Pichia pastoris

CA Batt, Cornell University, Ithaca, NY, USA

Ó 2014 Elsevier Ltd. All rights reserved.
This article is a revision of the previous edition article by Chitkala Kalidas, volume 3, pp 1686–1692, Ó 1999, Elsevier Ltd.

Characteristics of the Species on methanol in continuous cultures. With the increase in the
price of methane due to the oil crisis in the 1970s, however,
Pichia pastoris is a methylotrophic yeast that belongs to the interest in P. pastoris for the production of single-cell proteins
class Ascomycetes. It normally exists in the vegetative waned. In the 1980s, Salk Institute Biotechnology/Industrial
haploid state. Vegetative reproduction is by multilateral Associates (SIBIA), located in La Jolla, California, under
budding. Nitrogen limitation stimulates mating and leads to contract with Phillips Petroleum Company, developed the
the formation of diploid cells. Pichia pastoris is considered to Pichia expression system for the production of foreign
be homothallic because cells of the same strain can mate proteins. In 1993, Phillips Petroleum Company released the
with each other. There may be more than one mating type in Pichia expression system to research laboratories in academic
the population, which switches at a high frequency so that institutions. Since then, P. pastoris has been used widely used
mating occurs between haploid cells of opposite mating as an expression system even in laboratories not routinely
type. Diploid cells maintained in standard vegetative growth working with yeasts. The Pichia expression system is
medium remain diploid. If they are moved to nitrogen- available commercially from Invitrogen (Carlsbad, California,
limited medium, they undergo meiosis and produce United States).
haploid spores.
Physiological regulation of mating in P. pastoris facilitates its
genetic manipulation. Because it is most stable in its vegetative Advantages of Using P. pastoris
haploid state, easy isolation and characterization of mutants
are possible. The use of P. pastoris as a protein expression system has gained
Currently, P. pastoris is one of the main expression systems rapid acceptance in the past decade. This can be attributed to
for the production of heterologous proteins. A number of high protein yields, very high levels of secretion with little or no
bacterial and mammalian proteins have been expressed in secretion of native proteins, easy scale-up, and ease of
P. pastoris (Table 1). handling.
Pichia pastoris can be manipulated genetically using the
same protocols as for Saccharomyces cerevisiae, one of the best-
History studied eukaryotes. It has a strong preference for respiratory
growth, and this trait enables it to be cultured to high cell
Pichia pastoris initially was chosen for the production of single- densities compared with other fermentative yeasts. Apart
cell proteins for feed stock. This was due to its ability to grow from these benefits, P. pastoris also is capable of post-
to very high cell densities in simple media containing meth- translationa modifications of proteins, such as proteolytic
anol. The production of single-cell proteins from P. pastoris processing, glycosylation, and disulfide bridge formation.
was considered a commercially viable option because the Many proteins, which form inactive inclusion bodies in
synthesis of methanol from natural gas (methane) was inex- Escherichia coli, are expressed in their biologically active state
pensive in the late 1960s. Phillips Petroleum Company, in P. pastoris.
Bartlesville, Oklahoma, developed protocols to grow P. pastoris The yields from the Pichia expression system generally are
better than those from higher eukaryotic systems, such as insect
and mammalian cell lines. Expression in excess of 10 g l
1 have
been reported. It is also cost-effective and less time-consuming
Table 1 Heterologous proteins expressed in P. pastoris than the higher eukaryotic systems. One of the challenges in
glycosylation that can be achieved more authentically using
Expression Mode of expression mammalian cell culture is being overcome by the engineering
Protein levels (g l
1) and Mut phenotype of strains whose glycosylation machinery yields a glycosylated
Invertase 2.3 Secreted, Mutþ protein, which is more like human glycosylated patterns than
a-Amylase 2.5 Secreted, MutS other hosts.
Spinach phosphoribulokinase 0.1 Intracellular, MutS The importance of P. pastoris as a recombinant host for the
Human serum albumin 3.4 Secreted, MutS production of heterologous proteins has driven a considerable
Bovine lysozyme C2 0.55 Secreted, Mutþ amount of energy toward the characterization of the genomics
Hepatitis B surface antigen 0.4 Intracellular, MutS and secretome of the organism. The P. pastoris genome is
HIV-1 gp120 1.25 Intracellular, Mutþ 9.43 Mbp with a total of 5313 protein coding genes. Important
Carboxypeptidase B 0.8 Secreted, Mutþ/MutS pathways for the metabolism of methanol and formaldehyde
Bovine b-lactoglobulin 1.5 Secreted, Mutþ
have been identified.
Tumor necrosis factor 10.0 Intracellular, MutS
The secretome of P. pastoris has also been characterized by
Human interferon (IFN)-a2b 0.4 Intracellular, MutS
Anti–A33 single-chain antibody 4.0 Secreted, Mutþ two-dimensional (2D) gel electrophoresis and mass spec-
trometry. A total of 75 different proteins have been identified in

