Appl Microbiol Biotechnol (2014) 98:5301–5317
`DOI 10.1007/s00253-014-5732-5
`
`MINI-REVIEW
`
`Protein expression in Pichia pastoris: recent achievements
`and perspectives for heterologous protein production
`
`Mudassar Ahmad & Melanie Hirz & Harald Pichler &
`Helmut Schwab
`
`Received: 27 January 2014 / Revised: 25 March 2014 / Accepted: 26 March 2014 / Published online: 18 April 2014
`# The Author(s) 2014. This article is published with open access at Springerlink.com
`
`Abstract Pichia pastoris is an established protein expression
`host mainly applied for the production of biopharmaceuticals
`and industrial enzymes. This methylotrophic yeast is a distin-
`guished production system for its growth to very high cell
`densities, for the available strong and tightly regulated pro-
`moters, and for the options to produce gram amounts of
`recombinant protein per litre of culture both intracellularly
`and in secretory fashion. However, not every protein of inter-
`est is produced in or secreted by P. pastoris to such high titres.
`Frequently, protein yields are clearly lower, particularly if
`complex proteins are expressed that are hetero-oligomers,
`membrane-attached or prone to proteolytic degradation. The
`last few years have been particularly fruitful because of nu-
`merous activities in improving the expression of such com-
`plex proteins with a focus on either protein engineering or on
`engineering the protein expression host P. pastoris. This re-
`view refers to established tools in protein expression in
`P. pastoris and highlights novel developments in the areas of
`expression vector design, host strain engineering and screen-
`ing for high-level expression strains. Breakthroughs in mem-
`brane protein expression are discussed alongside numerous
`commercial applications of P. pastoris derived proteins.
`
`Keywords Yeast . Pichia pastoris . Protein expression .
`Protein secretion . Protease-deficient strains . Chaperone
`
`Mudassar Ahmad and Melanie Hirz contributed equally to this work.
`M. Ahmad : M. Hirz : H. Pichler : H. Schwab (*)
`Institute of Molecular Biotechnology, Graz University of
`Technology, Petersgasse 14/5, 8010 Graz, Austria
`e-mail: helmut.schwab@tugraz.at
`H. Pichler : H. Schwab
`Austrian Centre of Industrial Biotechnology (ACIB), Petersgasse 14,
`8010 Graz, Austria
`
`Introduction
`
`The methylotrophic yeast Pichia pastoris, currently
`reclassified as Komagataella pastoris, has become a substan-
`tial workhorse for biotechnology, especially for heterologous
`protein production (Kurtzman 2009). It was introduced more
`than 40 years ago by Phillips Petroleum for commercial
`production of single cell protein (SCP) as animal feed additive
`based on a high cell density fermentation process utilizing
`methanol as carbon source. However, the oil crisis in 1973
`increased the price for methanol drastically and made SCP
`production uneconomical. In the 1980s, P. pastoris was devel-
`oped as a heterologous protein expression system using the
`strong and tightly regulated AOX1 promoter (Cregg et al.
`1985). In combination with the already developed fermenta-
`tion process for SCP production, the AOX1 promoter provided
`exceptionally high levels of heterologous proteins. One of the
`first large-scale industrial production processes established in
`the 1990s was the production of the plant-derived enzyme
`hydroxynitrile lyase at >20 g of recombinant protein per litre
`of culture volume (Hasslacher et al. 1997). This enzyme is
`used as biocatalyst for the production of enantiopure m-
`phenoxybenzaldehyde cyanohydrin — a building block of
`synthetic pyrethroids — on the multi-ton scale.
`Through a far-sighted decision this expression system,
`initially patented by Phillips Petroleum, was made available
`to the scientific community for research purposes. A major
`breakthrough was the publication of detailed genome se-
`quences of the original SCP production strain CBS7435
`(Küberl et al. 2011), the first host strain developed for heter-
`ologous protein expression GS115 (De Schutter et al. 2009),
`as well as of the related P. pastoris DSMZ 70382 strain
`(Mattanovich et al. 2009b). Equally important breakthroughs
`for the commercial application of the P. pastoris cell factory
`were the Food and Drug Administration (FDA) GRAS (gen-
`erally recognized as safe) status for a protein used in animal
`
`~ Springer
`
`Motif Exhibit 1029, Page 1 of 17
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
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`Appl Microbiol Biotechnol (2014) 98:5301–5317
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`feed, phospholipase C (Ciofalo et al. 2006), and the FDA
`approval of a recombinant biopharmaceutical product,
`Kalbitor®, a kallikrein inhibitor (Thompson 2010).
