Journal of Biotechnology 202 (2015) 118–134

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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Expression of enzymes for the usage in food and feed industry with Pichia pastoris Sebastian C. Spohner a,b , Hagen Müller a,b , Hendrich Quitmann b , Peter Czermak a,b,c,d,∗ a

Faculty of Biology and Chemistry, Justus Liebig University Giessen, Giessen Germany Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen Germany Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Giessen Germany d Department of Chemical Engineering, Kansas State University, Manhattan USA b c

a r t i c l e

i n f o

Article history: Received 12 August 2014 Received in revised form 28 December 2014 Accepted 7 January 2015 Available online 14 February 2015 Keywords: Yeast Pichia pastoris Protein expression Food and feed industry Fructooligosaccharides Phytase

a b s t r a c t The methylotrophic yeast Pichia pastoris is an established protein expression host for the production of industrial enzymes. This yeast can be grown to very high cell densities and produces high titers of recombinant protein, which can be expressed intercellularly or be secreted to the fermentation medium. P. pastoris offers some advantages over other established expression systems especially in protein maturation. In food and feed production many enzymatically catalyzed processes are reported and the demand for new enzymes grows continuously. For instance the unique catalytic properties of enzymes are used to improve resource efficiency, maintain quality, functionalize food, and to prevent off-flavors. This review aims to provide an overview on recent developments in heterologous production of enzymes with P. pastoris and their application within the food sector. © 2015 Elsevier B.V. All rights reserved.

1. Recombinant proteins for industrial use By the end of the 20th century, researchers were able to produce the first recombinant proteins for industrial use. Since then, industry has continually searched for new components to improve the benefit of this development along with its cost-efficiency. The progress in the optimisation of bioprocesses and the development of recombinant DNA technology offers a wide variety of alternatives in the production of proteins with new and better properties. Yeast has proven to be an efficient host for recombinant protein expression (Buckholz and Gleeson, 1991), and has become one of the most abundant alternatives for large-scale protein production. 2. Introduction The methylotrophic yeast Pichia pastoris, currently reclassified as Komagataella pastoris (Kurtzman, 2009; Yamada et al., 1994, 1995), was introduced by Phillips Petroleum more than four decades ago for the commercial production of single cell protein

∗ Corresponding author at: Justus Liebig University, Faculty of Biology and Chemistry, Giessen, Germany. E-mail address: [email protected] (P. Czermak). http://dx.doi.org/10.1016/j.jbiotec.2015.01.027 0168-1656/© 2015 Elsevier B.V. All rights reserved.

(SCP) as an animal feed additive. It has since become a substantial expression system in biotechnological processes, especially for heterologous protein production (Kurtzman, 2009). In 1973 the price for methanol increased drastically as a consequence of the oil crisis, which made SCP production uneconomical. P. pastoris was developed as a protein expression system in the 1980s using the tightly regulated alcohol oxidase promoter AOX1 (Cregg et al., 1985) which provided exceptionally high levels of heterologous protein expression. The production of the plant-derived enzyme hydroxynitrile lyase at 20 g of recombinant protein per litre was the first large-scale industrial production process established in the 1990s (Hasslacher et al., 1997). The first host strains for heterologous protein expression were GS115 (Schutter et al., 2009) and P. pastoris DSMZ 70382 (Mattanovich et al., 2009). For long time P. pastoris was not considered to be as genetically amenable as Saccharomyces cerevisiae. However, the publication of detailed genome sequences was a major breakthrough (Küberl et al., 2011). Since then, genetic advances including the development of a P. pastoris strain, which has the capacity to produce “humanized” glycoproteins, have been made. This permits the production of active recombinant erythropoietin (Hamilton et al., 2006). The most important breakthrough for the usage of the P. pastoris in food technology was the GRAS (generally recognized as safe) status by the Food and Drug Administration (FDA) and

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the FDA approval of recombinant proteins (Ciofalo et al., 2006; Thompson, 2010). Today, more than 500 pharmaceutical compounds and recombinant proteins are known to be produced with P. pastoris (Macauley-Patrick et al., 2005a). In the last decade, this expression system has been reviewed extensively with the focus being on genetics and its usage in drug and pharmaceutical industries (Ahmad et al., 2014; Cereghino and Cregg, 2000; Daly and Hearn, 2005; Jin et al., 2006; Macauley-Patrick et al., 2005a). In this article, we focus on recent developments in heterologous production of enzymes with P. pastoris for the usage in food and feed industry.

3. Comparison of the expression system P. pastoris with E. coli and S. cerevisiae E. coli has been used extensively as a host for recombinant protein expression in the last four decades. Since this simple microorganism lacks the intracellular machinery to achieve posttranslational modification, the application of this system with eukaryotic proteins has been problematic. Therefore, the successful expression in E. coli depends on the protein sequence, secondary and tertiary fold, and the functional characteristics of the recombinant protein. The inability to fold heterologous proteins correctly and perform further post-translational modifications limits the protein expression. The protein product may be obtained as insoluble, miss-folded inclusion bodies. As a result, subsequent solubilisation and re-folding are mandatory (Makrides, 1986, 1996). The reducing environment of the cytoplasm and inadequate chaperones can lead to incorrect folding (Bardwell, 1994; Cole, 1996; Li et al., 2001; White et al., 1994). For these reasons E. coli is not generally suitable for the expression of proteins containing disulphide bridges (White et al., 1994) or proteins requiring other post-translational modifications like disulphide isomerization, lipidation, glycosylation, proline cis/trans, phosphorylation or sulfation (Lueking et al., 2000). The stability of proteins expressed in E. coli is also affected by the presence of their amino-terminal methionine (Chaudhuri et al., 1999; Takano et al., 1999). Due to the lack of proper glycosylation of proteins in E. coli, the function of certain recombinant proteins can be altered (Jenkins et al., 1996; Meldgaard and Svendsen, 1994). A difficult and time-consuming refolding of a recombinant protein can result in significant losses, lower productivities and increased manufacturing costs of the expressed protein (Tsujikawa et al., 1996; Wang et al., 2000). Proteins that cannot be expressed in E. coli with correct post-translational maturation have been successfully produced with the methylotrophic yeast, P. pastoris (King et al., 1995; Lueking et al., 2000). Further E. coli is unable to secrete proteins beyond its periplasm into the medium. Since P. pastoris has the capability to secrete the authentic protein to the medium in a soluble form, like S. cerevisiae, it has the capacity to produce large quantities of enzymes via economically attractive downstream processing without laborious purification. Many of the secreted proteins of S. cerevisiae are not found free in the medium, but rather in the periplasmic space, what leads to problems with purification resulting in decreased product yield (Buckholz and Gleeson, 1991). There are several reasons that make P. pastoris a more amiable expression system. For one, the developed bioprocesses for S. cerevisiae can be easily applied to P. pastoris. Furthermore, the strong, inducible AOX1 promoter can be used for protein production. In contrast to S. cerevisiae, the lower levels of host protein secretion in P. pastoris facilitate the isolation of the recombinant protein. The above mentioned progress in glycoengineering enables the production of further functional proteins for the food industry. For example, bulky high-mannose-type N-glycan blocks the taste-modifying activity of miraculin, a sweet tasting

