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` Heterologous Protein Expression in Yeasts
`and Filamentous Fungi
` NINGYAN ZHANG AND ZHIQIANG AN
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`11
`
`INTRODUCTION
` 11.1.
` Development of recombinant DNA technologies in the
`late 1970s laid the foundation for the advancement of
`various heterologous protein expression systems, including
`those based on microbial platforms. In the early 1980s,
` Escherichia coli and Saccharomyces cerevisiae expression
`systems were developed and used for heterologous protein
`expression (47). Many comprehensive reviews of these
`two expression systems are available (12, 29, 41, 48, 60,
`72, 74, 110, 134). Development of other microbial ex-
`pression systems, such as the methylotrophic yeast Pichia
`pastoris (72) and filamentous fungi (74), followed quickly,
`and the broad utility of these systems is becoming increas-
`ingly apparent.
` Heterologous protein expression technologies are widely
`used in both academic research and industrial applications.
`In basic research, heterologous gene expression serves as
`an important tool to prepare proteins and enzymes that are
`either at limited levels or difficult to purify in their native
`context. Heterologously expressed proteins and enzymes are
`used across the entire spectrum of biology. Many enzymes
`and proteins are of commercial importance, and therefore
`improved processes for large-scale production of industrial
`enzymes and proteins are of great interest. Since different
`enzymes and proteins possess different molecular and bio-
`chemical characteristics, different expression systems with
`specific features are required to meet the growing need for
`heterologous protein expression at the level of both basic
`and industrial research.
` With the steady advancement in gene expression tech-
`nology, such as new vectors for gene delivery, improvement
`in throughput of screening for strain selection, and geneti-
`cally engineered hosts, production of recombinant proteins
`has become more efficient with broader applications. In
`addition, introduction of fusion tags such as hexa-histi-
`dine (6× His) and inducible promoters have allowed rapid
`detection and purification of recombinant proteins and
`increased expression titers. Since enzymes and proteins
`have diverse physical and biochemical properties, expres-
`sion yields vary widely from microgram to low-milligram
`(37, 57) to multi-gram levels (62). Choosing a suitable
` expression system for a protein of interest is one of the
`most critical steps for the development of a high- expression
`process. Some advantages and drawbacks for commonly
`used heterologous protein expression systems are listed in
`
`Table 1 (138). E. coli expression systems provide quick and
`easy molecular manipulation and, in general, require low-
`cost growth media. However, the protein expression in E.
`coli cannot be used if foreign proteins require transcriptional
`and/or posttranslational processing and modifications. By
`contrast, fungal protein expression systems including yeast
`and filamentous fungi can provide high expression levels
`and posttranscriptional and posttranslational processes
`and modifications of eukaryotic proteins. Similar to E. coli
`protein expression systems, fungal expression systems need
`low-cost culture media, the technologies needed for scale-
`up of fungal fermentation processes are well developed, and
`the organisms are Generally Regarded As Safe (GRAS) (24,
`127). In contrast, insect and mammalian expression systems
`require special culture conditions and relatively high-cost
`culture media and often have lower expression titers.
` When choosing a heterologous protein expression sys-
`tem, the following general criteria should be considered: (i)
`requirement of translational modification and processing
`of the protein; (ii) authenticity of the expressed proteins;
`(iii) protein expression level and scale-up requirement;
`(iv) localization, e.g., intracellular, membrane-bound, or
`secreted proteins; (v) cofactor requirement; and (vi) cost,
`safety, and other regulatory-related issues. Each protein
`expression system offers a unique set of properties, and
`for successful production of a heterologous protein, it is
`often required to tailor a process specifically based on the
`properties of the protein to be produced and the expression
`system used.
` Many filamentous fungi and yeast species have been
`used for heterologous protein expression (32, 34, 53, 54,
`89, 94, 101, 105, 110, 112, 132, 133). There are numer-
`ous reviews addressing foreign protein expressions in each
`of the fungal protein expression systems (14, 20, 21, 44,
`63, 101, 107, 110). Most reviews are focused on one spe-
`cific system. Furthermore, some address specific aspects
`of the expression system, such as protein secretion in S .