`The classical P. pastoris expression system has been exten-
`sively reviewed over the years (Cereghino and Cregg 2000;
`Daly and Hearn 2005; Gasser et al. 2013; Jin et al. 2006;
`Macauley-Patrick et al. 2005). In this review, we focus on
`recent developments for heterologous protein production and
`describe examples for the commercial use of this expression
`system. In the first chapter, we refer to the established basic
`vector systems and elaborate on developments thereof with an
`emphasis on newly developed promoter systems. Herein, also
`some aspects of secretion will be summarized. The second
`part is devoted to the most recent developments regarding host
`strain development. As a specific novelty, a new platform
`based on the CBS7435 strain is described, for which patent
`protection has ceased and no specific material rights are
`pending. In the third chapter, we describe specific strategies
`for obtaining high-level expression strains and summarize
`important applications of P. pastoris for production of
`biopharmaceuticals, membrane proteins and industrial pro-
`teins. The last section provides an outlook on future perspec-
`tives covering recent progress in molecular and cell biology of
`P. pastoris and possibilities for implementing new strategies in
`expression strain development.
`
`Basic systems for cloning and expression in P. pastoris
`
`When devising strategies for cloning and expression of heter-
`ologous proteins in P. pastoris some points need to be consid-
`ered from the start, that is, the choice of promoter–terminator
`combinations, suitable selection markers and application of
`vector systems for either intracellular or secreted expression
`including selection of proper secretion signals (Fig. 1). The
`choice of the proper expression vector and complementary
`host strain are a most important prerequisite for successful
`recombinant protein expression.
`
`Promoters
`
`The use of tightly regulated promoters such as the alcohol
`oxidase (AOX1) promoter holds advantages for overexpres-
`sion of proteins. By uncoupling the growth from the produc-
`tion phase, biomass is accumulated prior to protein expres-
`sion. Therefore, cells are not stressed by the accumulation of
`recombinant protein during growth phase, and even the pro-
`duction of proteins that are toxic to P. pastoris is possible.
`Furthermore, it may be desirable to co-express helper proteins
`like chaperones at defined time points, for example, before the
`actual target protein is formed. On the other hand, use of
`constitutive promoters may ease process handling.
`Constitutive promoters are usually also applied to express
`
`~ Springer
`
`selection markers. Metabolic pathway engineering strategies
`might further take advantage of fine-tuned constitutive pro-
`moters to ensure a controlled flux of metabolites. An extensive
`summary of promoters used for heterologous expression in
`P. pastoris has recently been published by Vogl and Glieder
`(2013). An overview of broadly used and extensively studied
`as well as recently examined promoters is given in Table 1.
`
`Inducible promoters
`
`The tightly regulated AOX1 promoter (PAOX1), which was first
`employed for heterologous gene expression by Tschopp et al.
`(1987a), is still the most commonly used promoter (Lünsdorf
`et al. 2011; Sigoillot et al. 2012; Yu et al. 2013). PAOX1 is
`strongly repressed when P. pastoris is grown on glucose,
`glycerol or ethanol (Inan and Meagher 2001). Upon depletion
`of these carbon sources, the promoter is de-repressed, but is
`fully induced only upon addition of methanol. Several studies
`have identified multiple regulatory elements in the PAOX1
`sequence (Hartner et al. 2008; Kranthi et al. 2006, 2009; Ohi
`et al. 1994; Parua et al. 2012; Staley et al. 2012; Xuan et al.
`2009). Positively and negatively acting elements have been
`described (Kumar and Rangarajan 2012; Lin-Cereghino et al.
`2006; Polupanov et al. 2012), but the molecular details of
`PAOX1 regulation are still not completely elucidated.
`Methanol is a highly flammable and hazardous substance
`and, therefore, undesirable for large-scale fermentations.
