alters oleaginicity of the Sterol compounds play a variety of important roles in bio recombinant fungus, confers to the recombinant fungus ole logical and industrial systems. For example, certain sterol aginy, confers to the recombinant fungus the ability to pro compounds are provitamins or vitamins and/or lubricants or duce the at least one sterol compound to a level at least about moisturizers; sterol compounds are also key synthetic inter 1% of its dry cell weight, or confers to the recombinant fungus mediates for Saponins and steroid hormones. Given the rap the ability to produce at least one sterol compound which the idly growing market for nutritional Supplements and other parental fungus does not produce. products containing sterol compounds, there remains a need In other embodiments, the recombinant fungus is oleagi for improved systems for enabling cost effective production, nous in that it can accumulate lipid to at least about 20% of its isolation, and/or formulation of Sterol compounds. 25 dry cell weight; and the recombinant fungus produces at least one sterol compound selected from the group consisting of SUMMARY OF THE INVENTION squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho lesterol (provitamin D3), and combinations thereof, and can The present invention provides improved systems for the accumulate the produced sterol compound to at least about biological production of certainsterol compounds. In particu 30 1% of its dry cell weight; wherein the recombinant fungus lar, the present invention provides improved systems for the comprises at least one modification as compared with a biological production of sterol compounds such as squalene, parental fungus, the at least one modification being selected lanosterol, Zymosterol, ergosterol, 7-dehydrocholesterol from the group consisting of sterologenic modifications, ole (provitamin D3) and/or related compounds (e.g., metabolite aginic modifications, and combinations thereof, and wherein derivatives, and particularly vitamin D compound(s)). 35 the at least one modification alters oleaginicity of the recom In one aspect, the invention encompasses the discovery that binant fungus, confers to the recombinant fungus oleaginy, it is desirable to produce certain sterols, and particularly to confers to the recombinant fungus the ability to produce the at produce squalene, lanosterol, Zymosterol, ergosterol, 7-dehy least one sterol compound to a level at least about 1% of its drocholesterol (provitamin D3) and/or vitamin D compounds dry cell weight, or confers to the recombinant fungus the in oleaginous organisms. In some embodiments, the present 40 ability to produce at least one sterol compound which the invention thus provides biological systems able to accumu parental fungus does not naturally produce. latesterols in lipid bodies. In some embodiments, the biologi In certain embodiments, the recombinant fungus is oleagi cal systems may produce higher levels of sterol compounds nous in that it can accumulate lipid to at least about 20% of its when Such compounds are sequestered in lipid bodies. dry cell weight; and the recombinant fungus produces at least Regardless of whether absolute levels are higher; however, 45 one sterol compound, and can accumulate the produced sterol compounds that are accumulated within lipid bodies in ole compound to at least about 1% of its dry cell weight; wherein aginous organisms are readily isolatable through isolation of the recombinant fungus is a member of a genus selected from the lipid bodies. the group consisting of Aspergillus, Blakeslea, Bottytis, Can The present invention therefore provides oleaginous fungi dida, Cercospora, Cryptococcus, Cunninghamella, (including, for example, yeast or other unicellular fungi) that 50 Fusarium (Gibberella), Kluyveromyces, Lipomyces, Mortier produce certain sterols, and in particular that produce ella, Mucor, Neurospora, Penicillium, Phycomyces, Pichia squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho (Hansenula), Puccinia, Pythium, Rhodosporidium, Rhodot lesterol (provitamin D3) and/or one or more vitamin D com orula, Saccharomyces, Sclerotium, Trichoderma, Trichos pounds. The present invention also provides methods of con poron, Xanthophyllomyces (Phafia), and Yarrowia; or is a structing Such yeast and fungi, methods of using such yeast 55 species selected from the group consisting of Aspergillus and fungi to produce the sterol compounds. The present terreus, Aspergillus nidulans, Aspergillus niger, Blakeslea invention further provides methods of preparing certainsterol trispora, Botrytis cinerea, Candida japonica, Candida pull compounds, and particularly of preparing squalene, lanos cherrima, Candida revkaufi, Candida tropicalis, Candida uti terol, Zymosterol, ergosterol, 7-dehydrocholesterol (provita lis, Cercospora nicotianae, Cryptococcus curvatus, Cun min D3) and/or one or more vitamin D compounds, as well as 60 ninghamella echinulata, Cunninghamella elegans, Fusarium compositions containing them, such as food or feed additives, fiujikuroi (Gibberella zeae), Kluyveromyces lactis, Lipomyces nutritional Supplements, machine oil products (e.g., lubri Starkeyi, Lipomyces lipoferus, Mortierella alpina, Mortier cants), or compositions for nutraceutical, pharmaceutical ella ramanniana, Mortierella isabellina, Mortierella vina and/or cosmetic applications. In particular, the present inven cea, Mucor circinelloides, Neurospora crassa, Phycomyces tion provides systems and methods for generating yeast and 65 blakesleanus, Pichia pastoris, Puccinia distincta, Pythium fungi containing one or more oleaginic and/or sterologenic irregulare, Rhodosporidium toruloides, Rhodotorula gluti modifications that increase the oleaginicity and/or alter their nis, Rhodotorula graminis, Rhodotorula mucilaginosa, US 8,367,395 B2 3 4 Rhodotorula pinicola, Rhodotorula gracilis, Saccharomyces The present invention provides a strain of Yarrowia lipoly cerevisiae, Sclerotium rolfsii, Trichoderma reesei, Trichos tica comprising one or more modifications selected from the poron cutaneum, TrichospOron pullans, Xanthophyllomyces group consisting of an oleaginic modification, a sterologenic dendrorhous (Phafia rhodozyma), and Yarrowia lipolytica; modification, and combinations thereof. Such that the Strain wherein the recombinant fungus comprises at least one modi accumulates from 1% to 15% of its dry cell weight as at least fication as compared with a parental fungus, the at least one one sterol compound. modification being selected from the group consisting of The present invention provides an engineered Y lipolytica sterologenic modifications, oleaginic modifications, and strain that produces squalene or 7-dehydrocholesterol (pro combinations thereof, and wherein the at least one modifica Vitamin D3), the strain containing one or more sterologenic tion alters oleaginicity of the recombinant fungus, confers to 10 modifications selected from the group consisting of the recombinant fungus oleaginy, confers to the recombinant decreased expression or activity of a Y lipolytica GGPP syn fungus the ability to produce the at least one sterol compound thase polypeptide; increased expression or activity of a trun to a level at least about 1% of its dry cell weight, or confers to cated HMG CoA reductase polypeptide; increased expres the recombinant fungus the ability to produce at least one sion or activity of an FPP synthase polypeptide; increased sterol compound which the parental fungus does not naturally 15 expression or activity of an IPP isomerase polypeptide: produce. increased expression or activity of an HMG-CoA synthase In some embodiments, the recombinant fungus is oleagi polypeptide; increased expression or activity of a mevalonate nous in that it can accumulate lipid to at least about 20% of its kinase polypeptide; increased expression or activity of a dry cell weight; and the recombinant fungus produces at least phosphomevalonate kinase polypeptide; increased expres one sterol compound selected from the group consisting of sion or activity of a mevalonate pyrophosphate decarboxylate squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho polypeptide; increased expression or activity of a cytosolic lesterol (provitamin D3), and combinations thereof, and can malic enzyme polypeptide; increased expression or activity accumulate the produced sterol compound to at least about of a malate dehydrogenase polypeptide; increased expression 1% of its dry cell weight; wherein the recombinant fungus is 25 or activity of an AMP deaminase polypeptide; increased a member of a genus selected from the group, consisting of expression or activity of a glucose 6 phosphate dehydroge Aspergillus, Blakeslea, Botrytis, Candida, Cercospora, nase polypeptide; increased expression or activity of a malate Cryptococcus, Cunninghamella, Fusarium (Gibberella), dehydrogenase homolog2 polypeptide; increased expression Kluyveromyces, Lipomyces, Mortierella, Mucor, Neuro or activity of a GND1-6-phosphogluconate dehydrogenase spora, Penicillium, Phycomyces, Pichia (Hansenula), Puc 30 polypeptide; increased expression or activity of a isocitrate cinia, Pythium, Rhodosporidium, Rhodotorula, Saccharomy dehydrogenase polypeptide; increased expression or activity CeS, Sclerotium, Trichoderma, TrichospOron, of a IDH2-isocitrate dehydrogenase polypeptide; increased Xanthophyllomyces (Phafia), and Yarrowia; or is of a species expression or activity of a fructose 1.6 bisphosphatase selected from the group consisting of Aspergillus terreus, polypeptide; increased expression or activity of a Erg10 Aspergillus nidulans, Aspergillus niger, Blakeslea trispora, 35 acetoacetyl CoA thiolase polypeptide; increased expression Botrytis cinerea, Candida japonica, Candida pulcherrima, or activity of a squalene synthase polypeptide; and combina Candida revkaufi, Candida tropicalis, Candida utilis, Cer tions thereof. cospora nicotianae, Cryptococcus curvatus, Cunning The present invention provides an engineered Y lipolytica hamella echinulata, Cunninghamella elegans, Fusarium strain containing a truncated HMG CoA reductase polypep filiilicuroi (Gibberella zeae), Kluyveromyces lactis, Lipomy 40 tide. The present invention provides an engineered Y lipoly ces starkeyi, Lipomyces lipoferus, Mortierella alpina, Mor tica strain having increased expression or activity of a tierella ramanniana, Mortierella isabellina, Mortierella squalene synthase gene. vinacea, Mucor circinelloides, Neurospora Crassa, Phycomy The present invention provides a method of producing a ces blakesleanus, Pichia pastoris, Puccinia distincta, sterol compound, the method comprising steps of cultivating Pythium irregulare, Rhodosporidium toruloides, Rhodot 45 the fungus of any one of the preceding claims under condi orula glutinis, Rhodotorula graminis, Rhodotorula mucilagi tions that allow production of the sterol compound; and iso nosa, Rhodotorula pinicola, Rhodotorula gracilis, Saccharo lating the produced sterol compound. myces cerevisiae, Sclerotium rolfsii, Trichoderma reesei, In some embodiments, a recombinant fungus accumulates TrichospOron cutaneum, TrichospOron pullans, Xanthophyl lipid in the form of cytoplasmic oil bodies. lomyces dendrorhous (Phafia rhodozyma), and Yarrowia 50 The present invention provides a composition comprising: lipolytica; wherein the recombinant fungus comprises at least lipid bodies; at least one sterol compound; and intact fungal one modification as compared with a parental fungus, the at cells. least one modification being selected from the group consist The present invention provides a composition comprising: ing of sterologenic modifications, oleaginic modifications, an oil Suspension comprising: lipid bodies; at least one sterol and combinations thereof, and wherein the at least one modi 55 compound; and intact fungal cells; a binder or filler. fication alters oleaginicity of the recombinant fungus, confers The present invention provides a composition comprising: to the recombinant fungus oleaginy, confers to the recombi an oil Suspension comprising: lipid bodies; at least one sterol nant fungus the ability to produce the at least one sterol compound; and intact fungal cells; and one or more other compound to a level at least about 1% of its dry cell weight, or agents selected from the group consisting of chelating agents, confers to the recombinant fungus the ability to produce at 60 pigments, salts, Surfactants, moisturizers, viscosity modifi least one sterol compound which the parental fungus does not ers, thickeners, emollients, fragrances, preservatives, and naturally produce. combinations thereof. In some embodiments, a recombinant fungus accumulates The present invention provides an isolated Sterol com at least one sterol compound to a level selected from the group pound composition, prepared by a method comprising steps consisting of above about 1%, above about 2%, above about 65 of cultivating a fungus under conditions that allow produc 3%, above about 5%, and above about 10% of the fungus' dry tion of a sterol compound; and isolating the produced Sterol cell weight. compound. US 8,367,395 B2 5 6 The present invention provides a sterol compound compo FIG. 12 depicts the mevalonate-independent isoprenoid sition comprising a Y lipolytica cell containing at least 1% biosynthesis pathway, also known as the DXP pathway, sterol compounds by weight. which typically operates in bacteria and in the plastids of The present invention provides a sterol compound compo plants. sition comprising Y lipolytica lipid bodies; and at least one FIG. 13 depicts the ubiquinone and vitamin K biosynthesis sterol compound, wherein the at least one sterol compound is pathways. present at a level that is at least 1% by weight of the lipid FIG. 14 illustrates biosynthetic pathways of aromatic bodies. amino acids, and the shikimate pathway for production of The present invention provides a feedstuff comprising a chorismate. A depiction of how these pathways feed into sterol compound in lipid bodies. 10 ubiquinone biosynthesis is depicted. Boxed numerical refer Additional aspects of the present invention will be apparent ences are IUBMB Enzyme Nomenclature EC numbers for to those of ordinary skill in the art from the present descrip enzymes catalyzing the relevant reaction. tion, including the appended claims. DEFINITIONS 15 BRIEF DESCRIPTION OF THE DRAWING Aromatic amino acid biosynthesis polypeptide: The term 'aromatic amino acid biosynthesis polypeptide' refers to any FIG. 1 depicts various biological pathways involved in polypeptide that is involved in the synthesis of aromatic steroid biosynthesis, including the sterol biosynthesis path amino acids in yeast and/or bacteria through chorismate and way that branches off at farnesyl pyrophosphate and includes the shikimate pathway. For example, as discussed herein, squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho anthranilate synthase, enzymes of the shikimate pathway, lesterol (provitamin D3) as well as various vitamin D com chorismate mutase, chorismate synthase, DAHP synthase, pounds. The enzymes involved in these pathways are and transketolase are all aromatic amino acid biosynthesis depicted either as abbreviations of their names or by their EC polypeptides. Each of these polypeptides is also a ubiquinone numbers. 25 biosynthesis polypeptide or a ubiquinone biosynthesis com FIG. 2 depicts how sufficient levels of acetyl-CoA and petitor for purposes of the present invention, as production of NADPH may be accumulated in the cytosol of oleaginous chorismate is a precursor in the synthesis of para-hydroxy organisms to allow for production of significant levels of benzoate for the biosynthesis of ubiquinone. Representative cytosolic lipids. Enzymes: 1. pyruvate decarboxylase; 2. examples of some of these enzymes are provided in Tables malate dehydrogenase; 3, malic enzyme; 4, pyruvate dehy 30 32-37. drogenase; 5, citrate synthase; 6, ATP-citrate lyase; 7, citrate/ Aromatic amino acid pathway: The 'aromatic amino acid malate translocase. pathway” is understood in the art to refer to a metabolic FIG. 3 depicts the mevalonate isoprenoid biosynthesis pathway that produces or utilizes shikimate pathway pathway, which typically operates in eukaryotes, including enzymes and chorismate in the production of phenylalanine, 35 tryptophan or tyrosine. As discussed herein, two different fungi, as well as the mevalonate-independent isoprenoid bio pathways can produce the ubiquinoid precursor para-hy synthesis pathway, also known as the DXP pathway, which droxybenzoate the first, the “shikimate pathway' is utilized in typically operates in bacteria and in the plastids of plants and prokaryotes and induces conversion of chorismate to para production ofisoprenoid precursors. hydroxybenzoate through the action of chorismate pyruvate FIG. 4 depicts a sterol biosynthesis pathway that converts 40 lyase; the second is utilized in mammalian systems and IPP (e.g., produced via the mevalonate or the non-mevalonate induces induction of para-hydroxybenzoate by derivation of pathway depicted in FIG. 3) into various sterol compounds tyrosine or phenylalanine. The term 'aromatic amino acid including, for example squalene, lanosterol, Zymosterol, pathway encompasses both of these pathways. Lower ergosterol, 7-dehydrocholesterol (provitamin D3), etc. eukaryotes such as yeast can utilize either method for pro FIG. 5 illustrates how intermediates in the isoprenoid bio 45 duction of para-hydroxybenzoate. synthesis pathway can be processed into biomolecules, Biosynthesis polypeptide: The term “biosynthesis including ubiquinones, carotenoids, sterols, etc. polypeptide' as used herein (typically in reference to a par FIG. 6 illustrates how intermediates in isoprenoid biosyn ticular compound or class of compounds), refers to polypep thesis feed into the biosynthetic pathway, and how some tides involved in the production of the compound or class of intermediates can be processed into other molecules. 50 compounds. In some embodiments of the invention, biosyn FIGS. 7A-I show an alignment of certain representative thesis polypeptides are synthetic enzymes that catalyze par fungal HMG-CoA reductase polypeptides. As can be seen, ticular steps in a synthesis pathway that ultimately produces a these polypeptides show very high identity across the cata relevant compound. In some embodiments, the term “biosyn lytic region, and also have complex membrane spanning thesis polypeptide' may also encompass polypeptides that do domains. In some embodiments of the invention, these mem 55 not themselves catalyze synthetic reactions, but that regulate brane-spanning domains are disrupted or are removed, so expression and/or activity of other polypeptides that do so. that, for example, a hyperactive version of the polypeptide Cso quinone biosynthesis polypeptide: The term "Cso may be produced. quinone biosynthesis polypeptide' refers to any polypeptide FIGS. 8A-C depicts Genbank and sequence information that is involved in the synthesis of a Cso quinone, for example for various Y lipolytica genes. 60 a polyprenyldiphosphate synthase polypeptide. To mention FIGS. 9A-M present schematic diagrams of certain plas but a few, these include, for example, pentaprenyl, hexapre mids used herein. nyl, heptaprenyl, octaprenyl, and/or Solanesyl (nonaprenyl) FIGS. 10A-B depict various vitamin D compounds. diphosphate synthase polypeptides (i.e., polypeptides that FIGS. 11 A-C depicts various quinone-derived compounds perform the chemical reactions performed by the pentapre including ubiquinone/Coenzyme Q10 in its various oxidated 65 nyl, hexaprenyl, heptaprenyl, octaprenyl, and Solanesyl forms (Panel A); vitamin K (Panel B); and vitamin E (Panel (nonaprenyl) polypeptides, respectively, listed in Tables C). 57-61 (see also Okada et al., Biochim. Biophys. Acta 1302: US 8,367,395 B2 7 8 217, 1996; Okada et al., J. Bacteriol. 179:5992, 1997). As will diapocarotenoids) and tetraterpenes (Cao carotenoids) as well be appreciated by those of ordinary skill in the art, in some as their oxygenated derivatives and other compounds that are, embodiments of the invention, Cso quinone biosynthesis for example, Css. Cso, Co, C7o, Cso in length or other lengths. polypeptides include polypeptides that affect the expression Many carotenoids have strong light absorbing properties and and/or activity of one or more other Cso quinone biosynthesis may range in length in excess of Coo. Co diapocarotenoids polypeptides. typically consist of six isoprenoid units joined in Such a Carotenogenic modification: The term "carotenogenic manner that the arrangement ofisoprenoid units is reversed at modification', as used herein, refers to a modification of a the center of the molecule so that the two central methyl host organism that adjusts production of one or more caro groups are in a 1.6-positional relationship and the remaining tenoids, as described herein. For example, a carotenogenic 10 modification may increase the production level of one or non-terminal methyl groups are in a 1.5-positional relation more carotenoids, and/or may alter relative production levels ship. Such Co carotenoids may be formally derived from the of different carotenoids. In principle, an inventive caroteno acyclic CoH structure, having a long central chain of con genic modification may be any chemical, physiological, jugated double bonds, by: (i) hydrogenation (ii) dehydroge genetic, or other modification that appropriately alters pro 15 nation, (iii) cyclization, (iv) oxidation, (v) esterification/gly duction of one or more carotenoids in a host organism pro cosylation, or any combination of these processes. Cao duced by that organism as compared with the level produced carotenoids typically consist of eight isoprenoid units joined in an otherwise identical organism not subject to the same in Such a manner that the arrangement of isoprenoid units is modification. In most embodiments, however, the caroteno reversed at the center of the molecule so that the two central genic modification will comprise a genetic modification, methyl groups are in a 1.6-positional relationship and the typically resulting in increased production of one or more remaining non-terminal methyl groups are in a 1.5-positional selected carotenoids. In some embodiments, the caroteno relationship. Such Cao carotenoids may be formally derived genic modification comprises at least one chemical, physi from the acyclic CHs structure, having along central chain ological, genetic, or other modification; in other embodi of conjugated double bonds, by (i) hydrogenation, (ii) dehy ments, the carotenogenic modification comprises more than 25 drogenation, (iii) cyclization, (iv) oxidation, (v) esterifica one chemical, physiological, genetic, or other modification. tion/glycosylation, or any combination of these processes. In certain aspects where more than one modification is uti The class of Cao carotenoids also includes certain compounds lized, such modifications can comprise any combination of that arise from rearrangements of the carbon skeleton, or by chemical, physiological, genetic, or other modification (e.g., the (formal) removal of part of this structure. More than 600 one or more genetic, chemical and/or physiological modifi 30 different carotenoids have been identified in nature; caro cation(s)). In some embodiments, the selected carotenoid is tenoids include but are not limited to: antheraxanthin, one or more of astaxanthin, B-carotene, canthaxanthin, lutein, adonirubin, adonixanthin, astaxanthin, canthaxanthin, cap lycopene, phytoene, Zeaxanthin, and/or modifications of Sorubrin, B-cryptoxanthin, C-carotene, B-carotene, B. p-caro Zeaxanthin or astaxanthin (e.g., glucoside, or other ester of tene, Ö-carotene, e-carotene, echinenone, 3-hydrox Zeaxanthin or astaxanthin). In some embodiments, the 35 yechinenone, 3'-hydroxyechinenone, Y-carotene, up-carotene, selected carotenoid is one or more Xanthophylls, and/or a 4-keto-Y-carotene, C-carotene, C-cryptoxanthin, deoxyflex modification thereof (e.g., glucoside, esterified Xantho ixanthin, diatoxanthin, 7,8-didehydroastaxanthin, didehy phylls). In certain embodiments, the selected xanthophyll is drolycopene, fucoxanthin, fucoxanthinol, isorenieratene, selected from the group consisting of astaxanthin, lutein, B-isorenieratene, lactucaxanthin, lutein, lycopene, myxobac Zeaxanthin, lycopene, and modifications thereof. In some 40 tone, neoxanthin, neurosporene, hydroxyneurosporene, peri embodiments, the selected carotenoid is one or more of astax dinin, phytoene, rhodopin, rhodopin glucoside, 4-keto-rubix anthin, B-carotene, canthaxanthin, lutein, lycopene, and anthin, siphonaxanthin, spheroidene, spheroidenone, Zeaxanthin and/or modifications of Zeaxanthin or astaxan spirilloxanthin, torulene, 4-keto-torulene, 3-hydroxy-4-keto thin. In some embodiments, the carotenoid is B-carotene. In torulene, uriolide, uriolide acetate, violaxanthin, Zeaxanthin Some embodiments, the selected carotenoid is astaxanthin. In 45 B-diglucoside, zeaxanthin, and C30 carotenoids. Addition Some embodiments, the selected carotenoid is other than ally, carotenoid compounds include derivatives of these B-carotene. molecules, which may include hydroxy-, methoxy-, oxo-. Carotenogenic polypeptide: The term "carotenogenic epoxy-, carboxy-, or aldehydic functional groups. Further, polypeptide', as used herein, refers to any polypeptide that is included carotenoid compounds include ester (e.g., glycoside involved in the process of producing carotenoids in a cell, and 50 ester, fatty acid ester) and Sulfate derivatives (e.g., esterified may include polypeptides that are involved in processes other Xanthophylls). than carotenoid production but whose activities affect the Carotenoid biosynthesis polypeptide: The term “caro extent or level of production of one or more carotenoids, for tenoid biosynthesis polypeptide' refers to any polypeptide example by Scavenging a Substrate or reactant utilized by a that is involved in the synthesis of one or more carotenoids. To carotenoid polypeptide that is directly involved in carotenoid 55 mention but a few, these carotenoid biosynthesis polypep production. Carotenogenic polypeptides include carotenoid tides include, for example, polypeptides of phytoene Syn biosynthesis polypeptides, as defined herein. thase, phytoene dehydrogenase (or desaturase), lycopene Carotenoid: The term "carotenoid' is understood in the art cyclase, carotenoid ketolase, carotenoid hydroxylase, astax to refer to a structurally diverse class of pigments derived anthin synthase, carotenoid epsilon hydroxylase, lycopene from isoprenoid pathway intermediates. The commitment 60 cyclase (beta and epsilon Subunits), carotenoid glucosyltrans step in carotenoid biosynthesis is the formation of phytoene ferase, and acyl CoA:diacy glycerol acyltransferase. In some from geranylgeranyl pyrophosphate. Carotenoids can be acy instances, a single gene may encode a protein with multiple clic or cyclic, and may or may not contain oxygen, so that the carotenoid biosynthesis polypeptide activities. Representa term carotenoids include both carotenes and Xanthophylls. In tive examples of carotenoid biosynthesis polypeptide general, carotenoids are hydrocarbon compounds having a 65 sequences are presented in Tables 17a-21 and 38-41. As will conjugated polyene carbon skeleton formally derived from be appreciated by those of ordinary skill in the art, in some the five-carbon compound IPP including triterpenes (Cso embodiments of the invention, carotenoid biosynthesis US 8,367,395 B2 10 polypeptides include polypeptides that affect the expression when the gene sequence encoding a given polypeptide is and/or activity of one or more other carotenoid biosynthesis difficult to optimize to conform to the codon preference of the polypeptides. host cell due to experimental (e.g., cloning) and/or other Effective amount. The term “effective amount” is used reasons. In certain embodiments, the gene sequence encoding herein to describe concentrations or amounts of compounds a given polypeptide is optimized even when Such a gene and/or compositions that, when administered to a Subject, sequence is derived from the host cell itself (and thus is not achieve a desired therapeutic or physiological effect. heterologous). For example, a gene sequence encoding a FPP biosynthesis polypeptides: The term “FPP biosynthe polypeptide of interest may not be codon optimized for sis polypeptide' refers to any polypeptide that is involved in expression in a given host cell even though Such a gene the synthesis of farnesyl pyrophosphate. As discussed herein, 10 sequence is isolated from the host cell Strain. In such embodi farnesyl pyrophosphate represents the branchpoint between ments, the gene sequence may be further optimized to the sterol biosynthesis pathway and the carotenoid and other account for codon preferences of the host cell. Those of biosynthesis pathways. One specific example of an FPP bio ordinary skill in the art will be aware of host cell codon synthesis polypeptide is FPP synthase. Representative preferences and will be able to employ inventive methods and examples of FPP synthase polypeptide sequences are pre 15 compositions disclosed herein to optimize expression of a sented in Table 14. As will be appreciated by those of ordinary given polypeptide in the host cell. skill in the art, in some embodiments of the invention, FPP Host cell: As used herein, the “host cell is a fungal cell or biosynthesis polypeptides include polypeptides that affect the yeast cell that is manipulated according to the present inven expression and/or activity of one or more other FPP biosyn tion to accumulate lipid and/or to express one or more sterol thesis polypeptides. compounds as described herein. A “modified host cell’, as Gene: The term “gene’’, as used herein, generally refers to used herein, is any host cell which has been modified, engi a nucleic acid encoding a polypeptide, optionally including neered, or manipulated in accordance with the present inven certain regulatory elements that may affect expression of one tion as compared with a parental cell. In some embodiments, or more gene products (i.e., RNA or protein). the modified host cell has at least one sterologenic and/or at Heterologous: The term "heterologous', as used herein to 25 least one oleagenic modification. In some embodiments, the refer to genes or polypeptides, refers to a gene or polypeptide modified host cell containing at least one oleaginic modifica that does not naturally occur in the organism in which it is tion and/or one sterologenic modification further has at least being expressed. It will be understood that, in general, when one carotenogenic modification and/or at least one quinono a heterologous gene or polypeptide is selected for introduc genic modification. In some embodiments, the parental cell is tion into and/or expression by a host cell, the particular source 30 a naturally occurring parental cell. organism from which the heterologous gene or polypeptide Isolated: The term "isolated’, as used herein, means that may be selected is not essential to the practice of the present the isolated entity has been separated from at least one com invention. Relevant considerations may include, for example, ponent with which it was previously associated. When most how closely related the potential source and host organisms other components have been removed, the isolated entity is are in evolution, or how related the source organism is with 35 “purified’ or “concentrated”. Isolation and/or purification other source organisms from which sequences of other rel and/or concentration may be performed using any techniques evant polypeptides have been selected. Where a plurality of known in the art including, for example, fractionation, extrac different heterologous polypeptides are to be introduced into tion, precipitation, or other separation. and/or expressed by a host cell, different polypeptides may be Isoprenoid biosynthesis polypeptide: The term “iso from different source organisms, or from the same source 40 prenoid biosynthesis polypeptide' refers to any polypeptide organism. To give but one example, in Some cases, individual that is involved in the synthesis of isoprenoids. For example, polypeptides may represent individual Subunits of a complex as discussed herein, acetoacetyl-CoA thiolase, HMG-CoA protein activity and/or may be required to work in concert synthase, HMG-CoA reductase, mevalonate kinase, phos with other polypeptides in order to achieve the goals of the phomevalonate kinase, mevalonate pyrophosphate decar present invention. In some embodiments, it will often be 45 boxylase, and IPP isomerase are all involved in the meva desirable for such polypeptides to be from the same source lonate pathway for isoprenoid biosynthesis. Each of these organism, and/or to be sufficiently related to function appro proteins is also an isoprenoid biosynthesis polypeptide for priately when expressed together in a host cell. In some purposes of the present invention, and sequences of represen embodiments, such polypeptides may be from different, even tative examples of these enzymes are provided in Tables 7-13. unrelated source organisms. It will further be understood that, 50 As will be appreciated by those of ordinary skill in the art, in where a heterologous polypeptide is to be expressed in a host Some embodiments of the invention, isoprenoid biosynthesis cell, it will often be desirable to utilize nucleic acid sequences polypeptides include polypeptides that affect the expression encoding the polypeptide that have been adjusted to accom and/or activity of one or more other isoprenoid biosynthesis modate codon preferences of the host cell and/or to link the polypeptides (e.g., of one or more enzymes that participate(s) encoding sequences with regulatory elements active in the 55 in isoprenoid synthesis). Thus, for instance, transcription fac host cell. For example, when the host cell is a Yarrowia strain tors that regulate expression of isoprenoid biosynthesis (e.g., Yarrowia lipolytica), it will often be desirable to alter enzymes can be isoprenoid biosynthesis polypeptides for pur the gene sequence encoding a given polypeptide Such that it poses of the present invention. conforms more closely with the codon preferences of Such a Isoprenoid pathway: The term "isoprenoid pathway' is Yarrowia Strain. In certain embodiments, a gene sequence 60 understood in the art to refer to a metabolic pathway that encoding a given polypeptide is altered to conform more either produces or utilizes the five-carbon metabolite isopen closely with the codon preference of a species related to the tyl pyrophosphate (IPP). As discussed herein, two different host cell. For example, when the host cell is a Yarrowia strain pathways can produce the common isoprenoid precursor (e.g., Yarrowia lipolytica), it will often be desirable to alter IPP the “mevalonate pathway' and the “non-mevalonate the gene sequence encoding a given polypeptide Such that it 65 pathway”. The term "isoprenoid pathway' is sufficiently gen conforms more closely with the codon preferences of a eral to encompass both of these types of pathway. Biosynthe related fungal strain. Such embodiments are advantageous sis of isoprenoids from IPP occurs by polymerization of sev US 8,367,395 B2 11 12 eral five-carbon isoprene subunits. Isoprenoid metabolites and 1.6.1.2). Alternative or additional strategies to promote derived from IPP are of varying size and chemical structure, oleaginy may include one or more of the following: (1) including both cyclic and acyclic molecules. Isoprenoid increased or heterologous expression of one or more of acyl metabolites include, but are not limited to, monoterpenes, CoA:diacylglycerol acyltransferase (e.g., DGA1; sesquiterpenes, diterpenes, Sterols, and polyprenols such as YALIOE32769g); phospholipid:diacylglycerol acyltrans carotenoids. ferase (e.g., LRO1; YALIOE 16797g); and acyl-CoA:choles Oleaginic modification: The term "oleaginic modifica terol acyltransferase (e.g., ARE genes such as ARE1, ARE2. tion', as used herein, refers to a modification of a host organ YALIOF06578g), which are involved in triglyceride synthesis ism that adjusts the desirable oleaginy of that host organism, (Kalscheuer et al. Appl Environ Microbiol p. 7119-7125, as described herein. In some cases, the host organism will 10 2004; Oelkers et al. J Biol Chem 277:8877-8881, 2002; and already be oleaginous in that it will have the ability to accu Sorger et al. J Biol Chem 279:31190-31196, 2004), (2) mulate lipid to at least about 20% of its dry cell weight. It may decreased expression of triglyceride lipases (e.g., TGL3 and/ nonetheless be desirable to apply an oleaginic modification to or TGL4:YALIOD17534g and/or YALIOF10010g (Kurat et Such an organism, in accordance with the present invention, al. J Biol Chem 281:491-500, 2006); and (3) decreased for example to increase (or, in Some cases, possibly to 15 expression of one or more acyl-coenzyme A oxidase activi decrease) its total lipid accumulation, or to adjust the types or ties, for example encoded by POX genes (e.g. POX1, POX2, amounts of one or more particular lipids it accumulates (e.g., POX3, POX4, POX5; YALIOC238599, YALIOD24750g, to increase relative accumulation of triacylglycerol). In other YALIOE06567g, YALIOE27654g, YALIOE32835g, cases, the host organism, may be non-olleaginous (though YALIOF10857g; see, for example, Mlickova et al. Appl Envi may contain some enzymatic and/or regulatory components rom Microbiol 70: 3918-3924, 2004; Binns et al. J Cell Biol used in other organisms to accumulate lipid), and may require 173:719, 2006). Each of these proteins is an oleaginic peptide oleaginic modification in order to become oleaginous in for the purposes of the present invention, and sequences of accordance with the present invention. The present invention representative examples of these enzymes are provided in also contemplates application of oleaginic modification to Tables 66-81. non-olleaginous host strains such that their oleaginicity is 25 Oleaginous: The term "oleaginous, as used herein, refers increased even though, even after being modified, they may to ability of an organism to accumulate lipid to at least about not be oleaginous as defined herein. In principle, the oleaginic 20% of its dry cell weight. In certain embodiments of the modification may be any chemical, physiological, genetic, or invention, oleaginous yeast or fungi accumulate lipid to at other modification that appropriately alters oleaginy of a host least about 25% of their dry cell weight. In other embodi organism as compared with an otherwise identical organism 30 ments, inventive oleaginous yeast or fungi accumulate lipid not subjected to the oleaginic modification. In most embodi within the range of about 20-45% (e.g. 20%, 21%. 22%, 23%, ments, however, the oleaginic modification will comprise a 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, genetic modification, typically resulting in increased produc 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, tion and/or activity of one or more oleaginic polypeptides. In 44%, 45%) of their dry cell weight. In some embodiments, Some embodiments, the oleaginic modification comprises at 35 oleaginous organisms may accumulate lipid to as much as least one chemical, physiological, genetic, or other modifica about 70% of their dry cell weight. In some embodiments of tion; in other embodiments, the oleaginic modification com the invention, oleaginous organisms may accumulate a large prises more than one chemical, physiological, genetic, or fraction of total lipid accumulation in the form of triacylglyc other modification. In certain aspects where more than one erol. In certain embodiments, the majority of the accumulated modification is utilized, such modifications can comprise any 40 lipid is in the form of triacylglycerol. Alternatively or addi combination of chemical, physiological, genetic, or other tionally, the lipid may accumulate in the form of intracellular modification (e.g., one or more genetic, chemical and/or lipid bodies, or oil bodies. In certain embodiments, the physiological modification(s)). present invention utilizes yeast or fungi that are naturally Oleaginic polypeptide: The term "oleaginic polypeptide'. oleaginous. In some aspects, naturally oleaginous organisms as used herein, refers to any polypeptide that is involved in the 45 are manipulated (e.g., genetically, chemically, or otherwise) process of lipid accumulation in a cell and may include so as to father increase the level of accumulated lipid in the polypeptides that are involved in processes other than lipid organism. In other embodiments, yeast or fungi that are not biosynthesis but whose activities affect the extent or level of naturally oleaginous are manipulated (e.g., genetically, accumulation of one or more lipids, for example by Scaveng chemically, or otherwise) to accumulate lipid as described inga Substrate or reactant utilized by an oleaginic polypeptide 50 herein. For example, for the purposes of the present invention, that is directly involved in lipid accumulation. For example, Saccharomyces cerevisiae, Xanthophyllomyces dendrorhous as discussed herein, acetyl-CoA carboxylase, pyruvate decar (Phafia rhodozyma), and Candida utilis are not naturally boxylase, isocitrate dehydrogenase, ATP-citrate lyase, malic oleaginous fungi. enzyme, malate dehydrogenase, and AMP deaminase, among PHB polypeptide or PHB biosynthesis polypeptide: The other proteins, are all involved in lipid accumulation in cells. 55 terms “PHB polypeptide' or “PHB biosynthesis polypep In general, reducing the activity of pyruvate decarboxylase or tide' as used herein refers to a polypeptide that is involved in isocitrate dehydrogenase, and/or increasing the activity of the synthesis of para-hydroxybenzoate from chorismate. In acetyl CoA carboxylase, ATP-citrate lyase, malic enzyme, prokaryotes and lower eukaryotes, synthesis of para-hy malate dehydrogenase, and/or AMP deaminase is expected to droxybenzoate occurs by the action of chorismate pyruvate promote oleaginy. Each of these proteins is an oleaginic pep 60 lyase. Biosynthesis of para-hydroxybenzoate from tyrosine tide for the purposes of the present invention, and sequences or phenylalanine occurs through a five-step process in mam of representative examples of these enzymes are provided in malian cells. Lower eukaryotes Such as yeast can utilize either Tables 1-6, and 65. Other peptides that can be involved in method for production of para-hydroxybenzoate. For regenerating NADPH may include, for example, 6-phospho example, enzymes of the shikimate pathway, chorismate Syn gluconate dehydrogenase (gnd); Fructose 1.6 bisphosphatase 65 thase, DAHP synthase, and transketolase are all PHB biosyn (fbp); Glucose 6 phosphate dehydrogenase (g6pd); NADH thesis polypeptides. Each of these polypeptides is also a kinase (EC 2.7.1.86); and/or transhydrogenase (EC 1.6.1.1 ubiquinone biosynthesis polypeptide or a ubiquinone biosyn US 8,367,395 B2 13 14 thesis competitor polypeptide for purposes of the present quinone derived compounds (e.g., ubiquinone, Vitamin K invention. Exemplary PHB polypeptides are provided in compounds, vitamin E compounds, etc.), as described herein. Tables 33 and 35-37. For example, a quinonogenic modification may increase the Polypeptide: The term “polypeptide', as used herein, gen production level of a particular quinone derived compound, erally has its art-recognized meaning of a polymer of at least 5 or of a variety of different quinone derived compounds. In three amino acids. However, the term is also used to refer to Some embodiments of the invention, production of a particu specific functional classes of polypeptides, such as, for lar quinone derived compound may be increased while pro example, biosynthesis polypeptides, Cso quinone biosynthe duction of other quinone derived compounds is decreased. In sis polypeptides, carotenogenic polypeptides, carotenoid bio Some embodiments of the invention, production of a plurality synthesis polypeptides, FPP biosynthesis polypeptides, iso- 10 of different quinone derived compounds is increased. In prin prenoid biosynthesis polypeptides, oleaginic polypeptides, ciple, an inventive quinonogenic modification may be any sterol biosynthesis polypeptides, sterologenic polypeptides, chemical, physiological, genetic, or other modification that ubiquinogenic polypeptides, ubiquinone biosynthesis appropriately alters production of one or more quinone polypeptides, Vitamin D biosynthesis polypeptides, Vitamin derived compounds in a host organism produced by that E biosynthesis polypeptides, Vitamin K biosynthesis 15 organism as compared with the level produced in an other polypeptides, etc. For each Such class, the present specifica wise identical organism not subject to the same modification. tion provides several examples of known sequences of Such In most embodiments, however, the quinonogenic modifica polypeptides. Those of ordinary skill in the art will appreci tion will comprise a genetic modification, typically resulting ate, however, that the term “polypeptide' is intended to be in increased production of one or more quinone derived com Sufficiently general as to encompass not only polypeptides 20 pounds (e.g., ubiquinone, Vitamin K compounds, vitamin E having the complete sequence recited herein (or in a reference compounds). In some embodiments, the quinonogenic modi or database specifically mentioned herein), but also to encom fication comprises at least one chemical, physiological, pass polypeptides that represent functional fragments (i.e., genetic, or other modification; in other embodiments, the fragments retaining at least one activity) of Such complete quinonogenic modification comprises more than one chemi polypeptides. Moreoever, those of ordinary skill in the art 25 cal, physiological, genetic, or other modification. In certain understand that protein sequences generally tolerate some aspects where more than one modification is utilized. Such Substitution without destroying activity. Thus, any polypep modifications can comprise any combination of chemical, tide that retains activity and shares at least about 30-40% physiological, genetic, or other modification (e.g., one or overall sequence identity, often greater than about 50%, 60%, more genetic, chemical and/or physiological modification(s)) 70%, or 80%, and further usually including at least one region 30 of much higher identity, often greater than 90% or even 95%, Quinonogenic polypeptide: The term "quinonogenic 96%, 97%, 98%, or 99% in one or more highly conserved polypeptide', as used herein, refers to any polypeptide whose regions (e.g., isocitrate dehydrogenase polypeptides often activity in a cell increases production of one or more quinone share a conserved AMP-binding motif HMG-CoA reductase derived compounds (e.g., ubiquinone, Vitamin K compounds, polypeptides typically include a highly conserved catalytic 35 Vitamin E compounds) in that cell. The term encompasses domain see, for example, FIG. 7; acetyl coA carboxylase both polypeptides that are directly involved in quinone typically has a carboxyl transferase domain; see, for example, derived compound synthesis and those whose expression or Downing et al., Chem. Abs. 93:484, 1980; Gil et al., Cell activity affects the extent or level of production of one or more 41:249, 1985; Jitrapakdee et al., Curr Protein Pept Sci. 4:217, quinone derived compounds, for example by scavenging a 2003: U.S. Pat. No. 5,349,126, each of which is incorporated 40 Substrate or reactant utilized by a quinone biosynthetic herein by reference in its entirety), usually encompassing at polypeptide that is directly involved in quinone derived com least 3-4 and often up to 20 or more amino acids, with another pound production. Quinonologenic polypeptides include iso polypeptide of the same class, is encompassed within the prenoid biosynthesis polypeptides, ubiquinone biosynthesis relevant term “polypeptide' as used herein. Other regions of polypeptides, Cso quinone biosynthesis polypeptides, Vita similarity and/or identity can be determined by those of ordi- 45 min E biosynthesis polypeptides, and vitamin K biosynthesis nary skill in the art by analysis of the sequences of various polypeptides. Quinonogenic polypeptides may also include polypeptides presented in the Tables herein. ubiquinogenic polypeptides, etc. The term also encompasses Quinone biosynthesis polypeptide: A "quinone biosynthe polypeptides that may affect the extent to which one or more sis polypeptide', as that term is used herein, refers to any quinone derived compounds is accumulated in lipid bodies. polypeptide involved in the synthesis of one or more quinone 50 Small Molecule: In general, a small molecule is understood derived compounds, as described herein. In particular, in the art to be an organic molecule that is less than about 5 quinone biosynthesis polypeptides include ubiquinone bio kilodaltons (Kd) in size. In some embodiments, the Small synthesis polypeptides, Cso quinone biosynthesis polypep molecule is less than about 3 Kid, 2 Kid, or 1 Kid. In some tides, vitamin K biosynthesis polypeptides, and vitamin E embodiments, the small molecule is less than about 800 dal biosynthesis polypeptides. 