` cerevisia e (121) and glycosylation of proteins in P. pastoris
`(11). Significant progress has been made in recent years
`in the production of therapeutic proteins and antibodies
`using humanized yeast (40). This chapter covers the most
`commonly used fungal expression systems ( S. cerevisiae ,
` P. pastoris , and Aspergillus species), with a focus on vector
`systems, promoter and leader sequences, posttranslational
`modifications, and fermentation scalability.
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`Motif Exhibit 1022, Page 2 of 14
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`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
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`146 ■ FERMENTATION AND CELL CULTURE
`TABLE 1 Pros and cons for some commonly used heterologous protein expression systems
`
`Expression hosts
`
`Pros
`
`Cons
`
`E. coli
`
`S. cerevisiae
`
`P. pastoris
`
`Filamentous fungi
`
`Insect cells
`
`Easy genetic manipulation, rapid growth, simple me-
`dia requirements and low cost to grow, no human
`virus carried
`Low-cost culture and easy to grow, genetic regulation
`well-known, secretion protein, no risk for human
`virus infection
`High-level expression, low cost and easy to grow, ge-
`netic regulation well-known, secretion protein, no
`risk for human virus infection
`High level of expression, good secretion system, some
`level of posttranslational modification, culture me-
`dium is inexpensive
`
`Easy to grow up to 400 liters, easy to manipulate
`genetically for baculovirus, can grow in the same-
`style tanks as mammalian cells
`
`Mammalian cell lines
`
`Authentic protein expression and proper
` posttranslational processing and assembly for
` therapeutic proteins and other mammalian proteins
`of interest, extensively used, reasonable expression
`level, endotoxin is not a concern
`
`Inclusion bodies, no secretion into media, lack of
`posttranslational process
`
`Overglycosylation at N-linked sites, cell difficult to
`break, expression level limited
`
`Royalties can be expensive, potential for
` overglycosylation, cell difficult to break
`
`No good commercial vectors are available, longer
`culture time, spores are concerns for health
`problems, not well documented for production of
`therapeutic proteins
`Relatively low expression, serum-free medium
`is expensive, royalties for the commercially
` available systems can be costly, few contractors
`have current Good Manufacturing Practice
` experience
`Medium is expensive, special tank, require CO2,
`potential risk of human virus in culture, longer
`expression time
`
` S. CEREVISIAE FOR HETEROLOGOUS
` 11.2.
`PROTEIN EXPRESSION
` S. cerevisiae , a unicellular yeast, is one of the most studied
`eukaryotic microorganisms. Its genetics, physiology, bio-
`chemistry, metabolism, and fermentation are well researched
`and a wealth of information is readily available. Thousands
`of genes from S. cerevisiae have been characterized (43) and
`its genome sequence has been determined (33). S. cerevisiae
`has been extensively used as a host for heterologous gene
`expression of eukaryotic proteins for research and industrial
`applications (44, 110).
` S. cerevisiae is a GRAS organism and has been used for
`centuries in the brewing and baking industries. Similar to
` E. coli , S. cerevisiae requires simple culture media for growth
`and is easily scaled up for large-volume fermentations. On
`the other hand, unlike E. coli , S. cerevisiae is capable of
` accomplishing many posttranslational modifications, such
`as proteolytic processing, disulfide bond formation, glyco-
`sylation, and other posttranscriptional and translational
`processing unique to eukaryotic organisms. Many vector
`systems containing a variety of promoters and auxotrophic or
`dominant selectable markers have been developed allowing
`constitutive or regulated gene expression. Several methods
`for transformation of foreign DNA into S. cerevisiae with
`high efficiency have also been developed (31, 85). As a
`result, S. cerevisiae is one of the most widely used microbial
`hosts for heterologous gene expression. However, there are
`limitations to using S. cerevisiae for heterologous gene expres-
`sion; for example, the specific rate of production can vary
`from protein to protein, ranging from as low as 0.03 g/kg/h
`to as high as 4.5 g/kg/h (44). The use of episomal vector sys-
`tems for heterologous protein expression in S. cerevisiae often
`results in plasmid instability during fermentation, which may
`lead to a lower growth rate and reduced overall protein yield.
`Furthermore, since many proteins of therapeutic interest are
`
`secreted glycoproteins, the tendency of S. cerevisiae to hyper-
`glycosylate proteins not only reduces the efficiency of protein
`secretion but also may lead to undesired changes in the im-
`munogenic properties or biological activities of the expressed
`proteins (23, 39, 40, 44, 84).