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protein (Ito et al., 2010). Here glycoengineered P. pastoris can be a very suitable host for high-level production of active protein. Further information on the comparison between P. pastoris and S. cerevisiae can be found in literature (Näätsaari et al., 2012). Due to all these reasons, P. pastoris expression systems offer significant advantages for the production of many heterologous eukaryotic proteins.

4. Enzymes in food technology Food enzymes can be divided into the following categories depending on the intended use: food ingredients, food additives and processing aids. Food ingredients are enzymes added for nutritional reasons, but this is rarely the case. More commonly, enzymes are added for technical reasons. Enzymes which are still present in an active form in the end product are defined as food additives. Otherwise the proteins in the product commonly belong to the category of processing aids. In 2008 the European Commission (EC) made the so-called Food Improvement Agents Package, defining all food enzymes with technological purposes as a separate group (Regulation (EC) No. 1332/2008), regulating food additives (Regulation (EC) No. 1333/2008), flavourings (Regulation (EC) No. 1334/2008) and a common authorization procedure (Regulation (EC) No. 1331/2008). Although not in isolated form, enzymes have been used traditionally for dairy, baking, brewing, and winemaking for centuries. Enzymes are needed for cheese production and a wide variety of other dairy goods. For example, their application keeps bread soft and fresh longer, leads to crispy crusts, increases dough volume and can compensate for variations in flour and malt quality. Additionally, enzymes are used to lower alcohol concentration and calories in beer. In winemaking, the sulphur content can be reduced, clarity and wine colour can be maintained, flavours can be enhanced and the filterability can be improved with enzymes. They are also utilised to improve the quality, stability, clarity and yield of fruit juices. The starch and sugar industry has been revolutionized by the usage of enzyme catalysis to hydrolyse starch and to rearrange glucose into fructose. In new approaches even cellulose can be transformed into fructose by enzyme catalysis (Lee et al., 2013). Galactooligosaccharides (GOS) and fructooligosaccharides (FOS) are well-established non-digestible oligosaccharides, which provide several health benefits and have excellent technological properties that make them attractive food ingredients. FOS can be produced by the degradation of fructan using Inulinase (described below) or by transglycosylation of sucrose, while GOS can be produced from Lactose using ␤-galactosidase (Czermak et al., 2004; Engel et al., 2007; Gonzalez et al., 2009; Ebrahimi et al., 2010; Kovács et al., 2014). Enzymes are highly valuable for the food and feed industry on account of their wide range of applications and cost-efficiency. In 2013 the world industrial enzyme market had a value of approximately D 3.27 billion, according to the global market leader Novozymes with 47% market share (Novozymes, 2013). Further important enzyme producers are Danisco with 21% market share, DSM with 6% market share, AB Enzymes with 5% market share and BASF with 4% market share. Food and beverage enzymes make 29% of sales in enzyme business, which is outnumbered only by household care enzymes with 31%. Feed and other technical enzymes accounted for 13% of sales. The first five of the six different enzyme classes (EC 1: oxidoreductases, EC 2: transferases, EC 3: hydrolases, EC 4: lyases, EC 5: isomerases, and EC 6: ligases) are sold commercially for food and feed production. So fare ligases are not used in food and feed production. In the European Union, approximately 260 different enzymes are available (cf. Table 4). They are isolated from fungi

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Table 1 Commercial P. pastoris strains. Strain

Genotype

Source

Wild-type strains X-33 CBS704 (DSMZ 70382)

WT WT

CBS7435 (NRRLY-11430)

WT

BG10

WT

Life TechnologiesTM Centraalbureau voor Schimmelcultures, Netherlands Centraalbureau voor Schimmelcultures, Netherlands BioGrammatics

Auxotrophic strains GS115 PichiaPinkTM KM71 KM71H BG09 GS190, GS200, JC220, JC227, JC254, JC300-JC308

CBS7435 his4a CBS7435 MutS his4a CBS7435 MutS arg4a Protease-deficient strains SMD1163 SMD1165 SMD1168 SMD1168H BG21

his4 ade2 his4, aox1::ARG4, arg4 aox1::ARG4, arg4 arg4::nourseoR lys2::hygR Combinations of ade1 arg4 his4 ura3

Life TechnologiesTM Life TechnologiesTM Life TechnologiesTM Life TechnologiesTM BioGrammatics James Cregg’s laboratory (Cregg et al., 1998; Waterham et al., 1996) (Lin-Cereghino et al., 2001)

his4 aox1, his4 aox1, arg4

(Näätsaari et al., 2012) (Näätsaari et al., 2012) (Näätsaari et al., 2012)

his4 pep4 prb1 his4 prb1 his4 pep4::URA3 ura3 pep4 sub2

(Gleeson et al., 1998) (Gleeson et al., 1998) Life TechnologiesTM Life TechnologiesTM BioGrammatics