`Alternative inducible promoters or PAOX1 variants, which can
`be induced without methanol but still reach high expression
`levels, are desired. A recently published patent application
`describes such a method, wherein expression is controlled
`by methanol-inducible promoters, such as AOX1, methanol
`oxidase (MOX) or formate dehydrogenase (FMDH), without
`the addition of methanol (Takagi et al. 2008). This was
`achieved by constitutively co-expressing the positively acting
`transcription factor Prm1p from either of the GAP, TEF or
`PGK promoters. The relative activity of a phytase reporter
`protein was 3-fold increased without addition of methanol as
`compared to a control strain with PRM1 under its native
`promoter. However, phytase expression levels were not com-
`pared for standard methanol induction and constitutive Prm1p
`expression conditions. Hartner et al. have constructed a syn-
`thetic AOX1 promoter library by deleting or duplicating tran-
`scription factor binding sites for fine-tuned expression in
`P. pastoris (Hartner et al. 2008). Using EGFP as reporter,
`some promoter variants were found to confer even higher
`expression levels than the native PAOX1 spanning a range
`between 6 % and 160 % of the native promoter activity.
`These PAOX1 variants have also proven to behave similarly
`when industrially relevant enzymes such as horseradish per-
`oxidase and hydroxynitrile lyases were expressed.
`Numerous further controllable promoters are currently be-
`ing investigated for their ability to promote high-level
`
`Motif Exhibit 1029, Page 2 of 17
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`

`

`Appl Microbiol Biotechnol (2014) 98:5301–5317
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`5303
`
`Selection of host strain:
`• wild types
`• protease-deficient strains
`• auxotrophic strains
`
`Protein secretion
`
`• glyco-engineered strains --
`
`/
`
`/
`
`........
`/
`~
`Expression cassette
`!s-HRI P 100, j nl--J3'HR! \
`Genomic integration:
`• single-/multicopy
`• homologous recombination
`• ectopic integration
`
`Promoter:
`• constitutive
`• inducible
`Selectable marker:
`• drug resistance
`• auxotrophy
`
`Intracellular protein expression
`
`~ %& ~
`
`0
`
`0
`
`Secretory pathway:
`• secretion signals:
`S.c. a-MF, Pp. PH01
`• proteolytic processing, e.g.:
`Kex2p, Ste13p
`• glycosylation :
`N- or O-linked
`• membrane-associated proteins
`• surface display anchors
`
`Fig. 1 General considerations for heterologous gene expression in
`P. pastoris. Expression plasmids harbouring the gene(s) of interest
`(GOI) are linearized prior to transformation. Selectable markers (e.g.,
`AmpR) and origin of replication (Ori) are required for plasmid propaga-
`tion in E. coli. The expression level of the protein of interest may depend
`on (i) the chromosomal integration locus, which is targeted by the 5′ and
`
`3′ homologous regions (5′HR and 3′HR), and (ii) on the gene copy
`number. A representative promoter (P) and transcription terminator (TT)
`pair are shown. Proper signal sequences will guide recombinant protein
`for intracellular or secretory expression, and will govern membrane
`integration or membrane anchoring
`
`expression (Table 1). For example, a recently published patent
`application describes the use of three novel inducible pro-
`moters from P. pastoris, ADH1 (alcohol dehydrogenase),
`GUT1 (glycerol kinase) and ENO1 (enolase), showing
`
`interesting regulatory features (Cregg and Tolstorukov
`2012). However, due to a lack of absolute expression values
`the performance of these novel promoters cannot be compared
`to the widely used AOX1 and GAP promoters.
