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Struktol J673 (Strucktol Company of America, Stow, Ohio, USA) or Mazu DF 37C. (Mazer Chemicals, Inc., Gurnee, Illinois, USA). The cell yield expected for the.
CHAPTER 6

Pichia pastoris Koti Sreekrishna and Keith E. Kropp

1

History of Pichia pastoris

Interest in the study of nonconventional yeasts (yeasts other than Saccharomyces cerevisiae and Schizosaccharomyces pombe) has increased dramatically in the past few years (Reiser et al. 1990). One such category is methylotrophic yeasts (Wegner and Harder 1986; Harder et al. 1986), e.g., Pichia pastoris, Hansanula polymorpha, Candida boidinii, etc. Methylotrophic yeasts have the ability to use methanol as a sole source of carbon and energy. Adaptation to growth on methanol is associated with induction of methanol oxidase, MOX (also referred to as alcohol oxidase, AOX), dihydroxy acetone synthase DAS, and several other enzymes involved in methanol metabolism. The most spectacular increase, however, is seen with alcohol oxidase, which is virtually absent in glucose-grown cells, but can account for over 30% of the cell protein in methanol-grown cells. Extensive proliferation of peroxisomes, accounting for over 80% of the cell volume, is also observed in methanol-grown cells (Veenhuis et al. 1983). Due to these characteristics, methylotrophic yeasts have gained the attention of biochemists, molecular biologists, cell biologists, biotechnologists, microbiologists, and chemists in academics and industry. The present chapter focuses on one of the methylotrophic yeasts, namely P. pastoris. This yeast was initially developed by Phillips Petroleum Company for the production of single-cell protein for feed stock. A very efficient ultra-high cell density (>130g dry cell weight per liter) fermentation process with high biomass productivity (>lOgIliter-hour) was developed through meticulous fermentation research (Wegner 1983). Unfortunately, the economics of this process, while impressive from a fermentation standpoint (approximately $5 per pound of protein), was clearly an order of magnitude higher in comparison to the cost of a pound of soybean. Following this setback, Phillips Petroleum Company invested its efforts in developing this yeast as an expression system for the production of recombinant proteins, and this has proved to be a worthwhile endeavor (Wegner 1990; Romanos et al. 1992; Cregg et al. 1993).

Hoechst Marion Roussel Inc., 2210 E. Galbraith Road, Cincinnati, Ohio 45215, USA

K. Wolf, Nonconventional Yeasts in Biotechnology © Springer-Verlag Berlin Heidelberg 1996

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Since 1988, several pharmaceutical and biotechnology companies have licensed the P. pastoris expression technology. At least three products produced with this technology (Hepatitis B surface antigen, human serum albumin, and insulin-like growth factor-I) are being pursued for commercialization and many more are in the pipeline. Since 1990, Phillips Petroleum Company has made this technology available at no actual cost for research use to universities and nonprofit organizations through a materials transfer agreement. More recently, however, the system can be readily obtained for a nominal fee from Invitrogen Corporation, San Diego, California, USA. However, for commercial purposes, this technology can be licensed from Research Corporation Technologies, Tucson, Arizona, USA. In addition to its extensive use for the expression of heterologous proteins, P. pastoris is also being developed as a model organism for molecular analysis of peroxisome biogenesis (Gould et al. 1992; Liu et al. 1992). The primary intent of this chapter is to introduce investigators to practical techniques for manipulating P. pastoris with emphasis on its use for expression of heterologous proteins. 2