(filamentous ascomycetes and basidiomycetes: 58%, yeasts: 5%) and bacteria (28%). Few enzymes are isolated from plants (3%) and animals (6%). For the production of one-third of these enzymes, genetically modified organisms are used. In feed manufacturing, enzymes are used primarily to increase the availability of essential nutrients (e.g. phytase). They are used to complement the spectra of activities of enzymes, which are already present in feed or the animals’ own digestive enzymes. With the use of enzymes, feedstuff becomes more efficient; resources are conserved and waste is avoided. In the feed sector, the most important enzymes are phytase, xylanase and ␤-glucanase. More detailed information on the application of enzymes in the food and feed industry are given in (Fernandes, 2010; Fraatz et al., 2014; Panesar et al., 2010b; Tucker and Woods, 1995; Whitehurst and van Oort, 2009). 5. P. pastoris expression strains The choice of a specific P. pastoris strain depends on the desired application. The most common strains are summarised in Table 1 with their genotype and phenotype characteristics. Besides wild-type strains like P. pastoris X-33, strains with modifications like auxotrophy or protease deficiency are also available. The strains SMD1168 and SMD1168H are defective in the vacuole peptidase A (Pep4), which is responsible for activating carboxypeptidase Y and protease B1. Hence, both strains are also defective in these proteases. The strains KM71, GS115 and SMD1168 are defective in the histidine dehydrogenase gene (his4), which allows a selection based on the reconstruction of histidine biosynthesis in recombinants. The strains GS115 and SMD1168 have a wild-type methanol utilization phenotype designated as Mut+ (Inan and Meagher, 2001). They contain a functional copy of the

alcohol oxidase 1 gene (AOX1). Approximately 85% of the utilization of methanol is done by the alcohol oxidase enzyme. The strain KM71 contains a non-functional AOX1 gene (aox1). Here the alcohol oxidase enzyme is produced by an alternative gene called AOX2. The enzymes AOX1p and AOX2p have the same specific activity, but the expression level of AOX2p is lower due to the weaker promoter. Therefore, aox1 strains can only consume methanol slowly. As a result, this phenotype has been termed ‘methanol utilization slow’ (MutS ) (Cregg et al., 1993; Inan and Meagher, 2001). Further detailed information on available expression strains can be found in literature (Ahmad et al., 2014). Depending on the protein of interest, the desired modifications and the planed process for protein production, all of the strains above provide useful competencies and can be used in discrete applications. Additionally, according to the following cloning strategy, multi-copy protein expression or multi-protein co-expression can be archived.

6. Expression vectors The standard vectors used in yeast genetics are shuttle vectors, which can be replicated in E. coli and maintained in P. pastoris using a selection marker. Selection can be done via auxotrophic genetic markers (e.g. HIS4, MET2, ADE1, ARG4, URA3, URA5, GUT1). A set of plasmids for protein secretion and intracellular expression in P. pastoris that is based on different auxotrophic genetic markers has been developed in the James Cregg’s laboratory at the Keck Graduate Institute, Claremont, CA, USA. These vectors contain the strong AOX1 promoter and different selection markers (Lin-Cereghino et al., 2001). Selection can be conveyed via genes conferring resistance to drugs such as geneticin (G418), blasticidin S, hygromycine and ZeocinTM . In our lab we use the novel antibiotic BleocinTM (CALBIOCHEM© ) instead of ZeocinTM . BleocinTM is another member of the bleomycin family of antibiotics including bleomycin, hleomycin, tallysomycin, pepleomycin, and ZeocinTM . Bleomycins are DNA-cleaving glycopeptides with toxic effects on prokaryotes and eukaryotes at low concentrations. BleocinTM is considered to be most toxic in the G2 phase of the cell cycle and can be used at concentrations similar to phleomycin (Mulsant et al., 1988). BleocinTM is 8-fold more potent than ZeocinTM . The standard vector systems for intracellular and secretory expression are provided by Life Technologies (Carlsbad, CA, USA). It includes the constitutive glycerine aldehyde dehydrogenase promoter (GAP), the inducible alcohol oxidase 1 promoter (AOX1) and the signal sequence of the S. cerevisiae ␣-Mating Factor (␣MF). Using the PichiaPinkTM expression kit, multi-copy integration clones can easily be divided by differences in colour. This is based on an ade2 strain (Du et al., 2012; Nett et al., 2005). BioGrammatics (Carlsbad, CA, USA) provides P. pastoris expression vectors and strains for humanized glycosylation of target proteins. Additionally, several other vectors for mammalian-type N-glycan structures in P. pastoris are available. Detailed protocols and information for humanized glycolstructures can be found in literature (Jacobs et al., 2009; Vogl et al., 2013). A list of commercially available plasmids for protein expression in P. pastoris can be found in Table 2. For heterologous protein expression and the screening of mutant libraries in P. pastoris, some reports used episomal plasmids (Lee et al., 2005a; Uchima and Arioka, 2012), but nevertheless the most preferred method is stable genome integration. In S. cerevisiae, homologous recombination predominates, whereas in P. pastoris genetics, non-homologous end-joining is the most frequent procedure. Homologous recombination can also be archived in P. pastoris through elongation of the flanking regions in the expression cassettes and additional suppression of non-homologous endjoining (Näätsaari et al., 2012).

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Table 2 Commercial expression vectors for P. pastoris. Supplier

Promoter

Signal sequences

Selection in E. coli

Selection in P. pastoris

BioGrammatics BioGrammatics – GlycoSwitch®

AOX1 GAP

S. cerevisiae ␣-MF

Ampicillin ZeocinTM , Ampicillin, Kanamycin, Nurseothricin

DNA2.0

GAP, AOX1

ZeocinTM , Ampicillin

Life TechnologiesTM

AOX1, FLD1, GAP AOX1

10 different signal sequences S. cerevisiae ␣-MF; P. pastoris PHO1 S. cerevisiae ␣-MF; set of eight different signal sequences S. cerevisiae ␣-MF

ZeocinTM , G418, Nourseothricin ZeocinTM , G418, Hygromycin, HIS4, Nourseothricin ZeocinTM , G418

ZeocinTM , Ampicillin, Blasticidin Ampicillin

Blasticidin, G418, ZeocinTM , HIS4 ADE2

ZeocinTM

ZeocinTM

Life Technologies – PichiaPinkTM Lonza

GAP, G1, AOX1,

Further detailed information on expression plasmids and transformation of P. pastoris can be found in literature (Ahmad et al., 2014; Daly and Hearn, 2005; Jamshad and Darby Richard, 2012; Logez et al., 2012).