`
`Table 1 The most prominently used and very recently established promoters for heterologous expression in P. pastoris
`
`Inducible
`AOX1
`DAS
`FLD1
`ICL1
`
`PHO89
`THI11
`ADH1
`
`ENO1
`
`GUT1
`
`Corresponding gene
`Alcohol oxidase 1
`Dihydroxyacetone synthase
`Formaldehyde dehydrogenase 1
`Isocitrate lyase
`
`Putative Na+/phosphate symporter
`Thiamine biosynthesis gene
`Alcohol dehydrogenase
`
`Enolase
`
`Glycerol kinase
`
`Regulation
`Inducible with MeOH
`Inducible with MeOH
`Inducible with MeOH or methylamine
`Repressed by glucose, induction in absence
`of glucose/by addition of ethanol
`Induction upon phosphate starvation
`Repressed by thiamin
`Repressed on glucose and methanol, induced
`on glycerol and ethanol
`Repressed on glucose, methanol and ethanol,
`induced on glycerol
`Repressed on methanol, induced on glucose,
`glycerol and ethanol
`
`Reference
`(Tschopp et al. 1987a)
`(Ellis et al. 1985; Tschopp et al. 1987a)
`(Shen et al. 1998)
`(Menendez et al. 2003)
`
`(Ahn et al. 2009)
`(Stadlmayr et al. 2010)
`(Cregg and Tolstorukov 2012)
`
`(Cregg and Tolstorukov 2012)
`
`(Cregg and Tolstorukov 2012)
`
`Constitutive
`GAP
`
`Corresponding gene
`Glyceraldehyde-3-P dehydrogenase
`
`TEF1
`PGK1
`
`GCW14
`
`G1
`
`G6
`
`Translation elongation factor 1
`3-Phosphoglycerate kinase
`
`Potential glycosyl phosphatidyl
`inositol (GPI)-anchored protein
`High affinity glucose transporter
`
`Putative aldehyde dehydrogenase
`
`Regulation
`Constitutive expression on glucose, to a lesser
`extent on glycerol and methanol
`Constitutive expression on glycerol and glucose
`Constitutive expression on glucose, to a lesser
`extent on glycerol and methanol
`Constitutive expression on glycerol, glucose
`and methanol
`Repressed on glycerol, induced upon glucose
`limitation
`Repressed on glycerol, induced upon glucose
`limitation
`
`Reference
`(Waterham et al. 1997)
`
`(Ahn et al. 2007)
`(de Almeida et al. 2005)
`
`(Liang et al. 2013b)
`
`(Prielhofer et al. 2013)
`
`(Prielhofer et al. 2013)
`
`~ Springer
`
`Motif Exhibit 1029, Page 3 of 17
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`

`

`5304
`
`Constitutive promoters
`
`Constitutive expression eases process handling, omits the use
`of potentially hazardous inducers and provides continuous
`transcription of the gene of interest. For this purpose, the
`glyceraldehyde-3-phosphate promoter (PGAP) is commonly
`used, which — on glucose — reaches almost the same ex-
`pression levels as methanol-induced PAOX1 (Waterham et al.
`1997). Expression levels from PGAP drop to about one half on
`glycerol and to one third when cells are grown on methanol
`(Cereghino and Cregg 2000). Alternative constitutive pro-
`moters and promoter variants have been described recently
`(Table 1). The constitutive PGCW14 promoter, for example,
`was described to be a stronger promoter than the GAP and
`TEF1 promoters, which was assessed by secretory expression
`of EGFP (Liang et al. 2013b). It was found that EGFP expres-
`sion from PGCW14 yielded in a 10-fold increase compared to
`PGAP driven expression when cells were cultivated on glycerol
`or methanol, and a 5-fold increase on glucose.
`A recent DNA microarray study identified novel promoters
`that are repressed on glycerol, but are being induced upon shift
`to glucose-limited media (Prielhofer et al. 2013). Supposedly,
`the most interesting promoters discovered by this approach
`control expression of a high-affinity glucose transporter,
`HGT1, and of a putative aldehyde dehydrogenase. The former
`promoter was reported to drive EGFP expression to even
`higher levels than could be reached with PGAP. In glycerol
`fed-batch fermenter cultures, human serum album was
`expressed from the novel promoter to a 230 % increase in
`specific product yield as compared to PGAP driven expression.
`In some cases, it is desired that expression levels can be
`fine-tuned in order to (1) co-express accessory proteins facil-
`itating recombinant protein expression and secretion or (2)
`provide protein post-translational modifications as well as to
`(3) engineer whole metabolic pathways consisting of a cas-
`cade of different enzymatic steps. For such applications, a
`library of GAP promoter variants with relative strengths rang-
`ing from 0.6 % to 16.9-fold of the wild type promoter activity
`was developed and tested using three different reporter pro-
`teins, yEGFP, β-galactosidase and methionine acetyltransfer-
`ase (Qin et al. 2011).
`
`Vectors
`
`The standard setup of vectors is a bi-functional system en-
`abling replication in E. coli and maintenance in P. pastoris
`using as selection markers either auxotrophy markers (e.g.,
`HIS4, MET2, ADE1, ARG4, URA3, URA5, GUT1) or genes
`conferring resistance to drugs such as Zeocin™, geneticin
`(G418) and blasticidin S. Although there are some reports of
`using episomal plasmids for heterologous protein expression
`or for the screening of mutant libraries in P. pastoris (Lee et al.