`
` 11.2.1. Vectors
` S. cerevisiae expression vectors usually are shuttle plasmids
`that contain sequences for propagation and selection in
`both S. cerevisiae and E. coli . Two types of vectors have been
`described based on their mode of replication: episomal and
`integrating vectors (Table 2) (16, 18, 27, 45, 73, 86, 108,
`109, 111, 138). Episomal vectors can be characterized ac-
`cording to their copy numbers, mode of replication, and sta-
`bility. There are three major types of episomal vectors: YRp,
`YCp, and YEp. YRp-type vectors contain an autonomous
`replication sequence from the S. cerevisiae genome and have
`an average copy number of 1 to 10 per cell, even though
`higher copy numbers (up to 100 copies per cell) have been
`reported (44). These vectors are unstable, and plasmid
`loss rates can be as high as 10% per generation (44). This
`instability greatly limits their use in large-scale fermenta-
`tion processes. YCp-type vectors are derived from the in-
`corporation of S. cerevisiae centromeres into YRp plasmids
`and have improved plasmid stability. However, YCp-type
`vectors have lower copy numbers, typically at 1 to 2 copies
`per cell. The most commonly used episomal vectors are of
`the YEp type derived from the naturally occurring plasmid
`called 2µ circle in S. cerevisiae . These vectors are present at
`an average of 40 copies per cell and exhibit higher stabil-
`ity than the YRp and YCp vectors (5, 16). Consequently,
`YEp vectors are the best-developed vectors for heterologous
`gene expression in S. cerevisiae . These vectors are especially
`useful for controlled expression of toxic proteins. More than
`80 compact expression vectors have been developed based
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`Motif Exhibit 1022, Page 3 of 14
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`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
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`TABLE 2 Vector systems used for heterologous protein
` expression in S. cerevisiae
`
`Vector
`
`Copy number
`per cell
`
`Reference
`
`Episomal
`YEp: 2␮-based
`YCp: Centromere
`YRp: Replicating
`Regulated copy number
`Integrating
`YIp
`rDNA-integrating
`Ty␦
`Transplacement
`
`25–200
`1–2
`1–20
`3–100
`
`⬎1
`100–200
`⬍20
`1
`
`27
`18
`86
`16
`
`45
`73
`111
`109
`
`on the pRS series of centromeric and 2µ plasmids (26). The
`GATEWAY vectors (Invitrogen Life Technologies, Carls-
`bad, CA) incorporated some of these features into their sys-
`tems in order to provide fast cloning and transfer of genes to
`different vectors for heterologous expression in most strains
`of S. cerevisiae . Other 2µ-based expression vectors available
`from commercial sources include YEpFLAG-1 from Sigma
`(St. Louis, MO), YES vectors from Invitrogen, and pESC
`vectors from Stratagene (La Jolla, CA).
` Integrative vectors contain selectable S. cerevisiae genes
`and lack sequences for autonomous replication. These
`vectors are highly stable but usually present at low copy
`numbers. They include the YIp-type vectors, and their
`integration can be directed by homologous recombina-
`tion between sequences carried on the plasmid and its
`homologous counterpart in the S. cerevisiae genome. Some
`integration vector systems use repetitive elements such as
`delta sequences, Ty elements, or tRNA genes for heter-
`ologous gene integration (119). A system for multiple-site
`integration of a heterologous gene into the ribosomal DNA
`cluster of 9.1-kb segment has also been developed (73).
`Stability of the genome-integrated expression cassette can
`be maintained under selection. However, a decrease of copy
`numbers of the integrated expression cassette was observed
`under nonselective culture conditions (44).
`
` 11.2.2. Promoters
` An array of S. cerevisiae promoters has been used for
`heterologous gene expression. There are constitutive pro-
`moters derived from genes such as CYC1 (cytochrome C
`oxidase), TEF2 (translation elongation factor), and GPD
`(glyceraldehyde-3-phosphate dehydrogenase), as well as
`regulated promoters such as GAL1 (galactokinase/galactose
`epimerase 1), ADH2 (alcohol dehydrogenase 2), CUP1
`(metallothionein), and PHO5 (acid phosphatase) (6, 10,
`15, 46, 51, 55, 56, 58, 65, 66, 80, 97, 98, 124). It is often
`advantageous for improving expression titer to couple the
`protein production phase with cell growth phases if there is
`no toxicity issue for the protein of interest. Some inducible
`promoter systems that work well at the shake flask scale,
`such as temperature-regulated promoters, do not always
`function well in large-scale fermentation due to operational
`restraints (44). Hybrid promoters such as GAP/GAL and
` GAL10/CYC1 have also been reported (6). Several reviews
`on promoters and their applications in S. cerevisiae are
`available (108, 128, 138).