7. Choice of promoter For the expression of heterologous genes, several stronger or weaker endogenous promoters of P. pastoris can be used. These promoters include inducible promoters as well as constitutive promoters (cf. Table 3). The inducible expression of genes is usually favoured, since it allows control over the experimental conditions before the expression is started and it allows expression of the proteins that are toxic to the host. The provided promoters can be induced by various carbon or nitrogen sources (Zhang et al., 2009a). In P. pastoris, the expression of heterologous genes is usually controlled by the promoter of the alcohol oxidase I gene (AOX1). The AOX1 promoter controls the expression of the first enzyme in the pathway of methanol utilisation and is tightly regulated (Cregg and Madden, 1989; Tschopp et al., 1987). Carbon sources like glucose fully repress expression with the AOX1 promoter. A maximum expression is reached via induction with methanol. The advantage of this regulation is: the growing of strains expressing toxic heterologous proteins can be maintained under repressing conditions, and later the expression can be induced. Induction with methanol sometimes yields inappropriate or inconvenient expressions. The induction can be too strong, which causes too much stress and leads to non-desirable results. Induction with methanol is also not appropriate for the production of food products. Here, different inducible or constitutive promoters are favourable. Another common promoter for high-level expression is the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter, which was described in 1997 (Waterham et al., 1997). The GAP promoter has been tested on various carbon sources, such as glucose, glycerol, oleic acid and methanol. These expressions yielded strong and constitutive expression on glycerol and glucose. This promoter is particularly attractive for protein production based on large-scale fermenters, since it enables production without the need of methanol induction. In numerous cases, a constitutive expression yields higher expression levels, in particular when using the GAP promoter (Cos et al., 2006; Zhang et al., 2009a). For example, the expression levels of ␤-lactamase in P. pastoris under the control of the GAP promoter (grown on glucose) are higher than such under the AOX1 promoter (grown on methanol) (Waterham et al., 1997). Enzymes used in the food industry are usually not toxic to yeast host cells, hence constitutive promoters are favourable. To handle hazardous substrates or too strong induction with methanol, other promising promoters have been tested. An alternative promoter for high-level expression is the formaldehyde dehydrogenase (FDL1) promoter (Shen et al., 1998). Other

constitutive promoters like translation elongation factor 1-␣ (TEF1) promoter (Ahn et al., 2007) and the 3-phosphoglycerate kinase (PGK1) promoter (de Almeida et al., 2005) show growth-related expression behaviour. The peroxisomal matrix protein promoter (PEX8) (Liu et al., 1995) and the promoter of a GTPase (YPT1) involved in secretion (Sears et al., 1998) can be used for moderate expression as well. The use of the ethanol inducible promoters like the isocitrate lyase (ICL1) promoter (Menendez et al., 2003) has been described, as well as a promoter of putative sodium (Na+ )coupled phosphate symporter (PHO89). This promoter is highly active under phosphate limitation (Ahn et al., 2009). More detailed information on promoters for protein expression in P. pastoris and their regulation can be found in literature (Vogl and Glieder, 2013). As described above, many different promoters have been used for successful protein expression in P. pastoris. With these tools researchers are able to construct the suitable expression plasmid for the protein of interest.

8. Secretory protein expression In yeast, two ways of protein expression are common: intracellular expression and extracellular secretion. The choice between the two depends on the protein to be expressed. For example, the extracellular secretion of a protein that is not secreted within its native host system may result in altered glycosylation or the lack of other crucial post-translational modifications. In some cases, intracellular expression proves to be a more desirable method, since it usually does not result in glycosylation. On the other hand the purification of intracellular expressed proteins can be more difficult. One advantage of intracellular expression of recombinant proteins with P. pastoris is that the methionine amino-peptidase usually cleaves the amino-terminal methionine residue (Romanos et al., 1992). Protein folding usually begins with the formation of secondary structures. The disulphide bonds are formed in the endoplasmic reticulum (ER) (Holst et al., 1996). In particular, proteins that are naturally secreted contain a pro-region. This region is essential for correct folding and sometimes oligomerisation. To release the mature protein, the pro-region is removed proteolytically. The expression of proteins without their pro-region can result in slower release from the ER or in some cases, in misfolded products (Holst et al., 1996; Oka et al., 1999; Romanos et al., 1992). The pro-region is removed into the Golgi by dibasic endopeptidases, such as Kex2p. Following this, the recombinant protein is packaged into secretory vesicles and delivered to the cell surface (Romanos et al., 1992). A specific signal sequence is required to direct proteins into the secretory pathway. This signal sequence is a short amino-terminal pre-sequence. It typically contains a charged amino-terminal region, which is followed by 6–15 hydrophobic amino acid residues and a cleavage site (Paifer et al., 1994; Romanos et al., 1992). Due to the simple purification of secreted protein, this method is favourable for the production enzymes.

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Table 3 Promoters for heterologous expression in P. pastoris. Name

Gene

Regulation

Strength

Reference

pAOX1 pAOX2 pFLD1 pDHAS pPEX8 pICL pADH1 pPHO89 pENO1 pGUT1 pGAP pSDH pTEF1 pPGK1 pYPT1 pILV5 pAOD pGCW14

Alcohol oxidase 1 Alcohol oxidase 2 Formaldehyde dehydrogenase 1 Dihydroxyacetone synthase Peroxisomal matrix protein Isocitrate lyase Alcohol dehydrogenase 1 Putative Na+/phosphate symporter Enolase Glycerol kinase Glyceraldehyde-3-P dehydrogenase Sorbitol dehydrogenase Translation elongation factor 1 3-Phosphoglycerate kinase GTP-binding protein Acetohydroxyacid eductoisomerase Alternative oxidase Potential glycosyl phosphatidyl inositol (GPI)-anchored protein Thiamine biosynthesis gene High affinity glucose transporter Putative aldehyde dehydrogenase

Inducible (Methanol) Inducible (Methanol) Inducible (Methanol, Methylamine) Inducible (Methanol) Inducible (Methanol) Inducible (Ethanol) Inducible (Glycerol, Ethanol) Inducible (phosphate starvation) Inducible (Glycerol, Ethanol) Inducible (Glycerol) Constitutive Constitutive Constitutive Constitutive Constitutive Constitutive Constitutive Constitutive

Strong Weak Strong Strong Weak n.a. Strong Strong Moderate Strong Strong Strong Strong Weak Weak Weak Moderate Strong

(Tschopp et al., 1987) (Kobayashi et al., 2000) (Shen et al., 1998) (Ellis et al., 1985; Tschopp et al., 1987) (Liu et al., 1995) (Menendez et al., 2003) (Cregg and Tolstorukov, 2012) (Ahn et al., 2009) (Cregg and Tolstorukov, 2012) (Cregg and Tolstorukov, 2012) (Waterham et al., 1997) (Periyasamy et al., 2013) (Ahn et al., 2007) (de Almeida et al., 2005) (Sears et al., 1998) (Hubmann, 2005) (Kern et al., 2007) (Liang et al., 2013)