55 tons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In Quinone derived compounds: The term "quinone derived Some embodiments, Small molecules are non-polymeric. In compounds’ is used herein to refer to certain compounds that Some embodiments, Small molecules are not proteins, pep either contain a quinone moiety or are derived from a precur tides, oramino acids. In some embodiments, Small molecules Sor that contains a quinone moiety. In particular, quinone are not nucleic acids or nucleotides. In some embodiments, derived compounds according to the present invention are 60 Small molecules are not saccharides or polysaccharides. ubiquinones, Cso quinone compounds, vitamin E com Source organism: The term "source organism’, as used pounds, and/or vitamin K compounds. Structures of repre herein, refers to the organism in which a particular polypep sentative quinone derived compounds are presented in FIG. tide sequence can be found in nature. Thus, for example, if 11. one or more heterologous polypeptides is/are being expressed Quinonogenic modification: The term "quinonogenic 65 in a host organism, the organism in which the polypeptides modification, as used herein, refers to refers to a modification are expressed in nature (and/or from which their genes were of a host organism that adjusts production of one or more originally cloned) is referred to as the “source organism'. US 8,367,395 B2 15 16 Where multiple heterologous polypeptides are being one or more sterol compounds. For example, sterol biosyn expressed in a host organism, one or more source organism(s) thesis polypeptides include squalene synthase, which cataly may be utilized for independent selection of each of the ses conversion of farnesyl pyrophosphate to presqualene heterologous polypeptide(s). It will be appreciated that any pyrophosphate, and further catalyzes conversion of and all organisms that naturally contain relevant polypeptide presqualene pyrophosphate to squalene (i.e., enzyme 2.5.1.21 sequences may be used as source organisms in accordance in FIG. 1). In some embodiments of the invention, sterol with the present invention. Representative source organisms biosynthesis polypeptides further include one or more include, for example, animal, mammalian, insect, plant, fun polypeptides involved in metabolizing squalene into a vita gal, yeast, algal, bacterial, archaebacterial, cyanobacterial, min D compound. Thus, Sterol biosynthesis polypeptides can and protozoal source organisms. 10 include one or more of the 1.14.99.7, 5.4.99.7, 5.4.99.8, Subject. The term "subject' is used throughout the present 5.3.3.5, 1.14.21.6, 1.14.15.-, 1.14.13.13 enzyme polypep specification to describe an animal, in most instances a tides depicted in FIG.1, as well as other enzyme polypeptides human, to whom inventive compositions are administered. involved in the illustrated pathways. Furthermore, sterol bio Sterol biosynthesis competitor: The term “sterol biosyn synthesis polypeptides can include one or more of the enzyme thesis competitor, as used herein, refers to an agent whose 15 polypeptides depicted in FIG.4, including, for example, C-14 presence or activity in a cell either (1) reduces the level of demethylase (ERG9), squalene monooxygenase (ERG1), isopentenyl pyrophosphate (IPP) and/or farnesyl pyrophos 2,3-oxidosqualene-lanosterol synthase (ERG7), C-1 dem phate (FPP) available to enter the sterol biosynthesis path ethylase (ERG11), C-14 reductase (ERG24), C-4 methyloxi way; or (2) reduces the level or activity of one or more sterol dase (ERG25), C-4 decarboxylase (ERG26), 3-ketoreductase biosysthesis polypeptides; or both. The term “sterol biosyn (ERG27), C-24 methyltransferase (ERG6). A8-7 isomerase thesis competitor” encompasses both polypeptide and non (ERG2), C-5 desaturase (ERG3), C-22 desaturase (ERG5) polypeptide (e.g., Small molecule) inhibitoragents. Particular and/or C-24 reductase (ERG4) polypeptides, and/or other examples of sterol biosynthesis competitor agents act on iso polypeptides involved in producing one or more vitamin D prenoid intermediates prior to IPP or FPP, such that less IPP compounds (e.g., vitamin D2, Vitamin D3, or a precursor or FPP is generated (see, for example, FIG. 1). Other 25 thereof). As will be appreciated by those of ordinary skill in examples include agents that act downstream of IPP and/or the art, in Some embodiments of the invention, Sterol biosyn FPP and increase their partitioning into other metabolic path thesis polypeptides include polypeptides that affect the ways (e.g., toward carotenoid biosynthesis, ubiquinone bio expression and/or activity of one or more other Sterol biosyn synthesis, vitamin E biosyntheses, vitamin K biosynthesis, thesis polypeptides. Thus, for instance, transcription factors etc). Thus, Sterol biosynthesis competitor agents include, but 30 that regulate expression of sterol biosynthesis enzymes can be are not limited to, geranylgeranyl pyrophosphate (GGPP) sterol biosynthesis polypeptides for purposes of the present synthase polypeptides, carotenogenic polypeptides, ubiqui invention. To give but a couple of examples, the S. cerevisiae nogenic polypeptides, vitamin E biosynthesis polypeptides, Upc2 and YLR228c genes, and the Y lipolytica Vitamin K biosynthesis polypeptides, Cso quinone biosyn YALIOB00660g gene encode transcription factors that are thesis polypeptides, etc. Furthermore, those of ordinary skill 35 sterol biosynthesis polypeptides according to certain embodi in the art will appreciate that certain competitor agents that do ments of the present invention. For instance, the semidomi notact as inhibitors of sterol biosynthesis generally can none nant upc2-1 point mutation (G888D) exhibits increased sterol theless act as inhibitors of biosynthesis of a particular sterol levels (Crowley et al., J. Bacterial 180:4177-4183, 1998). compound. For instance, agents that inhibit conversion of CorrespondingYLR228c mutants have been made and tested squalene to squalene-2,3-epoxide may well increase produc 40 (Shianna et al., J Bacterial 183:830, 2001); such mutants may tion of one sterol compound (squalene) while inhibiting pro be useful in accordance with the present invention, as may be duction of one or more other sterol compounds (e.g., vitamin YALIOB00660g derivatives with corresponding upc2-1 D compound(s)). Similarly, agents that increase metabolism mutation(s). Representative examples of certain sterol bio of lanosterol to 14-demethyl lanosterol and/or decrease synthesis polypeptide sequences are presented in Tables 16 metabolism of lanosterol to 2, 25 dihydrolanosterol may act 45 and 83-95. as competitor agents with regard to vitamin D biosynthesis Sterol compound: The term “sterol compound, as used specifically but not with regard to sterol compounds gener herein, refers in general to squalene or any metabolite that is ally. Those of ordinary skill in the art, considering the known derived from squalene, either through a biochemical reaction metabolic pathways relating to sterol production and/or within a host cell, through whole-cell or enzymatic biocata metabolism (see, for example, FIG. 1 and other Figures and 50 lytic treatment of a distinct sterol compound, or through references herein) will readily appreciate a variety of other chemical or physical treatments (e.g. ultraviolet irradiation). particular sterol biosynthesis competitors, including sterol In particular, the term encompasses squalene, lanosterol, biosynthesis polypeptides. Representative examples of sterol Zymosterol, ergosterol, 7-dehydrocholesterol (provitamin biosynthesis polypeptide sequences are presented in Tables D3) and/or vitamin D compounds. 83-96. As will be appreciated by those of ordinary skill in the 55 Sterologenic modification: The term “sterologenic modi art, in Some embodiments of the invention, Sterol biosynthesis fication', as used herein, refers to a modification of a host polypeptides include polypeptides that affect the expression organism that adjusts production of one or more sterol and/or activity of one or more other sterol biosynthesis compounds (e.g., squalene, lanosterol, Zymosterol, ergos polypeptides. terol, 7-dehydrocholesterol (provitamin D3), vitamin D com Sterol biosynthesis polypeptide: The term “sterol biosyn 60 pound(s), etc.), as described herein. For example, a sterolo thesis polypeptide', as used herein, refers to any polypeptide genic modification may increase the production level of a that is involved in the synthesis of one or more sterol com particular sterol compound, or of a variety of different sterol pounds. Thus, Sterol biosynthesis polypeptides can include compounds. In some embodiments of the invention, produc isoprenoid biosynthesis polypeptides to the extent that they tion of a particular sterol compound may be increased while are involved in production ofisopentyl pyrophosphate. More 65 production of other sterol compounds is decreased. In some over, the term refers to any polypeptide that acts downstream embodiments of the invention, production of a plurality of offarnesyl pyrophosphate and is involved in the production of different sterol compounds is increased. In principle, an US 8,367,395 B2 17 18 inventive sterologenic modification may be any chemical, pounds and is based upon the length of the terpenoid side physiological, genetic, or other modification that appropri chain. Those which contain an isoprene side chain are also ately alters production of one or more sterol compounds in a referred to by the term coenzymes Q. A benzoquinone of this host organism produced by that organism as compared with family is therefore properly referred to as either “Coenzyme the level produced in an otherwise identical organism not 5 Qn, where n is an integer from one to twelve and designates Subject to the same modification. In most embodiments, how the number of isoprenoid units in the side chain, or alterna ever, the Sterologenic modification will comprise a genetic tively, “ubiquinone (x)' where x designates the total number modification, typically resulting in increased production of of carbon atoms in the side chain and is a multiple of five. one or more sterol compounds (e.g., squalene, lanosterol, Typically, n is an integer ranging from 0 to 12, in particular Zymosterol, ergosterol, 7-dehydrocholesterol (provitamin 10 D3) or vitamin D compound(s)). In some embodiments, the from 1 to 12, and more particularly 5, 6, 7, 8, 9, or 10. For sterologenic modification comprises at least one chemical, example, the most common ubiquinone in animals has a ten physiological, genetic, or other modification; in other isoprenoid side chain and is referred to as either Coenzyme embodiments, the sterologenic modification comprises more Q10 or ubiquinone (50). In other organisms (e.g. fungi, bac than one chemical, physiological, genetic, or other modifica 15 teria), ubiquinones, for example C (CoQ5), C (CoQ6), C, tion. In certain aspects where more than one modification is (CoQ7), Cs (CoQ8), or C (CoQ9) (collectively Cs) quino utilized. Such modifications can comprise any combination of nes are more prevalent than Coenzyme Q10 (CoQ10). As chemical, physiological, genetic, or other modification (e.g., mentioned, the ubiquinone may also lack an isoprene side one or more genetic modification and chemical and/or physi chain, and may be selected from alkylubiquinones in which ological modification(s)). the alkyl group may contain from 1 to 20 and preferably from Sterologenic polypeptide: The term “sterologenic 1 to 12 carbonatoms, such as, for example, decylubiquinones polypeptide', as used herein, refers to any polypeptide whose Such as 6-decylubiquinone or 2,3-dimethoxy-5-decyl-1,4- activity in a cell increases production of one or more sterol ubiquinone, derivatives thereof, and mixtures thereof. compounds (e.g., squalene, lanosterol, Zymosterol, ergos Ubiquinones may exist in reduced (ubiquinol), oxidized or terol, 7-dehydrocholesterol (provitamin D3), vitamin D com 25 Superoxidized States. For example, the oxidation states of pound(s), etc.) in that cell. The term encompasses both ubiquinone/Coenzyme Q10 are depicted in FIG. 11. polypeptides that are directly involved in sterol compound Ubiquinone biosynthesis polypeptide: The term synthesis and those whose expression or activity affects the "ubiquinone biosynthesis polypeptide' refers to any polypep extent or level of production of one or more sterol com tide that is involved in the synthesis of ubiquinone. To men pounds, for example by scavenging a Substrate or reactant 30 utilized by a sterol biosynthetic polypeptide that is directly tion but a few, these ubiquinone biosynthesis polypeptides involved in sterol compound production. Sterologenic include, for example, polypeptides of prenyldiphosphate syn polypeptides include isoprenoid biosynthesis polypeptides thase, PUB-polyprenyltransferase, and O-methyltransferase, and sterol biosynthesis polypeptides, and the term also as well as Cso quinone biosynthesis polypeptides. As will be encompasses polypeptides that may affect the extent to which 35 appreciated by those of ordinary skill in the art, in some sterol compounds are accumulated in lipid bodies. embodiments of the invention, ubiquinone biosynthesis Ubiquinogenic modification: The term "ubiquinogenic polypeptides include polypeptides that affect the expression modification', as used herein, refers to a modification of a and/or activity of one or more other ubiquinone biosynthesis host organism that adjusts production of ubiquinone (e.g., polypeptides. Representative examples of certain ubiquinone CoQ10), as described herein. For example, a ubiquinogenic 40 biosynthesis polypeptide sequences are presented in Tables modification may increase the production level of ubiquinone 23, 23b, 23c, 24-31. (e.g., CoQ10), and/or may alter relative levels of ubiquinone Vitamin D compound: The term “vitamin D compound', as and/or ubiquinol. In principle, an inventive ubiquinogenic used herein, refers to a group of steroid compounds including modification may be any chemical, physiological, genetic, or Vitamin D (cholecalciferol), vitamin D (ergocalciferol), other modification that appropriately alters production of 45 their provitamins, and certain metabolites. Vitamins D and ubiquinone (e.g., CoO10) in a host organism produced by that D. can be produced from their respective provitamins (e.g., organism as compared with the level produced in an other 7-dehydrocholesterol and ergosterol) by ultraviolet irradia wise identical organism not subject to the same modification. tion (e.g., by the action of Sunlight). The most biologically In most embodiments, however, the ubiquinogenic modifica active form of vitamin D is 1,25-dihydroxy vitamin D, which tion will comprise a genetic modification, typically resulting 50 is also known as calcitriol. Calcitriol is produced by hydroxy in increased production of ubiquinone (CoQ10). lation of vitamin D. at the 25 position, followed by hydroxy Ubiquinogenic polypeptide: The term "ubiquinogenic lation to generate calcitriol. FIGS. 10A-B present the struc polypeptide, as used herein, refers to any polypeptide that is tures of vitamins D, D, and other vitamin D compounds. involved in the process of producing ubiquinone (e.g., Vitamin D biosynthesis polypeptide: The term “vitamin D CoQ10, CoQ9, CoQ8, CoQ7, CoQ6, CoQ5) in a cell, and may 55 biosynthesis polypeptide' refers to any polypeptide that is include polypeptides that are involved in processes other than involved in the synthesis of one or more vitamin D com ubiquinone production but whose expression or activity pounds. To mention but a few, these include, for example, the affects the extent or level of production of ubiquinone and/or 1.14.99.7, 5.4.99.7, 5.4.99.8, 5.3.3.5, and/or 1.14.21.6, ubiquinol. Ubiquinogenic polypeptides include ubiquinone polypeptides depicted in FIG.1. They further can include the biosynthesis polypeptides and Cso quinone biosynthesis 60 hydroxylases that convert vitamin D to calcitriol (e.g., the polypeptides 1.14.15.- and 1.14.13.13 polypeptides depicted in FIG. 1). As Ubiquinone: The term “ubiquinone' is understood in the will be appreciated by those of ordinary skill in the art, in art to refer to a structural class of quinone derivatives with or some embodiments of the invention, vitamin D biosynthesis without isoprenoid side chains. Ubiquinones are described in polypeptides include polypeptides that affect the expression the Merck Index, 11th Edition, Merck & Co., Inc. Rahway, 65 and/or activity of one or more other vitamin D biosynthesis N.Y., USA, Abstr. 9751 (1989), which is incorporated herein polypeptides. Particular examples of certain vitamin D bio by reference. A dual nomenclature exists for these com synthesis polypeptides are presented in Tables 84-96. US 8,367,395 B2 19 20 Vitamin E biosynthesis polypeptide: The term “vitamin E fungus dry cell weight. Thus, the provided manipulated biosynthesis polypeptide' refers to any polypeptide that is strains can then be used to produce one or more sterol com involved in the synthesis of vitamin E. To mention but a few, pounds (e.g., squalene, lanosterol, Zymosterol, ergosterol, these include, for example, tyra, pds1(hppd), VTE1, HPT1 7-dehydrocholesterol (provitamin D3), one or more vitamin (VTE2), VTE3, VTE4, and/or GGH polypeptides (i.e., D compounds, etc). In some embodiments, the sterol com polypeptides that perform the chemical reactions performed pound(s) that partition(s) into the lipid bodies can readily be by tyra, pds1(hppd), VTE1, HPT1(VTE2), VTE3, VTE4, isolated. In some embodiments, the sterol compound is and/or GGH, respectively). As will be appreciated by those of squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho ordinary skill in the art, in some embodiments of the inven lesterol (provitamin D3); in some embodiments it is a vitamin tion, Vitamin E biosynthesis polypeptides include polypep 10 D compound. In some embodiments, it is vitamin D3 or a tides that affect the expression and/or activity of one or more provitamin thereof; in some embodiments, it is 7-dehydroc other vitamin E biosynthesis polypeptides. Particular holesterol. examples of certain vitamin E biosynthesis polypeptides are In some embodiments, it will be desirable to balance ole presented in Tables 50-56. aginy and sterol production in cells such that, as soon as a Vitamin K biosynthesis polypeptide: The term “vitamin K 15 minimum desirable level of oleaginy is achieved, Substan biosynthesis polypeptide' refers to any polypeptide that is tially all further carbon is diverted into a sterologenic produc involved in the synthesis of vitamin K. To mention but a few, tion pathway. In some embodiments of the invention, this these include, for example, MenF, Men), MenC, MenE, strategy involves engineering cells to be oleaginous; in other MenB, MenA, UbiE, and/or MenG polypeptides (i.e., embodiments, it involves engineering cells to accumulate a polypeptides that perform the chemical reactions performed higher level of lipid, particularly cytoplasmic lipid, than they by MenF, MenD, MenC, MenE, MenB, MenA, UbiE, and/or would do in the absence of such engineering even though the MenG, respectively). As will be appreciated by those of ordi engineered cells may not become "oleaginous” as defined nary skill in the art, in Some embodiments of the invention, herein. In other embodiments, the extent to which an oleagi Vitamin K biosynthesis polypeptides include polypeptides nous host cell accumulates lipid is actually reduced so that that affect the expression and/or activity of one or more other 25 remaining carbon can be utilized in sterol production. carotenoid biosynthesis polypeptides. Particular examples of To give but one example of adjustments that could be made certain vitamin K biosynthesis polypeptides are presented in to achieve a desired balance between oleaginy and sterol Tables 42-49. production, we note that, while increasing acetyl CoA car boxylase expression (and/or activity) promotes oleaginy, DETAILED DESCRIPTION OF CERTAIN 30 decreasing its expression and/or activity may promote sterol PREFERRED EMBODIMENTS OF THE production. Those of ordinary skill in the art will appreciate INVENTION that the expression and/or activity of acetyl CoA carboxylase, or of other polypeptides, may be adjusted up or down as The present invention embraces the reasoning that certain desired according to the characteristics of a particular host sterol compounds can effectively be produced in oleaginous 35 cell of interest. yeast and fungi. According to the present invention, strains We note that engineered cells and processes of using them that both (i) accumulate lipid, often in the form of cytoplas as described herein may provide one or more advantages as mic oil bodies; and (ii) producesterol compounds at a level at compared with unmodified (i.e., parental) cells. Such advan least about 1%, of their dry cell weight, are generated through tages may include, but are not limited to: increased yield (e.g., manipulation of host cells (i.e., strains, including, e.g., natu 40 sterol compound content expressed as either '% dry cell rally-occurring strains and strains which have been previ weight (mg/mg) or parts per million), titer (g sterol com ously modified). pound/L), specific productivity (mg sterol compoundg'bio In certain embodiments, strains can accumulate lipid typi mass hour'), and/or volumetric productivity (g sterol com cally to at least about 20% of their dry cell weight. In some pound liter' hour)) of the desired sterol compound (and/or embodiments, strains accumulate lipid to a level higher than 45 intermediates thereof), and/or decreased formation of unde at least about 20% of their dry cell weight (e.g., to a level at sirable side products (for example, undesirable intermedi least about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, ates). 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, Thus, for example, the specific productivity for one or 65%, 70%, 75%, or even 80%) in some embodiments, strains more desired sterol compound or total sterol compound con accumulate lipid to a level below at least about 20% but above 50 tent may be at or about 0.1, at or about 0.11, at or about 0.12. the level at which it is accumulated in the unmodified (i.e., at or about 0.13, at or about 0.14, at or about 0.15, at or about parental) Strain. In some Such embodiments, modified strains 0.16, at or about 0.17, at or about 0.18, at or about 0.19, at or accumulate lipid to a level about 5%, 6%, 7%, 8%, 9%, 10%, about 0.2, at or about 0.21, at or about 0.22, at or about 0.23, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% of their at or about 0.24, at or about 0.25, at or about 0.26, at or about dry cell weight. 55 0.27, at or about 0.28, at or about 0.29, at or about 0.3, at or In some embodiments sterol(s) can be produced in the about 0.31, at or about 0.32, at or about 0.33, at or about 0.34, strains to at least about 2%, at least about 3%, at least about at or about 0.35, at or about 0.36, at or about 0.37, at or about 4%, at least about 5%, at least about 6%, at least about 7%, at 0.38, at or about 0.39, at or about 0.4, at or about 0.41, at or least about 8%, at least about 9%, at least about 10%, at least about 0.42, at or about 0.43, at or about 0.44, at or about 0.45, about 11%, at least about 12%, at least about 13%, at least 60 at or about 0.46, at or about 0.47, at or about 0.48, at or about about 14%, at least about 15%, at least about 16%, at least 0.49, at or about 0.5, at or about 0.51, at or about 0.52, at or about 17%, at least about 18%, at least about 19%, at least about 0.53, at or about 0.54, at or about 0.55, at or about 0.56, about 20%, at least about 21%, at least about 22%, at least at or about 0.57, at or about 0.58, at or about 0.59, at or about about 23%, at least about 24%, at least about 25%, at least 0.6, at or about 0.61, at or about 0.62, at or about 0.63, at or about 26%, at least about 27%, at least about 28%, at least 65 about 0.64, at or about 0.65, at or about 0.66, at or about 0.67, about 29%, at least about 30%, at least about 35%, at least at or about 0.68, at or about 0.69, at or about 0.7, at or about about 40%, at least about 45%, at least about 50%, or more of 0.71, at or about 0.72, at or about 0.73, at or about 0.74, at or US 8,367,395 B2 21 22 about 0.75, at or about 0.76, at or about 0.77, at or about 0.78, pounds of interest Such as one or more vitamin D compounds at or about 0.79, at or about 0.8, at or about 0.81, at or about Such as 7-dehydrocholesterol or calcitriol. Any oleaginous 0.82, at or about 0.83, at or about 0.84, at or about 0.85, at or strain may be utilized as a host strain according to the present about 0.86, at or about 0.87, at or about 0.88, at or about 0.89, invention, and may be engineered or otherwise manipulated at or about 0.9, at or about 0.91, at or about 0.92, at or about to generate inventive oleaginous, Sterol-producing strains. 0.93, at or about 0.94, at or about 0.95, at or about 0.96, at or Alternatively, Strains that naturally are not oleaginous may be about 0.97, at or about 0.98, at or about 0.99, at or about 1, employed. Furthermore, even when a particular strain has a 1.05, at or about 1.1, at or about 1.15, at or about 1.2, at or natural capacity for oleaginy and for sterol production, its about 1.25, at or about 1.3, at or about 1.35, at or about 1.4, at natural capabilities may be adjusted as described herein for or about 1.45, at or about 1.5, at or about 1.55, at or about 1.6, 10 optimal production of one or more particular desired sterol at or about 1.65, at or about 1.7, at or about 1.75, at or about compounds. 1.8, at or about 1.85, at or about 1.9, at or about 1.95, at or In certain embodiments, engineering or manipulation of a about 2 mg hour' or more. strain results in modification of a type of lipid and/or sterol Thus, for example, the volumetric productivity for one or compound produced. For example, a strain may be naturally more desired sterol compound (e.g. squalene, Vitamin D com 15 oleaginous and/or may naturally produce one or more sterol pound) or total sterol compound content may be at or about compound(s). However, engineering or modification of the 0.01, at or about 0.011, at or about 0.012, at or about 0.013, at strain may be employed so as to change the type or amount of or about 0.014, at or about 0.015, at or about 0.016, at or about lipid that is accumulated and/or to adjust sterol compound 0.017, at or about 0.018, at or about 0.019, at or about 0.02, at production. In some embodiments, squalene production will or about 0.021, at or about 0.022, at or about 0.023, at or about be optimized; in Some embodiments, production of one or 0.024, at or about 0.025, at or about 0.026, at or about 0.027, more vitamin D compounds will be optimized; in some at or about 0.028, at or about 0.029, at or about 0.03, at or embodiments, production of 7-dehydrocholesterol will be about 0.031, at or about 0.032, at or about 0.033, at or about optimized; in some embodiments, production of calcitriol 0.034, at or about 0.035, at or about 0.036, at or about 0.037, will be optimized; in some embodiments, production of at or about 0.038, at or about 0.039, at or about 0.04, at or 25 lanosterol will be optimized; in some embodiments, produc about 0.041, at or about 0.042, at or about 0.043, at or about tion of Zymosterol will be optimized; in some embodiments, 0.044, at or about 0.045, at or about 0.046, at or about 0.047, production of ergosterol will be optimized. at or about 0.048, at or about 0.049, at or about 0.05, at or When selecting a particular yeast or fungal strain for use in about 0.051, at or about 0.052, at or about 0.053, at or about accordance with the present invention, it will generally be 0.054, at or about 0.055, at or about 0.056, at or about 0.057, 30 desirable to select one whose cultivation characteristics are at or about 0.058, at or about 0.059, at or about 0.06, at or amenable to commercial scale production. For example, it about 0.061, at or about 0.062, at or about 0.063, at or about will generally (though not necessarily always) be desirable to 0.064, at or about 0.065, at or about 0.066, at or about 0.067, avoid filamentous organisms, or organisms with particularly at or about 0.068, at or about 0.069, at or about 0.07, at or unusual or stringent requirements for growth conditions. In about 0.071, at or about 0.072, at or about 0.073, at or about 35 some embodiments of the invention, it will be desirable to 0.074, at or about 0.075, at or about 0.076, at or about 0.077, utilize edible organisms as host cells, as they may optionally at or about 0.078, at or about 0.079, at or about 0.08, at or be formulated directly into pharmaceutical compositions, about 0.081, at or about 0.082, at or about 0.083, at or about food or feed additives, or into nutritional Supplements, as 0.084, at or about 0.085, at or about 0.086, at or about 0.087, desired. Some embodiments of the invention utilize host cells at or about 0.088, at or about 0.089, at or about 0.09, at or 40 that are genetically tractable, amenable to molecular genetics about 0.091, at or about 0.092, at or about 0.093, at or about (e.g., can be efficiently transformed, especially with estab 0.094, at or about 0.095, at or about 0.096, at or about 0.097, lished or available vectors; optionally can incorporate and/or at or about 0.098, at or about 0.099, at or about 0.1, 0.105, at integrate multiple genes, for example sequentially; and/or or about 0.110, at or about 0.115, at or about 0.120, at or about have known genetic sequence; etc), devoid of complex 0.125, at or about 0.130, at or about 0.135, at or about 0.14, at 45 growth requirements (e.g., a necessity for light), mesophilic or about 0.145, at or about 0.15, at or about 0.155, at or about (e.g., prefer growth temperatures within the range of about 0.16, at or about 0.165, at or about 0.17, at or about 0.175, at 20-32° C.) (e.g. 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or about 0.18, at or about 0.185, at or about 0.19, at or about 32° C.), able to assimilate a variety of carbon and nitrogen 0.195, at or about 0.20 grams liter hour' or more. Sources and/or capable of growing to high cell density. Alter Host Cells 50 natively or additionally, various embodiments of the inven Those of ordinary skill in the art will readily appreciate that tion utilize host cells that grow as single cells rather than, for a variety of yeast and fungal strains exist that are naturally example, as mycelia. oleaginous, and all yeast and fungal strains that have been In general, when it is desirable to utilize a naturally oleagi analyzed have been shown to produce one or more sterol nous organism in accordance with the present invention, any compounds, at least under certain culturing conditions. Most 55 modifiable and cultivatable oleaginous organism may be yeast and fungal strains accumulate ergosterol, a sterol com employed. In certain embodiments of the invention, yeast or pound. In general, yeast and fungal strains accumulate ergos fungi of genera including, but not limited to, Blakeslea, Can terol as the predominant sterol compound. Most yeast and dida, Cryptococcus, Cunninghamella, Lipomyces, Mortier fungal strains accumulate ergosterol to several percent of the ella, Mucor, Phycomyces, Pythium, Rhodosporidium, dry cell weight, whereas other sterol compounds such as 60 Rhodotorula, Thraustochytrium, TrichospOron, and Yarrowia squalene, lanosterol, Zymosterol, episterol, and fecosterol are employed. In certain particular embodiments, organisms accumulate to approximately 1% dry cell weight or less. of species that include, but are not limited to, Blakeslea Specific yeast Strains have been modified to produce approxi trispora, Candida pulcherrima, C. revkaufi, C. tropicalis, mately 10% dry cell weight or more of the sterol compounds Cryptococcus curvatus, Cunninghamella echinulata, C. squalene and ergosterol (see, for example, EB0048629OB1 65 elegans, C. japonica, Lipomyces Starkeyi, L. lipoferus, Mor and UA20060088903A1). Yeast and fungal strains do not tierella alpina, M. isabellina, M. ramanniana, M. vinacea, typically accumulate significant quantities of sterol com Mucor circinelloides, Phycomyces blakesleanus, Pythium US 8,367,395 B2 23 24 irregulare, Rhodosporidium toruloides, Rhodotorula gluti Aspergillus niger; Candida utilis, Xanthophyllomyces den nis, R. gracilis, R. graminis, R. mucilaginosa, R. pinicola, drorhous (Phafia rhodozma) and Saccharomyces cerevi Thraustochytrium sp., TrichospOron pullans, T. cutaneum, and SCé. Yarrowia lipolytica are used. In some embodiments of the invention, it may be desirable All of these naturally oleaginous strains produce one or to utilize a host cell that is modified as compared with a more sterol compounds. In most cases, only low levels (less naturally-occurring host cell so that its natural ability to pro than about 1% dry cell weight) of sterol compound(s) other duce one or more particular sterol compounds and/or to gen than ergosterol are produced by naturally-occurring sterolo erate lipid bodies of a particular character, is modified. Thus, genic, oleaginous yeast or fungi. the engineering contemplated by the present invention may 10 include, for instance, disrupting an endogenous pathway for The present invention may utilize any naturally oleaginous, production of a particular sterol compound and/or lipid in sterol-compound-producing organism as a host cell. In gen favor of an engineered pathway for production of the same or eral, the present invention may be utilized to increase carbon a different sterol compound and/or lipid. To give but one flow into the isoprenoid pathway in naturally sterol-produc specific example, S. cerevisiae, like many yeasts, naturally ing organisms, and/or to shift production from one sterol 15 express ERG5 and ERG6 polypeptides, and naturally pro compound (e.g., squalene, lanosterol, Zymosterol, ergosterol, duce certain sterol compounds. ERG5 and ERG6 polypep 7-dehydrocholesterol (provitamin D3) or a vitamin D com tides are involved in synthesis of vitamin D. intermediates pound) to another (e.g., a vitamin D compound or squalene, and therefore are involved in a biosynthetic pathway that lanosterol, Zymosterol, ergosterol, 7-dehydrocholesterol competes with the Vitamin D. pathway. It is known that modi (provitamin D3), respectively). Introduction of one or more fied S. cerevisiae strains lacking either one of these polypep sterologenic modifications (e.g., increased expression of one tides are viable. The absence (or reduction in activity) of one or more endogenous or heterologous sterologenic polypep or both of ERG5 and ERG6 in S. cerevisiae or in other host tides), in accordance with the present invention, can achieve cells (e.g., Yarrowia lipolytica, C. utilis, Phafia rhodozyma) these goals. would be expected to reduce diversion of vitamin D interme In certain embodiments of the invention, the utilized ole 25 diates to production of vitamin D, and thereby to increase aginous, Sterol-producing organism is a yeast or fungus, for production of certain sterol compounds related to vitamin D. example of a genus Such as, but not limited to, Yarrowia; in (e.g., 7-dehydrocholesterol). In some embodiments of the Some embodiments, the organism is of a species such as invention, therefore, it may be desirable to utilize host cells Yarrowia lipolytica. that naturally produce vitamin D compounds but whose vita When it is desirable to utilize strains that are naturally 30 min D. biosynthetic pathway has been disrupted or inhibited. non-olleaginous as host cells in accordance with the present In some embodiments of the invention, it may be desirable to invention, genera of non-oleaginous yeast or fungi include, utilize host cells that naturally produce vitamin D compounds but are not limited to, Aspergillus, Botrytis, Cercospora, but whose vitamin D biosynthetic pathway has been dis Fusarium (Gibberella), Kluyveromyces, Neurospora, Peni rupted or inhibited, optionally in addition to disruption or cillium, Pichia (Hansenula), Puccinia, Saccharomyces, 35 inhibition of a vitamin D, biosynthetic pathway. Such host Schizosaccharomyces, Sclerotium, Trichoderma, and Xan cells might be particularly useful, for example, for production thophyllomyces (Phafia); in Some embodiments, the organ of squalene. ism is of a species including, but not limited to, Aspergillus Those of ordinary skill in the art will appreciate that the nidulans, A. niger, A. terreus, Botrytis cinerea, C. utilis, Cer selection of a particular host cell for use in accordance with cospora nicotianae, Fusarium filikuroi (Gibberella zeae), 40 the present invention will also affect, for example, the selec Kluyveromyces lactis, K lactis, Neurospora crassa, Pichia tion of expression sequences utilized with any heterologous pastoris, Puccinia distincta, Saccharomyces cerevisiae, polypeptide to be introduced into the cell, codon bias that can Schizosaccharomyces pombe, Sclerotium rolfsii, Tricho optionally be engineered into any nucleic acid to be expressed derma reesei, and Xanthophyllomyces dendrorhous (Phafia in the cell and will also influence various aspects of culture rhodozyma). 45 conditions, etc. Much is known about the different gene regu It will be appreciated that the term “non-olleaginous”, as latory requirements, and cultivation requirements, of differ used herein, encompasses both strains that naturally have ent host cells to be utilized in accordance with the present Some ability to accumulate lipid, especially cytoplasmically, invention. (see, for example, with respect to Yarrowia, Barth but do not do so to a level sufficient to qualify as “oleaginous et al. FEMS Microbiol Rev. 19:219, 1997: Madzak et al. J as defined herein, as well as strains that do not naturally have 50 Biotechnol. 109:63, 2004; see, for example, with respect to any ability to accumulate extra lipid, e.g., extra-membranous Xanthophyllomyces, Verdoes et al. Appl Environ Microbiol lipid. It will further be appreciated that, in some embodiments 69: 3728-38, 2003; Visser et al. FEMSYeast Res 4: 221-31, of the invention, it will be sufficient to increase the natural 2003; Martinez et al. Antonie Van Leeuwenhoek. 73(2): 147 level of oleaginy of a particular host cell, even if the modified 53, 1998; Kim et al. Appl Environ Microbiol. 64(5):1947-9, cell does not qualify as oleaginous as defined herein. In some 55 1998; Wery etal. Gene. 184(1):89-97, 1997: see, for example, embodiments, the cell will be modified to accumulate at least with respect to Saccharomyces, Guthrie and Fink Methods in about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, Enzymology 194:1-933, 1991). 10%, 9%, 8%, 7%, 6%, or 5% of dry cell weight as lipid, so As discussed above, any of a variety of organisms may be long as the accumulation level is more than that observed in employed as host cells in accordance with the present inven the unmodified parental cell. 60 tion. In certain embodiments of the invention, host cells will As with the naturally oleaginous organisms, all of the natu be Y lipolytica cells. Advantages of Y lipolytica include, for rally non-olleaginous fungi naturally produce one or more example, tractable genetics and molecular biology, availabil sterol compounds. Genera of naturally non-olleaginous fungi ity of genomic sequence (see, for example, Sherman et al. that may desirably be used as host cells in accordance with the Nucleic Acids Res. 32 (Database issue):D315-8, 2004), suit present invention include, but are not limited to, Aspergillus, 65 ability to various cost-effective growth conditions, ability to Kluyveromyces, Penicillium, Saccharomyces, Xanthophyllo grow to high cell density. In addition, Y lipolytica is naturally myces, and Pichia; species include, but are not limited to, oleaginous, such that fewer manipulations may be required US 8,367,395 B2 25 26 when using Y lipolytica as opposed to other candidate host sequences are known in the art, and include, but are not cell, to generate an oleaginous, strain that produces a particu limited to example, mitochondrial signal sequences provided lar sterol compound of interest. Furthermore, there is already in Table 22. In other embodiments, it may be desirable to extensive commercial experience with Y lipolytica. utilize genes from other source organisms such as animals, Saccharomyces cerevisiae is also a useful host cell in plants, alga, or microalgae, fungi, yeast, insect, protozoa, and accordance with the present invention, particularly due to its mammals. experimental tractability and the extensive experience that Engineering Oleaginy researchers have accumulated with the organism. Although All living organisms synthesize lipids for use in their mem cultivation of Saccharomyces under high carbon conditions branes and various other structures. However, most organ may result in increased ethanol production, this can generally 10 isms do not accumulate in excess of about 10% of their dry be managed by process and/or genetic alterations. cell weight as total lipid, and most of this lipid generally The edible fungus, Candida utilis is also a useful host cell resides within cellular membranes. in accordance with the present invention. Molecular biology Significant biochemical work has been done to define the tools and techniques are available in C. utilis (for example, metabolic enzymes necessary to confer oleaginy on microor see Iwakiri et al. (2006) Yeast 23:23-34, Iwakiri et al. (2005) 15 ganisms (primarily for the purpose of engineering single cell Yeast 2005 22:1079-87, Iwakiri et al. (2005) Yeast 22:1049 oils as commercial sources of arachidonic acid and docosa 60, Rodriquez et al. (1998) Yeast 14:1399-406, Rodriquez et hexaenoic acid; see for example Ratledge Biochimie 86:807, al. (1998) FEMS Microbiol Lett. 165:335-40, and Kondo et al. 2004, the entire contents of which are incorporated herein by (1995).J Bacteriol. 177:7171-7). reference). Although this biochemical work is compelling, To give but a few specific examples of useful expression there only have been a limited number of reports of de novo sequences, promoters including, but not limited to the Leu2 oleaginy being established through genetic engineering with promoter and variants thereof (see, for example, see U.S. Pat. the genes encoding the key metabolic enzymes. It should be No. 5,786.212); the EF1alpha protein and ribosomal protein noted that oleaginous organisms typically accumulate lipid S7gene promoters (see, for example, PCT Application WO only when grown under conditions of carbon excess and 97/.44470); the Gpm (see US 2005/0014270), Xpr2 (see U.S. 25 nitrogen limitation. The present invention further establishes Pat. No. 4.937, 189), Tefl, Gpd1 (see, for example, US 2005/ that the limitation of other nutrients (e.g. phosphate or mag 0014270A1), Caml (YALIOC24420g), YALIOD16467g, nesium) can also induce lipid accumulation. The present TefA (YALIOB12562g), Yef3 (YALIOE13277g), Pox2, Yat 1 invention establishes, for example, that limitation of nutrients (see, for example US 2005/0130280; PCT Application WO Such as phosphate and/or magnesium can induce lipid accu 06/052754), Fbal (see, for example WO 05/049805), and/or 30 mulation, much as is observed under conditions of nitrogen Gpat (see WO 06/031937) promoters; the sequences repre limitation. Under these conditions, the organism readily sented by SEQID NO: 2, SEQID NO:3, SEQID NO:4, SEQ depletes the limiting nutrient but continues to assimilate the IDNO:5, SEQIDNO: 6, SEQIDNO:7, SEQIDNO:8, SEQ carbon source. The “excess' carbon is channeled into lipid ID NO:9, SEQID NO: 10, SEQID NO: 11, and SEQID NO: biosynthesis so that lipids (usually triacylglycerols) accumu 12, Subsequences thereof, and hybrid and tandem derivatives 35 late in the cytosol, typically in the form of bodies. thereof (e.g., as disclosed in US 2004/0146975); the In general, it is thought that, in order to be oleaginous, an sequences represented by SEQ ID NO: 1, 2, or 3 including organism must produce both acetyl-CoA and NADPH in the fragments (e.g. by 462-1016 and by 197-1016 of SEQID NO: cytosol, which can then be utilized by the fatty acid synthase 1; by 5-523 of SEQID NO:3) and complements thereof (e.g., machinery to generate lipids. In at least Some oleaginous as disclosed in U.S. Pat. No. 5,952,195); CYP52A2A (see, for 40 organisms, acetyl-CoA is generated in the cytosol through the example, US 2002/0034788); promoter sequences from fun action of ATP-citrate lyase, which catalyzes the reaction: gal (e.g., C. tropicalis) catalase, citrate synthase, 3-ketoacyl CoA thiolase A, citrate synthase, O-acetylhornserine Sulphy citrate--CoA-ATP-> drylase, protease, carnitine O-acetyltransferase, hydratase acetyl-CoA+Oxaloacetate--ADP+P. (1) dehydrogenase, epimerase genes; promoter sequences from 45 Of course, in order for ATP-citrate lyase to generate appro Pox4 genes (see, for example, US 2004/0265980); and/or priate levels of acetyl-CoA in the cytosol, it must first have an promoter sequences from Met2, Met3, Metó, Met25 and available pool of its substrate citric acid. Citric acid is gener YALIOD12903g genes. Any such promoters can be used in ated in the mitochondria of all eukaryotic cells through the conjunction with endogenous genes and/or heterologous tricarboxylic acid (TCA) cycle, and can be moved into the genes for modification of expression patterns of endogenous 50 cytosol (in exchange for malate) by citrate/malate translo polypeptides and/or heterologous polypeptides. CaSC. Alternatively or additionally, regulatory sequences useful In most oleaginous organisms, and in Some non-olleaginous in accordance with the present invention may include one or organisms, the enzyme isocitrate dehydrogenase, which more Xpr2 promoter fragments, for example as described in operates as part of the TCA cycle in the mitochondria, is U.S. Pat. No. 6,083,717 (e.g. SEQID NOS: 1-4 also including 55 strongly AMP-dependent. Thus, when AMP is depleted from sequences with 80% or more identity to these SEQID NOs) the mitochondria, this enzyme is inactivated. When isocitrate in one or more copies either in single or in tandem. Similarly, dehydrogenase is inactive, isocitrate accumulates in the mito exemplary terminator sequences include, but are not limited chondria. This accumulated isocitrate is then equilibrated to, Y lipolytica Xpr2 (see U.S. Pat. No. 4,937,189) and Pox2 with citric acid, presumably through the action of aconitase. (YALIOF 10857g) terminator sequences as well as the termi 60 Therefore, under conditions of low AMP, citrate accumulates nator sequences disclosed in example 7 herein. in the mitochondria. As noted above, mitochondrial citrate is In some embodiments of this invention, it may be desirable readily transported into the cytosol. to fused sequences encoding specific targeting signals to bac AMP depletion, which in oleaginous organisms is believed terial source genes. For example, in certain embodiments to initiate the cascade leading to accumulation of citrate (and mitochondrial signal sequences are useful in conjunction 65 therefore acetyl-CoA) in the cytoplasm, occurs as a result of with, e.g., bacterial polypeptides for effective targeting of the nutrient depletion mentioned above. When oleaginous polypeptides for proper functioning. Mitochondrial signal cells are grown in the presence of excess carbon Source but the US 8,367,395 B2 27 28 absence of nitrogen or other nutrient (e.g., phosphate or mag Chem 279:31190-31196, 2004), (2) decreased expression of nesium), the activity of AMP deaminase, which catalyzes the triglyceride lipases (e.g., TGL3 and/or TGL4: reaction: YALIOD17534g, and/or YALIOF 10010g (Kurat et al. J Biol Chem 281:491-500, 2006); and (3) decreased expression of AMP->inosine 5'-monophosphate--NH (2) one or more acyl-coenzyme A oxidase activities, for example is strongly induced. The increased activity of this enzyme encoded by POX genes (e.g. POX1, POX2, POX3, POX4, depletes cellular AMP in both the cytosol and the mitochon POX5; YALIOC23859g, YALIOD24750g, YALIOE06567g, dria. Depletion of AMP from the mitochondria is thought to YALIOE27654g, YALIOE32835g, YALIOF10857g: see for inactivate the AMP-dependent isocitrate dehydrogenase, example Mlickova et al. Appl Environ Microbiol 70: 3918 resulting in accumulation of citrate in the mitochondria and, 10 3924, 2004; Binns et al. J Cell Biol 173:719, 2006). therefore, the cytosol. This series of events is depicted dia Thus, according to the present invention, the oleaginy of a grammatically in FIG. 2. host organism may be enhanced by modifying the expression As noted above, oleaginy requires both cytosolic acetyl or activity of one or more polypeptides involved in generating CoA and cytosolic NADPH. It is believed that, in many ole cytosolic acetyl-CoA and/or NADPH and/or altering lipid aginous organisms, appropriate levels of cytosolic NADPH 15 levels through other mechanisms. For example, modification are provided through the action of malic enzyme (Enzyme 3 of the expression or activity of one or more of acetyl-CoA in FIG. 2). Some oleaginous organisms (e.g., Lipomyces and carboxylase, pyruvate decarboxylase, isocitrate dehydroge Some Candida) do not appear to have malic enzymes, how nase, ATP-citrate lyase, malic enzyme, AMP-deaminase, glu ever, so apparently other enzymes can provide comparable cose-6-phosphate dehydrogenase, 6-phosphogluconate activity, although it is expected that a dedicated source of dehydrogenase, fructose 1.6 bisphosphatase, NADH kinase, NADPH is probably required for fatty acid synthesis (see, for transhydrogenase, acyl-CoA:diacylglycerol acyltransferase, example, Wynn et al., Microbiol 145:1911, 1999; Ratledge phospholipid:diacylglycerol acyltransferase, acyl-CoA:cho Adv. Appl. Microbiol. 