`2005; Uchima and Arioka 2012), stable integration into the
`
`~ Springer
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`Appl Microbiol Biotechnol (2014) 98:5301–5317
`
`host genome is the most preferred method. Unlike in
`Saccharomyces cerevisiae, where homologous recombination
`(HR) predominates, non-homologous end-joining (NHEJ) is a
`frequent process in P. pastoris. The ratio of NHEJ and HR can
`be shifted towards HR by elongating the length of the homol-
`ogous regions flanking the actual expression cassettes and by
`suppressing NHEJ efficiency (Näätsaari et al. 2012).
`The standard vector systems for intracellular and secretory
`expression provided by Life Technologies (Carlsbad, CA,
`USA) include constitutive (PGAP) and inducible promoters
`triggered by methanol or methylamine (PAOX1, PFLD). The
`recently introduced PichiaPink™ expression kit for intracel-
`lular or secreted expression enables easy selection of
`multicopy integration clones by differences in colour forma-
`tion based on ade2 knockout strains and truncated ADE2
`promoters of varying strengths in front of the ADE2 marker
`gene (Du et al. 2012; Nett 2010).
`Additionally, BioGrammatics (Carlsbad, CA, USA) holds
`licences for selling standard P. pastoris expression vectors and
`strains and also provides GlycoSwitch® vectors for human-
`ized glycosylation of target proteins (Table 2). Several vectors
`for disruption of OCH1 and expression of different glycosi-
`dases or glycosyltransferases are available to achieve
`mammalian-type N-glycan structures in P. pastoris. These
`vectors harbour, for example, the human GlcNAc transferase
`I, the mannosidase II from rat, or the human galactosyl trans-
`ferase I. A detailed protocol for humanizing the glycosylation
`pattern using the GlycoSwitch® vectors is provided (Jacobs
`et al. 2009).
`James Cregg’s laboratory at the Keck Graduate Institute,
`Claremont, CA, USA, has developed a set of plasmids for
`protein secretion and intracellular expression in P. pastoris
`containing the strong AOX1 promoter. These vectors are based
`on different auxotrophy markers, such as ARG4, ADE1, URA3
`and HIS4, for selection necessitating the use of the appropriate
`host strains (see section “Host strain development”). The
`vectors contain restriction sites for linearization within the
`marker genes to target the expression cassettes to the desired
`locus as well as for multicopy integration (Lin-Cereghino
`et al. 2001). Moreover, a set of integration vectors for se-
`quential disruption of ARG1, ARG2, ARG3, HIS1, HIS2,
`HIS5 and HIS6 in P. pastoris was applied to provide the host
`strains for engineering the protein glycosylation pathway
`(Nett et al. 2005).
`The Institute of Molecular Biotechnology, Graz University
`of Technology, Austria, provides vectors and strains to the
`P. pastoris community through the so-called ‘Pichia Pool’.
`The pPp plasmids described by Näätsaari et al. (2012) com-
`prise vectors containing the GAP or AOX1 promoters and, for
`secretory expression, the S. cerevisiae α-mating factor
`(α-MF) secretion signal. The antibiotic selection marker cas-
`settes were placed under the control of ADH1 or ILV5 pro-
`moters in the pPpB1 and pPpT4 vectors, respectively. It is
`
`Motif Exhibit 1029, Page 4 of 17
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`

`

`Appl Microbiol Biotechnol (2014) 98:5301–5317
`
`Table 2 Commercial vector systems
`
`5305
`
`Supplier
`
`Promoter
`
`Signal sequences
`
`Selection in yeast
`
`Selection in
`bacteria
`
`Comments
`
`Life Technologies™ AOX1, FLD1,
`GAP
`
`S. cerevisiae α-MF;
`P. pastoris PHO1
`
`Blasticidin, G418,
`Zeocin™, HIS4
`
`Zeocin™, Ampicillin,
`Blasticidin
`
`Life Technologies
`–PichiaPink™
`
`AOX1
`
`BioGrammatics
`
`AOX1
`
`α-MF; set of eight different
`signal sequences
`– not ready to usea
`α-MF
`
`BioGrammatics
`– GlycoSwitch®
`
`GAP
`
`–
`
`DNA2.0
`
`AOX1
`
`Ten different signal
`sequences
`– ready to useb
`
`ADE2
`
`Ampicillin
`
`Zeocin™, G418,
`Nourseothricin
`Zeocin™, G418,
`Hygromycin, HIS4,
`Nourseothricin
`Zeocin™, G418
`
`Ampicillin
`
`Zeocin™, Ampicillin,
`Kanamycin,
`Nurseothricin
`Zeocin™, Ampicillin
`
`c-myc epitope, V5 epitope,
`C-terminal 6× His-tag
`available for
`detection/purification
`Low- and high-copy vectors
`available, TRP2 sequence
`for targeting
`Intracellular or secreted
`expression
`Human GlcNAc transferase I, rat
`Mannosidase II, human Gal
`transferase I
`Intracellular or secreted
`
`a The different secretion signals have to be cloned into the vector by a three-way ligation step
`b The α-MF secretion signal is provided once with Kex2p (KR) and Ste13p cleavage sites (EAEA), once lacking EA repeats, and once as truncated
`version (pre-region only)
`
`described that the pPpT4-based vectors usually lead to lower
`gene copies in the cell as compared to the pPpB1-based vectors.