`
`11. Protein Expression in Yeasts and Fungi ■ 147
` 11.2.3. Transcriptional and Translational Regulation
` In order to fully utilize the transcriptional, translational,
`and posttranslational regulatory machinery and to get effi-
`cient heterologous gene expression in S. cerevisiae , it is very
`important to match the expressed protein with the proper
`expression vector system. There have been many studies
`and extensive reviews on these aspects (22, 44, 82, 123).
`Generally, the use of strong promoters and robust transcrip-
`tional terminators of S. cerevisiae is essential for maximal
`expression since most prokaryotic or higher eukaryotic
`transcription terminators are not active in S. cerevisiae .
`More importantly, mRNA levels are controlled by the rate
`of transcription initiation and the transcript turnover rate
`(mRNA stability). In addition to upstream regulatory ele-
`ments, downstream activation sequences are also required
`for maximum transcription initiation in S. cerevisiae (123).
`Expression levels of some foreign genes with high AT
`content (⬎60%) in S. cerevisiae can be low or absent due
`to incomplete transcript elongation (108). Transcript-de-
`stabilizing sequence elements have been reported in the 5⬘
`nontranslated regions, coding regions, and 3⬘ nontranslated
`regions of different mRNAs (22). Therefore, if the titer is
`low due to the instability of mRNA, it may be necessary to
`use a stronger promoter, to delete the transcript-destabiliz-
`ing sequence elements, and to introduce alternative codons.
`It has been shown that secondary structure of mRNA is one
`of the most important factors affecting the rate of initiation
`and translational elongation (123). For secreted proteins,
`posttranslational processes such as proteolytic cleavage,
`N-terminal modification, and glycosylation are critical
`to both proper protein folding and high protein yields.
`Consequently, it is worthwhile to evaluate the effect of the
`posttranslational steps when strong promoters and different
`strains fail to produce desired product yields.
` S. cerevisiae has been used for expression of many eu-
`karyotic proteins in both intracellular and secreted forms.
`For production of secreted proteins, secretion signals can
`come from either native protein signal sequences or from
` S. cerevisiae secretion protein signal sequences. Due to the
`limited studies available in the literature on the effects of
`different signal peptides on the yield of heterologous pro-
`tein expression in S. cerevisiae (120), it is a good starting
`point to include the signal sequence of the foreign protein
`in the initial test of expression cassettes. Alternatively, an
` S. cerevisiae signal from such genes as invertase (SUC2, 19
`amino acids), acid phosphatase (PHO5, 17 amino acids),
`and the most widely used ␣-factor pheromone (MF␣1, 20
`amino acids) can be incorporated into the recombinant
`construct(s).
` An important step among posttranslational modifica-
`tion processes is glycosylation. Heterologous glycoproteins
`expressed in S. cerevisiae are glycosylated at both N-linked
`and O-linked sites (64). Little is known about O-linked
`glycosylation, but more information is available on the pro-
`cess of N glycosylation. In S. cerevisiae , a core sugar moiety
`consisting of two N -acetylglucosamines (GlcNAc), nine
`mannoses, and three glucoses is added to the N-amide of
`asparagine at the Asn-X-Ser/Thr sequence in the endoplas-
`mic reticulum (ER) in the early process of N glycosylation
`(67). Three glucose residues and one mannose residue are
`subsequently removed in the early Golgi apparatus. It is
`in the Golgi apparatus where further modification takes
`place that results in major differences between S. cerevi-
`siae and higher eukaryotes in oligosaccharide structures.