42 Encyclopedia of Food Microbiology, Volume 3 http://dx.doi.org/10.1016/B978-0-12-384730-0.00254-8


the supernatant of methanol grown P. pastoris. The identifica-
tion of these proteins along with their genome sequences
establishes a larger base of signal sequences to select from in
engineering strains for the secretion of heterologous proteins.
The Pichia Expression System
Pichia pastoris produces the enzyme alcohol oxidase that is
required for it to metabolize methanol. The first step in the
metabolism of methanol is the oxidation of methanol to
formaldehyde, resulting in the formation of hydrogen
peroxide. This reaction is catalyzed by alcohol oxidase and
takes place in a specialized membrane-bound organelle called
the peroxisome. Strong proliferation of peroxisomes is seen
during methanol utilization in P. pastoris. Peroxisomes
sequester the toxic hydrogen peroxide from the rest of the cell.
Alcohol oxidase (AOX) has poor affinity for oxygen. Pichia
pastoris compensates for this deficiency by expressing large
amounts of this enzyme. Two genes, AOX1 and AOX2, code for
alcohol oxidase activity. The former accounts for more than
90% of the enzyme activity, while the latter accounts for less
than 5% of the activity. The AOX1 promoter is regulated tightly
and induced by methanol, but it remains repressed under other
conditions, including carbon starvation. This protein consti-
tutes up to 5% of the total soluble protein during methanol
induction in shake flask cultures. It can constitute more than
30% of the total soluble protein during growth on methanol in
the fermenter. Thus, this promoter is especially suited for the
controlled expression of foreign genes. By placing the foreign
gene under the control of the AOX1 promoter, it is possible to
grow the culture on a noninducing carbon source like glycerol
until a suitable cell density is attained and then induced with
methanol.
Other inducible and constitutive promoters have also been
utilized for heterologous gene expression in P. pastoris, such as
the promoter of the glutathione-dependent formaldehyde
dehydrogenase gene (pFLD1), which is independently induc-
ible by methylamine and methanol, and the promoter of
glyceralde-hyde-3-phosphate dehydrogenase gene–constitutive
expression.
Pichia Strains and Plasmids
All strains of P. pastoris are derivatives of the wild-type strain
NRRL-11430 (Northern Regional Research Laboratories,
Peoria, Illinois). Most strains are deficient in the enzyme his-
tidinol dehydrogenase (Table 2). This aids in the selection of
transformants that harbor the expression vector containing the
HIS4 gene. Auxotrophically marked strains are useful in the
selection of diploid strains. Biosynthetic genes, such as arg4-
argininosuccinate lyase and ura3-orotidine 50 -phosphate
decarboxylase, are some of the other commonly used auxo-
trophic markers in the Pichia system.
Three types of host strains are categorized according to their
ability to utilize methanol, resulting from mutations in one or
both AOX genes. The most commonly used strain is GS115.
This strain has both the AOX1 and AOX2 genes and grows on
methanol at wild-type rate. This phenotype is termed Mutþ
Pichia pastoris 43
Table 2 Pichia pastoris expression host strains
Strain name Genotype Phenotype
Y-11430 Wild-type NRRLa
GS115 his4 Mutþ His