`Further vectors based on either the GAP or the AOX1
`promoter and a series of strains have recently been added to
`this pool, both for intracellular and secretory protein expres-
`sion (M. Ahmad, unpublished results). For intracellular ex-
`pression, cloning of the target genes is accomplished by using
`EcoRI and NotI, whereby the Kozak consensus sequence has
`to be restored for efficient translation initiation (Fig. 2a). A
`special characteristic of these vectors is that the EcoRI site has
`been introduced by a single point mutation directly into the
`AOX1 promoter sequence without changing the promoter
`activity. Thereby, the gene of interest may be fused to the
`promoter without having additional nucleotides between the
`promoter and the start codon. Another advantage is the use of
`the short ARG4 promoter for the expression of the selection
`markers. The weaker ARG4 promoter used for selection mark-
`er cassettes enables selection at lower concentrations of
`Zeocin™ (i.e., 25 instead of 100 μg/ml) without obtaining
`false-positive clones. For secretory expression governed by
`the S. cerevisiae α-MF signal sequence, XhoI and/or NotI sites
`are used for cloning the genes of interest (Fig. 2b).
`
`Aspects of secretory expression
`
`One of the main advantages of using P. pastoris as a protein
`production host is its ability to secrete high titres of properly
`folded, post-translationally processed and active recombinant
`proteins into the culture media. As a rule of thumb, proteins
`secreted in their native hosts will also be secreted in
`P. pastoris. However, there are also some reports of successful
`
`secretion of typically intracellular proteins such as GFP or
`human catalase (Eiden-Plach et al. 2004; Shi et al. 2007). The
`most commonly employed secretion signals in P. pastoris are
`derived from S. cerevisiae α-MF, S. cerevisiae invertase
`(SUC2) and the P. pastoris endogenous acid phosphatase
`(PHO1) (Daly and Hearn 2005). As listed in Table 2, com-
`mercial kits also provide vectors with different secretion sig-
`nals, which allows for screening of the best-suited signal
`sequence.
`The α-MF signal sequence is composed of a pre- and pro-
`region and has proven to be most effective in directing protein
`through the secretory pathway in P. pastoris. The pre-region is
`responsible for directing the nascent protein post-
`translationally into the endoplasmic reticulum (ER) and is
`cleaved off subsequently by signal peptidase (Waters et al.
`1988). The pro-region is thought to play a role in transferring
`the protein from ER to Golgi compartment and is finally
`cleaved at the dibasic KR site by the endo-protease Kex2p
`(Julius et al. 1984). The two EA repeats are subsequently
`trimmed by the STE13 gene product (Brake et al. 1984).
`One of the common problems encountered while using the
`α-MF secretion signal is non-homogeneity of the N-termini of
`the recombinant proteins due to incomplete STE13 process-
`ing. Constructs without the EA repeats may enhance homo-
`geneity at the N termini of recombinant proteins. However,
`the removal of these sequences may affect protein yield.
`While no reports on enhanced co-expression of STE13 are
`available, co-overexpression of HAC1, a transcription factor
`in the unfolded protein response (UPR) pathway, with the
`membrane protein adenosine A2 receptor had a positive effect
`on proper processing of the α-MF signal sequence (Guerfal
`
`~ Springer
`
`Motif Exhibit 1029, Page 5 of 17
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`

`

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`Appl Microbiol Biotechnol (2014) 98:5301–5317
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`Gene of interest
`
`5'-GAATTCTTCGAAACGATG NNN.
`3' -CTTAAGAAGCTTTGCTAC NNN.