51:1, 2002, each of which is incorpo lesterol acyltransferase, triglyceride lipase, acyl-coenzyme A rated herein by reference in its entirety). oxidase can enhance oleaginy in accordance with the present Other activities which can be involved in regenerating 25 invention. Exemplary polypeptides which can be utilized or NADPH include, for example, 6-phosphogluconate dehydro derived so as to enhance oleaginy in accordance with the genase (gnd); Fructose 1.6 bisphosphatase (fbp); Glucose 6 present invention include, but are not limited to those acetyl phosphate dehydrogenase (gGpd); NADH kinase (EC CoA carboxylase, pyruvate decarboxylase, isocitrate dehy 2.7.1.86); and/or transhydrogenase (EC 1.6.1.1 and 1.6.1.2). drogenase, ATP-citrate lyase, malic enzyme, AMP-deami Gnd is part of the pentose phosphate pathway and catalyses 30 nase, glucose-6-phosphate dehydrogenase, the reaction: 6-phosphogluconate dehydrogenase, fructose 1, 6 bisphos 6-phospho-D-gluconate+NADP+->D-ribulose 5-phosphate-- phatase, NADH kinase, transhydrogenase, acyl-CoA:dia CO+NADPH cylglycerol acyltransferase, phospholipid:diacylglycerol Fbp is a hydrolase that catalyses the gluconeogenic reac acyltransferase, acyl-CoA:cholesterol acyltransferase, trig 35 lyceride lipase, acyl-coenzyme A oxidase polypeptides pro tion: vided in Tables 1-6 and 65-81 respectively. D-fructose 1,6-bisphosphate--HO-> In some embodiments of the invention, where an oleagi D-fructose 6-phosphate--phosphate nous host cell is employed, enzymes and regulatory compo Fbp redirects carbon flow from glycolysis towards the pen nents relevant to oleaginy are already in place but could be tose phosphate pathway. The oxidative portion of the pentose 40 modified, if desired, by for example altering expression or phosphate pathway, which includes glucose 6 phosphate activity of one or more oleaginic polypeptides and/or by dehydrogenase and 6-phosphogluconate dehydrogenase, introducing one or more heterologous oleaginic polypep enables the regeneration of NADPH. tides. In those embodiments of the invention where a non G6pd is part of the pentose phosphate pathway and cataly oleaginous host cell is employed, it is generally expected that ses the reaction: 45 at least one or more heterologous oleaginic polypeptides will be introduced. D-glucose 6-phosphate--NADP-> D-glucono-1,5-lactone 6-phosphate--NADPH-i- The present invention contemplates not only introduction H of heterologous oleaginous polypeptides, but also adjustment of expression oractivity levels of heterologous or endogenous NADH kinase catalyzes the reaction: 50 oleaginic polypeptides, including, for example, alteration of ATP-NADH-e ADP-NADPH constitutive or inducible expression patterns. In some Transhydrogenase catalyzes the reaction: embodiments of the invention, expression patterns are adjusted Such that growth in nutrient-limiting conditions is NADPH-i-NAD" () NADP'+NADH not required to induce oleaginy. For example, genetic modi Thus, enhancing the expression and/or activity of any of 55 fications comprising alteration and/or addition of regulatory these enzymes can increase NADPH levels and promote ana sequences (e.g., promoter elements, terminator elements) bolic pathways requiring NADPH. and/or regulatory factors (e.g., polypeptides that modulate Alternative or additional strategies to promote oleaginy transcription, splicing, translation, modification, etc.) may be may include one or more of the following: (1) increased or utilized to confer particular regulation of expression patterns. heterologous expression of one or more of acyl-CoA:diacylg 60 Such genetic modifications may be utilized in conjunction lycerol acyltransferase (e.g., DGA1:YALIOE32769g); phos with endogenous genes (e.g., for regulation of endogenous pholipid:diacylglycerol acyltransferase (e.g., LRO1; oleagenic polypeptide(s)), alternatively, such genetic modi YALIOE 16797g); and acyl-CoA:cholesterol acyltransferase fications may be included so as to conferregulation of expres (e.g., ARE genes such as ARE1, ARE2, YALIOF06578g), sion of at least one heterologous polypeptide (e.g., oleagenic which are involved in triglyceride synthesis (Kalscheuer et al. 65 polypeptide(s)). Appl Environ Microbiolp. 7119-7125, 2004; Oelkers et al. J In Some embodiments, at least one oleaginic polypeptide is Biol Chem 277:8877-8881, 2002; and Sorger et al. J Biol introduced into a host cell. In some embodiments of the US 8,367,395 B2 29 30 invention, a plurality (e.g., two or more) of different oleaginic cinelloides). In other embodiments, Candida utilis and Phaf polypeptides is introduced into the same host cell. In some fia rhodozyma can be similarly modified. Modified S. cerevi embodiments, the plurality of oleaginic polypeptides con siae, C. utilis, and P. rhodozyma strains can be further tains polypeptides from the same source organism; in other modified as described herein to increase production of one or embodiments, the plurality includes polypeptides indepen more sterol compounds. dently selected from different source organisms. In certain embodiments, host cells are engineered to be Representative examples of a variety of oleaginic polypep olegaginous by introducing one or more oleaginic polypep tides that may be introduced into or modified within host cells tides. In general, any oleaginic polypeptide can be introduced according to the present invention, include, but are not limited into any host cell of the present invention. In certain embodi to, those provided in Tables 1-6, and Tables 65-81. As noted 10 ments, such oleaginic polypeptides are codon-optimized to above, it is expected that at least some of these polypeptides accommodate the codon preferences of the host cell. In cer (e.g., malic enzyme and ATP-citrate lyase) should desirably tain embodiments, an oleaginic polypeptide introduced into a act in concert, and possibly together with one or more com host cell is from the same organism as the host cell and/or a ponents of fatty acid synthase, such that, in Some embodi related organism. For example, without limitation, the ments of the invention, it will be desirable to utilize two or 15 present invention encompasses the recognition that it may be more oleaginic polypeptides from the same source organism. desirable to introduce a fungal oleaginic polypeptide into a In certain embodiments, the oleaginy of a host organism is fungal host cell (e.g., from the same and/or a related fungal enhanced by growing the organism on a carbon Source com species). In certain embodiments, the host cell is a Y lipoly prising one or more oils. For example, an organism may be tica host cell. In certain aspects of Such embodiments, a Y. grown on a carbon source comprising one or more oils lipolytica olegainic polypeptide is introduced into the Y. selected from the group consisting of for example, olive, lipolytica host cell. In certain aspects, a S. cerevisiae ole canola, corn, Sunflower, soybean, cottonseed, rapeseed, etc., gainic polypeptide is introduced into the Y lipolytica host and combinations thereof. In certain embodiments, the ole cell. In certain aspects, any of a variety of fungal olegainic aginy of a host organism is enhanced by growing the organ polypeptides is introduced into the Y lipolytica host cell. ism on a carbon Source comprising one or more oils in com 25 Engineering Sterol Compound Biosynthesis bination with modifying the expression or activity of one or The present invention encompasses the recognition that more polypeptides such as any of those described above (e.g., lipid-accumulating systems are useful for the production and/ oleaginic polypeptides such as polypeptides involved in gen or isolation of certain sterol compounds, and particularly of erating cytosolic acetyl-CoA and/or NADPH) and/or altering squalene, lanosterol, Zymosterol, ergosterol and/or one or lipid levels through other mechanisms. 30 more vitamin D compounds (e.g., 7-dehydrocholesterol and/ In general, Source organisms for oleaginic polypeptides to or calcitriol). Without wishing to be bound by theory, the be used in accordance with the present invention include, but present inventors propose that the higher intracellular mem are not limited to, Blakeslea, Candida, Cryptococcus, Cun brane content may facilitate increased sterol compound pro ninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, duction and/or accumulation. The present invention therefore Pythium, Rhodosporidium, Rhodotorula, Trichosporon, Yar 35 encompasses the discovery that certainsterol compounds can rowia, Aspergillus, Botrytis, Cercospora, Fusarium (Gibber desirably be produced in oleaginous yeast and fungi. Accord ella), Kluyveromyces, Neurospora, Penicillium, Pichia ing to the present invention, strains that both (i) accumulate (Hansenula), Puccinia, Saccharomyces, Sclerotium, Tricho lipid, often in the form of cytoplasmic lipid bodies and typi derma, and Xanthophyllomyces (Phafia). In some embodi cally to at least about 20% of their dry cell weight, and (ii) ments, the source species for acetyl CoA carboxylase, ATP 40 produce one or more sterol compounds at a level at least about citrate lyase, malic enzyme and/or AMP deaminase 1%, and in some embodiments at least about 3-20%, of their polypeptides include, but are not limited to, Aspergillus nidu dry cell weight, are generated through manipulation of gen lans, Cryptococcus neoformans, Fusarium filikuroi, erally available strains (e.g., naturally-occurring strains and Kluyveromyces lactis, Neurospora crassa, Saccharomyces strains which have been previously genetically modified, cerevisiae, Schizosaccharomyces pombe, Ustilago maydis, 45 whether via recombinant DNA techniques or mutagenesis and Yarrowia lipolytica; in some embodiments, source spe directed modification). These manipulated Strains are then cies for pyruvate decarboxylase or isocitrate dehydrogenase used to produce one or more sterol compounds, so that com polypeptides include, but are not limited to Neurospora pound(s) that partitions into the lipid bodies can readily be crassa, Xanthophyllomyces dendrorhous (Phafia isolated. rhodozyma), Aspergillus niger, Saccharomyces cerevisiae, 50 In certain embodiments of the invention, host cells are Mucor circinelloides, Rhodotorula glutinis, Candida utilis, Yarrowia lipolytica cells. Advantages of Y lipolytica include, Mortierella alpina, and Yarrowia lipolytica. for example, tractable genetics and molecular biology, avail Aspergillus niger accumulates large amounts of citric acid, ability of genomic sequence, Suitability to various cost-effec whereas Mortierella alpina and Thraustochytrium sp. accu tive growth conditions, and ability to grow to high cell den mulate large amounts of fatty acid, respectively; Mortierella 55 sity. In addition, Y lipolytica is naturally oleaginous, Such that alpina and Thraustochytrium are also oleaginous. fewer manipulations may be required to generate an oleagi To give but one particular example of a host cellengineered nous, Sterol-producing Y lipolytica strain than might be to be oleaginous (or at least to accumulate increased levels of required for other organisms. Furthermore, there is already lipid) in accordance with the present invention, S. cerevisiae extensive commercial experience with Y lipolytica. In other can be engineered to express one or more oleaginic polypep 60 embodiments, the host cells are C. utilis, S. cerevisiae or P. tides, e.g., from heterologous source organisms. In some rhodozyma cells. embodiments, a plurality of different oleaginic polypeptides As mentioned, Sterol compounds are produced from the are expressed, optionally from different source organisms. isoprenoid compound isopentyl pyrophosphate (IPP). IPP For instance, in some embodiments, S. cerevisiae cells are can be generated through one of two different isoprenoid engineered to express (and/or to increase expression of) ATP 65 biosynthesis pathways. The most common isoprenoid bio citrate lyase (e.g., from N. crassa), AMP deaminase (e.g., synthesis pathway, sometimes referred to as the “mevalonate from S. cerevisiae), and/or malic enzyme (e.g., from M. cir pathway', is generally depicted in FIG. 3. As shown, acetyl US 8,367,395 B2 31 32 CoA is converted, via hydroxymethylglutaryl-CoA (HMG R167T/K169Y, I17T/R167F/K169Q, I17T/R167I/K169N, CoA), into mevalonate. Mevalonate is then phosphorylated I17T/R167H/K169Y, I17T/G47D/K93E or I17T/G47D/ and converted into the five-carbon compound isopentenyl K93Q). pyrophosphate (IPP). Thus, for example, a modified mevalonate kinase polypep An alternative isoprenoid biosynthesis pathway, that is uti tide may include one or more mutation(s) (particularly Sub lized by Some organisms (particularly bacteria) and is some stitutions), as compared with a parent mevalonate kinase times called the “mevalonate-independent pathway', is also polypeptide, at one or more amino acid position (s) selected depicted in FIG. 3. This pathway is initiated by the synthesis from the group consisting of amino acid positions corre of 1-deoxy-D-xyloglucose-5-phosphate (DOXP) from pyru sponding to positions 55, 59, 66, 83, 106, 111, 117, 142, 152, 10 158, 218, 231, 249,367 and 375 of the amino acid sequence vate and glyceraldehyde-3-phosphate. DOXP is then con of Saccharomyces cerevisiae mevalonate kinase as shown in verted, via a series of reactions shown in FIG. 12, into IPP. SEQ ID NO:1 of PCT application WO 06/063,752. For Various proteins involved in isoprenoid biosynthesis have example, such corresponding Substitutions may comprise one been identified and characterized in a number of organisms. or more of P55L, F59S, N66K, C117S, or I 152M. A modified Moreover, isoprenoids are synthesized in many, if not most, 15 mevalonate kinase may comprise a Substitution correspond organisms. Thus, various aspects of the isoprenoid biosyn ing to F59S substitution. A modified mevalonate kinase thesis pathway are conserved throughout the fungal, bacte polypeptide comprising 2 amino acid changes as compared rial, plant and animal kingdoms. For example, polypeptides with its parent mevalonate kinase polypeptide may, for corresponding to the acetoacetyl-CoA thiolase, HMG-CoA example, comprise changes at positions corresponding to the synthase, HMG-CoA reductase, mevalonate kinase, phos following positions 55/117,66/152, 83/249, 111/375 or 106/ phomevalonate kinase, mevalonate pyrophosphate decar 218 of to SEQ ID NO: 1 of WO 06/063,752 (e.g. P55L/ boxylase, IPP isomerase, shown in FIGS. 3, 5, and 6, have C117S, N66K/I152M, K83E/S249P, H111N/K375N or been identified in and isolated from a wide variety of organ L106P/S218P). A modified mevalonate kinase may comprise isms and cells; representative examples of a wide variety of a substitution corresponding to N66K/I152M. A modified such polypeptides are provided in Tables 7-13. One or more 25 mevalonate kinase polypeptide comprising 4 amino acid of the polypeptides selected from those provided in any one of changes as compared with its parent mevalonate kinase Tables 7-13 may be utilized or derived for use in the methods polypeptide may have changes at positions corresponding to and compositions in accordance with the present invention. one or more of the following positions 42/158/231/367 of Alternatively or additionally, modified mevalonate kinase SEQ ID NO:1 of WO 06/063,752 (e.g., I1421-111,158S/ polypeptides that exhibit decreased feedback inhibition prop 30 L2311/T367S). erties (e.g., to farnesyl pyrophosphate (FPP)) may be utilized According to the present invention, sterol compound pro in accordance with the present invention. Such modified duction in a host organism may be adjusted by modifying the mevalonate kinase polypeptides may be of eukaryotic or expression or activity of one or more proteins involved in prokaryotic origin. For example, modified versions of meva isoprenoid biosynthesis. In some embodiments, such modi lonate kinase polypeptides from animals (including humans), 35 fication involves introduction of one or more heterologous plants, algae, fungi (including yeast), and/or bacteria may be isoprenoid biosynthesis polypeptides into the host cell; alter employed; for instance, modified versions of mevalonate natively or additionally, modifications may be made to the kinase polypeptides disclosed in Table 10 herein may be expression or activity of one or more endogenous or heter utilized. ologous isoprenoid biosynthesis polypeptides. Given the con Particular examples of modified mevalonate kinase 40 siderable conservation of components of the isoprenoid bio polypeptides include “feedback-resistant mevalonate synthesis polypeptides, it is expected that heterologous kinases' disclosed in PCT Application WO 06/063,752. isoprenoid biosynthesis polypeptides will often function well Thus, for example, a modified mevalonate kinase polypeptide even in significantly divergent organisms. Furthermore, may include one or more mutation(s) at one or more amino should it be desirable to introduce more than one heterolo acid position(s) selected from the group consisting of amino 45 gous isoprenoid biosynthesis polypeptide (e.g., more than acid positions corresponding to positions 17, 47.93, 94, 132, one version of the same polypeptide and/or more than one 167, 169, 204, and 266 of the amino acid sequence of Para different polypeptides), in many cases polypeptides from dif coccus zeaxanthiinifaciens mevalonate kinase as shown in ferent Source organisms may function well together. In some SEQ ID NO:1 of PCT Application WO 04/111.214. For embodiments of the invention, a plurality of different heter example, the modified mevalonate kinase polypeptide may 50 ologous isoprenoid biosynthesis polypeptides is introduced contain one or more Substitutions at positions corresponding into the same host cell. In some embodiments, this plurality to one or more of I17T, G47D, K93E, V94I, R204H and contains only polypeptides from the same source organism; C266S in other embodiments the plurality includes polypeptides To give but a few specific examples, when a modified from different source organisms. mevalonate kinase polypeptide comprises 2 amino acid 55 In certain embodiments of the present invention that utilize changes as compared with a parent mevalonate kinase heterologous isoprenoid biosynthesis polypeptides, the polypeptide, it may comprise changes at positions corre Source organisms include, but are not limited to, fungi of the sponding to the following positions 132/375,167/169, 17/47 genera Blakeslea, Candida, Cryptococcus, Cunninghamella, and/or 17/93 of SEQ ID NO:1 of WO 04/111.214 (e.g. Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rho P132A/P375R, R167W/K169Q, I17T/G47D or I17T/K93E); 60 dosporidium, Rhodotorula, TrichospOron, Yarrowia, when a modified mevalonate kinase polypeptide comprises 3 Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), amino acid changes as compared with a parent mevalonate Kluyveromyces, Neurospora, Penicillium, Pichia kinase, it may comprise changes at positions corresponding to (Hansenula), Puccinia, Saccharomyces, Schizosaccharomy the following positions 17/167/169, 17/132/375,93/132/375, ces, Sclerotium, Trichoderms Ustilago, and Xanthophyllomy and/or 17/47/93 of SEQID NO: 1 of WO/2004/111214 (e.g., 65 ces (Phafia). In certain embodiments, the source organisms I717R167W/K169Q, I17T/P132A/P375R, K93E/P132A/ are of a species including, but not limited to, Cryptococcus P375R, I17T/R167W/K169H, I17T/R167T/K169M, I17T/ neoformans, Fusarium filiikuroi, Kluyverimyces lactis, Neu US 8,367,395 B2 33 34 rospora crassa, Saccharomyces cerevisiae, Schizosaccharo polypeptides, including, for example, alteration of constitu myces pombe, Ustilago maydis, and Yarrowia lipolytica. tive or inducible expression patterns so as to increase level The commitment step in sterol biosynthesis is the conver and/or activity of one or more sterologenic polypeptides and/ sion of farnesyl pyrophosphate into presqualene pyrophos or to decrease levels and/or activity of one or more sterol phate. Farnesyl pyrophosphate (FPP) is produced from iso- 5 biosynthesis competitor polypeptides. For example, genetic pentenyl pyrophosphate (IPP), for example in a process that modifications comprising alteration and/or addition of regu involves isomerization of IPP into dimethylallyl pyrophos latory sequences (e.g., promoter elements, terminator ele phate (DMAPP), followed by three sequential condensation ments) may be utilized to confer particular regulation of reactions with additional molecules of IPP generate the ten expression patterns. Such genetic modifications may be uti carbon molecule geranyl pyrophosphate (GPP), followed by 10 lized in conjunction with endogenous genes (e.g., for regula the fifteen-carbon molecule farnesyl pyrophosphate (FPP). tion of endogenous sterologenic polypeptide(s) and/or sterol FPP can either enter the sterol biosynthesis pathway by con biosynthesis competitor polypeptides); alternatively, Such version into presqualene puyrophosphate, or alternatively can genetic modifications may be included so as to confer regu be diverted toward biosynthesis of carotenoids and other lation of expression of heterologous polypeptides (e.g., Ster compounds (e.g., ubiquinone, Vitamin E. vitamin K, etc.) by 15 ologenic polypeptide(s)). conversion into the twenty-carbon compound geranylgeranyl In certain embodiments, host cells are engineered to pro pyrophosphate (GGPP). In many instances, FPP appears to be duce sterol compounds by introducing one or more sterol the predominant Substrate used by polyprenyldiphosphate biosynthesis polypeptides. In general, any sterol biosynthesis synthases (e.g. Coq 1 polypeptides) during ubiquinone bio polypeptide can be introduced into any host cell of the present synthesis. 2O invention. In certain embodiments, such sterol biosynthesis Once the sterol biosynthesis pathway has been entered, polypeptides are codon-optimized to accommodate the presqualene pyrophosphate is then converted to squalene by codon preferences of the host cell. In certain embodiments, a the same enzyme that performed the famesyl sterol biosynthesis polypeptide introduced into a host cell is pyrophosphate-epresqualene pyrophosphate conversion. from the same organism as the host cell and/or a related Squalene is then converted into a variety of different sterol 25 organism. For example, without limitation, the present inven compounds, including, but not limited to, lanosterol, Zymos tion encompasses the recognition that it may be desirable to terol, ergosterol, 7-dehydrocholesterol (provitamin D3), and introduce a fungal sterol biosynthesis polypeptide into a fun vitamin D compounds. The vitamin D biosynthetic pathway gal host cell (e.g., from the same and/or a related fungal and the vitamin D biosynthetic pathway share some common species). In certain embodiments, the host cell is a Y lipoly reactions, and there can be multiple points at which a vitamin 30 tica host cell. In certain aspects of Such embodiments, a Y. D. intermediate can be converted into a vitamin D interme lipolytica sterol biosynthesis polypeptide is introduced into diate (see, for example, FIG. 1). the Y lipolytica host cell. In certain aspects, a S. cerevisiae As noted herein, the isoprenoid biosynthesis pathway is sterol biosynthesis polypeptide is introduced into the Y. also involved in the production of non-sterol compounds, lipolytica host cell. In certain aspects, any of a variety of Such as carotenoids, ubiquinone, Steroids, and vitamins. Such 35 fungal sterol biosynthesis polypeptides is introduced into the as vitamin Korvitamin E. Polypeptides that action isoprenoid Y lipolytica host cell. biosynthesis pathway intermediates, and divert them into bio Squalene synthesis of non-sterol compounds are therefore indirect Squalene is a triterpene hydrocarbon Sterol compound that inhibitors of sterol biosynthesis (see, for example, FIG. 1, is naturally produced in all higher organisms, including which illustrates certain points at which isoprenoid interme- 40 humans. Squalene has found commercial utility as a machine diates are channeled into other biosynthesis pathways). Such lubricant, and as a component of various nutritional and skin polypeptides are therefore considered sterol biosynthesis care products. competitor polypeptides. Reductions of the level or activity of Squalene has traditionally been isolated from shark livers, Such sterol biosynthesis competitor polypeptides are but that route of production is expensive and impractical. expected to increase sterol compound production in host cells 45 There is a strong need for improved systems for producing according to the present invention. squalene. Produced squalene can be incorporated into any In some embodiments of the present invention, production number of a variety of products (for example, see below), or activity of endogenous sterol biosynthesis competitor and/or may be utilized as a synthetic precursor to any of a polypeptides may be reduced or eliminated in host cells. In variety of other chemical entities. Squalene is the natural some embodiments, this reduction or elimination of the activ- 50 biosynthetic precursor to the entire family of steroid com ity of a sterol biosynthesis competitor polypeptide can be pounds (see, for example, FIG. 1). achieved by treatment of the host organism with appropriate In some embodiments of the present invention, host cells small molecule inhibitors. are engineered to produce squalene and/or to accumulate it in Thus, in general, according to the present invention, pro lipid bodies. In some embodiments, squalene production is duction of one or more sterol compounds in a particular host 55 enhancedina cell by introduction of one or more sterologenic cell is increased by exposing the cell to a sterologenic modi modifications that increases levels of IPP, FPP and/or fication. In some embodiments of the present invention, the squalene itself. In some embodiments, squalene production is sterologenic modification comprises introducing or increas enhanced in a cell by increasing the level and/or activity of ing expression or activity of one or more sterologenic one or more squalene biosynthesis polypeptides (e.g., one or polypeptides (e.g., isoprenoid biosythesis polypeptides, Ste- 60 more isoprenoid biosynthesis polypeptides, an FPP synthase rol biosynthesis polypeptides) and/or removing or inhibiting polypeptide, and/or a squalene synthase polypeptide). Alter expression or activity of one or more sterol biosynthesis com natively or additionally, in Some embodiments, squalene pro petitor polypeptides (e.g., GGGP synthase). duction is enhanced in a cell by decreasing the level and/or The present invention contemplates not only introduction activity of one or more sterol biosynthesis competitor of heterologous sterologenic polypeptides, but also adjust- 65 polypeptides that diverts one or more intermediates away ment of expression or activity levels of heterologous or from squalene production and/or that metabolizes squalene endogenous sterologenic and/or sterol biosynthesis inhibitor itself. US 8,367,395 B2 35 36 For example, in Some embodiments of the present inven squalene biosynthesis competitor polypeptide and thereby to tion, squalene production in a host cell is increased by intro increase production of squalene, we note that it is known that ducing or increasing expression and/or activity of one or more fungal strains (e.g., S. cerevisiae) that lack any Erg5 or Erg6 squalene synthase polypeptides in the cell. Representative activity are viable. In some embodiments of the invention, examples of squalene synthase polypeptide sequences are 5 host cells are utilized that lack, or that are engineered to lack included in Table 16. In some embodiments of the invention (or to have reduced levels of), Erg5 and/or Erg6 polypeptide that utilize squalene synthase (or modifications of squalene activity. Absence or inhibition of Erg5 and/or Erg6 polypep synthase) source organisms include, but are not limited to, tide activity reduces diversion of carbon into vitamin D. Neurospora Crassa, Aspergillus niger; Saccharomyces cerevi production, and thereby allows increase in levels of squalene, siae, Mucor circinelloides, Candida utilis, Mortierella 10 lanosterol, Zymosterol, ergosterol, 7-dehydrocholesterol alpina, Phafia rhodozyma, and Yarrowia lipolytica. (provitamin D3) and/or other vitamin D compounds. In In some embodiments of the invention, squalene produc Some embodiments of the invention, in which increased pro tion in a host cell is increased by reducing the level or activity duction of squalene is particularly desired, one or more addi of one or more vitamin D biosynthesis polypeptides (e.g., tional sterologenic modifications may be combined with a which act to metabolize squalene). For instance, in some 15 reduction or inactivation of Erg5 and/or Erg6 activity in order embodiments, the level or activity of one or more polypep to reduce metabolism of squalene into Vitamin D com tides active in the ergosterol biosynthetic pathway (see, for pounds. example, FIG. 4) is reduced or eliminated. Lanosterol Enzymes of the ergosterol biosynthetic pathway include, Lanosterol is a tetracyclic triterpenoid that serves as a for example, squalene synthase (Erg9), squalene epoxidase synthetic intermediate for saponins and steroid hormones. (Erg1), 2.3-oxidosqualene-lanosterol cyclase (Erg7), cyto Lanosterol also functions as an emulsifier and active ingredi chrome P450 lanosterol 14C-demethylase (Erg11), C-14 ste ent in a variety of skin creams. rol reductase (Erg24), C-4 sterol methyl oxidase (Erg25), In some embodiments of the present invention, host cells SAM:C-24 sterol methyltransferase (Erg6), C-8 sterol are engineered to produce lanosterol and/or to accumulate it isomerase (Erg2), C-5 sterol desaturase (Erg3), C-22 sterol in lipid bodies. In some embodiments, lanosterol production desaturase (Erg5), and C-24 sterol reductase (Erg4) polypep 25 is enhanced in a cell by introduction of one or more sterolo tides. Each of these enzymes, other than squalene synthase, is genic modifications that increases levels of IPP, FPP and/or considered a squalene biosynthesis competitor polypeptide. lanosterol itself. In some embodiments, lanosterol production Regulators of these enzymes may also be considered to be is enhanced in a cell by increasing the level and/or activity of squalene biosynthesis competitor polypeptides (e.g., the one or more lanosterol biosynthesis polypeptides (e.g., one or yeast proteins Sut 1 (Genbank Accession JC4374 30 more isoprenoid biosynthesis polypeptides, an FPP synthase GI:2133159) and Mot3 (Genbank Accession NP 013786 polypeptide, squalene synthase polypeptide, squalene epoxi GI:6323715), which may or may not have homologs in other dase polypeptide, or 2.3-oxidosqualene-lanosterol cyclase organisms. polypeptide). Alternatively or additionally, in some embodi Known Small molecule inhibitors of Some squalene bio ments, lanosterol production is enhanced in a cell by decreas synthesis competitor enzymes include, but are not limited to 35 ing the leveland/or activity of one or more sterol biosynthesis terbinafine (e.g., LAMISIL(R), naftifine (NAFTINR), S-al competitor polypeptides that diverts one or more intermedi lylcysteine, garlic, resveratrol, NB-598 (e.g., from Banyu ates away from lanosterol production and/or that metabolizes Pharmaceutical Co), and/or green tea phenols that inhibit lanosterol itself. squalene epoxidase (see, for example, J. Biol Chem 265: For example, in Some embodiments of the present inven 18075, 1990: Biochem. Biophys. Res. Commun. 268:767, tion, lanosterol production in a host cell is increased by intro 2000); various azoles that inhibit cytochrome P450 lanosterol 40 ducing or increasing expression and/or activity of one or more 14C-demethylase; and fenpropimorph that inhibits the C-14 squalene synthase polypeptide, squalene epoxidase polypep sterol reductase and the C-8 sterol isomerase. In other tide, or 2.3-oxidosqualene-lanosterol cyclase polypeptide in embodiments, heterologous squalene biosynthesis competi the cell. Representative examples of squalene synthase tor polypeptides may be utilized (whether functional or non polypeptide, squalene epoxidase polypeptide, and 2.3-oxi functional; in Some embodiments, dominant-negative 45 dosqualene-lanosterol cyclase polypeptide sequences are mutants are employed). included in Table 16, 83, and 85. In some embodiments of the Alternatively or additionally, in some embodiments of the invention that utilize squalene synthase polypeptide, present invention, squalene production is enhanced by squalene epoxidase polypeptide, or 2.3-oxidosqualene decreasing the level and/or activity of one or more squalene lanosterol cyclase polypeptide (or modifications of these biosynthesis competitor polypeptides that diverts one or more 50 polypeptides) source organisms include, but are not limited intermediates away from the isoprenoid biosynthesis path to, Neurospora crassa, Aspergillus niger; Saccharomyces cer way, thereby reducing levels of IPP. For instance, in some evisiae, Phafia rhodozyma, Mucor circinelloides, Candida particular embodiments of the invention, the level and/or utilis, Mortierella alpina, and Yarrowia lipolytica. activity of acetyl CoA carboxylase, which diverts acetyl CoA In some embodiments of the invention, lanosterol produc from the isoprenoid synthesis pathway into the fatty acid tion in a host cell is increased by reducing the level or activity synthesis pathway, is inhibited. Such inhibition may be 55 of one or more vitamin D biosynthesis polypeptides (e.g., accomplished, for example, through one or more genetic which act to metabolize squalene). For instance, in some modifications and/or through use of one or more Small mol embodiments, the level or activity of one or more polypep ecule inhibitors. In some embodiments, an inhibitor selected tides active in the ergosterol biosynthetic pathway (see, for from the group consisting of aryloxyphenoxyproprionate, example, FIG. 4) is reduced or eliminated. cyclohexanedione (CHD), and combinations thereof (see also 60 Enzymes of the ergosterol biosynthetic pathway include, other inhibitors described, for example, in Shukla et al., J for example, squalene synthase (Erg9), squalene epoxidase Agric Food Chem. 52:5144, 2004: Webb et al., J AOAC Int (Erg1), 2.3-oxidosqualene-lanosterol cyclase (Erg7), cyto 0.84:143, 2001; Webb et al., J Agric Food Chem, 48:1219, chrome P450 lanosterol 14C-demethylase (Erg11), C-14 ste 2000; and Webb et al., JAgric Food Chem, 48:1210, 2000). rol reductase (Erg24), C-4 sterol methyl oxidase (Erg25), To give but one particular example of a sterologenic modi 65 SAM:C-24 sterol methyltransferase (Erg6), C-8 sterol fication that can be employed in accordance with the present isomerase (Erg2), C-5 sterol desaturase (Erg3), C-22 sterol invention to reduce or inhibit expression or activity of a desaturase (Erg5), and C-24 sterol reductase (Erg4) polypep US 8,367,395 B2 37 38 tides. Each of these enzymes, other than squalene synthase, ity of one or more Zymosterol biosynthesis polypeptides (e.g., squalene epoxidase, and 2.3-oxidosqualene-lanosterol one or more isoprenoid biosynthesis polypeptides, an FPP cyclase, is considered a lanosterol biosynthesis competitor synthase polypeptide, squalene synthase polypeptide, polypeptide. Regulators of these enzymes may also be con squalene epoxidase polypeptide, 2.3-oxidosqualene-lanos sidered to be squalene biosynthesis competitor polypeptides terol cyclase polypeptide, cytochrome P450 lanosterol 14C.- (e.g., the yeast proteins Sut1 (Genbank Accession JC4374 demethylase polypeptide, C-14 sterol reductase polypeptide, GI:2133159) and Mot3 (Genbank Accession NP 013786 or C-4 sterol methyl oxidase polypeptide). Alternatively or GI:6323715), which may or may not have homologs in other additionally, in some embodiments, Zymosterol production is organisms. enhanced in a cell by decreasing the level and/or activity of Known small molecule inhibitors of some lanosterol bio 10 one or more sterol biosynthesis competitor polypeptides that synthesis competitor enzymes include, but are not limited to diverts one or more intermediates away from lanosterol pro various azoles that inhibit cytochrome P450 lanosterol 14C.- duction and/or that metabolizes lanosterol itself. demethylase; and fempropimorph that inhibits the C-14 sterol For example, in Some embodiments of the present inven reductase and the C-8 sterol isomerase. In other embodi tion, Zymosterol production in a host cell is increased by ments, heterologous lanosterol biosynthesis competitor 15 introducing or increasing expression and/or activity of one or polypeptides may be utilized (whether functional or non more squalene synthase polypeptides, squalene epoxidase functional; in Some embodiments, dominant negative polypeptides, 2.3-oxidosqualene-lanosterol cyclase polypep mutants are employed). tides, cytochrome P450 lanosterol 14C-demethylase Alternatively or additionally, in some embodiments of the polypeptides, C-14 sterol reductase polypeptides, or C-4 ste present invention, lanosterol production is enhanced by rol methyl oxidase polypeptides in the cell. Representative decreasing the level and/or activity of one or more lanosterol examples of squalene synthase polypeptide, squalene epoxi biosynthesis competitor polypeptides that diverts one or more dase polypeptide, 2.3-oxidosqualene-lanosterol cyclase intermediates away from the isoprenoid biosynthesis path polypeptide, cytochrome P450 lanosterol 14C-demethylase way, thereby reducing levels of IPP. For instance, in some polypeptide, C-14 sterol reductase polypeptide, or C-4 sterol particular embodiments of the invention, the level and/or 25 methyl oxidase polypeptide sequences are included in Tables activity of acetyl CoA carboxylase, which diverts acetyl CoA 16, 83-85, and 93-95. In some embodiments of the invention from the isoprenoid synthesis pathway into the fatty acid that utilize squalene synthase polypeptides, squalene epoxi synthesis pathway, is inhibited. Such inhibition may be dase polypeptides, 2.3-oxidosqualene-lanosterol cyclase accomplished, for example, through one or more genetic polypeptides, cytochrome P450 lanosterol 14C-demethylase modifications and/or through use of one or more Small mol 30 polypeptides, C-14 sterol reductase polypeptides, or C-4 ste ecule inhibitors. In some embodiments, an inhibitor selected rol methyl oxidase polypeptides (or modifications of these from the group consisting of aryloxyphenoxyproprionate, polypeptides) source organisms include, but are not limited cyclohexanedione (CHD), and combinations thereof (see also to, Neurospora crassa, Aspergillus niger; Saccharomyces cer other inhibitors described, for example, in Shukla et al., J evisiae, Phafia rhodozyma, Mucor circinelloides, Candida Agric Food Chem. 52:5144, 2004: Webb et al., J AOAC Int 35 utilis, Mortierella alpina, and Yarrowia lipolytica. 0.84:143, 2001; Webb et al., J Agric Food Chem, 48:1219, In some embodiments of the invention, Zymosterol produc 2000; Webb et al., JAgric Food Chem, 48:1210, 2000). tion in a host cell is increased by reducing the level or activity To give but one particular example of a sterologenic modi of one or more vitamin D biosynthesis polypeptides (e.g., fication that can be employed in accordance with the present which act to metabolize squalene). For instance, in some invention to reduce or inhibit expression or activity of a 40 embodiments, the level or activity of one or more polypep lanosterol biosynthesis competitor polypeptide and thereby tides active in the ergosterol biosynthetic pathway (see, for to increase production of lanosterol, we note that it is known example, FIG. 4) is reduced or eliminated. that fungal strains (e.g., S. cerevisiae) that lack any Erg5 or Enzymes of the ergosterol biosynthetic pathway include, Erg6 activity are viable. In some embodiments of the inven for example, squalene synthase (Erg9), squalene epoxidase tion, host cells are utilized that lack, or that are engineered to 45 (Erg1), 2.3-oxidosqualene-lanosterol cyclase (Erg7), cyto lack (or to have reduced levels of), Erg5 and/or Erg6 polypep chrome P450 lanosterol 14C-demethylase (Erg11), C-14 ste tide activity. Absence or inhibition of Erg5 and/or Erg6 rol reductase (Erg24), C-4 sterol methyl oxidase (Erg25), polypeptide activity reduces diversion of carbon into Vitamin SAM:C-24 sterol methyltransferase (Erg6), C-8 sterol D. production, and thereby allows increase in levels of isomerase (Erg2), C-5 sterol desaturase (Erg3), C-22 sterol squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho 50 desaturase (Erg5), and C-24 sterol reductase (Erg4) polypep lesterol (provitamin D3) and/or other vitamin D compounds. tides. SAM:C-24 sterol methyltransferase (Erg6), C-8 sterol In some embodiments of the invention, in which increased isomerase (Erg2), C-5 sterol desaturase (Erg3), C-22 sterol production of lanosterol is particularly desired, one or more desaturase (Erg5), and C-24 sterol reductase (Erg4) polypep additional sterologenic modifications may be combined with tides are considered Zymosterol biosynthesis competitor a reduction or inactivation of Erg5 and/or Erg6 activity in 55 polypeptides. Regulators of these enzymes may also be con order to reduce metabolism of lanosterol into vitamin D, sidered to be squalene biosynthesis competitor polypeptides compounds. (e.g., the yeast proteins Sut1 (Genbank Accession JC4374 Zymosterol GI:2133159) and Mot3 (Genbank Accession NP 013786 Zymosterol also functions as a key synthetic intermediate GI:6323715), which may or may not have homologs in other for saponins and steroid hormones. 60 organisms. In some embodiments, heterologous Zymosterol In some embodiments of the present invention, host cells biosynthesis competitor polypeptides may be utilized are engineered to produce Zymosterol and/or to accumulate it (whether functional or non-functional; in Some embodi in lipid bodies. In some embodiments, Zymosterol production ments, dominant negative mutants are employed). is enhanced in a cell by introduction of one or more sterolo Alternatively or additionally, in some embodiments of the genic modifications that increases levels of IPP, FPP and/or 65 present invention, Zymosterol production is enhanced by Zymosterol itself. In some embodiments, Zymosterol produc decreasing the leveland/or activity of one or more Zymosterol tion is enhanced in a cell by increasing the level and/or activ biosynthesis competitor polypeptides that diverts one or more US 8,367,395 B2 39 40 intermediates away from the isoprenoid biosynthesis path tide, 2.3-oxidosqualene-lanosterol cyclase polypeptide, cyto way, thereby reducing levels of IPP. For instance, in some chrome P450 lanosterol 14C-demethylase polypeptide, C-14 particular embodiments of the invention, the level and/or sterol reductase polypeptide, C-4 sterol methyl oxidase activity of acetyl CoA carboxylase, which diverts acetyl CoA polypeptide, SAM:C-24 sterol methyltransferase polypep from the isoprenoid synthesis pathway into the fatty acid tide, C-8 sterol isomerase polypeptide, C-5 sterol desaturase synthesis pathway, is inhibited. Such inhibition may be polypeptide, C-22 sterol desaturase polypeptide, or C-24 ste accomplished, for example, through one or more genetic rol reductase polypeptide in the cell. Representative modifications and/or through use of one or more Small mol examples of squalene synthase polypeptide, squalene epoxi ecule inhibitors. In some embodiments, an inhibitor selected dase polypeptide, 2.3-oxidosqualene-lanosterol cyclase from the group consisting of aryloxyphenoxyproprionate, 10 polypeptide, cytochrome P450 lanosterol 14C-demethylase cyclohexanedione (CHD), and combinations thereof (see also other inhibitors described, for example, in Shukla et al., J polypeptide, C-14 sterol reductase polypeptide, C-4 sterol Agric Food Chem. 52:5144, 2004: Webb et al., J AOAC Int methyl oxidase polypeptide, SAM:C-24 sterol methyltrans 0.84:143, 2001; Webb et al., J Agric Food Chem, 48:1219, ferase polypeptide, C-8 sterol isomerase polypeptide, C-5 2000; Webb et al., JAgric Food Chem, 48:1210, 2000). 15 sterol desaturase polypeptide, C-22 sterol desaturase To give but one particular example of a sterologenic modi polypeptide, or C-24 sterol reductase polypeptide sequences fication that can be employed in accordance with the present are included in Table 16, 83-90, and 93-96. In some embodi invention to reduce or inhibit expression or activity of a ments of the invention that utilize squalene synthase polypep Zymosterol biosynthesis competitor polypeptide and thereby tide, squalene epoxidase polypeptide, 2.3-oxidosqualene to increase production of Zymosterol, we note that it is known lanosterol cyclase polypeptide, cytochrome P450 lanosterol that fungal strains (e.g., S. cerevisiae) that lack any Erg5 or 14C-demethylase polypeptide, C-14 sterol reductase Erg6 activity are viable. In some embodiments of the inven polypeptide, C-4sterol methyl oxidase polypeptide, SAM:C- tion, host cells are utilized that lack, or that are engineered to 24 sterol methyltransferase polypeptide, C-8 sterol isomerase lack (or to have reduced levels of), Erg5 and/or Erg6 polypep polypeptide, C-5 sterol desaturase polypeptide, C-22 sterol tide activity. Absence or inhibition of Erg5 and/or Erg6 25 desaturase polypeptide, or C-24 sterol reductase polypeptide polypeptide activity reduces diversion of carbon into Vitamin (or modifications of these polypeptides) source organisms D. production, and thereby allows increase in levels of include, but are not limited to, Neurospora crassa, Aspergil squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho lus niger; Saccharomyces cerevisiae, Mucor circinelloides, lesterol (provitamin D3) and/or other vitamin D compounds. Candida utilis, Mortierella alpina, and Yarrowia lipolytica. In some embodiments of the invention, in which increased 30 production of Zymosterol is particularly desired, one or more In some embodiments of the invention, ergosterol produc additional sterologenic modifications may be combined with tion in a host cell is increased by reducing the level or activity a reduction or inactivation of Erg5 and/or Erg6 activity in of one or more vitamin D biosynthesis polypeptides (e.g., order to reduce metabolism of Zymosterol into vitamin D. which act to metabolize squalene). For instance, in some compounds. 