Growth and Storage 2.1

Shake Flask, Shake Tube, Plate, and Slant Cultures P. pastoris grows well both in liquid and on solid media, on a wide variety of simple

carbon sources including glucose, glycerol, fructose, sorbitol, ethanol, methanol, alanine, lysine, succinate, ethyl amine, cadaverine, glucitol, mannitol, L-rhamnose, and trehalose. The doubling time is dependent on the carbon source used and is typically 90 min on glucose and approximately 6 h on methanol. In solid media it forms white or cream-colored nonfilamentous colonies. Multilateral buds are noticed under light microscopy. The natural habitats of P. pastoris are the oak tree and packaged foods. The compositions of the various P. pastoris growth media (MD, MDH, MGy, MGyH, MM, MMH, YPD) are given in Sects. 2.2.2 and 2.2.3. The growth temperature is 30°C with shaking (250rpm) for liquid cultures and with incubation for plates and slants. When minimal methanol (MM) plates are used as growth medium, 100,ul of 100% methanol (filter-sterilized) is added to the plate lid once every day to compensate for the methanol lost due to evaporation (the plate is placed inverted in the incubator). When MM liquid medium is used, methanol is added to a final concentration of 0.5% (v/v) every 2 days to compensate for methanol lost due to evaporation. Because P. pastoris grows well under a wide pH range of 3-6.5, buffering the growth medium is generally unnecessary, except under some special circumstances, as noted under Sect. 7.4. For large-scale, high cell-density cultivation of P. pastoris, refer to Sect. 5.

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P. pastoris as such does not grow on galactose, arabinose, ribose, maltose, sucrose, lactose, raffinose, melibiose, cellulose, or starch. However, it will grow on autoclaved sucrose, due to breakdown of sucrose into glucose and fructose. Also, P. pastoris strains transformed with the sucrase (invertase) gene SUC2 of Saccharomyces cerevisiae efficiently grow on sucrose with high growth yields (Sreekrishna et al. 1987).

2.2 Media 2.2.1 Stock Solutions

Note. For filter sterilization of various solutions and liquids, filter wares (disposable or reusable types) equipped with cellulose acetate or cellulose nitrate membranes (pore size 0.2 to 0.22,urn) from one of the several manufacturers (N algene Company, Rochester, New York, USA; Costar Corporation, Cambridge, Massachusetts, USA; Corning Glass Works, Corning, New York, USA) can be used. For filter sterilization of methanol and methanol-containing media, only cellulose acetate membranes (0.2-0.22.um) are suitable, because methanol does not filter through cellulose nitrate membranes of pore size 0.2.um. lOx YNB: Dissolve 13.4 g of yeast nitrogen base without amino acids (YNB, Difco labs., Detroit, Michigan, USA) in 100 ml of water (heat if necessary) and filtersterilize. This solution can be stored for over a year at 4°C. B: Dissolve 20 mg of d-biotin (Sigma Chemicals, St. Louis, Missouri, USA) in 100 ml of water and filter sterilize.

~OOx

IOOx H: Dissolve 400 mg L-histidine in 100 ml of water (heat if necessary) and filter sterilize. lOx D: Dissolve 20 g of D-glucose in 100 ml water. Autoclave for 15 min or filter sterilize. Stores well for years at room temperature. lOx GY: Mix 10 ml of glycerol with 90 ml of water. Filter sterilize. Stores well for years at room temperature. lOx M: Mix 5ml of methanol (100%) with 95ml of water. Filter sterilize and store at 4°C. 100% methanol: Filter sterilize pure methanol (100%). Store at room temperature in a fireproof cabinet.

2.2.2 Minimal Media Compositions

MD: Mix 100ml of lOx YNB, 2ml of 500x B, and 100ml of lOx D with 800ml of autoclaved water (include 15 g Bacto agar for plates).

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MM: Mix lOOml of lOx YNB, 2ml of 500x B, and 100ml of lOx M with 800ml of autoclaved water (include 15g Bacto agar for plates). MGY: Mix lOOml of lOx YNB, 2ml of 500x B, and 100ml of lOx GY with 800ml autoclaved water (include l5g Bacto agar for plates). All these liquid media and plates store well for several weeks at 4°C. Minimal media with other carbon sources (such as D-sorbitol, D,L-alanine) are prepared by using the desired carbon source at 10 gil in place of glucose in MD. Minimal media containing a mixture of carbon sources can also be prepared by combining two or more desired substrates in the growth medium.