`In higher eukaryotes, additional mannose residues are
`removed and several other sugars such as galactose, sialic
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`Case No.: IPR2023-00321
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`148 ■ FERMENTATION AND CELL CULTURE
`acid, and fucose are added (39, 40). S. cerevisiae maintains
`the core mannose scaffold and is often extended with a
`large number of additional mannose residues. This often
`results in a hyperglycosylated outer chain containing more
`than 50 mannose residues with many branch chains. Many
`examples of hyperglycosylation of heterologous proteins in
` S. cerevisiae have been reported (23, 68, 84). Since specific
`glycosylation patterns are important for the immune re-
`sponse in mammalian systems, wild-type S. cerevisiae is not
`a good system for the production of therapeutic proteins
`such as monoclonal antibodies that have complex sugar
`structures.
`
`Industrial Protein Production in
` 11.2.4.
` S. cerevisiae
` Many heterologous proteins have been produced in S.
`cerevisiae and large-scale fermentation processes have been
`developed (43, 108). Nevertheless, most heterologous
`protein production in S. cerevisiae remains in shaker flask
`cell cultures. The first therapeutic recombinant protein
`expressed in S. cerevisiae was human alpha interferon (47).
`Since then, many other therapeutic proteins have been
`expressed in S. cerevisiae . These include the hepatitis B sur-
`face antigen (126), human insulin (122), the human papil-
`lomavirus (HPV) vaccine Gardasil™ (93, 135), and many
`others. In the case of Gardasil, the four vaccine components
`were expressed in S. cerevisiae , which were transformed by
`pGAL110-based yeast expression vectors, each containing
`the gene of interest coding for HPV L1 types 6, 11, 16,
`or 18 (75, 104). An excellent review by Vasavada (128)
`details a comprehensive list of foreign proteins expressed
`in S. cerevisiae at small and large industrial scale. Various
`strategies have been reported to improve the expression
`levels of different heterologous proteins in S. cerevisiae , such
`as screening of various host strains with different expression
`vectors, altering host genetics by molecular engineering,
`and optimization of fermentation conditions (83, 115, 128).
`Since there are no defined conditions that can be used for
`all protein production, it is a general rule of thumb to exam-
`ine different parameters to develop an optimized process for
`heterologous protein expression in S. cerevisiae .
`
` P. PASTORIS FOR HETEROLOGOUS
` 11.3.
`PROTEIN EXPRESSION
` Pichia expression systems have been widely used for ex-
`pression of heterologous proteins from a diverse array of
`organisms, including bacteria, fungi, protists, plants, inver-
`tebrates, nonhuman vertebrates, and humans (72). Similar
`to S. cerevisiae , protein expression systems in Pichia have the
`advantages of well-established, cost-effective fermentation
`technology and comprehensive understanding of the host
`molecular genetics. P. pastoris is a methylotrophic yeast
`and has strong preference for respiratory growth (72). Cell
`cultures can reach extremely high density, with dry weight
`mass reaching as high as 100 g/liter (44). Most of the Pichia -
`based protein expression systems use a tightly controlled
`and highly inducible promoter derived from the alcohol
`oxidase 1 gene ( AOX1 ). Under methanol-induced condi-
`tions, a single protein such as alcohol oxidase can reach
`as high as 30% of the total cell proteins (44, 72). Similar
`methods used for the molecular genetic manipulation for
` S. cerevisiae can be applied to P. pastoris . Extensive studies
`have been published on the use of Pichia systems in heterol-
`ogous protein expression. A recent review article by Cregg
`
`et al. (14, 20) listed 241 individual studies of heterologous
` proteins expressed in P. pastoris , more than 80 of which were
`human proteins. Expression levels for heterologous proteins
`differ greatly, ranging from microgram-per-liter to gram-
`per-liter levels. For example, a hydroxynitrile lyase from a
`tropical rubber plant ( Hevea brasiliensis ) was expressed at
`levels as high as 22 g/liter (42). There are numerous reports
`on heterologous protein expression in P. pastoris (9, 91, 94,
`107, 113, 114, 130, 138).
`
` 11.3.1. Expression Vectors and Host Strains
` A fully developed P. pastoris expression system is com-
`mercially available (Invitrogen). The Pichia vectors can
`be grouped into two categories based on their promoters
`used: (i) inducible promoters derived from the AOX1
`gene or the formaldehyde dehydrogenase gene ( FLD1 )
`(117), and (ii) a strong constitutive promoter derived
`from the P. pastoris glyceraldehyde-3-phosphate dehy-
`drogenase gene ( GAP ). The methanol-inducible AOX1
`promoter provides tighter control of gene expression. The
` FLD1 promoter can be induced by either methanol as the
`sole carbon source or certain methylated amines such as
`methylamine as the sole nitrogen source. Alternatively,
`the constitutive GAP promoter does not require cultures
`to be shifted from one carbon source to another for the
`induction of heterologous gene expression. However, if
`the foreign proteins to be expressed are toxic to the host
`cells, constitutive expression of the enzyme or proteins
`may cause inhibition of cell growth or result in loss of the
`expression vector from the cell.