35 embodiments, the level or activity of one or more polypep Ergosterol tides (see, for example, FIG. 4) is reduced or eliminated. Ergosterol is of commercial importance because it is a Alternatively or additionally, in some embodiments of the precursor for the production of Steroid hormones and ultra present invention, ergosterol production is enhanced by violet irradiation of ergosterol can result in the production of decreasing the level and/or activity of one or more ergosterol vitamin D. 40 biosynthesis competitor polypeptides that diverts one or more In some embodiments of the present invention, host cells intermediates away from the isoprenoid biosynthesis path are engineered to produce ergosterol and/or to accumulate it way, thereby reducing levels of IPP. For instance, in some in lipid bodies. In some embodiments, ergosterol production particular embodiments of the invention, the level and/or is enhanced in a cell by introduction of one or more sterolo activity of acetyl CoA carboxylase, which diverts acetyl CoA genic modifications that increases levels of IPP, FPP and/or 45 from the isoprenoid synthesis pathway into the fatty acid ergosterol itself. In some embodiments, ergosterol produc synthesis pathway, is inhibited. Such inhibition may be tion is enhanced in a cell by increasing the level and/or activ accomplished, for example, through one or more genetic ity of one or more ergosterol biosynthesis polypeptides (e.g., modifications and/or through use of one or more Small mol one or more isoprenoid biosynthesis polypeptides, an FPP ecule inhibitors. In some embodiments, an inhibitor selected synthase polypeptide, squalene synthase polypeptide, 50 from the group consisting of aryloxyphenoxyproprionate, squalene epoxidase polypeptide, 2.3-oxidosqualene-lanos cyclohexanedione (MD), and combinations thereof (see also terol cyclase polypeptide, cytochrome P450 lanosterol 14C.- other inhibitors described, for example, in Shukla et al., J demethylase polypeptide, C-14 sterol reductase polypeptide, Agric Food Chem. 52:5144, 2004: Webb et al., J AOAC Int C-4 sterol methyl oxidase polypeptide, SAM:C-24 sterol 0.84:143, 2001; Webb et al., J Agric Food Chem, 48:1219, methyltransferase polypeptide, C-8 sterol isomerase 55 2000; Webb et al., JAgric Food Chem, 48:1210, 2000). polypeptide, C-5 sterol desaturase polypeptide, C-22 sterol Vitamin D desaturase polypeptide, or C-24 sterol reductase polypep As noted above, vitamin D compounds are a group of tide). Alternatively or additionally, in some embodiments, steroid compounds including vitamin D (cholecalciferol), ergosterol production is enhanced in a cell by decreasing the Vitamin D (ergocalciferol), their provitamins, and certain level and/or activity of one or more sterol biosynthesis com 60 metabolites (see, for example, FIG. 10A-B). Vitamins D and petitor polypeptides that diverts one or more intermediates D. can be produced from their respective provitamins (e.g., away from ergosterol production and/or that metabolizes 7-dehydrocholesterol and ergosterol) by ultraviolet irradia ergosterol itself. tion (e.g., by the action of Sunlight). The most biologically For example, in Some embodiments of the present inven active form of vitamin D is 1,25-dihydroxy vitamin D, which tion, ergosterol production in a host cell is increased by intro 65 is also known as calcitriol. Calcitriol is produced by hydroxy ducing or increasing expression and/or activity of one or more lation of vitamin D. at the 25 position, followed by hydroxy squalene synthase polypeptide, squalene epoxidase polypep lation to generate calcitriol. US 8,367,395 B2 41 42 Vitamin D acts in the body as a hormone involved in terol 14C.-demethylase; and fempropimorph that inhibits the mineral metabolism and bone growth. Vitamin D binds to C-14 sterol reductase and the C-8 sterol isomerase. intracellular receptors; such binding activates the receptors Production and Isolation of Sterol Compounds activity as a transcriptional regulator, for example activating As discussed above, accumulation of lipid bodies in ole transcription of various proteins involved in intestinal absorp aginous organisms is generally induced by growing the rel tion of calcium (and, to some degree, phosphate and magne evant organism in the presence of excess carbon and limiting sium) and of various bone matrix proteins. nitrogen and/or other nutrients (e.g., phosphate and magne Vitamin D deficiency causes rickets in children and osteo sium). Specific conditions for inducing Such accumulation malacia in adults. Both of these disorders result from defec have previously been established for a NUMBER of different 10 oleaginous organisms (see, for example, Wolf (ed)) Noncon tive mineralization of newly synthesized bone matrix. ventional yeasts in Biotechnology Vol. 1, Springer-Verlag, Although a Recommended Dietary Allowance has not been Berlin, Germany, pp. 313-338; Lipids 18(9):623, 1983, established for Vitamin D, the recommended Adequate Intake Indian J. Exp. Biol. 35(3):313, 1997, J. Ind. Microbiol. Bio is 5 g/day for individuals under 50 years of age; 10 ug/day technol.30(1):75, 2003, Bioresour Technol. 95(3):287, 2004; for individuals 51-70 years of age, and 15 g/day for indi 15 each of which is incorporated herein by reference in its viduals over 71 year old. entirety). In some embodiments of the present invention, host cells In general, it will be desirable to cultivate inventive modi are engineered to produce one or more vitamin D compounds fied host cells under conditions that allow accumulation of at and/or to accumulate it/them in lipid bodies. In some embodi least about 20% of their dry cell weight as lipid. In other ments, vitamin D production is enhanced in a cell by intro embodiments, the inventive modified host cells are grown duction of one or more sterologenic modifications that under conditions that permit accumulation of at least about increases levels of IPP, FPP and/or squalene. In some embodi 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, ments, vitamin D production is enhanced in a cell by increas 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, ing the level and/or activity of one or more vitamin D biosyn 75%, or even 80% or more of their dry cell weight as lipid. In thesis polypeptides (e.g., one or more isoprenoid biosynthesis 25 certain embodiments, the host cells utilized are cells which polypeptides, an FPP synthase polypeptide, a squalene Syn are naturally oleaginous and induced to produce lipid to the thase polypeptide, and/or one or more polypeptides involved desired levels. In other embodiments, the host cells are cells in converting squalene into a particular vitamin D compound which naturally produce lipid, but have been engineered to of interest e.g., 7-dehydrocholesterol and/or calcitriol). increase production of lipid such that desired levels of lipid Alternatively or additionally, in Some embodiments, vitamin 30 production and accumulation are achieved. D production is enhanced in a cell by decreasing the level In certain embodiments, the host cells of the invention are and/or activity of one or more sterol biosynthesis competitor not naturally oleaginous, but have been engineered to produce polypeptides that diverts one or more intermediates away lipid such that desired levels of lipid production are obtained. from vitamin D production. In some embodiments of the Those of ordinary skill in the art will appreciate that, in invention, production of a particular vitamin D compound 35 general, growth conditions that are effective for inducing (e.g., 7-dehydrocholesterol and/or calcitriol) is enhanced by lipid accumulation in a source organism, may well also be decreasing the level and/or activity of one or more polypep useful for inducing lipid accumulation in a host cell into tides that diverts a relevant intermediate toward an alternative which the Source organism’s oleaginic polypeptides have Vitamin D compound (e.g., ergosterol, Vitamin D). been introduced. Of course, modifications may be required in To give but one particular example of a sterologenic modi 40 light of characteristics of the host cell, which modifications fication that can be employed in accordance with the present are within the skill of those of ordinary skill in the art. invention to increase production of one or more vitamin D. It will also be appreciated by those of ordinary skill in the compounds (e.g., 7-dehydrocholesterol and/or calcitriol), in art that it will often be desirable to ensure that production of accordance with some embodiments of the present invention, the desired sterol compound by the inventive modified host the level and/or activity of one or more polypeptides that 45 cell occurs at an appropriate time in relation to the induction diverts a relevant intermediate toward a vitamin D com ofoleaginy Such that the sterol compound(s) accumulate(s) in pound (e.g., ergosterol, Vitamin D), and away from vitamin the lipid bodies. In some embodiments, it will be desirable to D compounds, can be inhibited or destroyed. induce production of one or more sterol compounds (e.g., For example, fungal Strains (e.g., S. cerevisiae) that lack squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho any Erg5 or Erg6 activity are viable. In some embodiments of 50 lesterol (provitamin D3), Vitamin D compounds, etc.) in a the invention, host cells are utilized that lack, or that are host cell which does not naturally produce the compound engineered to lack (or to have reduced levels of), Erg5 and/or such that detectable levels of the compound are produced. In Erg6 polypeptide activity. Absence or inhibition of Erg5 and/ certain aspects the host cells which do not naturally produce or Erg6 polypeptide activity reduces diversion of carbon into a particular sterol compound of interest (e.g., squalene, lanos Vitamin D. production, and thereby allows increase in levels 55 terol, Zymosterol, ergosterol, 7-dehydrocholesterol (provita of squalene, lanosterol, Zymosterol, and/or vitamin D com min D3), vitamin D compounds, etc.) are capable of produc pounds (e.g., 7-dehydrocholesterol and/or calcitriol). In some ing other sterol compounds. In other embodiments, it will be embodiments, heterologous vitamin D biosynthesis competi desirable to increase production levels of a particular sterol tor polypeptides may be utilized (whether functional or non compound (e.g., squalene, lanosterol, Zymosterol, ergosterol, functional; in Some embodiments, dominant-negative 60 7-dehydrocholesterol (provitamin D3), vitamin D com mutants are employed). pounds, etc.) in a host cell which does naturally produce low Alternatively or additionally, one or more small molecule levels of the compound, such that increased detectable levels inhibitors that reduce the level and/or activity of one or more of the compound are produced. In certain aspects, the host vitamin D biosynthesis polypeptides that diverts a relevant cells which do naturally produce the particular sterol com intermediate away from vitamin D. production may be 65 pound of interest (e.g., squalene, lanosterol, Zymosterol, employed. Representative such inhibitors include, for ergosterol, 7-dehydrocholesterol (provitamin D3), vitamin D example, various azoles that inhibit cytochrome P450 lanos compounds, etc.) also produce additional sterol compounds; US 8,367,395 B2 43 44 in other embodiments, the cells which naturally produce a at least about 40%, at least about 45% or more of the total dry particular sterol compound of interest (e.g., squalene, lanos weight of the cells and in some cases to a level above about terol, Zymosterol, ergosterol, 7-dehydrocholesterol (provita 50% the total dry weight of the cells. min D3), Vitamin D compounds, etc.) do not produce addi In some particular embodiments of the present invention, tional sterol compound(s). squalene is accumulated in Y lipolytica cells to a level above In certain embodiments of the invention, it will be desirable 2%, at least about 2.5%, at least about 3%, at least about 3.5%, to accumulate one or more sterol compounds (i.e., consider at least about 4%, at least about 4.5%, at least about 5%, at ing the total amount of all produced sterol compounds least about 5.5%, at least about 6%, at least about 6.5%, at together or considering a particular sterol compound e.g., least about 7%, at least about 7.5%, at least about 8%, at least squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho 10 about 8.5%, at least about 9%, at least about 9.5%, at least lesterol (provitamin D3), vitamin D compounds, etc.) to about 10%, at least about 10.5%, at least about 11%, at least levels that are greater than at least about 1% of the dry weight about 11.5%, at least about 12%, at least about 12.5%, at least of the cells. In some embodiments, the total sterol compound about 13%, at least about 13.5%, at least about 14%, at least accumulation will be to a level at least about 1.5%, at least about 14.5%, at least about 15%, at least about 15.5%, at least about 2%, at least about 2.5%, at least about 3%, at least about 15 about 16%, at least about 16.5%, at least about 17%, at least 3.5%, at least about 4%, at least about 4.5%, at least about 5%, about 17.5%, at least about 18%, at least about 18.5%, at least at least about 5.5%, at least about 6%, at least about 6.5%, at about 19%, at least about 19.5%, at least about 20% or more least about 7%, at least about 7.5%, at least about 8%, at least of the total dry weight of the cells and in some cases to a level about 8.5%, at least about 9%, at least about 9.5%, at least above about 25% the total dry weight of the cells. about 10%, at least about 10.5%, at least about 11%, at least In some particular embodiments of the present invention, about 11.5%, at least about 12%, at least about 12.5%, at least lanosterol is accumulated to a level above 1%, at least about about 13%, at least about 13.5%, at least about 14%, at least 1.5%, at least about 2%, at least about 2.5%, at least about 3%, about 14.5%, at least about 15%, at least about 15.5%, at least at least about 3.5%, at least about 4%, at least about 4.5%, at about 16%, at least about 16.5%, at least about 17%, at least least about 5%, at least about 5.5%, at least about 6%, at least about 17.5%, at least about 18%, at least about 18.5%, at least 25 about 6.5%, at least about 7%, at least about 7.5%, at least about 19%, at least about 19.5%, at least about 20% or more about 8%, at least about 8.5%, at least about 9%, at least about of the total dry weight of the cells. 9.5%, at least about 10%, at least about 10.5%, at least about In some particular embodiments of the present invention, 11%, at least about 11.5%, at least about 12%, at least about squalene is accumulated to a level above 2%, at least about 12.5%, at least about 13%, at least about 13.5%, at least about 2.5%, at least about 3%, at least about 3.5%, at least about 4%, 30 14%, at least about 14.5%, at least about 15%, at least about at least about 4.5%, at least about 5%, at least about 5.5%, at 15.5%, at least about 16%, at least about 16.5%, at least about least about 6%, at least about 6.5%, at least about 7%, at least 17%, at least about 17.5%, at least about 18%, at least about about 7.5%, at least about 8%, at least about 8.5%, at least 18.5%, at least about 19%, at least about 19.5%, at least about about 9%, at least about 9.5%, at least about 10%, at least 20% or more of the total dry weight of the cells and in some about 10.5%, at least about 11%, at least about 11.5%, at least 35 cases to a level above about 25% the total dry weight of the about 12%, at least about 12.5%, at least about 13%, at least cells. about 13.5%, at least about 14%, at least about 14.5%, at least In some particular embodiments of the present invention, about 15%, at least about 15.5%, at least about 16%, at least Zymosterol is accumulated to a level above 1%, at least about about 16.5%, at least about 17%, at least about 17.5%, at least 1.5%, at least about 2%, at least about 2.5%, at least about 3%, about 18%, at least about 18.5%, at least about 19%, at least 40 at least about 3.5%, at least about 4%, at least about 4.5%, at about 19.5%, at least about 20% or more of the total dry least about 5%, at least about 5.5%, at least about 6%, at least weight of the cells and in some cases to a level above about about 6.5%, at least about 7%, at least about 7.5%, at least 25% the total dry weight of the cells. about 8%, at least about 8.5%, at least about 9%, at least about In some particular embodiments of the present invention, 9.5%, at least about 10%, at least about 10.5%, at least about squalene is accumulated to a level above 6%, at least about 45 11%, at least about 11.5%, at least about 12%, at least about 6.5%, at least about 7%, at least about 7.5%, at least about 8%, 12.5%, at least about 13%, at least about 13.5%, at least about at least about 8.5%, at least about 9%, at least about 9.5%, at 14%, at least about 14.5%, at least about 15%, at least about least about 10%, at least about 10.5%, at least about 11%, at 15.5%, at least about 16%, at least about 16.5%, at least about least about 11.5%, at least about 12%, at least about 12.5%, at 17%, at least about 17.5%, at least about 18%, at least about least about 13%, at least about 13.5%, at least about 14%, at 50 18.5%, at least about 19%, at least about 19.5%, at least about least about 14.5%, at least about 15%, at least about 15.5%, at 20% or more of the total dry weight of the cells and in some least about 16%, at least about 16.5%, at least about 17%, at cases to a level above about 25% the total dry weight of the least about 17.5%, at least about 18%, at least about 18.5%, at cells. least about 19%, at least about 19.5%, at least about 20% or In some particular embodiments of the present invention, more of the total dry weight of the cells and in some cases to 55 ergosterol is accumulated to a level above 1%, at least about a level above about 25% the total dry weight of the cells. 1.5%, at least about 2%, at least about 2.5%, at least about 3%, In some particular embodiments of the present invention, at least about 3.5%, at least about 4%, at least about 4.5%, at squalene is accumulated in S. cerevisiae to a level above 17%, least about 5%, at least about 5.5%, at least about 6%, at least at least about 17.5%, at least about 18%, at least about 18.5%, about 6.5%, at least about 7%, at least about 7.5%, at least at least about 19%, at least about 19.5%, at least about 20%, 60 about 8%, at least about 8.5%, at least about 9%, at least about at least about 20.5%, at least about 21%, at least about 21.5%, 9.5%, at least about 10%, at least about 10.5%, at least about at least about 22%, at least about 22.5%, at least about 23%, 11%, at least about 11.5%, at least about 12%, at least about at least about 23.5%, at least about 24%, at least about 24.5%, 12.5%, at least about 13%, at least about 13.5%, at least about at least about 25%, at least about 25.5%, at least about 26%, 14%, at least about 14.5%, at least about 15%, at least about at least about 26.5%, at least about 27%, at least about 27.5%, 65 15.5%, at least about 16%, at least about 16.5%, at least about at least about 28%, at least about 28.5%, at least about 29%, 17%, at least about 17.5%, at least about 18%, at least about at least about 29.5%, at least about 30%, at least about 35%, 18.5%, at least about 19%, at least about 19.5%, at least about US 8,367,395 B2 45 46 20% or more of the total dry weight of the cells and in some mallow; meadowfoam seed; coffee; emu, mink; grape seed; cases to a level above about 25% the total dry weight of the thistle; tea tree; pumpkin seed; kukui nut; and mixtures cells. thereof. In some particular embodiments of the present invention, Also, it should be noted that, the absolute and/or relative ergosterol is accumulated to a level above 2%, at least about amounts of particular sterol compounds produced in accor 2.5%, at least about 3%, at least about 3.5%, at least about 4%, dance with the present invention, and/or the absolute and/or at least about 4.5%, at least about 5%, at least about 5.5%, at relative amounts of other compounds derived from iso least about 6%, at least about 6.5%, at least about 7%, at least prenoids can sometimes be altered by adjustment of growth about 7.5%, at least about 8%, at least about 8.5%, at least conditions. For example, it has been reported that controlling about 9%, at least about 9.5%, at least about 10%, at least 10 the concentration of dissolved oxygen in a culture during about 10.5%, at least about 11%, at least about 11.5%, at least cultivation can regulate relative production levels of certain about 12%, at least about 12.5%, at least about 13%, at least sterol compounds (see, for example, J. Cell. Comp. Physiol. about 13.5%, at least about 14%, at least about 14.5%, at least Vol. 41, p 23, 1953). Such condition alterations can therefore about 15%, at least about 15.5%, at least about 16%, at least be utilized in accordance with the present invention to favor about 16.5%, at least about 17%, at least about 17.5%, at least 15 partition of carbons into pathways that produce one or more about 18%, at least about 18.5%, at least about 19%, at least sterol compounds of interest, and/or to disfavor partition of about 19.5%, at least about 20% or more of the total dry carbon into pathways that compete with Such sterol biosyn weight of the cells and in some cases to a level above about thesis pathways. 25% the total dry weight of the cells. In certain embodiments, fungal or yeast sterologenic genes In some embodiments of the invention, a particular sterol (when fungi make the sterol of interest) may be transferred compound (e.g. a vitamin D compound) may not accumulate from one organism to another, and are therefore useful in to a level as high as 1% of the total dry weight of the cells; accordance with the present invention Similarly, bacterial appropriately engineered cells according to the present inven sterologenic genes may be transferred from one organism to tion, and any lipid bodies and/or sterol compound(s) they another. In other embodiments, it may be desirable to utilize produce, remain within the scope of the present invention. 25 genes from other source organisms such as plant, alga, or Thus, in some embodiments, the cells accumulate a given microalgae. Still additional useful source organisms include sterol compound to a level below about 1% of the dry weight insect, protozoal, and mammalian sources of polypeptides. of the cells. In some embodiments, the sterol compound accu In some embodiments of the present invention, isoprenoid mulates to a level below about 0.9%, 0.8%, 0.7%, 0.6%, production is increased in host cells (e.g., in Y lipolytica 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%, or lower, of the dry 30 cells) through expression of a truncated variant of a cell weight of the cells. hydroxymethylglutaryl-CoA (HMG CoA) reductase In some embodiments of the invention, one or more sterol polypeptide. In some embodiments, the truncated variant is a compound(s) accumulate both within lipid bodies and else truncated variant of a Y lipolytica HMG CoA reductase where in the cells. In some embodiments, one or more sterol polypeptide. According to the present invention, expression compound(s) accumulate primarily within lipid bodies. In 35 of such a truncated HMG CoA reductase polypeptide can Some embodiments, one or more sterol compound(s) accu result in increased isoprenoid and/or sterol compound pro mulate substantially exclusively within lipid bodies. In some duction in host cells (e.g., Y lipolytica cells). embodiments, at least about 10%, 15%, 20%, 25%, 30%, Alternatively or additionally, in some embodiments of the 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, present invention, isoprenoid production is increased in host 85%, 90%, 95% or more of desired produced sterol com 40 cells (e.g., in Y lipolytica, S. cerevisiae cells, or C. utilis pound(s) accumulates in lipid bodies. cells) through application of one or more sterologenic modi In some embodiments of the invention, modified host cells fication(s) that increase(s) level and/or activity of a polypep are engineered to produce one or more sterol compound(s) tide selected from the group consisting of squalene synthase, characterized by negligible solubility in water (whether hot or squalene epoxidase, and combinations thereof. In some cold) and detectable solubility in one or more oils. In some 45 embodiments, the Source organism for the selected polypep embodiments, such compounds have a solubility in oil below tide is Y lipolytica. In some specific embodiments of the about 0.2%. In some embodiments, such compounds have a present invention, Y lipolytica cells are engineered to express solubility in oil within the range of about <0.001%-0.2%. elevated amounts of endogenous or exogenous squalene Syn The present invention therefore provides engineered host thase and/or squalene oxidase polypeptides. In some embodi cells (and methods of making and using them) that contain 50 ments, such cells further express a truncated HMG CoA lipid bodies and that further contain one or more sterol com reductase polypeptide (e.g., a truncated Y lipolytica HMG pounds accumulated in the lipid bodies, where the com CoA reductase polypeptide. pounds are characterized by a negligible solubility in water Inventive modified cells, that have been engineered to pro and a solubility in oil within the range of about <0.001%- ducesterol compounds and/or to accumulate lipid (including 0.2%; 0.004%-0.15%; 0.005-0.1%; or 0.005-0.5%. For 55 to be oleaginous), can be cultured under conditions that example, in some embodiments, such compounds have a achieve Sterol production and/or oleaginy. In some embodi solubility in oil below about 0.15%, 0.14%, 0.13%, 0.12%, ments, it will be desirable to control growth conditions in 0.11%, 0.10%. 0.09, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, order to maximize production of a particular sterol compound 0.03%, 0.02%, 0.01%, 0.05%, or less. In some embodiments, or set of sterol compounds (including all sterol compounds) the compounds show such solubility in an oil selected from 60 and/or to optimize accumulation of the particular sterol com the group consisting of Sesame; soybean, apricot kernel; pound(s) in lipid bodies. In some embodiments it will be palm; peanut, safflower, coconut, olive; cocoa butter, palm desirable to control growth conditions to adjust the relative kernel; shea butter, Sunflower, almond; avocado; borage; car amounts of different sterol compound products produced. nauba; hazel nut, castor, cotton seed; evening primrose; In some embodiments, it will be desirable to limit accumu orange roughy, rapeseed; rice bran; walnut, wheat germ; 65 lation of a particular intermediate, for example ensuring that peach kernel; babassu; mango seed; black current seed; Substantially all of a particular intermediate compound is jojoba, macademia nut, sea buckthorn; Sasquana; tsubaki; converted so that accumulation is limited. For example, par US 8,367,395 B2 47 48 ticularly in situations where a downstream enzyme may be of culture and not controlled, or controlled at a different point, less efficient than an upstream enzyme and it is desirable to during others. In some embodiments, oxygen concentration is limit accumulation of the product of the upstream enzyme not controlled. In some embodiments, cultures are grown at (e.g., to avoid its being metabolized via a competitive path an oxygen concentration within the range of about 5-30%, way and/or converted into an undesirable product), it may be 5-20%, 10-25%, 10-30%, 15-25%, 15-30%, including at desirable to grow cells under conditions that control (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, slow) activity of the upstream enzyme so that the downstream 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, enzyme can keep pace. 25%, 26%, 27%, 28%, 29%, 30%, or more. In some embodi Those of ordinary skill in the art will appreciate that any of ments, oxygen concentration is maintained above about 20%, a variety of growth parameters, including for example amount 10 of a particular nutrient, pH, temperature, pressure, oxygen at least for some period of the culture. concentration, timing of feeds, content of feeds, etc can be In some embodiments, cells are grown via fed-batch fer adjusted as is known in the art to control growth conditions as mentation. In some embodiments, feed is continued until feed desired. exhaustion and/or the feed is controlled to initiate or increase To give but a few examples, in Some embodiments, growth 15 once a certain level of dissolved oxygen is detected in the and/or metabolism is/are limited by limiting the amount of culture medium (e.g., about 5%, 6%, 7%, 8%.9%, 10%, 11%, biomass accumulation. For example, growth and/or metabo 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, lism can be limited by growing cells under conditions that are 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, limiting for a selected nutrient. The selected limiting nutrient 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or more can then be added in a regulated fashion, as desired. In some dissolved oxygen). The feed rate can be modulated to main embodiments, the limiting nutrient is carbon, nitrogen (e.g., tain the dissolved oxygen at a specific level (e.g., about 5%, via limiting ammonium or protein), phosphate, magnesium, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, one or more trace metals, or combinations thereof. In some 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, embodiments, the limiting nutrient is carbon. In some 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, embodiments, the limiting nutrient is one or more trace met 25 37%, 38%, 39%, 40%, or more dissolved oxygen). als. In some embodiments, inventive modified cells are grown In some embodiments, use of a limiting nutrient can by in a two-phase feeding protocol in which the first phase is utilized to control metabolism of a particular intermediate designed to maintain conditions of excess carbon and limiting and/or to adjust relative production of particular sterol com oxygen, and the second phase results in conditions of excess pounds. In some embodiments, this result can be achieved by 30 oxygen and limiting carbon. The carbon sources in each controlling metabolism of a particular intermediate as dis phase can be the same (e.g., both glucose) or different (e.g., cussed above; in some embodiments, it can be achieved, for glucose then glucose-oil mixture, oil then glucose, or glu example, by limiting progress through the sterol biosynthesis cose-oil mixture then glucose). pathway so that a desired sterol compound product (e.g., In some embodiments, inventive modified cells are culti squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho 35 vated at constant temperature (e.g., between about 20-40, or lesterol (provitamin D3) and/or vitamin D compounds, etc.) 20-30 degrees, including for example at about 20, 20.5, 21, is not converted to a downstream compound. 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, In some embodiments, cells are grown in the presence of 28, 28.5, 29, 29.5, 30° C. or above) and/or pH (e.g., within a excess carbon Source and limiting nitrogen, phosphate, and/ range of about 4-7.5, or 4-6.5, 3.5-7, 3.5-4, 4-4.5, 4.5-5.5-5.5, or magnesium to induce oleaginy. In some embodiments cells 40 5.5-6, 6-6.5, 6.5-7, 7-7.5, 7-8, etc., including at about 4.0, 4.1, are grown in the presence of excess carbon Source and limit 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, ing nitrogen. In some embodiments, the carbon: nitrogen ratio 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4., 6.5, 6.6, 6.7, 6.8, 6.9, is within the range of about 200:1, 150:1, 125:1, 100:1.95:1, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 90:1,85:1, 8.0:1, 75:1,70:1, 65:1, 60:1, 55:1, 50:1, 45:1,40:1, 8.4, 8.5 or above); in other embodiments, temperature and/or 35:1, 30:1, 25:1, 20:1, 15:1, 10:1, or less. Those of ordinary 45 pH may be varied during the culture period, either gradually skill in the art are aware of a wide variety of carbon sources, or in a stepwise fashion. including, for example, glycerol, glucose, galactose, dex For example, in Some embodiments, the pH at inoculation trose, any of a variety of oils (e.g., olive, canola, corn, Sun is lower than the pH during the course of the fermentation. For flower, soybean, cottonseed, rapeseed, etc., and combinations example, the pH may be 7.0 at inoculation and increased to thereof) that may be utilized in accordance with the present 50 pH 8.0 during the course of the fermentation. The pH may be invention. Combinations of such may also be utilized. For increased either continuously or in discrete steps. In certain example, common carbon Source compositions contain oil: embodiments, the pH in increased continuously by increasing glucose in a ratio within the range of about 5:95 to 50:50 (e.g. the pH at a rate of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, about 5:95, about 10:90, about 15:85, about 20:80, about 0.007, 0.008, 0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 25:75, about 30:70, about 35:65, about 40:60, about 45:55, 55 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, about 50:50). 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, 0.032, 0.033, Those of ordinary skill in the art are also aware of a variety 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, of different nitrogen sources (e.g., ammonium sulfate, pro 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049,0.050 or more line, Sodium glutamate, soy acid hydrolysate, yeast extract units/hour. peptone, yeast nitrogen base, corn steep liquor, etc., and com 60 In certain embodiments, the pH in increased in discrete binations thereof) that can be utilized in accordance with the steps by increasing the pH by 0.001, 0.002, 0.003, 0.004, present invention. 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.011, 0.012, 0.013, In some embodiments, cultures are grown at a selected 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, oxygen concentration (e.g., within a selected range of oxygen 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, concentrations). In some embodiments, oxygen concentra 65 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039,0.040, tion may be varied during culture. In some embodiments, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, oxygen concentration may be controlled during some periods 0.050 or more at each step. US 8,367,395 B2 49 50 In certain embodiments, the pH is increased employing a 343, U.S. Pat. No. 6,166,231, U.S. Pat. No. 6,750,048, U.S. combination of continuous increase and discrete steps. Pat. No. 6,812,001, U.S. Pat. No. 6,818,239, U.S. Pat. No. In certain embodiments, increasing the pH during the 7,015,014, US2003/0054070, US2005/0266132, each of course of fermentation results in one or more beneficial which is incorporated herein by reference). effects such as, without limitation, an increase in total biom 5 In many typical isolation procedures, cells are disrupted ass accumulation and/or an increase in the percentage of (e.g., mechanically (for example using a bead mill, mashing), biomass representing sterol accumulation. Those of ordinary enzymatically (e.g. using Zymolyase or a B-1.3 glucanase skill in the art will be able to select without undue experimen such as Glucanex 200G (Novozyme), chemically (e.g., by tation an appropriate rate of increase, an appropriate type of exposure to a mild caustic agent such as a detergent or 0.1 N increase (e.g. continuous, discrete steps or a combination of 10 the two), and/or an optimum pH within the selected range to NaOH, for example at room temperature or at elevated tem maximize these and/or other beneficial effects. perature), using a reducing agent (e.g. dithiothreitol, 3-mer In some embodiments, the temperature at which inventive captoethanol), using high pressure homogenization/shearing, cells are cultivated is selected so that production of one or by changing pH, etc. and combinations thereof) to allow more particular sterol compound(s) is adjusted (e.g., so 15 access of intracellular sterol compound(s) to an extraction that production of one or more particular compound(s) is Solvent, and are then extracted one or more times. In certain increased and/or production of one or more other com embodiments, cells are disrupted mechanically using a bead pound(s) is decreased). In some embodiments, the tempera mill/mashing at high pressure (e.g. at 25K, 10K-30K, 15K ture at which inventive cells are cultivated is selected so that 25K, or 20-25K, pound-force per square inch (psi)). Cells the ratio of one sterol compound to another, is adjusted. To may optionally be concentrated (e.g., to at least about 100 g/L give but one example, in Some embodiments, a temperature is or more, including to at least about 120 g/l. 150 g/l. 175 g/L, selected to be sufficiently low that levels of one sterol com 200 g/L or more) and/or dried (e.g., with a spray dryer, double pound levels are reduced and the level of at least one sterol drum dryer (e.g. Blaw Knox double drum dryer), single drum compound is increased. vacuum dryer, etc.), prior to exposure to extraction solvent In some embodiments, cultures are grown at about pH 5.5, 25 (and/or prior to disruption or homogenization). Disruption at about pH 7.0, and or at a temperature between about 28-30° can, of course, be performed prior to and/or during exposure C. In some embodiments, it may be desirable to grow inven to extraction solvent. After extraction, Solvent is typically tive modified cells under low pH conditions, in order to mini removed (e.g., by evaporation, for example by application of mize growth of other cells. In some embodiments, it will be vacuum, change of temperature, etc.). desirable to grow inventive modified cells under relatively 30 In some instances, cells are disrupted and then Subjected to higher temperature conditions in order to slow growth rate Supercritical liquid extraction or solvent extraction. Typical and/or increase the ultimate dry cell weight output of sterols. liquids or solvents utilized in such extractions include, for In some embodiments, it will be desirable to grow inventive example, organic or non-organic liquids or solvents. To give modified cells under conditions in which the pH in increased but a few specific examples, such liquids or solvents may (e.g. continuously, in discrete steps, or both) during the course 35 include acetone, Supercritical fluids (e.g. carbon dioxide, pro of fermentation (e.g. increased from pH 7.0 to pH 8.0). In pane, Xenon, ethane, propylene, methane, ethylene, ethanol), some embodiments, it will be desirable to grow inventive carbon dioxide, chloroform, ethanol, ethyl acetate, heptane, modified cells under two or more of these conditions. For hexane, isopropanol, methanol, methylene chloride, octane, example, inventive modified cells can be grown under rela tetrahydrofuran (THF), cyclohexane, isobutyl acetate, methyl tively higher temperature conditions while simultaneously 40 ketone, ethyl ketone, toluene, cyclohexanone, benzene, pro increasing the pH over the course of the fermentation. Those pylene glycol, vegetable oils (e.g. soybeen soybean oil, rape of ordinary skill in the art will be able to select appropriate seed oil, corn oil, cottonseed oil, canola oil, etc.) and combi growth conditions to achieve their experimental, production nations thereof (e.g. hexane:ethyl acetate, combination of a and/or other cell culture goals. polar and non-polar solvent, combination of an alcohol with One advantage provided by the present invention is that, in 45 either hexane or ethyl acetate). Particular solvents may be addition to allowing the production of high levels of one or selected, for example, based on their ability to solubilize more sterol compound(s), certain embodiments of the present particular sterol compounds, or sets of Sterol compounds invention allow produced compounds to be readily isolated (e.g., all Sterols), and/or based on regulatory or other consid because they accumulate in the lipid bodies withinoleaginous erations (e.g., toxicity, cost, ease of handling, ease of removal, organisms. Methods and systems for isolating lipid bodies 50 ease of disposal, etc.). have been established for a wide variety of oleaginous organ In some embodiments, combinations of solvents may be isms (see, for example, U.S. Pat. Nos. 5,164.308; 5,374,657: utilized In some embodiments, combinations of a relatively 5,422,247; 5,550,156; 5,583,019; 6,166,231; 6,541,049; polar solvent (e.g., alcohols, acetone, chloroform, methylene 6,727,373; 6,750,048; and 6,812,001, each of which is incor chloride, ethyl acetate, etc.) and a relatively non-polar solvent porated herein by reference in its entirety). In brief, cells are 55 (e.g., hexane, cyclohexane, oils, etc.) are utilized for extrac typically recovered from culture, often by spray drying, fil tion. Those of ordinary skill in the art will readily appreciate tering or centrifugation. that different ratios of polar to non-polar solvent may be Ofcourse, it is not essential that lipid bodies be specifically employed as appropriate in a particular situation. Just to give isolated in order to collect sterol compounds produced a few examples, common ratios include 1:1, 2:1, 3:1, 3:2. according to the present invention. Any of a variety of 60 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, 65:45, 60:40, 55:45, approaches can be utilized to isolate and/or purify sterol and 50:50. It will be appreciated that solvents or solvent compounds. Many useful extraction and/or purification pro mixtures of different polarities may be more effective at cedures for particular sterol compounds, and/or for sterols extracting particular sterols (e.g., based on their polarities generally, are known in the art (see, for example, EP670306, and/or as a function of other attributes of the host cell material EP719866, U.S. Pat. No. 4,439,629, U.S. Pat. No. 4,680,314, 65 from which they are being extracted). Those of ordinary skill U.S. Pat. No. 5,310,554, U.S. Pat. No. 5,328,845, U.S. Pat. in the art are well able to adjust the overall polarity of the No. 5,356,810, U.S. Pat. No. 5,422,247, U.S. Pat. No. 5,591, extracting solvent, for instance by adjusting the relative US 8,367,395 B2 51 52 amounts of polar and non-polar solvents in a solvent blend, in and/or other purification strategies. In some embodiments, order to achieve more efficient extraction. sterol crystals are collected by filtration and/or centrifuga Extraction may be performed under any of a variety of tion. Isolated or purified sterols may be dried and/or formu environmental conditions, including any of a variety of tem lated for storage, transport, sale, and/or ultimate use. To give peratures. For example, extraction may be performed on ice but a few specific examples, Sterols may be prepared as a (for example at 4°C., 0°C., less than 0°C.), at room tem water (e.g. cold-water) dispersible powder (e.g., 1%-20% perature, or at any of a variety of other temperatures. For sterol: microencapsulation ingredient), as a Suspension of example, a solvent may be maintained at a selected tempera crystals in oil (e.g., vegetable oil, e.g., about 1%-30%, ture (e.g., about less than 0, 0, 4, 25, 28, 30, 37, 68,70, 75,80, 5%-30%, 10%-30% w/w), etc. 85,90, 95, or 100°C.) in order to improve or adjust extraction 10 Uses of a particular desired sterol compound. Sterol compounds of interest (e.g., squalene, lanosterol, Extraction typically yields a crude oil suspension. In some Zymosterol, ergosterol, 7-dehydrocholesterol (provitamin embodiments, the crude oil suspension contains some intact D3), Vitamin D compounds e.g., 7-dehydrocholesterol, cal host cells but is at least about 95% free of intact host cells. In citriol) produced according to the present invention can be Some embodiments, the crude oil suspension is at least about 15 utilized in any of a variety of applications, for example 96%, 97%, 98%, or 99% or more free of intact host cells. In exploiting biological or nutritional (e.g., metabolic, anti-oxi Some embodiments, the Suspension is Substantially free of dant, anti-proliferative, etc.) properties. water-soluble cell components (e.g., nucleic acids, cell wall For example, according to the present invention, one or or storage carbohydrates, etc.). In some embodiments, the more vitamin D compounds (and particularly 7-dehydrocho suspension contains less than about 5%, 4%. 3%, 2%, or 1% lesterol, and/or calcitriol) may be used in pharmaceutical or less water-soluble cell components. and/or nutraceutical applications for treatment and/or preven Extraction conditions that yield a crude oil suspension will tion of disorders associated with vitamin D deficiency. Alter enrich for lipophilic components that accumulate in the lipid natively or additionally, one or more vitamin D compounds bodies within oleaginous organisms. In general, the major (and/or optionally one or more other Sterol compounds. Such components of the lipid bodies consist of triacylglycerols, 25 as squalene, lanosterol, Zymosterol, ergosterol, 7-dehydroc ergosteryl esters, other steryl esters, free ergosterol, phospho holesterol (provitamin D3)) may be incorporated into a nutri lipids, and some proteins, which often function in the synthe tional Supplement or a cosmetic product (e.g., a skin cream, sis or regulation of the levels of other lipid body components. etc.). Sterol compound(s) produced herein can also be co C16 and C18 (e.g. C16:0, C16:1, C18:0, C18:1, and C18:2) administered with an HMG CoA reductase inhibitor (e.g. a are generally the major fatty acids present in lipid bodies, 30 statin Such as atorvastatin, simvastatin, rosuvastatin, etc.). mainly as components of triacylglycerol and steryl esters. It will be appreciated that, in some embodiments of the In some embodiments of the invention, the crude oil sus invention, one or more sterol compound(s) (e.g., squalene, pension contains at least about 2.5% by weight sterol com lanosterol, Zymosterol, ergosterol, 7-dehydrocholesterol pound(s); in some embodiments, the crude oil suspension (provitamin D3), produced by manipulated host cells as contains at least about 5% by weight Sterol compound(s), at 35 described herein are incorporated into a final product (e.g., least about 10% by weight sterolog compound(s), at least food or feed supplement, pharmaceutical, cosmetic, etc.) in about 20% by weight sterol compound(s), at least about 30% the context of the host cell. For example, host cells may be by weight sterol compound(s), at least about 40% by weight lyophilized, freeze dried, frozen or otherwise inactivated, and sterol compound(s), or at least about 50% by weight sterol then whole cells may be incorporated into or used as the final compound(s). 40 product. The host cells may also be processed prior to incor The crude oil Suspension may optionally be refined as poration in the product to, e.g., increase bioavailability (e.g., known in the art. Refined oils may be used directly as feed or via lysis). Alternatively or additionally, a final product may food additives. Alternatively or additionally, sterols can be incorporate only a portion of a host cell (e.g., fractionated by isolated from the oil using conventional techniques. size, solubility), separated from the whole. For example, in Given the sensitivity of sterols generally to oxidation, 45 some embodiments of the invention, lipid bodies are isolated many embodiments of the invention employ oxidative stabi from host cells and are incorporated into or used as the final lizers (e.g., ascorbyl palmitate, tocopherols, vitamin C (e.g., product. For instance, inventive sterol compound-containing sodium ascorbate), ethoxyquin, vitamin E. BHT, BHA, lipid bodies (e.g., from engineered cells, and particularly TBHQ, etc., or combinations thereof) during and/or after from engineered fungal cells) may be substitutes for the plant sterol isolation. Alternatively or additionally, nitrogen or an 50 oil bodies described in U.S. Pat. No. 6,599,513 (the entire inert gas can be utilized to purge oxygen from the process contents of which are hereby incorporated by reference) and lines of any tanks or equipment. Alternatively or additionally, incorporated into emulsions or emulsion formulations, as microencapsulation, for example with a microencapsulation described thereon. In other embodiments, ubiquinone itself is ingredients such as proteins, carbohydrates (e.g. maltodex isolated and reformulated into a final product. trin, gum acacia, Xanthan gum, starches/sugars like Sucrose), 55 It will further be understood that, in some embodiments of or gelatins, or any other Substance which creates a physical the present invention, host cells are engineered to produce barrier to air and/or light), may be employed to add a physical and/or accumulate a compound that is an intermediate to a barrier to oxidation and/or to improve handling (see, for commercial product. For instance, in some embodiments, example, U.S. Patent Applications 2004/0191365 and 2005/ squalene, lanosterol, Zymosterol, or ergosterol can be utilized 0.169999). For example, sterol compounds produced accord 60 as an intermediate to production of various steroids. Also, in ing to the present invention may be microencapsulated after Some embodiments, a vitamin D compound is produced (e.g., isolation during the formulation of commercial products (e.g. 7-dehydrocholestrol) that is a precursor to an ultimate active pharmaceuticals, food Supplements, electro-optic applica compound (e.g., calcitriol). In other embodiments, the inven tions, animal feed additives, cosmetics, etc.) to minimize or tive system is utilized to prepare commercial end products. eliminate oxidation during production, storage, transport, etc. 65 Preparations of sterol compound(s) produced according to Extracted sterols may be further isolated and/or purified, the present invention, including formulations, dosing, Supple for example, by crystallization, washing, recrystallization, ments, have been described and are known in the art. For US 8,367,395 B2 53 54 example, see citations relating to pharmaceutical prepara the host cell in which they were produced (see section entitled tions, nutritional Supplement, and food additives described “Production and Isolation of Sterol Compounds”). above. Additionally, see, for example, International Patent In some embodiments, feedstuffs containing sterol com Publication No. WO 06/072175, U.S. Patent Publication No. pounds produced inaccordance with the present invention are US 2006/0067960, U.S. Patent Publication No. US 2006/ not substantially free of intact host cells. For example, feed 0.134095, International Patent Publication No. WO stuffs of the present invention may comprise greater than 91/016914, and International Patent Publication No. WO about 95% intact host cells. In certain embodiments, feed O2/O55,060. stuffs of the present invention comprise greater than about The amount of sterol compound(s) produced according to 70%, 75%, 85%, or 90% intact host cells. In certain embodi 10 ments, feedstuffs of the present invention comprise nearly the present invention that is incorporated into a given product intact host cells. For example, feedstuffs of the present inven may vary dramatically depending on the product, and the tion may comprise greater than about 70%, 75%, 85%, 90%, particular compound(s) involved. Amounts may range, for or 95% nearly intact host cells. As will be appreciated by example, from less than 0.01% by weight of the product, to those of ordinary skill in the art, Sterol compound-containing more than 1%, 10%, 20%, 30% or more; in some cases the 15 feedstuffs of the present invention that contain intact cells sterol compound may comprise 100% of the product. Thus, and/or nearly intact cells will have great utility in providing the amount of Sterol compound incorporated into a given the sterol compounds of interest present in Such host cells to product may be, for example, 0.01%, 0.02%, 0.03%, 0.04%, an animal. Such embodiments are advantageous when host 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, cells that produce the sterol compounds of interest contain 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, additional vitamins, nutrients, etc. that benefit the animal. 