Constitutive Constitutive Constitutive

Strong Strong Weak

(Stadlmayr et al., 2010) (Prielhofer et al., 2013) (Prielhofer et al., 2013)

pTHI11 pG1 pG6

More detailed information on protein secretion by yeast and possible signal sequences can be found in literature (Ahmad et al., 2014; Hou et al., 2012; Logez et al., 2012). 9. Codon optimised genes Even if recombinant genes are often cloned and expressed in host strains like P. pastoris in their native form, several adjustments can optimise the gene expression. These improvements in the sequence can be made to best fit the transcriptional and translational machineries of the yeast. The codon optimisation of the gene of interest and its fusion partners often results in a dramatic increase of protein expression levels. Some sequence parameters were shown to notably influence the expression level; these include translation initiation sequence, adaption to the codon usage of yeast (Sinclair and Choy Francis, 2002; Woo et al., 2002), adaption of GC-content and the adaption of the isoelectric point of the desired protein (Boettner et al., 2007). For example, the expression of Trichoderma viride endochitinase was increased by 35% when compared to a strain bearing the wild-type endochitinase cDNA (Yu et al., 2013). Also the expression levels of inulinase (He et al., 2014), keratinase (Hu et al., 2013), phytase (Xiong et al., 2005) and lignocellulolytic enzymes (Mellitzer et al., 2012) were significantly improved by the usage of codon optimised gens. 10. Fermentation techniques In P. pastoris the fermentation techniques that are utilized mainly depend on which promoter is used. The standard fermentation procedure with the commonly used AOX1 promoter is three staged fed-batch fermentation. Since the AOX1 promoter is repressed by glucose, the first stage (“growth phase”) is batch fermentation with glycerol as a carbon source. In this stage, a wet cell weight up to 100 g/L can be obtained (Zhang et al., 2000). The second stage (“transition phase”) is a fed-batch phase with two purposes: the first purpose is the production of more biomass and the second is the inhibition-free transition from the glycerol, to methanol metabolism, which introduces the “production phase”. The maximum of wet cell weight generated during this phase is limited by the medium that is used, the oxygen supply during the fermentation and the necessary heat transfer (Zhang et al., 2000). The “transition phase” is started right after all glycerol is metabolized, which is indicated by a peak in dissolved

oxygen. In the transition phase, sole glycerol feed or a mixed feed of glycerol and methanol can be applied. In the case of mixed feed fermentations, the glycerol amount in the transition feed is slowly reduced to zero, whereas the methanol amount is increased to up to 100% to induce protein production. Especially for the “production phase”, different feeding profiles have been reported including: constant oxygen feeding, constant specific growth rate feeding, constant methanol concentration feeding, oxygen limited fed-batch and temperature limited fed-batch (Potvin et al., 2012). Sometimes continuous protein production with the AOX1 promoter is applied. Here the “production phase” consists of continuous fed-batch cultivation. The critical factors are the dilution rate as well as the feed composition (d’Anjou and Daugulis, 2001; Hélène et al., 2001). The fermentation strategy for the constitutive protein production using the GAP promoter differs significantly from these strategies. No specific transition and production phases are necessary, which allows for easier management of the process. Without the need of methanol induction, the amount of produced protein solely depends on the produced cell mass, therefore making the fed-batch and the continuous fermentation the modes of choice. Due to longer production periods in continuous fermentations, the protein yields are five to six-fold higher when compared to fed-batch processes (Goodrick et al., 2001). An example for a successfully developed large scale fed-batch process using the GAP promoter system is the production of Candida rugosa lipase, generating up to 500 g/L wet cell weight and stable lipase activity of 14,000 U/mL (Zhao et al., 2008). This demonstrates how the GAP promoter system should be considered when it comes to large-scale fermentation processes with P. pastoris. More detailed information on different fermentation strategies can be found in literature (Potvin et al., 2012; Zhang et al., 2000). 11. Application of P. pastoris expression system within the food and feed industry The application of enzymes to industrial processes has been reviewed in great detail (Kirk et al., 2002; Sanchez and Demain, 2011). Current trends in the feed industry have been reviewed by (Ravindran and Son, 2011). Due to the enormous variety, it is not possible to cover all potential applications of enzymes in the food and feed industry. An overview of possible applications of enzymes in the food and feed industry is given in Table 4. Hence, a number

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Table 4 Enzymes used in food and feed industry and recombinant proteins expressed in P. pastoris. Accepted name

EC number

Common production strains

Application areas

Source of recombinant protein

Yield

Oxidoreductases Glucose oxidase

EC 1.x.x.x EC 1.1.3.4

Aspergillus niger, A. oryzae (GM), Penicillium chrysogenum Hansenula polymorpha (GM)

Bakery, eggs, starch processing Bakery, starch processing

Aspergillus niger

(Meng et al., 2014)

Hexose oxidase

EC 1.1.3.5

Chondrus crispus

(Hansen and Stougaard, 1997) (Bey et al., 2011) (Kittl et al., 2012)

Cellobiose dehydrogenase Laccase

EC 1.1.99.18

Fusarium venenatum (GM)

7.8 U/mL 0.35 g/L 51 U/mL 0.495 g/L

EC 1.10.3.2

Catalase

EC 1.11.1.6

Aspergillus niger, A. oryzae (GM), Trametes hirsuta, T. versicolor, Trichoderma longibrachiatum Aspergillus niger, A. oryzae (GM), Micrococcus luteus

Peroxidase

EC 1.11.1.7

Lactoserum, soy

Linoleate 13Slipoxygenase Transferases Protein-glutamine ␥glutamyltransferase Dextransucrase

EC 1.13.11.12

soy, Escherichia coli (GM)

EC 2.x.x.x EC 2.3.2.13

Streptomyces mobaraensis

EC 2.4.1.5

Leuconostoc mesenteroides

1,4-␣-glucan branching enzyme Cyclomaltodextrin

EC 2.4.1.18

Bacillus amyloliquefaciens (GM)

EC 2.4.1.19

1,4-␣-glucan 6-␣glucosyltransferase

EC 2.4.1.24

Hydrolases Carboxylesteras

EC 3.x.x.x EC 3.1.1.1

Triacylglycerol lipase

EC 3.1.1.3

Phospholipase A2

EC 3.1.1.4

Lysophospholipase

EC 3.1.1.5

Pectinesterase

EC 3.1.1.11

Tannase

EC 3.1.1.20

Aspergillus niger (GM), A. oryzae (GM), Bacillus licheniformis (GM), calf, Candida lipolytica, C. rugosa, goat, Hansenula polymorpha (GM), lamb, Mucor javanicus, Penicillium roqueforti, Pichia angusta (GM), Rhizomucor miehei, Rhizopus niveus (non-GM and GM), R. oryzae Aspergillus oryzae (GM), A. niger (GM), ox, pig, Streptomyces violaceoruber (non-GM and GM), Trichoderma longibrachiatum (GM) Aspergillus niger (non-GM and GM), Trichoderma longibrachiatum (GM) Aspergillus niger (non-GM and GM), A. oryzae (GM), A. sojae, Penicillium funiculosum, Rhizopus oryzae, Trichoderma longibrachiatum (GM) Aspergillus oryzae, A. niger