` There are no stable episomal expression vectors available
`in P. pastoris , and the expression genes need to be integrated
`into the genome to obtain stable expression strains. The
`simplest way of integration is single crossover homologous
`recombination by digesting the vector at a unique site with
`a restriction enzyme within either the marker gene (e.g.,
` HIS4 ) or the promoter sequences (e.g., AOX1 ) (117). In
`the case of HIS4 as the selectable marker, transformants of
`auxotrophic mutant strain (his4) can be selected for His +
`cell growth. Alternatively, expression strains can be gen-
`erated with AOX1 flanking sequences at both the 5⬘ and
`3⬘ terminals by cutting the vector in the AOX1 sequence
`region. The flanking AOX1 sequences at both ends can
`stimulate gene replacement events at the AOX1 site in the
` Pichia genome, and this can result in a gene replacement
`integration. Heterologous proteins expressed in P. pastoris
`can be retained inside cells (intracellular expression) or
`secreted into medium depending on signal sequences in the
`expression vector systems. Commercial vectors for secreted
`protein expression often carry a leader sequence from ␣-MF
`(mating factor) of S. cerevisiae as a secretion signal (117).
`Other leader sequences, such as the alkaline phosphatase
`(PHO) signal sequence from P. pastoris and some native
`protein leader sequences, have also been used for secre-
`tion of foreign proteins in Pichia (79). To facilitate protein
`detection and purification, most vectors have 6× His (his-
`tidine) or c-myc tag sequences fused at the C-terminal or
`N-terminal end.
` A variety of selection markers are available for Pichia
`protein expression. Some can be used for selecting multi-
`copy gene integration, such as the drug resistance markers
`zeocin and blasticidin, while others provide auxotrophic
`selection such as his4 − mutant strains. Commercially avail-
`able Pichia host strains for protein expression include X-33,
`GS115, SMD1168, and KM71. Additional host strains for
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`Case No.: IPR2023-00321
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` P. pastoris heterologous protein expression are reviewed by
`Cregg et al. (20).
`
` 11.3.2. Protein Folding and Glycosylation in
` P. pastoris
` P. pastoris , similar to S. cerevisiae and other fungi species,
`can perform protein posttranslational modifications, such
`as cleavage of signal sequences for secreted proteins, N- and
`O-linked glycosylation, and formation of disulfide bonds
`(71, 72). It has been well documented that overexpression
`of heterologous proteins in P. pastoris can have a deleterious
`effect on its secretion pathway. Protein folding in the ER
`and Golgi is often the rate-limiting step for heterologous
`protein expression. Overloading the secretion pathway can
`lead to accumulation of partial or misfolded heterologous
`proteins in cells. Existence of high levels of unfolded or mis-
`folded proteins in cells may induce a stress reaction termed
`the “unfolded protein response” (UPR), which triggers the
`expression of folding chaperones and enzymes in addition
`to other physiological responses. Both heat shock protein
`70-type chaperone (BiP) and a protein disulfide isomerase
`(Pdi1) were reported to be induced when cells face UPR in
` Pichia (44, 72). BiP and Pdi1 showed synergistic effects on
`improving secretory capacity (136).