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, In some embodiments of the invention, produced sterol 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, compound(s) is/are incorporated into a cosmetic product. 95%, or 100%. In some embodiments of the invention, one or Examples of Such cosmetics include, for instance, skin cos more produced sterol compound(s) (e.g., squalene, lanos metics (e.g., lotions, emulsions, liquids, creams and the like), terol, Zymosterol, ergosterol, 7-dehydrocholesterol (provita 25 lipsticks, anti-Sunburn cosmetics, makeup cosmetics, fra min D3), is incorporated into a component of food or feed grances, products for daily use (e.g., toothpastes, mouth (e.g., a food Supplement, food additive). Types of food prod washes, bad breath preventive agents, Solid soaps, liquid ucts into which SSch compound(s) can be incorporated Soaps, shampoos, conditioners), etc. according to the present invention are not particularly limited, In some embodiments, produced sterol compound(s) is/are and include beverages such as milk, water, sports drinks, 30 incorporated into a pharmaceutical. Examples of Such phar energy drinks, teas, juices, and liquors; confections such as maceuticals include, for instance, various types of tablets, jellies and biscuits; fat-containing foods and beverages such capsules, drinkable agents, troches, gargles, etc. In some as dairy products; processed food products such as rice and embodiments, the pharmaceutical is suitable for topical Soft rice (or porridge); infant formulas; breakfast cereals or application. Dosage forms are not particularly limited, and the like. In some embodiments, one or more produced Sterol 35 include capsulae, oils, granula, granula Subtilae, pulveres, compounds is incorporated into a dietary Supplements. Such tabellae, pilulae, trochisci, or the like. Oils and oil-filled cap as for example a multi-vitamin. In certain embodiments, Sules may provide additional advantages both because of their squalene, lanosterol, Zymosterol, ergosterol, 7-dehydrocho lack of ingredient decomposition during manufacturing, and lesterol (provitamin D3) and/or one or more vitamin D com because inventive sterol-compound-containing lipid droplets pounds produced according to the present invention are 40 may be readily incorporated into oil-based formulations. included in a dietary Supplement. In some embodiments of Pharmaceuticals according to the present invention may be this aspect of the invention, it may be useful to one or more prepared according to techniques established in the art sterol compounds within bodies of edible lipids as it may including, for example, the common procedure as described facilitate incorporation into certain fat-containing food prod in the United States Pharmacopoeia, for example. ucts. Thus, for example, when the edible fungus, Candida 45 In still other embodiments, produced sterol compound(s) utilis is used as a host, its sterol compound containing lipids is/are incorporated into a nutritional Supplement or nutraceu may be directly incorporated into a component of food or feed tical. Examples of such nutraceuticals, include, for instance, (e.g., a food Supplement). various types of tablets, capsules, drinkable agents, troches, Examples offeedstuffs into which sterol compound(s) pro gargles, etc. In some embodiments, the nutraceutical is Suit duced in accordance with the present invention may be incor 50 able for topical application. Dosage forms are, as in pharma porated include, for instance, pet foods such as cat foods, dog ceutical products, not particularly limited and include any of foods and the like, feeds for aquarium fish or cultured fish, the same types of dosages as pharmaceuticals. etc., feed for farm-raised animals (including livestock and Sterol compound(s) produced according to the present further including fish raised in aquaculture). Food or feed invention (whether isolated or in the context of lipid droplets material into which Such compound(s) is/are incorporated is 55 or of cells, e.g., fungal cells) may be incorporated into prod preferably palatable to the organism which is the intended ucts as described herein by combination with any of a variety recipient. Such food or feed material may have any physical of agents. For instance, such compound(s) may be combined properties currently known for a food material (e.g., Solid, with one or more binders or fillers. In some embodiments, liquid, Soft). inventive products will include one or more chelating agents, In some embodiments, feedstuffs containing sterol com 60 pigments, salts, Surfactants, moisturizers, viscosity modifi pounds produced in accordance with the present invention are ers, thickeners, emollients, fragrances, preservatives, etc., substantially free of intact host cells. For example, feedstuffs and combinations thereof. of the present invention may be at least about 95% free of Useful Surfactants include, for example, anionic Surfac intact host cells. In some embodiments, feedstuffs of the tants such as branched and unbranched alkyl and acyl hydro present invention are at least about 96%, 97%, 98%, or 99% 65 carbon compounds, sodium dodecyl sulfate (SDS); sodium or more free of intact host cells. Such embodiments are typi lauryl sulfate (SLS); sodium lauryl ether sulfate (SLES); cal when the sterol compounds are highly purified away from sarconisate; fatty alcohol Sulfates, including sodium, potas US 8,367,395 B2 55 56 sium, ammonium or triethanolamine salts of Co to Cs satu ane triol; aloe Vera in any of its forms, for example aloe Vera rated or unsaturated forms thereof; ethoxylated fatty alcohol gel, Sugars and starches; Sugar and starch derivatives, for Sulfates, including alkyl ether Sulfates; alkyl glyceryl ether example alkoxylated glucose: hyaluronic acid; lactamide Sulfonate, alpha Sulpho fatty acids and esters; fatty acid esters monoethanolamine; acetamide monoethanolamine; glycolic of isethionic acid, including Igepon A; acyl (fatty) N-meth acid; alpha and beta hydroxy acids (e.g. lactic, glycolic Sali yltaurides, including Igepon T: dialkylsulfo Succinate esters, cylic acid); glycerine; pantheol; urea; Vaseline; natural oils; including Cs Co and C forms thereof; Miranot BT also oils and waxes (see: the emollients section herein) and mix referred to as lauroamphocarboxyglycinate and Sodium tride tures thereof.) cath Sulfate; N-acylated amino acids, such as sodium N-lau Viscosity modifiers that may be used in accordance with royl sarconisate or gluconate; sodium coconut monoglycer 10 the present invention include, for example, cetyl alcohol; ide Sulfonate; and fatty acid soaps, including Sodium, glycerol, polyethylene glycol (PEG); PEG-stearate; and/or potassium, DEA or TEA soaps. Keltrol. Among the cationic Surfactants that are useful are Appropriate thickeners for use in inventive products monoalkyl trimethyl quartenary salts; dialkyl dimethyl quar include, for example, gelling agents such as cellulose and tenary salts; ethoxylated or propoxylated alkyl quaternary 15 derivatives; Carbopol and derivatives; carob; carregeenans ammonium salts, also referred to in the art as ethoduats and and derivatives; Xanthane gum, Sclerane gum, long chain propoquats; cetyl benzylmethylalkyl ammonium chloride; alkanolamides; bentone and derivatives; Kaolin USP; Vee quaternized imidazolines, which are generally prepared by gum Ultra; Green Clay; Bentonite NFBC; etc. reacting a fat or fatty acid with diethylenetriamine followed Suitable emollients include, for example, natural oils, by quaternization, and non-fat derived cationic polymers esters, silicone oils, polyunsaturated fatty acids (PUFAs), such as the cellulosic polymer, Polymer JR (Union Carbide). lanoline and its derivatives and petrochemicals. Further useful cationic surfactants include lauryl trimethyl Natural oils which may be used in accordance with the ammonium chloride; cetyl pyridinium chloride; and alkyltri present invention may be obtained from sesame: Soybean: methylammonium bromide. Cationic Surfactants are particu apricot kernel; palm; peanut, safflower, coconut, olive; cocoa larly useful in the formulation of hair care products. Such as 25 butter, palm kernel; shea butter; Sunflower, almond; avocado; shampoos, rinses and conditioners. borage; carnauba; hazel nut, castor; cotton seed; evening Useful nonionic Surfactants include polyethoxylated com primrose; orange roughy, rapeseed; rice bran; walnut; wheat pounds and polypropoxylated products. Examples of ethoxy germ; peach kernel; babassu; mango seed; black current seed; lated and propoxylated non-ionic Surfactants include ethoxy jojoba, macademia nut, sea buckthorn; Sasquana; tsubaki; lated anhydrohexitol fatty esters, for example Tween-20; 30 mallow; meadowfoam seed; coffee; emu, mink; grape seed; mono- and di-ethanolamides; Steareth-20, also known as thistle; tea tree; pumpkin seed; kukui nut; and mixtures Volpo20; polyethylene glycol fatty esters (PEGs), such as thereof. PEG-8-stearate, PEG-8 distearate; block co-polymers, which Esters which may be used include, for example, Cs-Co are essentially combinations of hydrophilic polyethoxy alkyl esters of Cs-Co carboxylic acids; C-C diol chains and lipophilic polypropoxy chains and generically 35 monoesters and diesters of Cs-Cocarboxylic acids; Co-Co known as Poloaxamers. alcohol monosorbitan esters, Co-Co alcohol Sorbitan di- and Still other useful non-ionic surfactants include fatty esters tri-esters; Co-Co alcohol Sucrose mono-, di-, and tri-esters of polyglycols or polyhydric alcohols, such as mono and and Co-Co fatty alcoholesters of C-C2-hydroxy acids and diglyceride esters; mono- and di-ethylene glycol esters; mixtures thereof. Examples of these materials include isopro diethylene glycol esters; sorbitol esters also referred to as 40 pyl palmitate; isopropyl myristate; isopropyl isononate; C/ Spans; Sucrose esters; glucose esters; Sorbitan monooleate, Cabenzoate ester (also known as Finesolve); Sorbitan palmi also referred to as Span&0; glyceryl monostearate; and Sorbi tate, Sorbitan oleate; sucrose palmitate; Sucrose oleate; tan monolaurate, Span20 or Arlacel 20. isostearyl lactate; sorbitan laurate; lauryl pyrrolidone car Yet other useful nonionic surfactants include polyethylene boxylic acid; panthenyl triacetate; and mixtures thereof. oxide condensates of alkyl phenols and polyhydroxy fatty 45 Further useful emollients include silicone oils, including acid amide Surfactants which may be prepared as for example non-volatile and Volatile silicones. Examples of silicone oils disclosed in U.S. Pat. No. 2,965,576. that may be used in the compositions of the present invention Examples of amphoteric Surfactants which can be used in are dimethicone; cyclomethycone; dimethycone-copolyol; accordance with the present invention include betaines, aminofunctional silicones; phenyl modified silicones; alkyl which can be prepared by reacting an alkyldimethyl tertiary 50 modified silicones; dimethyl and diethyl polysiloxane: mixed amine, for example lauryl dimethylamine with chloroacetic C-Clso alkyl polysiloxane; and mixtures thereof. Addition acid. Betaines and betaine derivatives include higher alkyl ally useful silicones are described in U.S. Pat. No. 5,011,681 betaine derivatives including coco dimethyl carboxymethyl to Ciotti et al., incorporated by reference herein. betaine; Sulfopropyl betaine; alkyl amido betaines; and A yet further useful group of emollients includes lanoline cocoamido propyl betaine. Sulfosultaines which may be used 55 and lanoline derivatives, for example lanoline esters. include for example, cocoamidopropylhydroxy Sultaine. Still Petrochemicals which may be used as emollients in the other amphoteric Surfactants include imidazoline derivatives compositions of the present invention include mineral oil; and include the products sold under the trade name “Miranol petrolatum; isohexdecane; permethyl 101; isododecanol: described in U.S. Pat. No. 2,528,378 which is incorporated C-C Isoparrafin, also known as Isopar H. herein by reference in its entirety. Still other amphoterics 60 Among the waxes which may be included in inventive include phosphates for example, cocamidopropyl PG-dimo products are animal waxes such as beeswax, plant waxes such nium chloride phosphate and alkyldimethylamine oxides. as carnauba wax, candelilla wax, ouricurry wax, Japan wax or Suitable moisturizers include, for example, polyhydroxy waxes from cork fibres or Sugar cane. Mineral waxes, for alcohols, including butylene glycol, hexylene glycol, propy example paraffin wax, lignite wax, microcrystalline waxes or lene glycol, Sorbitol and the like; lactic acid and lactate salts, 65 oZokerites and synthetic waxes may also be included. Such as sodium or ammonium salts; C and C diols and triols Exemplary fragrances for use in inventive products including hexylene glycol, 1.4 dihydroxyhexane, 1.2.6-hex include, for instance, linear and cyclic alkenes (i.e., terpenes); US 8,367,395 B2 57 58 primary, secondary and tertiary alcohols; ethers; esters; organisms is described in U.S. Provisional Application No. ketones; nitrites; and Saturated and unsaturated aldehydes; 60/663,621, filed Mar. 18, 2005, and is also described in U.S. etc. patent application Ser. No. 1 1/385,580, entitled Production of Examples of synthetic fragrances that may be used in Carotenoids in Oleaginous Yeast and Fungi, filed Mar. 20. accordance with the present invention include without limi 2006. Each of these applications is incorporated herein by tation acetanisole; acetophenone; acetyl cedrene; methyl reference in its entirety. nonyl acetaldehyde; musk anbrette; heliotropin; citronellol; In some embodiments of the invention, host cells are engi Sandella; methoxycitranellal; hydroxycitranellal; phenyl neered to produce at least two sterol compounds, and particu ethyl acetate; phenylethylisobutarate; gamma methyl ionone; larly at least two compounds selected from the group consist geraniol; anethole; benzaldehyde; benzyl acetate; benzyl sali 10 ing of squalene, lanosterol, Zymosterol, ergosterol, cate; linalool, cinnamic alcohol; phenyl acetaldehyde; amyl 7-dehydrocholesterol, and calcitriol. In some such embodi cinnamic aldehyde; caphore; p-tertiairy butyl cyclohexyl ments, host cells are engineered to produce a least one com acetate; citral; cinnamyl acetate; citral diethyl acetal; cou pound selected from the group consisting of squalene, lanos marin; ethylene brasslate; eugenol; 1-menthol; Vanillin; etc. terol, Zymosterol, ergosterol, 7-dehydrocholesterol Examples of natural fragrances of use herein include with 15 (provitamin D3), and calcitriol, and at least one other com out limitation lavandin; heliotropin; Sandlewood oil; oak pound. Such host cells are particularly useful for producing moss; pathouly, ambergristincture; ambrette seed absolute; mixtures or combination products that contain squalene, angelic root oil; bergamont oil; benzoin Siam resin; buchu lanosterol, Zymosterol, ergosterol, 7-dehydrocholesterol leafoil; Cassia oil; cedarwood oil; Cassia oil; castoreum; civet (provitamin D3) and/or one or more vitamin D compounds. absolute; chamomile oil; geranium oil; lemon oil; lavender Such host cells are also useful for producing combination oil, YTang Ylang oil; etc. products including one or more ubiquinones, one or more A list of generally used fragrance materials can be found in ubiquinone-related compounds (e.g., vitamin E. vitamin K. various reference sources, for example, “Perfume and Flavor Cs quinones, etc) and/or one or more carotenoids. Chemicals'', Vols. I and II; Steffen Arctander Allured Pub.Co. In some embodiments of the invention, host cells are engi (1994) and “Perfumes: Art, Science and Technology': 25 neered to produce at least one sterol compound and at least Muller, P. M. and Lamparsky, D., Blackie Academic and one carotenoid. Carotenoids, which have an isoprene back Professional (1994) both incorporated herein by reference. bone consisting of 40 carbon atoms, have antioxidant effects Suitable preservatives include, among others, (e.g., sodium as well as use in coloring agents. Carotenoids such as B-caro metabisulfite: Glydant Plus; Phenonip; methylparaben; Ger tene, astaxanthin, and cryptoxanthin are believed to possess mall 115; Germaben II; phytic acid; sodium lauryl sulfate 30 cancer preventing and immunopotentiating activity. (SLS); sodium lauryl ether sulfate (SLES); Neolone; Kathon; The carotenoid biosynthesis pathway branches off from Euxyl and combinations thereof), anti-oxidants (e.g., buty theisoprenoid biosynthesis pathway at the point where GGPP lated hydroxytoluened (BHT); butylated hydroxyanisol is formed (see FIG. 1). The commitment step in carotenoid (BHA); ascorbic acid (vitamin C); tocopherol; tocopherol biosynthesis is the formation of phytoene by the head-to-head acetate; phytic acid; citric acid; pro-Vitamin A. 35 condensation of two molecules of GGPP catalyzed by phy In some embodiments, inventive products will comprise an toene synthase (often called crtB). A series of dehydrogena emulsion (e.g., containing inventive lipid bodies), and may tion reactions, each of which increases the number of conju include one or more emulsifying agents (e.g., Arlacel, such as gated double bonds by two, converts phytoene into lycopene Alacel 165: Glucamate; and combinations thereof) and/or via neurosporene. The pathway branches at various points, emulsion stabilizing agents. 40 both before and after lycopene production, so that a wide In some embodiments, inventive products will include one range of carotenoids can be generated. For example, action of or more biologically active agents other than the sterols(s). To a cyclase enzyme onlycopene generates Y-carotene; action of give but a few examples, inventive cosmetic or pharmaceuti a desaturase instead produces 3,4-didehydrolycopene. cal products may include one or more biologically active Y-carotene is converted to 0-carotene through the action of a agents such as, for example, Sunscreen actives, anti-wrinkle 45 cyclase. B-carotene can be processed into any of a number of actives, anti-aging actives, whitening actives, bleaching products, including astaxanthin (via echinenone, hydrox actives, Sunless tanning actives, anti-microbial actives, anti yechinenone, and phoenicoxanthin). acne actives, anti-psoriasis actives, anti-eczema actives, anti Carotenoid production in a host organism may be adjusted oxidants, anesthetics, vitamins, protein actives, etc. by modifying the expression or activity of one or more Engineering Production of Multiple Isoprenoid Compounds 50 polypeptides involved in carotenoid biosynthesis. In some In certain embodiments of the invention, it may be desir embodiments of the invention, it will be desirable to utilize as able to generate engineered organisms that accumulate one or host cells organisms that naturally produce one or more caro more other compounds in addition to the sterol compound(s), tenoids. In some such cases, the focus will be on increasing and further to accumulate such other compound, optionally production of a naturally-produced carotenoid, for example together with the sterol compound(s), in lipid bodies. For 55 by increasing the level and/or activity of one or more proteins example, certain inventive engineered organisms may accu involved in the synthesis of that carotenoid and/or by decreas mulate Sterol compound(s) together with at least one other ing the level or activity of one or more proteins involved in a compound derived from an isoprenoid precursor. In some competing biosynthetic pathway. Alternatively or addition embodiments, the other compound derived from an iso ally, in some embodiments it will be desirable to generate prenoid precursor will be one or more quinone-derived com 60 production of one or more carotenoids not naturally produced pounds as discussed in either or both of in U.S. patent appli by the host cell. cation 60/848,064, filed on Sep. 28, 2006 and U.S. patent In some embodiments of the invention, it will be desirable application 60/784,499 filed on Mar. 20, 2006 (e.g., Vitamin to introduce one or more carotenogenic modifications into a K. Vitamin E, and/or related quinone compounds (e.g., Cs)). host cell. In certain embodiments the carotenogenic modifi Alternatively or additionally, in some embodiments the other 65 cation may confer expression of one or more heterologous compound derived from an isoprenoid precursor will be one carotenogenic polypeptides into a host cell. As will be appar or more carotenoids. Production of carotenoids in oleaginous ent to those of ordinary skill in the art, any of a variety of US 8,367,395 B2 59 60 heterologous polypeptides may be employed; selection will In some embodiments of the invention, host cells are engi consider, for instance, the particular carotenoid whose pro neered to produce at least one sterol compound and at least duction is to be enhanced. Still further, selection will consider one quinone derived compound. Quinone derived com the complementation and/or ability of the selected polypep pounds are produced from the isoprenoid compound isopen tide to function in conjunction with additional oleaginic and/ tyl pyrophosphate (IPP), via geranylgeranyl pyrophosphate or sterologenic modifications of a cell Such that each of ole (see, for example, FIG.5. IPP can be generated through one of aginy, Sterol biosynthesis and carotenoid biosynthesis are two different isoprenoid biosynthesis pathways. The most effectuated to the desired extent. common isoprenoid biosynthesis pathway, sometimes Proteins involved in carotenoid biosynthesis include, but referred to as the “mevalonate pathway', is generally 10 depicted in FIG. 3. As shown, acetyl-CoA is converted, via are not limited to, phytoene synthase, phytoene dehydroge hydroxymethylglutaryl-CoA (HMG-CoA), into mevalonate. nase, lycopene cyclase, carotenoid ketolase, carotenoid Mevalonate is then phosphorylated and converted into the hydroxylase, astaxanthin synthase (a single multifunctional five-carbon compound isopentenyl pyrophosphate (IPP). enzyme found in Some source organisms that typically has An alternative isoprenoid biosynthesis pathway, that is uti both ketolase and hydroxylase activities), carotenoid epsilon 15 lized by Some organisms (particularly bacteria) and is some hydroxylase, lycopene cyclase (beta and epsilon Subunits), times called the “mevalonate-independent pathway', is also carotenoid glucosyltransferase, and acyl CoA:diacy glycerol depicted in FIG. 3. This pathway is initiated by the synthesis acyltransferase. Representative example sequences for caro of 1-deoxy-D-xyloglucose-5-phosphate (DOXP) from pyru tenoid biosynthesis polypeptides are provided in Tables 17a vate and glyceraldehyde-3-phosphate. DOXP is then con 21 and Tables 38-41. verted, via a series of reactions shown in FIG. 3, into IPP. Alternatively or additionally, modified carotenoid ketolase Various proteins involved in isoprenoid biosynthesis have polypeptides that exhibit improved carotenoid production been identified and characterized in a number of organisms. activity may be utilized in accordance with the present inven Moreover, isoprenoids are synthesized in many, if not most, tion. For example, carotenoid ketolase polypeptides compris organisms. Thus, various aspects of the isoprenoid biosyn ing one more mutations to corresponding to those identified 25 thesis pathway are conserved throughout the fungal, bacte Sphingomonas sp. DC18 which exhibited improved astaxan rial, plant and animal kingdoms. For example, polypeptides thin production (Tao et al., 2006 Metab Eng. 2006 Jun. 27) corresponding to the acetoacetyl-CoA thiolase, synthase, and Paracoccus sp. strain N81106 which exhibited altered HMG-CoA reductase, mevalonate kinase, phosphomeva carotenoid production (Ye et al., 2006 Appl Environ Micro lonate kinase, mevalonate pyrophosphate decarboxylase, IPP biol 72:5829-37). 30 isomerase, FPP synthase, and GGPP synthase, some of which In some embodiments of the invention, potential source are shown for example in FIGS. 5-6 have been identified in organisms for carotenoid biosynthesis polypeptides include, and isolated from a wide variety of organisms and cells; but are not limited to, genera of naturally oleaginous or non representative examples of a wide variety of Such polypep oleaginous fungi that naturally produce carotenoids. These tides are provided in Tables 7-15. One or more of the polypep include, but are not limited to. Botlytis, Cercospora, 35 tides selected from those provided in any one of Tables 7-15 Fusarium (Gibberella). Mucor, Neurospora, Phycomyces, may be utilized or derived for use in the methods and com Puccina, Rhodotorula, Sclerotium, Trichoderma, and Xan positions in accordance with the present invention. thophyllomyces. Exemplary species include, but are not lim Alternatively or additionally, modified mevalonate kinase ited to, Neurospora crassa, Xanthophyllomyces dendrorhous polypeptides that exhibit decreased feedback inhibition prop (Phafia rhodozyma), Mucor circinelloides, and Rhodotorula 40 erties (e.g., to farnesyl pyrophosphate (FPP)) may be utilized glutinis. Of course, carotenoids are produced by a wide range in accordance with the present invention. Such modified of diverse organisms such as plants, algae, yeast, fungi, bac mevalonate kinase polypeptides may be of eukaryotic or teria, cyanobacteria, etc. Any such organisms may be source prokaryotic origin. For example, modified versions of meva organisms for carotenoid biosynthesis polypeptides accord lonate kinase polypeptides from animals (including humans), ing to the present invention. 45 plants, algae, fungi (including yeast), and/or bacteria may be It will be appreciated that the particular carotenogenic employed; for instance, modified versions of mevalonate modification to be applied to a host cell inaccordance with the kinase polypeptides disclosed in Table 10 herein may be present invention will be influenced by which carotenoid(s) is utilized. desired to be produced. For example, isoprenoid biosynthesis Particular examples of modified mevalonate kinase polypeptides are relevant to the production of most caro 50 polypeptides include “feedback-resistant mevalonate tenoids. Carotenoid biosynthesis polypeptides are also kinases' disclosed in PCT Application WO 06/063,752. broadly relevant. Carotenoid ketolase activity is particularly Thus, for example, a modified mevalonate kinase polypeptide relevant for production of canthaxanthin, as carotenoid may include one or more mutation(s) at one or more amino hydroxylase activity is for production of lutein and Zeaxan acid position(s) selected from the group consisting of amino thin, among others. Both carotenoid hydroxylase and keto 55 acid positions corresponding to positions 17, 47.93, 94, 132, lase activities (and/or astaxanthin synthase) are particularly 167, 169, 204, and 266 of the amino acid sequence of Para useful for production of astaxanthin. coccus zeaxanthiinifaciens mevalonate kinase as shown in Bacterial carotenogenic genes have already been demon SEQ ID NO:1 of PCT Application WO 04/111,214. For strated to be transferable to other organisms, and are therefore example, the modified mevalonate kinase polypeptide may particularly useful in accordance with the present invention 60 contain one or more Substitutions at positions corresponding (see, for example, Miura et al., Appl. Environ. Microbiol. to one or more of I17T, G47D, K93E, V94I, R204H and 64:1226, 1998). In other embodiments, it may be desirable to C266S utilize genes from other source organisms such as plant, alga, To give but a few specific examples, when a modified or microalgae, these organisms provide a variety of potential mevalonate kinase polypeptide comprises 2 amino acid sources for ketolase and hydroxylase polypeptides. Still addi 65 changes as compared with a parent mevalonate kinase tional useful source organisms include fungal, yeast, insect, polypeptide, it may comprise changes at positions corre protozoal, and mammalian Sources of polypeptides. sponding to the following positions 132/375,167/169, 17/47 US 8,367,395 B2 61 62 and/or 17/93 of SEQ ID NO:1 of WO 04/111.214 (e.g. Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rho P132A/P375R, R167W/K169Q, I17T/G47D or I17T/K93E); dosporidium, Rhodotorula, TrichospOron, Yarrowia, when a modified mevalonate kinase polypeptide comprises 3 Aspergillus, Botrytis, Cercospora, Fusarium (Gibberella), amino acid changes as compared with a parent mevalonate Kluyveromyces, Neurospora, Penicillium, Pichia kinase, it may comprise changes at positions corresponding to (Hansenula), Puccinia, Saccharomyces, Schizosaccharomy the following positions 17/167/169, 17/132/375,93/132/375, ces, Sclerotium, Trichoderms Ustilago, and Xanthophyllomy and/or 17/47/93 of SEQID NO: 1 of WO/2004/111214 (e.g., ces (Phafia). In certain embodiments, the source organisms I17T/R167W/K169Q, I17T/P132A/P375R, K93E/P132A/ are of a species including, but not limited to, Cryptococcus P375R, I17T/R167W/K169H, I17T/R167T/K169M, I17T/ neoformans, Fusarium filiikuroi, Kluyverimyces lactis, Neu R167T/K169Y, I17T/R167F/K169Q, I17T/R1671/K169N, 10 rospora crassa, Saccharomyces cerevisiae, Schizosaccharo I17T/R167H/K169Y, I17T/G47D/K93E or I17T/G47D/ myces pombe, Ustilago maydis, and Yarrowia lipolytica. K93Q). In some embodiments of the invention, the quinone derived Thus, for example, a modified mevalonate kinase polypep compound whose production is engineered is ubiquinone. tide may include one or more mutation(s) (particularly Sub The commitment step in ubiquinone biosynthesis is the for stitutions), as compared with a parent mevalonate kinase 15 mation of para-hydroxybenzoate (PHB) from tyrosine orphe polypeptide, at one or more amino acid position(s) selected nylalanine in mammals or chorismate in bacteria, followed by from the group consisting of amino acid positions corre condensation of PHB and isoprene precursor, resulting in sponding to positions 55, 59, 66, 83, 106, 111, 117, 142, 152, addition of the prenyl group (see FIG. 13). Lower eukaryotes, 158, 218, 231, 249,367 and 375 of the amino acid sequence such as yeast, can synthesize PHB from either tyrosine or of Saccharomyces cerevisiae mevalonate kinase as shown in chorismate. The 3-decaprenyl-4-hydroxybenzoic acid result SEQ ID NO:1 of PCT application WO 06/063,752. For ing from the condensation reaction undergoes further modi example, Such corresponding Substitutions may comprise one fications, which include hydroxylation, methylation and or more of P55L, F59S, N66K, C117S, or I 152M. A modified decarboxylation, in order to form ubiquinone. Ubiquinone mevalonate kinase may comprise a Substitution correspond biosynthetic enzymes and genes encoding these proteins have ing to F59S substitution. A modified mevalonate kinase 25 been characterized in several organisms. The most extensive polypeptide comprising 2 amino acid changes as compared analysis has been performed in bacterial systems such as with its parent mevalonate kinase polypeptide may, for Escherichia coli and Rhodobacter sphaeroides as well as the example, comprise changes at positions corresponding to the yeast, Saccharomyces cerevisiae. At least 8 enzymes are following positions 55/117, 66/152, 83/249, 111/375 or 106/ required for the synthesis of CoQ10 from PHB and the iso 218 of to SEQ ID NO: 1 of WO 06/063,752 (e.g. P55L/ 30 prene precursors. C117S, N66KVI152M, K83E/S249P, H111N/K375N or As mentioned, ubiquinone is formed by the combination of L106P/S218P). A modified mevalonate kinase may comprise para-hydroxybenzoate and isoprenoid chains produced, for a substitution corresponding to N66K/I152M. A modified example, via the isoprenoid biosythesis pathways discussed mevalonate kinase polypeptide comprising 4 amino acid above. Once IPP is formed according to that pathway, it changes as compared with its parent mevalonate kinase 35 ismoerizes into dimethylallyl pyrophophate (DMAPP). polypeptide may have changes at positions corresponding to Three sequential condensation reactions with additional mol one or more of the following positions 42/158/231/367 of ecules of IPP generate the ten-carbon molecule geranyl pyro SEQID NO:1 of WO 06/063,752 (e.g., I142N/L158S/L2311/ phosphate (GPP), followed by the fifteen-carbon molecule T367S). farnesyl pyrophosphate (FPP), which can be used to form the According to the present invention, quinone derived com 40 twenty-carbon compound geranylgeranyl pyrophosphate pound production in a host organism may be adjusted by (GGPP). In many instances, FPP appears to be the predomi modifying the expression or activity of one or more proteins nant Substrate used by polyprenyldiphosphate synthases involved in isoprenoid biosynthesis. In some embodiments, (e.g., Coq1 polypeptides) during ubiquinone biosynthesis. such modification involves introduction of one or more het According to the present invention, ubiquinone production in erologous isoprenoid biosynthesis polypeptides into the host 45 a host organism may be adjusted by modifying the expression cell; alternatively or additionally, modifications may be made or activity of one or more proteins involved in isoprenoid to the expression or activity of one or more endogenous or biosynthesis as discussed above. heterologous isoprenoid biosynthesis polypeptides. Given As discussed herein, two different pathways can produce the considerable conservation of components of the iso the ubiquinoid precursor para-hydroxybenzoate—the first, prenoid biosynthesis polypeptides, it is expected that heter 50 the “shikimate pathway' is utilized in prokaryotes and yeast, ologous isoprenoid biosynthesis polypeptides will often involves synthesis of para-hydroxybenzoate (PHB) through function well even in significantly divergent organisms. Fur chorismate. Biosynthesis of para-hydroxybenzoate from cho thermore, should it be desirable to introduce more than one rismate occurs by the action of chorismate pyruvate lyase. For heterologous isoprenoid biosynthesis polypeptide (e.g., more example, as discussed herein, enzymes of the shikimate path than one version of the same polypeptide and/or more than 55 way, chorismate synthase, DAHP synthase, and transketolase one different polypeptides), in many cases polypeptides from are involved in this process. Representative examples of these different source organisms may function well together. In enzymes are provided in Table 33 and Tables 35 through 37. some embodiments of the invention, a plurality of different In the second possible pathway, para-hydroxybenzoate is pro heterologous isoprenoid biosynthesis polypeptides is intro duced by derivation of tyrosine or phenylalanine. Biosynthe duced into the same host cell. In some embodiments, this 60 sis of para-hydroxybenzoate from tyrosine or phenylalanine plurality contains only polypeptides from the same source occurs through a five step process in mammalian cells (see, organism; in other embodiments the plurality includes for example FIG. 14). polypeptides from different source organisms. Accordingly, ubiquinone production in a host organism In certain embodiments of the present invention that utilize may be adjusted by modifying the expression or activity of heterologous isoprenoid biosynthesis polypeptides, the 65 one or more proteins involved in PHB biosynthesis. In some Source organisms include, but are not limited to, fungi of the embodiments, such modification involves introduction of one genera Blakeslea, Candida, Cryptococcus, Cunninghamella, or more heterologous PHB polypeptides into the host cell; US 8,367,395 B2 63 64 alternatively or additionally, modifications may be made to In Some embodiments of the present invention, the quinone the expression or activity of one or more endogenous or derived compound that host cells are engineered to produce heterologous additional PHB polypeptide, and/or isoprenoid along with one or more sterol compound(s) is Vitamin K. biosynthesis polypeptides. Given the considerable conserva Vitamin K is a generic term that refers to derivatives of tion of components of the PHB biosynthesis polypeptides, it 2-methyl-1,4-naphthoquinone that have coagulation activity. is expected that heterologous PHB biosynthesis polypeptides The two natural forms of vitamin K differ in the identity of will often function well even in significantly divergent organ their side chains at position 3. Vitamin K, also known as isms. Further, given the conservation of the pathways among phylloquinone (based on its presence in plants), has a phyty1 organism, it is anticipated that heterologous polypeptides side chain in position 3; vitamin K, also known as throughout the ubiquinone biosynthetic pathway will func 10 menaquinone, has an isoprenyl side chain at position 3. Dif tion together effectively. Furthermore, should it be desirable ferent forms of menaquinone, having side chains with differ to introduce more than one heterologous PHB polypeptide ent numbers of isoprene units (typically 4-13) are found in and/or isoprenoid biosynthesis polypeptide, in many cases different types of cells. The present invention provides engi polypeptides from different source organisms will function neered host cells, e.g., fungal cells, that produce vitamin Kas well together. In some embodiments of the invention, a plu 15 a result of the engineering. That is, the present invention rality of different heterologous PHB and/or isoprenoid bio provides engineered host cells containing a Vitamin K pro synthesis polypeptides is introduced into the same host cell. duction modification. Such a modification may comprise, for In some embodiments, this plurality contains only polypep instance, introduction or activation of one or more Vitamin K tides from the same source organism; in other embodiments biosynthetic polypeptides within a host cell. Exemplary such the plurality includes polypeptides from different source polypeptides include, for example, MenF, Men), MenC, organisms. In still other embodiments, modification of MenE, MenB, MenA, UbiE, and/or MenG polypeptides. endogenous PHB and/or isoprenoid biosynthesis polypep In Some embodiments of the present invention, the quinone tides are also utilized, either alone or in combination with derived compound that host cells are engineered to produce heterologous polypeptides as discussed herein. along with at least one sterol compound is Vitamin E. Vitamin Particularly for embodiments of the present invention 25 E is a generic term for a family of structurally related com directed toward production of CoQ10, it will often be desir pounds that have a 6-chromanol ring, an isoprenoid side able to utilize one or more genes from a natural CoQ10 chain, and the biologic activity of C-tocopherol. The term producing organism. In general, where multiple heterologous encompasses the eight known naturally occurring vitamin E polypeptides are to be expressed, it may be desirable to utilize 30 compounds, the four tocopherols (C, B, Y. Ö) and four tocot the same source organism for all, or to utilize closely related rienols (C., B, Y. Ö), which all contain a hydrophilic chromanol Source organisms. ring and a hydrophobic side chain. The C, B, Y, and 8 forms Bacterial ubiquinogenic genes have already been demon differ from one another in the number of methyl groups on the strated to be transferrable to other organisms, and are there chromanol ring. Several synthetic vitamin E compounds have fore useful in accordance with the present invention (see, for 35 also been prepared, and still others are possible (see, for example, Okada et al., FEMS Lett. 431:241-244 (1998)). In example, Bramley et al., J. Sci Food Agric 80:913, 2000). some embodiments of this invention, it may be desirable to C-tocopherol is a potent antioxidant, and is generally consid fused sequences encoding specific targeting signals to bacte ered to be the most active vitamin E compound in humans. rial ubiquinogenic genes. For example, in certain embodi 40 Vitamin E compounds are synthesized by higher plants and ments mitochondrial signal sequences are useful in conjunc cyanobacteria by two pathways: the isoprenoid pathway and tion with, e.g., bacterial ubiquinogenic polypeptides for the homogentisic acid formation pathway. The first step is effective targeting of polypeptides for proper functioning. formation of the homogentisic head group (HGA), which is Mitochondrial signal sequences are known in the art, and produced from p-hydroxyphenylpyruvic acid (HPP) by the include, but are not limited to example, mitochondrial signal 45 enzyme p-hydroxyphenylpyruvic acid dioxygenase (HPP sequences provided in Table 22. In other embodiments, it may Dase). This is a complex reaction involving the addition of be desirable to utilize genes from other source organisms such two oxygen atoms as well as the decarboxylation and rear as animals, plants, alga, or microalgae, fungi, yeast, insect, rangement of the HPP side chain. protozoa, and mammals. In the next step, HGS is prenylated and decarboxylated to The present invention contemplates not only introduction 50 form 2-methyl-6-phytylplastoquinol. This step also repre of heterologous ubiquinogenic polypeptides, but also adjust sents the commitment step for production of tocopherols ment of expression or activity levels of heterologous or and/or tocotrienols, as 2-methyl-6-phytylplastoquinol repre endogenous ubiquinogenic polypeptides, including, for sents the common intermediate in the synthesis of all toco pherols. The present invention provides host cells that have example, alteration of constitutive or inducible expression 55 patterns so as to increase activity of ubiquinogenic polypep been engineered to accumulate 2-methyl-6-phytylplasto quinol. In some embodiments, the host cells are (or have been tides. For example, genetic modifications comprising alter engineered to be) oleaginous or lipid-accumulating. In some ation and/or addition of regulatory sequences (e.g., promoter embodiments, produced 2-methyl-6-phytylplastoquinol elements, terminator elements) may be utilized to confer par 60 accumulates in lipid bodies within the engineered host cells. ticular regulation of expression patterns. Such genetic modi In the final steps of tocopherol synthesis, methylation and fications may be utilized in conjunction with endogenous ring cyclization reactions convert the 2-methyl-6-phytylplas genes (e.g., for regulation of endogenous ubiquinogenic toquinol into various tocopherols. polypeptide(s)): alternatively, Such genetic modifications It is expected in accordance with the present invention that may be included so as to confer regulation of expression of 65 availability of tocopherol precursors and/or intermediates heterologous polypeptides (e.g., ubiquinogenic poly may well affect the rate and/or extent of tocopherol (or other peptide(s)). Vitamin E compound) production and/or accumulation by US 8,367,395 B2 65 66 and/or within cells. The present invention therefore encom Example 1 passes engineering host cells to adjust the rate or amount of one or more tocopherol precursors and/or intermediates. Production of Plasmids for Sterol Strain The present invention provides engineered host cells, e.g., Construction fungal cells, that produce vitamin E as a result of the engi neering. The present invention specifically provides engi 1A. Production of pMB4637 (ADE1 tef-Y lipolytica neered host cells, e.g., fungal cells, that produce larger Hmg1'"), Encoding a Truncated Y lipolytica HMG-CoA amounts of vitamin E that an otherwise identical non-engi Reductase. neered host cell. That is, the present invention provides engi The native HMG1 gene from Y lipolytica was modified neered host cells containing a Vitamin E production modifi 10 cation. Such a modification may comprise, for instance, using primers M04658 (described above) and M04657, to introduction or activation of one or more Vitamin E biosyn create pMB4637: thetic polypeptides within a host cell. Exemplary such polypeptides include, for example, tyra, pds 1 (hppd), VTE1. MO 4657 5 - CACACTCTAGACACAAAAATGACCCAGTCTGTGAAGGTGG HPT1(VTE2), VTE3, VTE4, and/or GGH polypeptides. Spe 15 cific examples of each of these can be found, for example, in MO4658 5 - CACACACGCGTACACCTATGACCGTATGCAAAT Tables 50-56. It will be appreciated by those of ordinary skill in the art Primers M04657, which encodes a start methionine, and that, in some embodiments of the invention, where multiple M04658 were used to amplify a 1.5 kb fragment encoding the heterologous polypeptides are to be expressed (e.g., because C-terminal 499 residues of the Hmg1 protein of Y lipolytica, one or more activities of interest require two or more polypep using genomic DNA as template. The resulting fragment was tide chains and/or because multiple activities of interest are blunt-end cloned into pBluescript SKII-, which had been being engineered), it may be desirable to utilize the same EcoRV digested and treated with calf intestinal phosphatase. Source organism for all, or to utilize closely related Source Following sequence verification, the resulting plasmid organisms; in other embodiments, heterologous polypeptides 25 pMB4623 was digested with Xbal and Mlul, and the 1.5 kb may be from different source organisms. In some embodi fragment was ligated to Nhel/Mlul digested pMB4629. ments, two or more versions of a particular heterologous pMB4629 is a Yarrowia expression vector containing the polypeptide, optionally from different source organisms, may ADE 1 auxotrophic marker, the nativeTEF1 promoter, and the native XPR2 terminator. be introduced into and/or engineered within, a single host The resulting nucleic acid coding sequence in pMB4637 cell. 30 encoded Hmg1' protein, which was comprised of a start Having now generally described the invention, the same methionine followed by the carboxy-terminal 499 amino will be more readily understood through reference to the acids of the Y lipolytica HMG-CoA reductase protein are as following exemplification which is provided by way of illus follows: tration, and is not intended to be limiting of the present DNA: invention. 35