KM71 aox1D::SARG4 his4 arg4 MutS His

MC 100–3 aox1D::SARG4 his4 arg4 Mut
His

aox2D::Phis4 his4 arg4
SMD1168 pep4Dhis4 Mutþ His
, protease-deficient
SMD1165 prb1 his4 MutþHis
, protease-deficient
SMD1163 pep4 prb1 his4 MutþHis
, protease-deficient
a
Northern Regional Research Laboratories, Peoria, Illinois, United States.
Reproduced with permission from Higgins and Cregg, 1998.
(methanol utilization). KM71 is a strain in which the chro-
mosomal AOX1 gene is replaced with the S. cerevisiae ARG4
gene. Therefore, this strain has lower alcohol oxidase activity
from AOX2. It grows very slowly on methanol and this
phenotype is termed MutS (methanol utilization slow). The
strain MC 100–3 has a Mut
phenotype as it has deletions at
both the AOX1 and AOX2 loci. This strain is unable to grow on
methanol, but the AOX1 promoter is inducible by methanol.
Mut
strains, and therefore, it requires an alternative carbon
source such as glycerol for growth. Excess glycerol, however, has
a negative effect on expression and has to be fed at growth-
limiting rates.
For the large-scale production of secreted proteins, Mutþ
strains often are used because they grow much faster on
methanol compared with the AOX-defective strains. MutS and
Mut
strains are more tolerant, however, to residual methanol
in the fermenter than the Mutþ strains. For this reason, the
AOX-defective strains sometimes are preferred over Mutþ
strains for the production of secreted proteins. For intracellular
expression, the AOX-defective strains are preferred because low
levels of alcohol oxidase expression increase the specific yield
of the heterologous protein.
Some secreted foreign proteins are unstable in the P. pastoris
culture medium due to the action of endogenous proteases.
The strain SMD1168 is similar to GS115 except that it lacks
proteinase A activity. Other protease-deficient strains are
SMD1163 and SMD1165. These strains are used in cases in
which proteolytic cleavage results in low yields of expressed
proteins.
The schematic of a typical Pichia expression vector is shown
in Figure 1. The vector, pPIC9, consists of the AOX1 promoter
fragment, the AOX1 transcription terminator region and the 30
AOX1 region. The AOX1 promoter is followed by the
S. cerevisiae a-mating factor (a-MF) signal sequences and
a multiple cloning site. This plasmid also carries the histidinol
dehydrogenase gene (HIS4), which is used to select for
recombinant P. pastoris clones. The ColE1 sequence and the
gene for ampicillin resistance on the plasmid are useful for
subcloning into E. coli.
Construction of Recombinant Strains
To obtain stable recombinant strains of P. pastoris, the expres-
sion vectors are integrated into the host genome. Linear DNA
44 Pichia pastoris
Figure 1 Pichia expression vector. Reproduced with permission from
Invitrogen Corporation, 1996.
can generate stable transformants because of homologous
recombination between the plasmid DNA and homologous
regions within the genome. Selection of transformants is based
on histidine prototrophy.
Integration of the expression vector can occur in three ways.
This generates transformants with different methanol utiliza-
tion phenotypes (Mutþ, MutS), depending on the host strain
used (Figure 2). It is possible to stimulate either single or
double crossover events by linearizing the plasmid. If the
plasmid is cut at one of the restriction sites within the 50 AOX
sequences, it will stimulate single crossovers, leading to the
insertion of the expression cassette at either the AOX1 locus as
in GS115 or aox1::ARG4 locus as in KM71. The phenotype of
such transformants would be HisþMutþ if the host strain is
GS115 and HisþMutS if the host strain is KM71.
Insertion of the plasmid also can occur at the his4 locus on
the host genome. This results from a single crossover event
between the his4 on the chromosome and the HIS4 on the
plasmid. Because the genomic AOX1 locus is not involved, the
Mut phenotype of the Hisþ transformant would be the same as
the host strain used.
Double crossover events can be generated in the strain
GS115 by cutting the plasmid at the BglII site. This leads to the
formation of a fragment with the AOX1 sequences at its termini
and the gene of interest and HIS4 in between. This would
stimulate gene replacement events at AOX1. The resulting
strains would lack AOX1 and would have to depend on the
weak AOX2 gene for methanol utilization. The phenotype of
such a transformant would be HisþMutS.
Multiple gene insertion events can occur at the his4 or the
AOX1 loci, leading to the generation of multicopy recombinant
strains. Such events have been found to occur spontaneously at
a low frequency of 1–10% of all selected Hisþ transformants.
The Mut phenotype of such strains would be the same as the
host strain used.
Even though high yields have been obtained from single-
copy recombinants, yields from multicopy strains often have
been found to be significantly higher. As a result, methods of
generating multicopy strains have been developed. These are
based on three different approaches. The first approach
involves identification of multicopy strains that occur naturally
within the population of transformants. A large number of
transformants can be screened for protein expression using
SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis). Multicopy transformants can be identified based
on their protein yields, which typically are higher than those
from single-copy transformants. Alternatively, immunoblot-
ting or colony hybridization can be carried out to detect
multiple copies of the heterologous gene.
The second approach is to detect multicopy strains within
a population based on their level of antibiotic resistance. For
this, plasmids carrying the Tn903kanR gene are used. This gene
confers resistance to G418. The level of antibiotic resistance
depends on the number of copies of this gene. Multicopy
strains will be resistant to higher concentrations of G418 and
thereby can be identified.
The third approach involves transformation of the host cells
with a vector carrying multiple copies of the expression cassette.
By this method, a single gene insertion event would be suffi-
cient to generate a multicopy strain.
Protein Expression
Once stable recombinant strains are obtained, test tube cultures
are used to screen for protein expression. To maximize protein
yields, the cells are grown under noninducing conditions until
the culture reaches the log phase (OD600 ¼ 2–6). Then the
culture is induced with methanol and the regiment depends on
the type of methanol utilizer. For the most common Mutþ
strains, methanol is added every 24 h until maximum produc-
tion is obtained, which varies from 2 days to more than 10 days.
One of the advantages in using P. pastoris is the ease with
which small cultures can be scaled up to larger volumes
without any decrease in yield. Therefore, when optimum
conditions for expression are determined, the culture volume
can be scaled up using large shake flasks or by fermentation.
Protein yields are typically higher in fermenter cultures than
in shake flasks. One of the reasons for this is that high cell
densities (>300 g l
1 dry cell wt) can be reached in fermenter
cultures. The level of product yield is directly proportional to
the cell density, especially in the case of secreted proteins. The
other reason for higher yields in the fermenter cultures is that
optimum levels of oxygen and methanol can be maintained.
Both excess oxygen and methanol have a negative effect on
protein expression.
A number of fedbatch and continuous culture schemes have
been developed for the high-cell-density fermentation of
expression strains. The process of fermentation using P. pastoris
can be divided into three stages. The first is the continuous
phase, using glycerol as the carbon source, which lasts for about
24 h. The second stage is the glycerol fedbatch phase. In this