2.2.3 Supplemental Minimal Media Compositions

Minimal media are supplemented with necessary supplemental nutrients such as amino acids, depending on the specific requirement of a given strain. For example, P. pastoris strains GSl15 and KM71, commonly used in molecular genetic manipulations, are auxotrophic for histidine. Such strains will grow in minimal media only in the presence of supplemental histidine. However, once transformed with HIS4 (histidinol dehydrogenase gene), they readily grow in the absence of histidine. The composition of supplemental minimal histidine media (suitable for histidine auxotrophic strains such as GS1l5) is as follows. Other supplemental media can be prepared depending on the need of a particular strain in use. MDH: Mix 100mi of lOx YNB, 2ml of 500x B, 100ml of lOx D, and 10mi of 100x H with 790ml of autoclaved water (include 15 g agar for plates). MMH: Mix 100 ml of lOx YNB, 2 ml of 500x B, 100 ml of lOx M, and 10 ml of 100x H with 790ml of autoclaved water (include 15 g agar for plates). MGyH: Mix 100mi of lOx YNB, 2ml of 500x B, 100mi of lOx GY, 10ml of 100x H with 790ml of autoclaved water (include 15 g agar for plates). All of these liquid media and plates store well for several weeks at 4°C. Supplemental minimal histidine media with other carbon sources is prepared by adding a similar amount of histidine as above to the minimal media with the desired carbon source.

2.2.4 Complex Medium Composition

YPD: Dissolve 109 of Bacto yeast extract, 20 g of peptone, and 20 g of glucose in lOOOml of water (also include 15 g Bacto agar for slants and plates) and autoclave for 20 min.

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2.3 Storage For medium-term storage, P. pastoris should be kept at 4°C in complex (YPD) liquid medium or YPD agar slants. Cells are cultured initially on a desired media (selective minimal methanol, dextrose, or glycerol media such as MM, MD, or MGY for transformants) and then transferred to YPD. Most of the commonly used strains can be stored in such media for over 1 year at 4°C. However, the protease-deficient strains (SMD1163, SMD1165, and SMD1168) should be restreaked or regrown every 2-4 weeks, because they do not keep well. For long-term storage, cells are suspended at an O.D60onm of 50-100 in YPD containing 50% glycerol. The cells are frozen at -80°C or preferably kept in a liquid nitrogen freezer. Cells stored in these ways have remained viable for several years. 3

Available Strains NRRL Y-11430-SC5 (wild type; Sreekrishna et al. 1987) GS115 (his4) - this strain is also known as GTS115 (Sreekrishna et al. 1987) KM71 (his4, aoxl::ARG4; Cregg and Madden 1988) PPFI (his4, arg4; J.M. Cregg, pers. comm.) Protease-deficient strains (derived by protease A (PEP4) and/or Protease B (PRB) gene disruption (M.A. Gleeson, pers. comm.): SMD1163 (his4, pep4, prBl) SMD1165 (his4, prBl) SMD1168 (his4, pep4) 4

Genetic Techniques 4.1

Life Cycle The members of the ascomycetous genus Pichia Hansen are distinguished from most other yeasts by the occurrence of hat-shaped spores. Investigations of the DNA/DNA reassociation demonstrated a narrow relatedness to members of the genus Hansenula. Therefore, many of these yeasts are included now also in the genus Pichia (Kurtzman 1984a,b; Barnett et al. 1990). In most cases, the life cycle of the Pinus species is unknown. The protocols given here are for the genetic analysis of P. pastoris (M.E. Digan, pers. comm.; see also Digan and Lair 1986).