EXEMPLIFICATION atgacc cagtctgttgaaggtggttgagalagcacgttcct at C9tcatt All basic molecular biology and DNA manipulation pro gaga agcc.ca.gc.gagaaggaggagga cacct Cttctgaagact coatt 40 cedures described herein are generally performed according gagdtigactgtcggaaagcagcc caa.gc.ccgtgaccgaga.ccc.gttct to Sambrook et al., or Ausubel et al., (Sambrook J. Fritsch E F. Maniatis T (ed.) 1989, Molecular Cloning: A Laboratory Ctggacgacctagaggctatcatgaaggcagg talagacCaagcttctg Manual, Cold Spring Harbor Laboratory Press: New York; Ausubel FM, Brent R. Kingston RE, Moore D D, Seidman J gaggaccacgaggttgttcaagct ct ct ct cagggcaa.gct tcctittg G, Smith J. A. Struhl K (ed.) 1998 Current Protocols in 45 tatgct Cttgagalagcagcttggtgacaac accc.gagctgttggcatc Molecular Biology, Wiley: New York). The TEF1 promoteris from Y lipolytica as is the XPR2 terminator. cgacgatctatoat citcc.ca.gcagtictaataccalagactittagagacic Plasmids are generated for construction of sterol-derived tdaaagct tccttacctgcactacgacitacgaccgtgtttittggagcc metabolite producing strains, including strains to produce metabolic precursors of vitamin D. The following subparts 50 tgttgcgagaacgtt attggitta catgcct ct coccgttggtgttgct describe production of plasmids used for construction of said strains. All PCR amplifications use NRRL Y-1095 genomic ggc.cccatgaacattgatggcaagaactaccacatt cotatggccacc DNA as template. The URA5 gene described below is allelic actgagggttgtc.ttgttgcct Caac catgcgaggttgcaaggcc at C with the ura2-21 auxotrophy below in Table 82. TABLE 82 arrowia lipolytica strains. NRRLY-1095 Wild type diploid ATCC76861 MATB ura2-21 lyc1-5 LYS1-5B ATCC76982 MATB ade1 leu2-35 lyc1-5 xpr2 ATCC201249 MATAura3-302 leu2-270 lys8-11 PEX17 HA MF346 MATAura2-21 ATCC76861 X ATCC2O1249 MF350 MATB ura-21 leu2-35 ade1 ATCC76982 x MF346