` Glycosylation is one of the most important posttrans-
`lational modifications for heterologous protein expression,
`since it not only affects proper folding and secretion of the
`protein but also has a direct impact on the biological activi-
`ties of proteins. Both N- and O-linked glycosylation have
`been reported using Pichia systems for heterologous protein
`expression (20). The consensus sequence for N-linked
`glycosylation in Pichia is the same as that in S . cerevisiae
`(Asn-X-Thr/Ser), in which Ser or Thr provides a hydroxyl
`group for glycosylation. Similar to S. cerevisiae and other
`fungal organisms, Pichia transfers a lipid-linked oligosac-
`charide unit, Glc 3 Man 9 GlcNAc 2 , to amino acid (Asn) in
`the ER. This oligosaccharide core is further modified in the
`Golgi. Unlike hyperglycosylation (⬎50 Man) occurring in
` S. cerevisiae , the most commonly found N-glycosylation
`structure in P. pastoris is Man 14 GlcNAc 2 . There has been
`no ␣-1,3-mannosyltransferase activity detected in P. pas-
`toris ; only ␣-1,6 and ␣-1,2 linkages were found in glycosyl-
`ated proteins in Pichia (11). The O-linked oligosaccharides
`in Pichia are also reported, but a consensus sequence for
`O-linked glycosylation is not available. The O-linked oli-
`gosaccharides in P. pastoris are generally short (⬍5 residues)
`and contain only ␣-1,2-linked mannose units. Phosphoryla-
`tion of oligosaccharide units has been found in P. pastoris
`expression systems, but the exact sites of phosphate link-
`age on saccharides are unknown (72). In order to produce
`antibodies for therapeutic use, extensive glycoengineering
`efforts have been carried out to make humanlike glycoforms
`in Pichia (39, 40). Fully humanized sialylated antibody was
`produced successfully in a humanized Pichia strain (39). It
`is noteworthy that unlike antibodies produced in mamma-
`lian cells, antibodies produced in humanized Pichia do not
`contain fucose in the core carbohydrate structures and the
`afucosylated antibodies showed increased binding affinity
`to Fc␥ receptor IIIA, which in turn results in higher anti-
`body-dependent cellular cytotoxicity (70).
`
` 11.3.3. Heterologous Protein Production in Pichia
` Expression levels of heterologous proteins in P. pastoris vary
`greatly depending on copy numbers of the foreign gene
` integrated and the stability of both mRNA and protein.
`
`11. Protein Expression in Yeasts and Fungi ■ 149
`Since expression levels are correlated with gene copy
`number, it is important to achieve high copy numbers of
`gene integration. To obtain multiple copies of the target
`gene integrated in Pichia , one can screen large numbers
`of transformants in a 96-well plate format, applying high
`concentrations of the selection agents (e.g., zeocin and blas-
`ticidin), or construct multiple copies of the gene in tandem
`repeats in vitro.
` In addition to the high-copy-number transformants,
`optimization of the expression process is vital to achieve
`high-level expression of heterologous proteins in Pichia .
`Although some proteins can be expressed well in flask
`cultures, it is common that expression levels are greatly en-
`hanced in a controlled environment. Cultures in fermentor
`vessels offer several advantages over flask cultures, such as
`control of pH and dissolved oxygen levels, and continuous
`feeding of carbon and other components during cell growth
`and protein expression (49, 72). Since P. pastoris cells have
`a preference for respiratory growth, cultures can be grown to
`extremely high cell densities (up to 500 absorbance units at
`600 nm) (20, 21). This is particularly important for secreted
`proteins since the concentration of the expressed proteins is
`often proportional to the concentration of cells in culture.
`Common defined media for P. pastoris protein expression
`are composed of glucose and glycerol as the carbon source
`and casamino acids and/or ammonium salts as the nitro-
`gen source in addition to biotin and trace elements. The
` Pichia fermentation for heterologous protein expression
`can be separated into two phases. The first phase is that of
`growth using glycerol as a carbon source, and at this stage
`the AOX1 promoter is fully repressed. After depletion of
`glycerol, a transitional phase may be added to feed more
`glycerol at a growth-limiting rate. The second phase of
`feeding methanol starts induction of the AOX1 promoter
`and heterologous gene expression. Detailed protocols for
`fermentation of P. pastoris can be found in various articles
`(30, 50, 72, 90, 96).
` Similar to other heterologous protein expression sys-
`tems, proteolytic hydrolysis often causes instability of
`foreign proteins in Pichia . In addition to use of proteinase-
`deficient strains, several fermentation strategies can be
`applied to control proteolysis of foreign proteins. Some
`common approaches that have been used successfully to
`reduce hydrolysis of heterologous proteins in Pichia include:
`(i) adding amino acid-rich supplements such as peptone
`or casamino acids; (ii) culturing at more acidic pH values
`( Pichia cultures grow well between pH 3 and 7); (iii) using
`a protease ( PEP4 , vacuolar protease gene)-deficient strain;
`and (iv) controlling culture temperature (44, 72). Another
`li