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4.2 Mating and Sporulation P. pastoris strains that are available are homothallic (switch mating types), thus it is essential to use selection plates against both parents used in the genetic cross prior to sporulation of mated cells. Appropriate minimal plates are used to select against parents based on amino acid or nucleotide or carbon source requirements. Cultures form four-spored asci, but the viability of the spores is low. Tetrad analysis is possible with a few asci. However, the segregation frequencies do not fit the expected 2+: 2- ratio. In most cases the spores are phenotypically wild type. The low spore viability and the aberrant segregation ratios suggest that the establishment of a mating system which can be used for genetic analysis requires further research, including a backcrossing program with several strains and mutants and, perhaps, the search for heterothallic strains.

4.2.1 Mating

1. Resuspend single colonies of each of the two parental strains in 100,u1 ofYPD. Mix the parental strains thoroughly together, and spread on presporulation plates (see below for composition). Incubate for 24h at 30°C. 2. Replica-plate onto sporulation plates (see below for composition). Incubate for 24 h at 30°C. 3. Replica-plate onto plates which select against both parental types and allows survival of only the progeny of cross-mated cells. Incubate at 30°C until colonies appear. YPD (see Sect. 2.2.4) Presporulation medium: Mix 50 g glucose, 20 g peptone, 10 g yeast extract, 5 g agar, and 23 g nutrient agar in 1000rnl water, and autoclave. Sporulation medium: Mix 5 g NaOAc (anhydrous), 10 g KCI (anhydrous), and 20 g agar in 1000 rnl water, and autoclave.

4.2.2 Sporulation

4. Disperse single colonies from step 3 in YPD. Plate on presporulation plates and after 24h of incubation at 30°C, replica-plate onto sporulation plates. Incubate at 30°C for 3-5 days. At this point, asci can be dissected directly, or the mixture of cells and spores on the SPA plate can be digested extensively with cell wall-degrading enzymes to kill the vegetative cells as described below.

4.2.3 Random Spore Preparation

5. Wash sporulation plates with sterile water to harvest tlle spores.

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6. Wash the spore suspension twice with 0.1 M sodium phosphate buffer, pH 7.4. 7. Resuspend spores in 3m! ofo.lM phosphate buffer, pH 7.4. Add Zymolyase lOOT, Glusulase, and j3-mercaptoethanol to final concentrations ofO.5mg/m!, 2%v/v, and O.I%v/v respectively. Incubate for 5h at 30 C with occasional shaking. D

8. Sonicate the spore suspension three times for 15 s and harvest by centrifugation at 3000 g for 10 min at room temperature. Note. Spores can be quantified under a light microscope by counting an aliquot of spore suspension placed in a counting chamber. 9. Examine spores microscopically for clumps. If clumping appears to be excessive, repeat step 4. 10. Plate the spores on YPD plates at approximately 200 spores/plate or use a micro-manipulator to separate single spores on YPD plates. 11. Let spores germinate at 30 DC on YPD and screen for the desired phenotype{ s) by replica-plating onto two selective plates, each of which selects against one or the other parent in the cross. 5

Fermentation Process The following paragraphs describe general methods for production of biomass as well as for the production of heterologous proteins using Mutt and Mut- cells in both continuous and batch modes of fermentation. The process described here can be scaled up (>1000 liters) or scaled down (0.2l) as desired. Fermentation can be conducted over a wide pH range (3.0-5.9) at 30 D C (Wegener 1983). Prolific growth at low pH (which reduces risk of microbial contamination) was considered as one of the advantages of using this yeast for production of single-cell protein. Interestingly, this has also been valuable in optimizing fermentation parameters for the production of recombinant proteins. For example, human serum albumin (HSA) secretion yield is improved over threefold by using pH 5.85 compared to the generally used pH of 5.0 (Sreekrishna et al. 1990). In the case of secretion of the VI domain of CD 4 (amino acid residues 1 to 106 of mature CD4 ), intact product was seen only at acidic pH (2.5-3.5) (Buchholz et al. 1991). As high as a two- to fourfold increase in secreted yields of human epidermal growth factor and human insulin-like growth factor-l are observed at pH 3.0 (Siegel et al. 1990; Brierley et al. 1992). Other kinds of media and growth conditions are also known to improve the production of specific proteins. Addition of yeast extract and peptone increased the level of secretion ofHSA and tissue plasminogen activator (TPA) (Sreekrishna et al. 1990; J.F. Tschopp, pers. comm.). Likewise, addition of 1% cas amino acids improved the yield of mouse epidermal growth factor (Clare et al. 1991). In the case of intracellular production of hepatitis B surface antigen, the addition of certain