Acylglycerol lipase

EC 3.1.1.23

Penicillium camemberti

Beverages, cork treatment

Pycnoporus cinnabarinus Trametes versicolor

99 U/mL 0.8 g/L

Beverages, eggs, starch processing, others Starch processing, others Bakery, flavour production

Homo sapiens

(Shi et al., 2007)

1380 U/mL 0.55 g/L

Armoracia rusticana

(Spadiut et al., 2012)

31 U/mL 0.4 g/L

Pisum sativum

(Veronico et al., 2006)

Meat and fish, bakery, milk and cheese Dextran production Starch processing

Zea mays

(Li et al., 2013a)

3.9 mU/mL 4.4 mg/L

Leuconostoc mesenteroides Aspergillus oryzae

(Kang et al., 2011) (Wu et al., 2010)

14 U/mL

Bacillus licheniformis (GM)

Production of cyclodextrins

Paenibacillus macerans

(Zhang et al., 2009b) [Article in Chinese]

4.5 U/mL

Aspergillus niger, Trichoderma longibrachiatum (GM)

Starch processing

Rhizomucor miehei

Hydrolysis of various carboxylic esters Bakery, milk and cheese, starch processing, others

Tribolium castaneum

(Delroisse et al., 2005)

0.44 U/mL 0.08 g/L

Aspergillus tamarii

(Shi et al., 2010)

20 U/mL

Bakery, eggs, starch processing, others

Cobra

(Lefkowitz et al., 1999)

Beverages, starch processing Beverages, fruits and vegetables

Aspergillus niger

(Zhu, 2008)

19.8 U/mL 0.026 g/L

Aspergillus niger

(Jiang et al., 2013)

1.39 U/mL 0.144 g/L

Aspergillus oryzae Rhizopus oryzae

(Zhong et al., 2004) (Minning et al., 1998)

7 U/mL 0.074 g/L 110 U/mL 7.97 g/L

vegetables Lipids

1.74 U/mL 0.23 g/L

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Table 4 (Continued) Accepted name

EC number

Common production strains

Application areas

Source of recombinant protein

Yield

Phospholipase A1

EC 3.1.1.32

Aspergillus oryzae (GM)

Vespula vulgaris

Feruloyl esterase

EC 3.1.1.73

Phytase (3-, 4-)

EC 3.1.3.8/26

(Borodina et al., 2011) (Moukouli et al., 2008) (Xiong et al., 2005)

Phosphodiesterase I Phospholipase C

EC 3.1.4.1 EC 3.1.4.3

Aspergillus niger, Streptomyces werraensis Aspergillus niger (non-GM and GM), A. oryzae (GM), Schizosaccharomyces pombe (GM), Trichoderma longibrachiatum (GM) Leptographium procerum, malt, Penicillium citrinum Pichia pastoris (GM)

Milk and cheese Biomass degradation Feed, bakery

Yeast extract production Emulsifiers

Triticum aestivum Bacillus cereus

Ribonuclease P

EC 3.1.26.5

Penicillium citrinum

Cattle

Amylase

EC 3.2.1.1/2/3

Barley, Penicillium multicolor, wheat, soy

Glucan 1,4-␣glucosidase

EC 3.2.1.3

Aspergillus niger (non-GM and GM),

Cellulase

EC 3.2.1.4

Endo-1,3(4)-␤glucanase

EC 3.2.1.6

Aspergillus niger, Humicola insolens, Hypocrea jecorina (GM), Penicillium funiculosum, Talaromyces emersonii, Trichoderma longibrachiatum (non-GM and GM), T. viride Aspergillus niger, A. oryzae (GM), Bacillus amyloliquefaciens (non-GM and GM), Cellulosimicrobium cellulans, Disporotrichum dimorphosporum, Humicola insolens, Penicillium funiculosum, Talaromyces emersonii, Trichoderma longibrachiatum (non-GM and GM)

Yeast extract production Beverages, starch processing Bakery, beverages, starch processing Bakery, beverages, starch processing

Inulinase

EC 3.2.1.7

Endo-1,4-␤xylanase

EC 3.2.1.8

Dextranase

EC 3.2.1.11

Chitinase

EC 3.2.1.14

Polygalacturonase

EC 3.2.1.15

Lysozyme ␣-Glucosidase

EC 3.2.1.17 EC 3.2.1.20

Aspergillus niger, A. oryzae (GM) Aspergillus niger (non-GM and GM), A. oryzae (GM), Bacillus amyloliquefaciens (GM), B. licheniformis (GM), Disporotrichum dimorphosporum, Humicola insolens, Penicillium funiculosum, Talaromyces emersonii, Trichoderma longibrachiatum (non-GM and GM), T. viride Chaetomium erraticum, C. gracile, Penicillum lilacinum Streptomyces violaceoruber (GM) Aspergillus niger (non-GM and GM), A. wentii, Rhizopus oryzae, Trichoderma longibrachiatum Chicken egg Aspergillus niger

␤-Glucosidase

EC 3.2.1.21

Aspergillus niger

␣-Galactosidase

EC 3.2.1.22

Aspegillus niger, A. oryzae (GM), Saccharomyces cerevisiae (GM)

Fusarium oxysporum Asperguillus niger

1.7 mg/L 0.83 U/mL 865 U/mL 6.1 g/L

(Joye et al., 2010) (Seo and Rhee, 2004) (Chatani et al., 2000) (Liu et al., 2012)

3 U/mL 0.059 g/L

Aspergillus awamori

(Fierobe et al., 1997)

7.8 U/mL 0.8 g/L

Trichoderma reesei

(Mellitzer et al., 2012)

6.55 g/L

Beverages

Penicillium sp.