Figure 2 Integration of expression vectors into the Pichia pastoris genome. (a) Single crossover integration at the his4 locus. (b) Integration of vector
fragment by replacement of AOX1 gene. Reproduced with permission from Higgins and Cregg (1998).
stage, glycerol is fed at a growth-limiting rate. During growth
on glycerol, the cells multiply rapidly but remain strongly
repressed. The third is the methanol fedbatch phase. The
optimum period of induction and the time of harvest have to
be determined empirically for each protein.
Posttranslational Modification of Expressed Proteins
Signal Sequence Processing
One of the main attractions of the Pichia expression system is
its ability to secrete heterologous proteins at high levels with
little or no secretion of native proteins. As a result of this,
downstream processing of secreted proteins becomes much
easier.
A signal sequence is required for the proper secretion of the
foreign protein. The native signal sequence of the foreign
protein has been used successfully in some cases, for example,
bovine lysozyme. In some cases, however, native signal
sequences have not worked well, for example, invertase. Yeast
signal sequences have been used as other alternatives. The most
commonly used yeast signal sequence is the prepro a-MF
sequence from S. cerevisiae. In S. cerevisiae, three enzymes are
Pichia pastoris 45
required for the proper processing of the a-MF signal sequence.
They are a signal peptidase that cleaves the pre region, KEX2
gene product that cleaves at the junction between a-MF prepro
region and the foreign protein, and the product of STE13 that
removes Glu–Ala spacer residues from the amino terminal of
the foreign protein. A number of foreign proteins have been
expressed in P. pastoris at high levels and in the fully processed
state using the a-MF. This indicates that the enzymes for pro-
cessing the signal sequence are present in sufficient quantities
in the P. pastoris secretory system as well. Incomplete processing
of the signal sequence also has been observed in some cases.
Conformational characteristics of the expressed protein could
prevent access to the signal sequence processing enzymes.
Glycosylation
Glycosylation is another posttranslational modification carried
out by P. pastoris. Both O- and N-linked glycosylation has been
observed in proteins expressed in this yeast. N-linked glyco-
sylation in P. pastoris is significantly different than in higher
eukaryotes. Oligosaccharides from P. pastoris lack the N-ace-
tylgalactosamine, galactose, and sialic acid residues found in
mammals. Carbohydrate side chains in mammals are
46 Pichia pastoris
composed of a mixture of different sugars (complex type) or of
Man5–6GlcNAc2 (high mannose type) or both. In Pichia, the
carbohydrate side chains usually are of the high mannose type.
Two distinct patterns of glycosylation, however, are seen with
regard to the number of mannose residues added. In some
cases, 8–15 mannose residues are added, for example, invertase
from S. cerevisiae. In other cases, hyperglycosylation has been
observed.
Core structures of P. pastoris oligosaccharide molecules have
been determined to be identical to those of S. cerevisiae. The
structure of the oligosaccharide side chains from P. pastoris
secreted invertase has been determined. The major species
found were Man8–11GlcNAc2 and all except Man11GlcNAc2
were identical to S. cerevisiae core structures. The terminal
mannose residues in P. pastoris were of the a-1,2 type, as
opposed to the more common a-1,3 type in S. cerevisiae. Apart
from the absence of a-1,3-linked mannose, little information is
available about the structure of outer chain oligosaccharides.
The mechanism underlying the addition of outer chains is also
not well understood.