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trace metals (KI, NaMoO 4· 2HP, CoC12 • 6H 20 at 0.8, 0.2, and 0.5 g per liter, respectively), plus allowing the cells to sit longer in the fermentor, improved the yield of antigen particles (J.A. Cruze, pers. comm.). Thus, it is evident that some experimentation with fermentation parameters may be necessary to establish productspecific optimal conditions. 5.1

Continuous Culture of Mut+ and Mut- Strains on Methanol

Fermentation is carried out in two steps. First in the batch mode on glycerol or glucose as the carbon source followed by continuous mode on methanol-containing medium. The process described here is typically with recombinant cells, where it is preferable to use glycerol rather than glucose. 5.1.1 Inoculum for the Fermentor

Grow cells to an O.D6oonm of 2-10 in a 2-1 shake flask containing 11 of one of the following growth media: MD, MGY, YPD (see Sect. 2.2.2 for composition), YMPD, YMPGy, or MGyB (see Sect. 5.1.2 for compositions). This volume of inoculum is adequate for inoculating a 20-1 fermentor with a 10-1 operating volume. 5.1.2 Media

YMPD: Dissolve 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, and 10 g of glucose in 11 of water, and autoclave. YMPGy: Same as YMPD with the exception that 10 ml of 100% glycerol is used instead of 10 g of glucose. MGyB: Dissolve 11.5g KH2P0 4, 2.66g K2HP0 4, 6.7g YNB, pH 6.0, and 20ml glycerol in 11 water, and autoclave. FM21 basal salt media Composition is for 11 final volume in water Phosphoric acid, H3P04 (85%) 3.5ml Calcium sulfate, CaS0 4 • 2HP 0.15 g Potassium sulfate, K2SO4 2.4 g Magnesium sulfate, MgSO 4· 7H20 1.95 g Potassium hydroxide, KOH 0.65 g Biotin stock solution Biotin

0.2 g per liter

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PTMI trace salts Composition is for II final volume in water Cupric sulfate (CuSO 4' 5H zO) 6.0 g Manganese sulfate (MnSO 4' HzO) 3.0 g Ferrous sulfate (FeSO 4' 7H zO) 65.0 g Zinc sulfate (ZnS04 • 7H zO) 20.0 g Silfuric acid (H ZS0 4 ) 5.0ml Cobalt chloride (CoClz ' 6H zO) 0.5 g Boric acid (H 3B03) 0.02 g Sodium molybdate (NaMo0 4 ·2HzO) 0.2g Potassium iodide (KI) 0.1 g BSM medium composition Composition is for II final volume in water Phosphoric acid, H3P0 4 (85%) 26.0ml Calcium sulfate, CaS04 ·2HzO 0.9g Potassium sulfate, K ZS04 18.0g Magnesium sulfate, MgSO 4' 7HzO 14.0 g Potassium hydroxide, KOH 4.0 g 5.1.3 Batch Phase