(Chen et al., 2012)

28 U/mL 2.61 g/L

Starch processing Bakery, beverages, starch processing, others

Aspergillus niger Thermomyces lanuginosus

(He et al., 2014)

1.3 U/mL 2.21 g/L 138 U/mL 1.2 g/L

Sugar refinery

Lipomyces starkey Homo sapiens

Barley

(Mellitzer et al., 2012)

5 mg/L 68 U/mL 0.125 mg/L

83.3 U/mL 0.46 g/L 0.3 g/L

Aspergillus niger

(Chen et al., 2008a) (Goodrick et al., 2001) (Liu et al., 2014)

Preservation Starch processing

Chicken Apis cerana indica

(Xu et al., 2014) (Kaewmuangmoon et al., 2013)

0.0123 g/L 13 U/mL 4.2 mg/L

Sugar specialties Medical applications

Trichoderma reesei Coffea

(Chen et al., 2011) (Zhu et al., 1995)

60.5 U/mL 0.55 g/L 1.5 U/mL 0.4 g/L

Nacetylglucosamine Beverages

205 U/mL 0.098 g/L

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125

Table 4 (Continued) Accepted name

EC number

Common production strains

Application areas

Source of recombinant protein

Yield

␤-Galactosidase (lactase)

EC 3.2.1.23

Aspergillus niger, A. oryzae (non-GM and GM), Bacillus circulans, Kluyveromyces fragilis, K. lactis (non-GM and GM)

Milk processing

Paecilomyces aerugineus

(Katrolia et al., 2011a)

9500 U/mL 22 g/L

Invertase

EC 3.2.1.26

␣-l-Rhamnosidase

EC 3.2.1.40

Aspegillus niger, Saccharomyces cerevisiae Aspergillus sp.

Sucrose processing Beverages

(Tschopp et al., 1987) (Gerstorferová et al., 2012)

7500 U/mL 2.5 g/L 9 U/mL 0.3 g/L

Pullulanase

EC 3.2.1.41

Beverages, starch processing

(Xu et al., 2006a)

350 U/mL

␤-l-N-acetyl hexosaminidase

EC 3.2.1.52

Bacillus acido-pullulyticus, B. amyloliquefaciens (GM), B. brevis, B. licheniformis (GM), B. subtilis (GM), Klebsiella planticola, Pullulanibacillus sp., Trichoderma longibrachiatum (GM) Streptomyces violaceoruber (GM)

Saccharomyces cerevisiae Aspergillus terreus, A. kawachii Bacillus naganoensis

Talaromyces flavus

(Slámová et al., 2012)

0.78 U/mL 0.022 g/L

␣-Narabinofuranosidase Glucan 1,3-␤glucosidase Glucan 1,4-␣maltotetraohydrolase Mycodextranase Isoamylase

EC 3.2.1.55

Alicyclobacillus sp. Pichia pastoris

(Alias et al., 2011) (Xu et al., 2006b)

21 U/mL

Bacillus lentus

Arabinan endo-1,5␣-l-arabinanase Glucan 1,4-␣maltohydrolase

EC 3.2.1.99

(Li et al., 2013b) (Huy et al., 2013)

318 U/mL 10.4 g/L 1.66 U/mL 8 mg/L

EC 3.2.1.58 EC 3.2.1.60 EC 3.2.1.61 EC 3.2.1.68

EC 3.2.1.133

Aspergillus niger (non-GM and GM) Penicillium funiculosum, Trichoderma harzianum Bacillus licheniformis (GM), B. subtilis (GM) Bacillus licheniformis (GM) Pseudomonas amyloderamosa Aspergillus niger Bacillus amyloliquefaciens (GM), Microbacterium imperiale Aspergillus niger, A. oryzae, Rhizopus oryzae

Aminopeptidase

EC 3.4.11.x

Leucyl aminopeptidase

EC 3.4.11.1

Aspergillus oryzae

Serine-type carboxypeptidase

EC 3.4.16.x

Aspergillus niger (GM)

Chymotrypsin

EC 3.4.21.1

Trypsin

EC 3.4.21.4

Bacillus licheniformis (GM), beef pancreas Fusarium venenatum (GM)

Thrombin Prolyl oligopeptidase Subtilisin

EC 3.4.21.5 EC 3.4.21.26

Cattle, pig Aspergillus niger (GM)

EC 3.4.21.62

Bacillus licheniformis

Oryzin

EC 3.4.21.63

Aspergillus oryzae

Aqualysin 1

EC 3.4.21.111

Papain

EC 3.4.22.2

Bacillus amyloliquefaciens (GM) Carica papaya

Bromelain

EC 3.4.22.32/33

Aspartic endopeptidases

EC 3.4.23.x

Pepsin

EC 3.4.23.1/2

Ananas comosus, Bromelia sp. Animal origin, Aspergillus niger (GM), A. oryzae, A. wentii, Bacillus licheniformis, Macrococcus caseolyticus Bovine rennet

Milk Fruit processing, industry Bakery Starch processing Flour processing Bakery Starch processing Processing of l-arabinan Bakery, starch processing

Phanerochaete chrysosporium

12 U/mL 0.175 g/L

Milk processing

Ustilago maydis

(JuárezMontiel et al., 2014)

Beverages, production of soy sauce Meat and fish, milk processing, flavoring preparations Protein hydrolysis Milk and cheese Meat and fish Beverages, others Protein hydrolysis Bakery

Trichophyton rubrum

(Monod et al., 2005)

0.1 g/L

Bacillus stearothermophilus

(Despreaux and Manning, 1993)

0.14 g/L

Metarhizium anisopliae Streptomyces griseus

(Screen and St Leger, 2000) (Ling et al., 2012)

Aspergillus niger Pichia pastoris

(Kang et al., 2013) (Salamin et al., 2010) (Guo and Ma, 2008) (Oledzka et al., 2003) (Dufour et al., 1998) (Mueller et al., 2009) (Caffrey et al., 2000)

Bakery Beverages, others Beverages, others Bakery, beverages, milk and cheese, protein hydrolysis Protein hydrolysis, Milk and cheese

Aspergillus oryzae Thermus aquaticus Carica papaya Ananas comosus Schistosoma mansoni

Pig

(Yoshimasu et al., 2002)

14.4 U/mL

0.5 U/mL 3 mg/L 47.6 U/mL 0.513 g/L 2825 U/mL 0.11 g/L 5 mg/L 12.5 mg/L

1 U/mL 14.7 g/L

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Table 4 (Continued) Accepted name

EC number

Common production strains

Application areas

Source of recombinant protein

Yield

Chymosin

EC 3.4.23.4

Milk and cheese, others

Buffalo

(Vallejo et al., 2008)

314 U/mL 0.0195 g/L

Aspergillopepsin I

EC 3.4.23.18

Endothiapepsin Mucorpepsin

EC 3.4.23.22 EC 3.4.23.23

Thermolysin

EC 3.4.24.27

Bacillolysin

EC 3.4.24.28

Calf, Aspergillus niger (GM), Escherichia coli (GM), Kluyveromyces lactis (GM) Aspergillus oryzae (non-GM and GM), A. wentii Cryphonectria parasitica Aspergillus oryzae (GM), Mucor pusillus, Rhizomucor miehei Geobacillus caldoproteolyticus Bacillus amyloliquefaciens (non-GM and GM)

Deuterolysin

EC 3.4.24.39

Aspergillus oryzae, A. wentii

Asparaginase

EC 3.5.1.1

Aspergillus niger (GM), A. oryzae (GM)

Saccharomyces cerevisiae

(Ferrara et al., 2006)

85.6 U/mL

Glutaminase

EC 3.5.1.2

Aspergillus niger, Bacillus sp.