US 8,367,395 B2 69 70 Protein: - Continued gt caac attct ct coatggcc ct ctact cqct ctitcgcc.gc.cgacticg gactacctg.cgaatcctgcagct cqgatgcttcaac tactt caagctg mgklielllhpsel saaihyklWrdplhprolskestelrricyelldvcs ggaggcatctgtgtggaccaccc.cgt catgctgttggctggagttctic cc cc gacc catgtacctgtttacgcatttctt cqtagtggc catctac rsfaavirelhpevrdavmlfylilraldtieddmtlsrdikipilrdft ggcggaatctgcaa.catgcaggccaacggcattgccaagctgc.ccgc.g togctact.gcaatttgtc.gc.ct citctggtcaccocttgcatcgtcatc kcmktpgwkftdisdpnerdrvvldefpvvmtefnklkpkydeviyalitar tt coct tacatttggagcgagctgacittag mgngmady vidddfnning votiaaydlychhvagivgeglitritilagfg Protein: tdvlhenprlde Smglflgkvniirdy redidynrafwpreiwhkyaeem 10 mvtcqsaaetsatotneydivvivgagiagpalavalignqgrkvlwverdl rdfkdpkyskkalhcts dilvanalghatdclidyldnvtdpstftfcaipo sepdrivgellopggvaalktlglgsciedlidaipcqgynviysgeecvil vmaliatildlyrnpdvfgknvklirkgttv.slilea snvsgvcdiftryark kypkvprodiggidynellyrsgksadis neapirgvsfhhgrfvmnlrraard vykksdpndpnyfrysvlcgkieqihaalikircrgppaktiaqlegerkem 15 tpnvtilleatvtevvknpytghiigvktfsktggakiykhiffapltvvcd alslivclavifsmsglmayilay vsgfrwspreifolskmfplrd gtfskfrkdfstnikt Svirshfaglilkdavlp spohghvil spins cp vlv 1C. Production of pMBERGI(LEU2 tef-Y lipolytica ERG1), Encoding a Y lipolytica Squalene Epoxidase. yovgaretrilcdiqgpvp snatcalkehmeknvmphlpksiqpsfcaal The native ERG1 gene from Y lipolytica may be modified using primers MOERG1XBA and MOERG1MLU, to create kedtirvmpnsfils askindhhgilillgdalnmrhpltgggmtvalndall a plasmid for expression of ERG1 in Y lsrlltgvnledtyavs swims sq.fhwgrkhildsiivnilsmally slfaads dylrildlg.cfnyklggicvdhp Vmillagylpromylfthffvvaiyggi MOERG1XBA 5 - CACACTCTAGACACAAAAATGGTCACCCAA 25 CAGTCTGCA cnmgangiaklpaslildfvasilvtacivifpyiwiselt