Sterilize a 20-1 fermentor with 91 of the basal salt medium FM21 (see Sect. 5.1.2 for composition) containing 5% v/v glycerol (higher levels may be toxic to the cells) or 5-10% glucose. Allow the system to cool to the set temperature of 30 cC. The pH of this medium will be 20%, with the agitator speed set between 500 to 1500rpm and a vessel pressure of 2 to 3 psi. Foaming is controlled through the addition of a 5% Struktol J673 (Strucktol Company of America, Stow, Ohio, USA) or Mazu DF 37C (Mazer Chemicals, Inc., Gurnee, Illinois, USA). The cell yield expected for the batch phase on 5% glycerol under these conditions is 20-25 g of washed dry cell weight per liter. 5.1.4 Continuous Phase

Continuous fermentation is established by feeding FM21-methanol (15%v/v) for Mut30% of the total soluble protein. We used TNF as an example, because its expression level in Pichia was known to be copy number-dependent. This approach for screening is useful in identifying transformants with presumably multicopy integrants. In several cases, it has been noted by us as well as others, that the gene dosage has a profound effect on the expression level in Pichia (Sreekrishna 1993) (e.g., TNF, EGF, salmon growth hormone, Clostridium tetani toxin fragment C, Bordetella pertussis p69 antigen. etc.)

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pPIC3K: pPIC3 with kanamycin resistance gene (M.A. Romanos, pers. comm.; Fig. 10).

pHIL-DS: pHIL-D2 with kanamycin resistance gene (K. Sreekrishna and S. Hopkins, unpubl. observ.; Fig. 11). pHIL-D6: pHIL-DS with unique ASUII site and multiple cloning sites (R. Belagaje, pers. comm.; Fig. 12). pHIL-D7: pHIL-DS with unique ASUII site (K. Kropp, unpubl. result; Fig. 13). pHIL-Sl: Secretion vector with P. pastoris acid phosphatase secretion signal; Fig. 14. Signal sequence including cloning junction of pPIC9 and pPIC9K (Fig. 15). pPIC9: Secretion vector with S. cerevisiae alpha mating factor pre-pro signal (Clare et al. 1991b; Fig. 16). pPIC9K: pPIC9 with kanamycin resistance gene (Scorer et al. 1994; Fig. 17).

AsuII(934) BamHI(939) SnaBI(960) EcoRI(964) AvrII(970) Notl(977)

pPIC3K 9017 bp

BglII(6615)

XhoI(5450) OaI(5359) SmaI(5178)

Fig. 10. pPIC3K

pHIL-D5 9462 bp

SaII(2887)

Notl(6589)

Fig. 11. pHIL-DS Asull(934) EcoRI(944) NdeI(951) SpeI(961) SmaI(969)

BamHI(971)

pHIL-D6 9474 bp

Bgill(6605)

SalI(2907)

XhoI(5438) CJaI(5347)

Fig. 12. pHIL-D6

pHIL-D7 9442 bp SaII(2875)

BglII(6573)

J

Fig. 13. pHIL-D7

AsuII(934)

XhoI(lOO6)

EcoRI(lOll) SmaI(1019) BamHI(I02I)

sequence

ClaI(!360)

NaeI(6693)

pHIL-Sl 8260 bp NdeI(5629) BglII(5393)

AsuII(4896) NaeI(4392)

A

Fig. 14. pHIL-SL Plasmid pHIL-S1 is a secretion vector and other features are similar to pHIL-DL This vector also contains fl origin of replication, similar to that in pHIL-D2. The

junction sequence for making fusions to the acid phosphatase secretion signal sequence is shown

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--/l-S'AOXl---TIATICGAAACG ATG TTC TCT CCA ATT TTG TCC TTG MFS P IL SL

< Sienal peptide c1eavau site EcoR I

S11UZ J

Bam HI

CQAGM'ITCCCCGGQATCCI'TAGA CAT......../I .... REFPG I L ---- Multi Cloning Site---x----3' AO-t----/l---

Fig.l4B

.....•/! ....................•.• .5'AOXl ........................... TTCGAAGGATCCAAACG

ATG AGA TIT CCTTCAATI TIT ACI'GCAGTITIA TIC GCAGCATCCTeC MR FPSIFTAVLFAASS