`EcoRI
`
`TAAGCGGCCGC-3'
`. .ATTCGCCGGCG-5'
`Nofi
`
`- -EcoR~
`
`Kex2 processing signal ,I, Gene of interest
`L E K R X
`5'-CTCGAGAAAAGANNN .... TAAGCGGCCGC-3'
`ATTCGCCGGCG-5'
`3'-GAGCTCTTTTCTN NN.
`Nofi
`Xhol
`Xho/
`
`\ Not/ ..
`
`PROMOTER X :
`A ➔ AOX1
`G ➔ GAP
`
`SELECTION MARKER Y:
`H ➔ H/S4
`Z ➔ Zeocin™
`A ➔ ARG4
`K ➔ Kan"!G418
`
`LINEARIZATION SITE Z:
`Bgl ➔ Bgm
`Sph ➔ Sphl
`Swa ➔ Swal
`
`SIGNAL SEQUENCE a:
`a-MF
`
`\ Not/ - - - - -
`
`pAaZBgl
`5205 bp
`
`z
`a
`Fig. 2 Novel ‘Pichia Pool’ plasmid sets for intracellular and secretory
`expression. a General features of pXYZ vector for intracellular expres-
`sion. Letters refer to the choice of promoters (X), selection markers (Y),
`and restriction enzymes (Z) for linearization. Available elements are
`shown in boxes. The vector backbone harbours an ampicillin resistance
`marker and origin of replication for maintenance of the plasmid in E. coli.
`The GOI is EcoRI–NotI cloned directly after the promoter of choice. The
`Kozak consensus sequence for yeast (i.e., CGAAACG), should be re-
`stored between the EcoRI cloning site and the start codon of the GOI in
`order to achieve optimal translation. In addition, sequence variation
`
`Bg/11
`
`b
`within this region will allow fine-tuning translation initiation efficiency.
`Expression in P. pastoris is driven either by the methanol inducible AOX1
`or the constitutive GAP promoter. Positive clones can be selected for by
`antibiotic resistance (i.e., to Zeocin™ or geneticin sulphate) or by selec-
`tion for His or Arg prototrophy. Selection marker expression is uniformly
`driven by the ARG4 promoter–terminator pair. b Plasmid pAaZBgl from
`‘Pichia Pool’ is shown as an example of a vector made for secretory
`expression encoding S. cerevisiae α-MF signal sequence in front of the
`GOI cloning site. The Kex2 processing site AAAAGA should be restored
`between the XhoI cloning site and the fusion point of the GOI
`
`et al. 2010). Recently, Yang et al. (2013) reported enhanced
`secretory protein production by optimizing the amino acid
`residues at the Kex2 P1′ site.
`Multiple strategies have been followed to enhance the
`secretory potential of the α-MF signal sequence including
`codon optimization (Kjeldsen et al. 1998), directed evolution
`(Rakestraw et al. 2009), insertion of spacers and deletion
`mutagenesis (Lin-Cereghino et al. 2013). Directed evolution
`of the α-MF signal sequence in S. cerevisiae resulted in up to
`16-fold enhanced full-length IgG1 secretion as compared to
`the wild type. Furthermore, when this improved leader se-
`quence was combined with strain engineering strategies com-
`prising PDI overexpression and elimination of proteins in-
`volved in vacuolar targeting, up to 180-fold enhanced secre-
`tion of the reporter protein was observed (Rakestraw et al.
`2009). Deletion mutagenesis based on a predicted structure
`model of α-MF signal peptide resulted in 50 % increased
`secretion of horseradish peroxidase and C. antarctica lipase
`B (CALB) in P. pastoris (Lin-Cereghino et al. 2013). It ap-
`pears that decreasing the hydrophobicity of the leader se-
`quence by deleting hydrophobic residues or substituting them
`with more polar or charged residues increased the flexibility of
`the α-MF signal sequence structure, which enhanced the
`overall secretory capacity of the pro-region. Alternative signal
`sequences used to direct protein secretion and their features
`and applications are summarized in Table 3.