N-linked glycosylation in P. pastoris poses a problem with
regard to the use of expressed proteins for therapeutic appli-
cations. The high mannose oligosaccharide could be highly
antigenic and could preclude therapeutic use. The other
problem, due to differences in glycosylation pattern between
P. pastoris and mammals, is that the long outer chains could
interfere with the proper folding of proteins. Remedies for
these challenges have been realized by the engineering of
strains that mimic human-like glycosylation of heterologous
proteins.
Importance to the Food Industry
Pichia pastoris initially was used to produce single-cell
proteins, several unusual enzymes (e.g., alcohol oxidase,
formate dehydrogenase), and metabolites such as adenosine
triphosphate, aldehydes, and amino acids. Since then,
a number of food proteins and enzymes have been expressed
in P. pastoris (Table 1). Recombinant amylases and sugar-
converting enzymes from different sources have been
expressed in P. pastoris. Examples of this class of proteins are
a-amylases 1 and 2 from barley. These enzymes from
P. pastoris were found to be similar in structure and function to
those from malt extracts. b-Lactoglobulin, the major whey
protein in bovine milk, has been expressed in P. pastoris at
the level of >1 g l
1. The physical characteristics of this
recombinant protein were found to be indistinguishable from
the native bovine form.
Pichia pastoris has also been used as a biocatalyst in the
conversion of glycolate and its derivatives to glyoxalate and
other corresponding 2-oxo-acids. This reaction produces
hydrogen peroxide that has to be metabolized for the efficient
conversion of the substrate. Recombinant strains of P. pastoris
carrying the glycolate oxidase gene from spinach and the
endogenous catalase have been used as catalysts for this
process.
Conclusion
Pichia pastoris has been used successfully for the production of
a number of heterologous proteins, including those scaled to
commercially viable processes.
See also: Saccharomyces: Saccharomyces cerevisiae;
Single-Cell Protein: Yeasts and Bacteria.
Further Reading
De Schutter, K., Lin, Y.-C., Tiels, P., Van Hecke, A., Glinka, S., Weber-Lehmann, J.,
Rouze, P., Van de Peer, Y., Callewaert, N., 2009. Genome sequence of the
recombinant protein production host Pichia pastoris. Nature Biotechnology 27,
561–566.
Hollenberg, C.P., Gellissen, G., 1997. Production of recombinant proteins by meth-
ylotrophic yeasts. Current Opinion in Biotechnology 8, 554–560.
Invitrogen Corporation, 1996. Invitrogen Corporation Pichia Expression Kit. Instruction
Manual. Carlsbad, CA.
Kim, T., Goto, Y., Hirota, N., Kuwata, K., Denton, H., Wu, S., Sawyer, L., Batt, C.A.,
1997. High level expression of bovine b-lactoglobulin in Pichia pastoris and
characterization of its physical properties. Protein Engineering 10 (11),
1339–1345.
Kurtzman, C.P., 2005. Description of Komagataella phaffii sp. nov. and the transfer of
Pichia pseudopastoris to the methylotrophic yeast genus Komagataella. Interna-
tional Journal of Systematic and Evolutionary Microbiology 55, 973–976.
Lin Cereghino, G.P., Cereghino, J.L., Ilgen, C., Cregg, J.M., 2002. Production of
recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Current
Opinion in Biotechnology 13, 329–332.
Potvin, G., Ahmad, A., Zhang, Z., 2012. Bioprocess engineering aspects of heterol-
ogous protein production in Pichia pastoris: A Review. Biochemical Engineering
Journal 64, 91–105.
Romanos, M., 1995. Advances in the use of Pichia pastoris for high-level gene
expression. Current Opinion in Biotechnology 6, 527–533.
Vogl, T., Hartner, F.S., Glieder, A., 2013. New opportunities by synthetic biology for
biopharmaceutical production in Pichia pastoris. Current Opinion in Biotechnology
24, 1–8.