MOERG1MLU 5 - CACACACGCGTACACCTAAGTCAGCTCGCT CCA Example 2

Primers MOERG1 XBA and MOERG1MLU are used to 30 Engineering Yarrowia lipolytica for Increased Sterol amplify a 1.47 kb fragment encoding the Erg1 protein of Y Production lipolytica, using genomic DNA or cDNA as template. Fol lowing T4 polynucleotide kinase treatment, the resulting 2A. Production of Y lipolytica Expressing a Truncated Y. fragment is blunt-end cloned into pBluescript SKII-, which is lipolytica HMG-CoA Reductase. EcoRV digested and treated with calf intestinal phosphatase. 35 MF350 (MATBura2-21 leu2-35adel) is transformed with Following sequence verification, the resulting plasmid is pMB4637 (ADE1 tef-Y lipolytica Hmg1") that has been digested with Xbal and Mlul, and the 1.47 kb fragment is cleaved with SnaBI, and Ade" colonies are selected. One such ligated to NheI/Mlul digested pMB4603.pMB4603 is a Yar colony. MFHMG, produces elevated (for example, in the rowia expression vector containing the LEU2 auxotrophic range of 2-fold or more) 5,7-diene-containing Sterols than marker, the native TEF1 promoter, and the native XPR2 ter 40 MF350 after 2 days of growth in YPD at 30° C. minator. 2B. Production of Y lipolytica Expressing a Y lipolytica The resulting nucleic acid coding sequence in pMBERG1 Squalene Synthase encodes Erg1 protein, a protein of 489 amino acids. The DNA MF350 (MATBura2-21 leu2-35adel) is transformed with and corresponding protein are as follows: pMBERG9 (URA5 tef-Y lipolytica ERG9), that has been DNA: 45 cleaved upstream of URA5 with SspI; a Ura transformant carrying the plasmid at the ura2 locus is identified and named atggtcacccaa.ca.gtctgcagcagagaccagcgcc acccagaccaac MFERG9. One such colony, MFERG9, produces elevated gagtacgacgtggt cattgtcggagctgg tattgc.cgggc.ccgct ctg (for example, in the range of about 1.5-fold or more) 5,7- gcc.gtggcticttggaaatcagggcagaaaggttcttgttgttggaacga diene-containing sterols than MF350 after 2 days of growth in gatctotc.cgaaccggaccgaatcgtgggaga.gctgct tcagoccgga 50 YPD at 30° C. ggagtc.gctgct ct caagacitctgggit ct cqgct Cttgttatcgaggat 2C. Production of Y lipolytica Expressing a Y lipolytica atcgacgcgatc.ccctgcc agggatacaacgtgatctact ctggagaa gagtgcgttct caaataccc.caagg to ccc.cgagacat coagcaggac Squalene Epoxidase. tacaacgagctgtacagaa.gcggaaagttctgc.cgaCatct coaacgag MF350 (MATBura2-21 leu2-35adel) is transformed with gctic ccc.gaggagtatic ct tccaccacgg.ccgatttgt catgaacttg SspI-cleaved pMBERG1 (LEU2tef-Y lipolytica ERG 1), and cgaagggcc.gcacgaga cacacic caatgtgactctgctggaggccaca 55 gtcaccgaggtggt caagaac cctt acaccggccacattattggagtic Leu" transformants are identified. One such colony is named aagacctt ct ctaaaactggaggcgc.caaaatctacaa.gcactitctitt MFERG9. MFERG1 produces elevated (for example, in the gctic ct ct caccgt.cgtctgttgatggaactttitt coaagttc.cgaaag range of about 1.3-fold or more) 5,7-diene-containing sterols gactittagcaccaacaagacgtctgtgcgttctgcattt cqc.cggtctg than MF350 after 2 days of growth in YPD at 30° C. attct caaggacgctgttctgcc ct coccc.ca.gcatggccacgtgatt 2D. Production of Y lipolytica Expressing a Truncated Y. Ctgtc.gcc caacticgtgtc.ccgttcttgtctaccaggttggagctica gaga.ccc.gaattctgttgttgacattcagggaccc.gtc.ccct ctaatgca 60 lipolytica HMG-CoA Reductase, Squalene Synthase, and accggagc cct Caaggalacacatggagaagaacgt.catgc.cccacctg Squalene Epoxidase. cctaagtic catc.ca.gc.cgt.ctitt coaa.gc.cgct citcaaggagcagacic MFHMG is transformed with pMBERG9 (URA5 tef-Y. attcagt catgcc caact ctitt cotgtcggcct coaagaacgat cac cacggtttgattctgctgggtgacgcact caa.catgcgacatccactt lipolytica ERG9), that has been cleaved upstream of URA5 accggaggaggaatgaccgttgct ct caatgatgcc cttctacticago with Ssp I; a Ura transformant carrying the plasmid at the agacittct caccggcgitta acctggaagacaccitatgcc.gtgtcct c c 65 ura2 locus is identified and named MFHMGERG9. MFH gtcatgagct cqcagttcc actggcagcgaaaac acct cqact coat c MGERG9 can be subsequently transformed with SspI cleaved pMBERG1 (LEU2 tef-Y lipolytica ERG1), and Leu" US 8,367,395 B2 71 72 transformants can be identified. One such colony is named as Xanthophyllomyces dendrorhous (Phafia rhodozyma), MFHMGERG1 ERG9. MFERG9 produces elevated (for using the same, homologous, or functionally similar ole example, in the range of about 3.5-fold or more) 5,7-diene aginic polypeptides. containing sterols than MF350 after 2 days of growth inYPD Qiagen RNAEasy kits (Qiagen, Valencia, Calif.) are used at 30° C. Alternatively, Y lipolytica strains expressing Y. to prepare messenger RNA from lyophilized biomass pre lipolytica HMG-CoA reductase and squalene synthase, or pared from cultures of N. crassa. Subsequently, RT-PCR is squalene epoxidase and HMG-CoA reductase, or squalene performed in two reactions containing the mRNA template synthase and squalene epoxidase can be made and tested. and either of the following primer pairs. Example 3 10 acl1: Extraction of Sterols from Yarrowia lipolytica Cells 1fwd: 5' CACACGGATCCTATAatgcct tcc.gcaacg accg 1rev: 5' CACACACTAGttaaatttggacct caac acgaccc 50 ml cultures of Y lipolytica strains are grown for 2 days 15 in YPD liquid medium at 30°C. prior to harvesting for sterol acl2: analysis. Analysis is performed upon the non-Saponifiable 2fwd: 5' CACACGGATCCAATATAAatgtctg.cgaagagcatcc tog fractions. Saponification and extraction are performed essen tially as described (Methods in Molecular Biology, Vol. 53, 2rev: 5' CACACGCATGCttaa.gcttggaactic caccgcac Yeast Protocols, pp 123-131, 1996; J. Bacteriol. Vol. 108, No. 1, pp 69-73, 1971). In brief, cells are harvested by centrifu The resulting fragment from the ac11 reaction is cleaved gation, and then Saponified in a screw cap bottle using a with Spel and BamHI, and that from the ac12 reaction is mixture of methanol (3 ml), 0.5% pyrogallol (2 ml), and 60% cleaved with BamHI and SphI, and both are ligated together potassium hydroxide (2 ml). The tubes are then incubated at into YEp24 that has been digested with NheI and SphI, cre 90°C. for 2 hours prior to extraction with n-heptane (3 extrac 25 ating the plasmid “p12. The bi-directional GAL 1-10 pro tions using 5 ml of n-heptane). Each extraction is centrifuged moter is amplified from S. cerevisiae genomic DNA using the at 500 g for 5 minutes to enable phase separation. If GC-MS primers. analysis is to be performed, then the heptane-containing frac tions are evaporated under nitrogen at 55° C. gal10: 30 Example 4 5' CACACGGATCCaattittcaaaaattct tacttittitttittggatggac gal1 : Quantification of Sterol Production 5' CACACGGATCCttttitt citccttgacgittaaagtatagagg, UV spectrophotometry scanning between 220-300 nm can 35 and the resulting 0.67 kb fragment is cleaved with BamHI and be used to estimate the relative content of 5,7-diene-contain ligated in either orientation to BamHI-digested “p 12 to cre ing sterols in the heptane fractions described above. 5,7- ate “plgal2 and “p2gall', containing GAL1-acl1/GAL10 diene-containing sterols such as ergosta-5.7.24(28)-treinol, acl2 and GAL10-acl1/GAL1-acl2, respectively (Genbank ergosta-5.7.22.24(28)-tetraenol, and ergosterol give a charac accession: ad11: CAB91740.2; ac12: CAB91741.2). teristic triple peak at 293,281.5, and 271 nm, whereas many 40 In order to amplify the S. cereviseae gene encoding AMP of the other yeast sterols display a peak at 250 nm. By using deaminase and a promoter Suitable for expressing this gene, S. an authentic ergosterol standard and extinction values pro cerevisiae genomic DNA is amplified using two primer pairs vided by Shaw and Jefferies (Analyst 28:509-528, 1953), it is in separate reactions: possible to estimate the sterol content in crude heptane eXtractS. 45 AMD1 ORF: More detailed analysis of Y lipolytica sterol content can be attained by Subjecting the same extracts to either gas-liquid AMD 1 FWD : chromatography or, more preferably, gas chromatography 5' CACACGAGCTCAAAAatgga caat caggctacacagag coupled to mass spectrometry, as described (e.g. Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 11173-11178, 1997). Specific 50 AMD 1 rew: metabolites to examine include squalene, 2,3-epoxysqualene, 5' CACACCCTAGGtcacttittcttcaatggttctottgaaattg lanosterol. 4.4-dimethylcholesta-8, 14.24-trienol, 4.4-dim ethylzymysterol, 4-methylzymosterol, Zymosterol, fecos GAL7p: terol, episterol, ergosta-5.7.24(28)-treinol, ergosta-5.7.22.24 55 (28)-tetraenol, and ergosterol. ga17prox: Example 5 5' CACACGAGCTCdgaat attcaactgtttittttittat catgttgatg ga17 dist: Constructing an Oleaginous Strain of Saccharomyces 5' CACACGGAtc ctitcttgaaaatatgcactictatat cittittag, cerevisiae 60 The resulting fragment from the AMD1 reaction (2.4 kb) is Genes encoding the two subunits of ATP-citrate lyase from cleaved with SacI and AVr, and that from the GAL7 reaction N. crassa, the AMP deaminase from Saccharomyces cerevi (0.7 kb) is cleaved with BamHI and SphI, and both are ligated siae, and the cytosolic malic enzyme from M. circinelloides together into YEp13 that has been digested with NheI and are overexpressed in S. cereviseae strains in order to increase 65 BamHI, creating the plasmid “pAMPD. This plasmid carries the total lipid content. Similar approaches to enhance lipid the S. cerevisiae gene, AMD1, encoding AMP deaminase, production could be employed in other host organisms such under the control of the galactose-inducible GAL7 promoter. US 8,367,395 B2 73 74 Messenger RNA is prepared from lyophilized biomass of given for each polypeptide in addition to oligo pairs which M. circinelloides, as described above, and the mRNA tem can be used to amplify the coding region for each gene from plate is used in a RT-PCR reaction with two primers: Y lipolytica genomic DNA or cDNA. Resulting PCR frag ments can be cleaved with restriction enzyme pairs (e.g. depending on what site is present within the oligo sequence, MAEfwc ; Xbal/Mlul or NheI/Mlul or Xbal/AscI or NheI/AscI) and 5' CACACGCTAGCTACAAAatgttgtcact caaacgcatagdaac inserted into expression vectors (e.g. fungal expression vec MAErew: tors including Y lipolytica expression vectors disclosed 5' CACACGTCGACttaatgat citcggtatacgagaggaac, herein such as MB4629). 10 The DNA and proteins they encode of the Y lipolytica and the resulting fragment is cleaved with NheI and SalI, and genes represented in FIG. 8 areas follows (intron sequence is ligated to Xbal- and XhoI-digested pRS413TEF (Mumberg, underlined): D. et al., (1995) Gene, 156:119-122), creating the plasmid YALIOF30481g “pTEFMAE, which contains sequences encoding the cyto DNA: solic NADP-dependant malic enzyme from M. circinel 15 loides (E.C. 1.1.1.40; mce gene; Genbank accession: AY209191) under the control of the constitutive TEF1 pro atgtc.gcaac cccagaacgttggaatcaaag.ccct Cagat Ctacgtgcc moter. The plasmids “plgal2”, “pAMPD, and “pTEFMAE are ttct caattgtcaacCaggctgagctic gagaag cacgacggtgtc.gctg sequentially transformed into a strain of S. cereviseae to Ctggcaagtacac cattggtcttggt cagaccala catggcCtttgtcgac restore prototrophy for uracil (p1 gal2), leucine (“pAMPD), and histidine (“pTEFMAE) (Guthrie and Fink gacagagaggacatc tatt cotttgc cctd accqc.cgtct citcgactgct Methods in Enzymology 194: 1-933, 1991). The resulting Caagaacaacaac at Caccctgcatct attggt caatcgaggttggta transformants are tested for total lipid content following shake flask testing (e.g., 20 ml cultures in 125 ml flasks grown 25 ctgaaac Catctggacaagtic caagt cc.gt Caagtctgtgct catgcagc at 30°C. for 72-96 hour cultures) in either synthetic complete totttggcgagaa.ca.gcaa.cattgagggtgtgga caacgtcaacgc.ctgc (SC) medium lacking uracil, leucine and histidine or in a 2-step fermentation process. In the 2-step process, 1.5 ml of tacggaggaaccalacgc.cctgttcaacgct at Caactgggttgagggtcg cells from an overnight 2 ml roll tube culture containing SC at Cttgggacggc.cgaaacgc.catcgt.cgttgc.cggtgacattgc cct ct medium lacking uracil, leucine and histidine are centrifuged, 30 washed in distilled water, and resuspended in 20 ml of a acgcaaagggcgctgc.ccgacccaccggaggtgc.cggctgttgttgcc atg nitrogen-limiting medium suitable for lipid accumulation (30 g/L glucose, 1.5 g/L yeast extract, 0.5 g/L NHCl, 7 g/L ct cattggcc.ccgacgctic Cectggttcttgacaacgt.ccacggat citta KHPO, 5 g/L NaHPO-12H2O, 1.5 g/L MgSO.7H2O, ctitcgagcatgcc tacgatttctacaa.gc.ctgatctgacct cogagtacc 0.08 g/L FeC1-6H2O, 0.01 g/L ZnSO-7H2O, 0.1 g/L CaCl 35 2HO, 0.1 mg/L MnSO.5H2O, 0.1 mg/L CuSO4.5H2O, 0.1 cc tatgttgatggccactact coctacctgttacacaaaggc cct coac mg/L Co(NO)-6H.O; pH 5.5 (JAm Oil ChemSoc. 70:891 894 (1993)). aaggcc tacgctgcctacaacgc.ccgagcc.gaga aggtoggtctgttcaa Intracellular lipid content of the modified and control S. ggactic.cgacaagaagggtgctgaccgatttgact actctgcct tccacg cerevisiae strains is analyzed using the fluorescent probe, 40 Nile Red (J Microbiol Meth (2004) 56:331-338). In brief, tgcc cacctgcaa.gcttgtcaccaagtc.ttacgct cqacttct ctacaac cells diluted in buffer are stained with Nile Red, excited at 488 gactacct Caacgacaa.gagcctgtacgagggcc aggtocccdaggaggit nm, and the fluorescent emission spectra in the wavelength region of 400-700 nm are acquired and compared to the tgctg.ccgtc.tcc tacgatgcct ct citcaccgacaagaccgt.cgaga aga corresponding spectra from cells not stained with Nile Red. 45 To confirm results from the rapid estimation method, the total cc titcc ttgg tattgccaaggct cagtc.cgc.cgagc gaatggctic cttct lipid content is determined by gas chromatographic analysis ct coagggacccaccaacaccgg taa catgtacaccgc.ctctgtgtacgc of the total fatty acids directly transmethylesterified from dried cells, as described (Appl Microbiol Biotechnol. 2002 ttct ct catctotctgctgactitttgtc.ccc.gctgagcagotgcagggca November; 60(3):275-80). Yeast strains expressing the mul 50 tiple oleaginic polypeptides produce elevated total lipid (for agcgaatctotct cittct cittacggatctggit cittgct tcc acticttitt c example, in the range of 17% and 25% dry cell weight basis) totctgaccgtcaagggagacatttctic ccatcgtcaaggcctg.cgacitt following growth in YPD and lipid accumulation medium when compared to non-transformed S. cerevisiae strains Caaggctaagct catgaccgatccaccgagact cocgt.cgactacgagg which may, for example, produce in the range of 6% and 10% 55 Ctgccaccgatct Cogagaga aggcc cacct Caagaagaactittgagcc C total lipid after growth in YPD and lipid accumulation medium. cagggaga catcaag cacatcaagtctgg.cgt.ctactacct caccaacat Example 6 cgatgacatgttc.cgacgaaagtacgagat Caag cagtag 60 Protein: Y. lipolytica Oleaginic and Isoprenoid Biosynthesis Genes Misqpqnvgikaleiyvpsrivindaelekhdgvaagkytiglgqtnmafvd FIG. 8 is a list of Y lipolytica genes representing various drediysfaltav Srillknninidpa sigrievgtetlldlksksviksvlmd polypeptides (e.g. oleaginic and isoprenoid biosynthesis pep 65 tides) useful in the fungal strains and methods described lfigensniegvdnvnacyggtnal finainWvegrswdgrnaivvagdial herein. The Genbank accession number and GI number is

US 8,367,395 B2 99 100 - Continued an addition control plasmid (pMB4691), which is a URA3 ttctgaacaa.ccgagtc.cgaatc.cgatttgtgggitat cacatgtc.tcaag plasmid that contains regulatory sequences but no gene of interest. tagctgaaatgt attitcgctagaataaaataaatgagattaagaattaaa Strain MF921 has genotype MATB erg9-3'UTR::URA3 aatattggaatatatttitcctagaatagaaactittggatttitttitt.cggc ura2::URA2/tef-GGS1 ChrA-1635618::tef-carE3 ura3-302 ade 1:2 ADE1 ftef-HMG1 trunc leu2::PLEU2/tef-carRP. tattacagtctgaactggacaaacggctgactatatataaatatt attgg Strain MF921 harbors the same four plasmids as MF760, and gtctgttittcttgtttatgtcgaaattatctgggttt tact actgtgtcg an addition URA3 plasmid, pMB4789, which contains sequences for insertion into the 3' UTR of the native ERG9. tcgagtatagagtggcctgactggagaaaatgcagtag tatggacagtag 10 Insertion into 3' UTR of ERG9 is presumed to result in a gtactgc.cago.ca.gagaagtttittggaattgatacttgagt catttitt.cc hypomorphic mutation to decrease squalene synthase activ ity. attic cc catt coccatt coaacacaat caactgtttctgaacatttitcca 8B. Lipid Accumulation in Media Containing Various Car aaacgcggagatgtatgtcacttgg cactgcaagtc.tc.gattcaaaatgc 15 bon:Nitrogen Ratios Shake flask testing was conducted using carbon to nitrogen atct ctitt cagaccaaagtgtcatcagctttgtttggc cc caaattaccg (C/N) ratios of 160, 80, 60, 40, 30, 20, and 10 with yeast caaatacttgtcgaaattgaagtgcaatacggcct cqtctgccatgaaac nitrogen base being the base medium providing vitamins, trace elements and salts. Ammonium sulfate (which contains ctgcct attct cittcaaattgg.cgt caggttt cacgtc.ca.gcatt cotcg 21% nitrogen) was used as the nitrogen source and glucose cc.ca.gacagagttgctatoggttgaatcgtgtactgttaatatatgtatgt (which contains 40% carbon) was used as the carbon source at a concentration of 30 g/L. The concentrations of ammo attatact cqtactacgatatactgttcaatagagt ct cittataatcgta nium sulfate corresponding to these ratios are: 0.36, 0.71, cgacgattctgggca 0.95, 1.43, 1.91, 2.86, and 4.6 g/L, respectively. Uracil was 25 Supplemented at 0.2 mM. As controls, strains were also grown in yeast extract-peptone with 50 g/L of glucose (media Example 8 in which lipids do not accumulate at high levels) and yeast extract-peptone with 5% olive oil (v/v) (media in which lipids Cultures Conditions, Such as Limitation for accumulate at high levels). Strain MF760 (10-14 ml of cul Nitrogen, Magnesium or Phosphate, can Promote 30 ture) was harvested after 4 days of growth at 30°C., during Lipid Accumulation in Y lipolytica which time the cultures were shaking at 250 rpm. Following harvesting, cells were washed three times with water, with the 8A. Strains Used to Analyze Lipid Accumulation During exception of the oil-grown cells which were washed three Growth Under Various Conditions. 35 times in 0.5% BSA and one time with water before lipid Strains MF760, MF858, and MF921 were grown under an extractions. Lipids were extracted as described in Folch J. array of culture conditions, and then harvested cells were Lees, M, and Stanley, G. H. S. J. Biol. Chem. 226: 497-509, extracted and analyzed for total lipid content and levels of 1957. In brief, cell pellets were resuspended in 6 ml of water. specific lipophilic metabolites. FIG.9 depicts schematic rep A 1 ml aliquot was transferred to a pre-weighed tube with a resentations of plasmids generated and described in this 40 hole on the lid, spun down and the cell pellet lyophilized example. Strain MF760 has genotype MATB ura2::URA2/ overnight to determine the dry cell weight. The remaining 5 tef-GGS1 ChrA-1635618::tef-carE ura3-302 ade1:2ADE1/ ml were placed in a 15 ml Falcon tube and spun down. Cell tef-HMG1 trunc leu2:?LEU2/tef-carRP (questions marks pellets were frozen at -20° C. until extractions were per denote presumed loci of chromosomal integration). Strain formed. Two to three volumes of a Zymolyase solution (2 MF760 harbors four insertion plasmids, pMB4637, 45 mg/ml Zymolyase 100T in 1M Sorbitol, 50 mM EDTA and pMB4591, pMB4705, and pMB4660, which encode native or 0.01% B-mercaptoethanol) was added to each cell pellet and heterologous genes required for synthesis of either isoprenoid placed at 37°C. with constant agiatation for 1 hr. Two vol metabolites in general, or carotenoid metabolites specifically. umes of cubic zirconia beads were added to each tube and In all insertion plasmids, except pMB4789, described in this vortexed for 15-20 min. Samples were viewed under a micro example, the Y lipolytica TEF1 promoter and XPR2 termi 50 Scope to ensure cell breakage before continuing with extrac nator were the regulatory sequences used to control expres tions. After cell breakage was complete, 6 ml of extraction sion of genes of interest. Also, in Some instances multiple solvent was added (a 2:1 mix of chloroform and methanol) URA3-containing plasmids can be utilized in the same strain, and mixed. The mixture was spun down for 5 min at 3000 rpm since 5-fluoroorotic acid can be used to select for Ura ser and the organic layer was transferred to a clean tube. NaCl geants following transformation with a URA3 plasmid. 55 was added to the remaining aqueous layer to make it a 0.29% pMB4637 is an ADE 1 plasmid that encodes a truncated vari NaCl solution. 6 ml of extraction solvent was added and ant of the Y lipolytica HMG-CoA reductase. pMB4591 is a mixed, and the mixture was spun down for 5 min. The organic URA5 plasmid that encodes the Y lipolytica geranylgera layers were pooled and filtered through a 0.2 um filter to get nylpyrophosphate synthase. pMB4705 is a LEU2 plasmid rid of any cell debris. The extract was washed with 0.2 vol that encodes the phytoene synthase/lycopene cyclase 60 umes of 0.29% NaCl solution and another 6 ml of extraction (CarRP) from Mucor circinelloides. pMB4660 is a URA3 Solvent added and mixed. Mixtures were spun and the organic plasmid that encodes a phytoene dehydrogenase from M. layer was placed in a pre-weighed glass Vial, the solvent was circinelloides. evaporated under a flow on nitrogen and the vial was weighed Strain MF858 has genotype MATBura2::URA2/tef-GGS1 again to determine the weight of the lipid extracted. The dry ChrA-1635618::tef-carE ura3-302::?URA3/tef-plasmid 65 cell weight is used to determine the percentage of lipid per dry ade 1::PADE1 ftef-HMG1 trunc leu2::PLEU2/tef-carRP. cell weight. The lipid accumulation results are in the Table 62 Strain MF858 harbors the same four plasmids as MF760, and below: US 8,367,395 B2 101 102 TABLE 62 Strains MF858 and MF921 were harvested after 4 days of growth at 30°C., during which time the cultures were shaking Lipid accumulation under various carbon: nitrogen at 250 rpm. Cells were washed three times with water before ratio growth conditions lipid extraction. Lipids were extracted as described above. CNRatio % lipid Lipid accumulation data is listed in the Table 64 below: YNB 160 61 3% Glucose 8O 49 TABLE 64 60 34 40 17 Lipid accumulation in phosphate or magnesium 30 16 10 limiting growth conditions 2O 14 10 15 % Lipid YEP 5%. Glucose 22 5% olive oil 38 g/L MF858 MF921 phosphate 1 14 14 15 O.0625 18 2O Other nitrogen Sources tested were proline (12% nitrogen), O.O313 34 41 Sodium glutamate (7% nitrogen), soy acid hydrolysate (12% O.O156 62 63 O.OO78 83 76 nitrogen), and yeast extract-peptone (26.8% nitrogen). All magnesium O.S 12 11 nitrogen sources tested at C/N ratios of 80 (with glucose as a O.O313 NA 16 carbon Source), had significantly larger lipid bodies than at O.O156 NA 25 O.OO78 NA 42 C/N ratios of 10 (also with glucose as a carbon source). O.OO39 48 48 Strains MF858 and MF921 were harvested after 4 days of growth at 30°C. (3% glucose was used as the carbon source). The following tables are referenced throughout the Cells were washed three times with water and lipids extracted 25 description. Each reference and information designated by as described above. Lipid accumulation data for Soy hydroly each of the Genbank Accession and GI numbers are hereby sate, yeast extract-peptone and yeast nitrogen base, used as a incorporated by reference in their entirety. The order of genes, control, are listed in Table 63 below. polypeptides and sequences presented in the tables is not indicative of their relative importance and/or suitability to any TABLE 63 30 of the embodiments disclosed herein. Lipid accumulation under different carbon and nitrogen conditions with various nitrogen sources % lipid Lengthy table referenced here 35 CNRatio MF858 MF921 USO8367395-20130205-TOOOO1 Soy hydrolysate 8O 36 36 Please refer to the end of the specification for access instructions. 60 36 35 10 14 15 Yeast Extract- 8O 37 37 Peptone 10 15 14 40 Yeast Nitrogen 8O 37 38 Base 10 13 11 Lengthy table referenced here 8C. Determination of Lipid Levels Under High Carbon and USO8367395-20130205-TOOOO2 Phosphate or Magnesium Limiting Conditions. 45 Please refer to the end of the specification for access instructions. To test whether other nutrient limitations, under high car bon conditions, will allow for higher lipid accumulation, phosphate or magnesium limiting conditions were tested. For phosphate limiting conditions, yeast nitrogen base medium without phosphate was prepared. Shake flask testing was 50 performed using carbon to phosphate ratios ranging from Lengthy table referenced here 5376 down to 42. This range corresponds to 7.8 mg/L up to 1 g/L, respectively, and the latter concentration corresponds to USO8367395-20130205-TOOOO3 that are commonly used in yeast nitrogen base medium. Glu 55 Please refer to the end of the specification for access instructions. cose, at 30 g/L, was used at the carbon Source. Potassium phosphate monobasic (containing 28.7% phosphate) was used as the phosphate source. For magnesium limiting conditions, yeast nitrogen base medium without magnesium was prepared. Shake flask test 60 ing was conducted using carbon to magnesium ratios ranging Lengthy table referenced here from 31360 down to 245. This range corresponds to 0.375 USO8367395-20130205-TOOOO4 mg/L up to 0.5g/L, and the latter magnesium concentration corresponds to that commonly used in yeast nitrogen base. Please refer to the end of the specification for access instructions. Glucose, at 30 g/L, was used as the carbon Source. Magne 65 sium sulfate (containing 9.8% magnesium) was used as the magnesium source. US 8,367,395 B2 103 104

Lengthy table referenced here Lengthy table referenced here

USO8367395-20130205-TOOOO5 USO8367395-20130205-TOOO12

Please refer to the end of the specification for access instructions. Please refer to the end of the specification for access instructions.

10

Lengthy table referenced here 15 Lengthy table referenced here USO8367395-20130205-TOOOO6 USO8367395-20130205-TOOO13 Please refer to the end of the specification for access instructions. Please refer to the end of the specification for access instructions.

Lengthy table referenced here 25 Lengthy table referenced here USO8367395-20130205-TOOOOT USO8367395-20130205-TOOO14 Please refer to the end of the specification for access instructions. Please refer to the end of the specification for access instructions.

30

Lengthy table referenced here Lengthy table referenced here USO8367395-20130205-TOOOO8 35 USO8367395-20130205-TOOO15 Please refer to the end of the specification for access instructions. Please refer to the end of the specification for access instructions.

40

Lengthy table referenced here Lengthy table referenced here USO8367395-20130205-TOOOO9 USO8367395-20130205-TOOO16 45 Please refer to the end of the specification for access instructions. Please refer to the end of the specification for access instructions.

50 Lengthy table referenced here Lengthy table referenced here USO8367395-20130205-TOOO 10 USO8367395-20130205-TOOO17

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EQUIVALENTS 2O Those skilled in the art will recognize, or be able to under Lengthy table referenced here stand that the foregoing description and examples are illus trative of practicing the provided invention. Those skilled in USO8367395-20130205-TOOO91 the art will be able to ascertain using no more than routine Please refer to the end of the specification for access instructions. 25 experimentation, many variatons of the detail presented herein may be made to the specific embodiments of the inven tion described herein without departing from the spirit and Scope of the present invention.

LENGTHY TABLES The patent contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US08367395 B2). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

What is claimed is: 40 g. increased expression or activity of a phosphomevalonate 1. An engineered Y lipolytica strain that produces at least kinase polypeptide compared to an otherwise compa one sterol compound selected from the group consisting of rable strain lacking the one or more sterologenic modi squalene, lanosterol, Zymosterol, ergosterol or 7-dehydroc fication; holesterol (provitamin D3), the strain containing one or more h. increased expression or activity of a mevalonate pyro sterologenic modifications selected from the group consist 45 phosphate decarboxylate polypeptide compared to an ing of otherwise comparable strain lacking the one or more a. decreased expression or activity of a Y lipolytica GGPP sterologenic modification; synthase polypeptide compared to an otherwise compa rable strain lacking the one or more sterologenic modi i. increased expression or activity of a cytosolic malic fication; 50 enzyme polypeptide compared to an otherwise compa b. increased expression or activity of a truncated HMG rable strain lacking the one or more sterologenic modi CoA reductase polypeptide compared to an otherwise fication; comparable strain lacking the one or more sterologenic j. increased expression or activity of a malate dehydroge modification; nase polypeptide compared to an otherwise comparable c. increased expression or activity of an FPP synthase 55 strain lacking the one or more sterologenic modification; polypeptide compared to an otherwise comparable k. increased expression or activity of an AMP deaminase strain lacking the one or more sterologenic modification; polypeptide compared to an otherwise comparable d. increased expression or activity of an IPP isomerase strain lacking the one or more sterologenic modification; polypeptide compared to an otherwise comparable 60 1. increased expression or activity of a glucose 6 phosphate strain lacking the one or more sterologenic modification; dehydrogenase polypeptide compared to an otherwise e increased expression or activity of an HMG-CoA syn comparable strain lacking the one or more sterologenic thase polypeptide compared to anotherwise comparable modification; strain lacking the one or more sterologenic modification; m. increased expression or activity of a malate dehydroge f increased expression or activity of a mevalonate kinase 65 nase homolog 2 polypeptide compared to an otherwise polypeptide compared to an otherwise comparable comparable strain lacking the one or more sterologenic strain lacking the one or more sterologenic modification; modification; US 8,367,395 B2 117 118 n. increased expression or activity of a GND1-6-phospho a. decreased expression or activity of a Y lipolytica GGPP gluconate dehydrogenase polypeptide compared to an synthase polypeptide compared to an otherwise compa otherwise comparable strain lacking the one or more rable strain lacking the one or more sterologenic modi sterologenic modification; fication; o. increased expression or activity of a isocitrate dehydro b. increased expression or activity of a squalene synthase genase polypeptide compared to an otherwise compa polypeptide compared to an otherwise comparable rable strain lacking the one or more sterologenic modi strain lacking the one or more sterologenic modification; fication; c. increased expression or activity of a truncated HMG p. increased expression or activity of a IDH2-isocitrate CoA reductase polypeptide compared to an otherwise dehydrogenase polypeptide compared to an otherwise 10 comparable strain lacking the one or more sterologenic modification; and combinations thereof. comparable strain lacking the one or more sterologenic 3. The engineered Y lipolytica strain of claim 1, wherein modification; the produced sterol compound accumulates to levels that are q. increased expression or activity of a fructose 1.6 bispho greater than at least about 1.0%, greater than at least about sphatase polypeptide compared to an otherwise compa 15 2.5%, or greater than at least about 5% of the dry weight of the rable strain lacking the one or more sterologenic modi cells. fication; 4. The engineered Y lipolytica strain of claim 1, wherein r. increased expression or activity of a Erg 10-acetoacetyl the sterol compound is squalene. CoA thiolase polypeptide compared to an otherwise 5. The engineered Y lipolytica strain of claim 1, wherein comparable strain lacking the one or more sterologenic the sterol compound is lanosterol. modification; 6. The engineered Y lipolytica strain of claim 1, wherein S. increased expression or activity of a squalene synthase the sterol compound is Zymosterol. polypeptide; and combinations thereof compared to an 7. The engineered Y lipolytica strain of claim 1, wherein otherwise comparable strain lacking the one or more the sterol compound is ergosterol. sterologenic modification; 25 8. The engineered Y lipolytica strain of claim 1, wherein wherein the engineered strain can accumulate lipid to at the sterol compound is 7-dehydrocholesterol. least about 20% of its dry cell weight; and 9. The engineered Y lipolytica strain of claim 1, further whereinas a result of genetic engineering the engineered comprising at least one, oleaginic modification, strain produces the at least one sterol compound to a wherein as a result of the at least one oleaginic modifica level at least about 1% of its dry cell weight. 30 tion, the strain produces the at least one sterol compound 2. The engineered Y lipolytica strain of claim 1, wherein to a level at least about 1% of its dry cell weight. the one or more sterologenic modifications is selected from the group consisting of k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,367,395 B2 Page 1 of 1 APPLICATIONNO. : 12/443350 DATED : February 5, 2013 INVENTOR(S) : Richard B. Bailey, Joshua Trueheart and Kevin T. Madden It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the title page of the patent, in the left-hand column at INID code 60, under the heading “Related U.S. Application Data: Insert the following text in front of the existing citation: --This application is a 371 of PCT/US2007/021091, filed September 28, 2007, which claims benefit of

Signed and Sealed this Ninth Day of April, 2013

Teresa Stanek Rea Acting Director of the United States Patent and Trademark Office UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,367,395 B2 Page 1 of 1 APPLICATIONNO. : 12/443350 DATED : February 5, 2013 INVENTOR(S) : Richard B. Bailey et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:

On the Title Page:

The first or Sole Notice should read --

Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 436 days.

Signed and Sealed this Twentieth Day of August, 2013

Teresa Stanek Rea Acting Director of the United States Patent and Trademark Office