`
`Beyond the choice of the secretion signals there are several
`other factors that govern efficient protein secretion. The newly
`synthesized proteins are translocated co- or post-
`translationally into the ER lumen through the Sec61p
`translocon. Then, proteins may undergo one or several post-
`translational modifications, folding into the native state,
`disulphide-bond formation, glycosylation and membrane-
`anchoring. When the recombinant protein fails to fold into
`its native state or protein expression exceeds the folding
`capacity of the ER (Sha et al. 2013), unfolded proteins may
`start to aggregate, triggering the UPR pathway. UPR is re-
`sponsible for induction of genes that are involved in protein
`folding. In parallel to UPR pathway, ER-associated degrada-
`tion (ERAD) by the proteasome may relieve blocks in protein
`secretion (recently reviewed by Idiris et al. 2010 and
`Damasceno et al. 2012). Inappropriate mRNA structure and
`gene copy numbers, limits in transcription, translation and
`protein translocation into the ER, incomplete protein folding
`and inefficient protein targeting to the exterior of the cell are
`major bottlenecks encountered in secretory expression of het-
`erologous proteins. Commonly used strategies to overcome
`such secretory bottlenecks comprise the overexpression of
`folding helper proteins like BiP/Kar2p, DnaJ, PDI, PPIs and
`Ero1p or, alternatively, overexpression of HAC1, a transcrip-
`tional regulator of the UPR pathway genes. Unlike in
`S. cerevisiae, Guerfal et al. (2010) reported that HAC1 is
`
`~ Springer
`
`Motif Exhibit 1029, Page 6 of 17
`
`Case No.: IPR2023-00321
`U.S. Patent No. 10,689,656
`
`

`

`Appl Microbiol Biotechnol (2014) 98:5301–5317
`
`5307
`
`Table 3 Signal sequences used to secrete the protein into the extracellular space
`
`Secretion signal
`
`Source
`
`Target protein(s)
`
`Length
`
`Reference
`
`α-MF
`
`PHO1
`
`SUC2
`
`PHA-E
`KILM1
`pGKL
`CLY and CLY-L8
`
`K28 pre-pro-toxin
`Scw, Dse and Exg
`
`Pp Pir1
`HBFI and HBFII
`
`S.c. α-mating factor
`
`P.p. acid phosphatase
`
`S.c. Invertase
`
`Phytohemagglutinin
`Kl toxin
`pGKL killer protein
`C-lysozyme and syn.
`leucin-rich peptide
`K28 virus toxin
`P.p. Endogenous signal
`peptides
`P.p. Pir1p
`Hydrophobins of
`Trichoderma reesei
`
`Most commonly used secretion
`signal in P. pastoris
`Mouse 5-HT5A, porcine
`pepsinogen,
`Human interferon, α-amylase,
`α-1-antitrypsin
`GNA, GFP and native protein
`CM cellulase
`Mouse α-amylase
`Human lysozyme
`
`85 aa, with or
`without EA repeats
`15 aa
`
`19 aa
`
`21 aa
`44 aa
`20 aa
`18 and 16 aa
`
`(Brake et al. 1984)
`
`(Payne et al. 1995; Weiss et al. 1995;
`Yoshimasu et al. 2002)
`(Moir and Dumais 1987; Paifer
`et al. 1994; Tschopp et al. 1987b)
`(Raemaekers et al. 1999)
`(Skipper et al. 1985)
`(Kato et al. 2001)
`(Oka et al. 1999)
`
`Green fluorescent protein
`CALB and EGFP
`
`36 aa
`19, 20 and 23 aa
`
`(Eiden-Plach et al. 2004)
`(Liang et al. 2013a)
`
`EGFP and Human α1-antitrypsin
`EGFP
`
`61 aa
`16 and 15 aa
`
`(Khasa et al. 2011)
`(Kottmeier et al. 2011)
`
`constitutively expressed and spliced in P. pastoris under nor-
`mal growth conditions, which may explain the higher titers of
`secreted proteins obtainable with this organism. A contradic-
`tory observation was reported by Whyteside et al. (2011). Un-
`spliced HAC1 mRNA was detected under normal growth
`conditions and splicing of HAC1 mRNA was only detected
`when cells were grown in presence of dithiothreitol (DTT) to
`activate the UPR. It should be mentioned, though, that some-
`times overexpression of folding helpers actually reduced protein
`secretion or did not have any effect (van der Heide et al. 2002).
`
`Host strain development
`
`Elucidation of full genome sequences and gene annotation
`were great steps toward rational strain engineering, identifying
`new promoters and progressing in the (systems) biology of
`P. pastoris (Küberl et al. 2011; Mattanovich et al. 2009a; De
`Schutter et al. 2009). Two online databases (http://
`bioinformatics.psb.ugent.be/orcae/overview/Picpa and http://
`www.pichiagenome.org) provide convenient access to
`genome sequences and annotations. Frequently used
`commercially available strains are the his4 strain GS115, the
`reconstituted prototrophic s