Urease Protein-glutamine glutaminase AMP deaminase

EC 3.5.1.5 EC 3.5.1.44

Lactobaillus fermentum Chryseobacterium proteolyticum Aspergillus melleus, A. oryzae

Lyases Pectate lyase

EC 4.x.x.x EC 4.2.2.2

Alginate lyase

EC 4.2.2.3

54 U/mL 0.041 g/L 0.2 g/L

Acetolactate decarboxylase

EC 4.1.1.5

Serratia marcescens

(Zhang et al., 2013) (Mertens and Bowman, 2011) (Wang et al., 2013)

Pectin lyase

EC 4.2.2.10

Penicillium purpurogenum

(Pérez-Fuentes et al., 2014)

1.16 U/mL 0.245 g/L

Isomerases Glucose isomerase

EC 5.x.x.x EC 5.3.1.x

Xylose isomerase

EC 5.3.1.5

Glucose-6phosphate isomerase

EC 5.3.1.9

EC 3.5.4.6

Milk and cheese, others Others Meat and fish, protein hydrolysis Protein hydrolysis Bakery, beverages, meat and fish, flour processing, protein hydrolysis Beverages, protein hydrolysis Potato products, bakery, fruits and vegetables, starch processing, others Protein hydrolysis Others Production of seasonings Yeast extract

Bacillus subtilis

Beverages

Bacillus subtilis

Sphingobacterium multivorum Bacillus amyloliquefaciens (GM), Saccharomyces cerevisiae (GM) Aspergillus niger (non-GM and GM), A. sojae, Penicillium funiculosum, Rhizopus oryzae, Trichoderma longibrachiatum (GM)

Processing of alginates Beverages

Rhizopus oryzae

Actinoplanes missouriensis, Bacillus coagulans, Streptomyces murinus, S. olivochromogenes Streptomyces murinus, S. olivochromogenes, S. rubiginosus (non-GM and GM) Streptomyces violaceoniger

of prominent examples are highlighted here. So far, many proteins used in the food and feed industry have been expressed in P. pastoris, but there are still several important proteins remaining (cf. Table 4). Some of these proteins have been expressed in other yeasts like S. cerevisiae, Yarrowia lipolytica or in the ascomycete Trichoderma reesei (e.g. Xylose isomerase, Mucorpepsin or Endothiapepsin).

Beverages

Sugar production

Sugar production

Sugar production

11.1. Amylase Due to the increasing availability of specific enzymes, more and more biotechnological processes replace common chemical processes. During the 19th century sugar syrup was produced by boiling starch with acids such as sulphuric acid. In the 20th century enzymes rapidly replaced this process. In the 1970s a process was

S.C. Spohner et al. / Journal of Biotechnology 202 (2015) 118–134

developed to produce table sugar from maize (High Fructose Corn Sugar). The usage of enzymes emerged as the method of choice due to greater specificity and mild use-conditions. Today, large quantities of starch are converted to useful sweeteners, which are used in beverages, baking, jams and many other foods applications. Amylases have become one of the most important industrial enzymes (Guzmán-Maldonado and Paredes-López, 1995). Amylases can be used in the detergent, textile, paper, and pharmaceutical industries (Akoh et al., 2008; Kirk et al., 2002; Sivaramakrishnan et al., 2006). Starch and similar carbohydrates can be degraded by ␣-Amylases (EC 3.2.1.1) by the endohydrolysis of their (1-4)-␣-dglucosidic bonds. The majority of ␣-amylases are metalloenzymes and thus require ions like Ca2+ . To maximise the performance and adapt its specific needs to the processing industry, several amylases were engineered through directional evolution (Kelly et al., 2009). Using DNA-shuffling techniques, the thermostability of amylase was enhanced (Tang et al., 2006). The baking industry and consumers might benefit from genetically optimized starchmodifying enzymes. The production of ␣-Amylase with P. pastoris yields 0.125 mg/L with an activity of 68 U/mL (Liu et al., 2012). 11.2. Bromelain Bromelain is a plant protease isolated from pineapple (Ananas comosus). This protease is derived from the stem and fruit. While ananain (EC 3.4.22.31) and stem bromelain (EC 3.4.22.32) are extracted from pineapple stems, fruit bromelain (EC 3.4.22.33) is derived mainly from the fruit juice (Rowan et al., 1990). Similar proteases are also present in pineapple core, crown, leaves and peel. The highest proteolytic activity and protein contents were detected in the extract of pineapple crown (Ketnawa et al., 2010). Due to its strong proteolytic activity, bromelain is of wide interest for numerous applications including: tenderization, bakery, protein hydrolysis, enzymatic browning inhibition, and animal feed, as well as application within the cosmetic and textile industries (Arshad et al., 2014). In the food industry, bromelain has been utilised to produce hypoallergenic flour that is suitable for wheat-allergic patients (Tanabe et al., 1996; Watanabe et al., 2000). Bromelain can hydrolyse peptide bonds near proline residues, which degrades the epitope structure of gluten that consists of two major proteins, gliadin and glutenin. Continuous interest in bromelain has motivated many researchers to extract the enzyme from pineapple juice and waste leftover from the pineapple processing industry (core, crown, leaves and peel). The by-products are rich in bromelain, but still underutilised. The extraction of bromelain from plant material is difficult due to low extraction efficiency and denaturation by oxidation. On account of the low concentration of bromelain, which is about 0.015 U/mL (Leite and Lima, 2012), the protein concentration has to be increased during the process of protein purification. Besides precipitation, adsorption, reverse micelle extraction and chromatography, ultrafiltration is the method of choice (Arshad et al., 2014). Owing to problems with the purification and applicability of bromelain, it is desirable to provide pure Bromelain as well as chemically stable proteins, which can be included in pharmaceutical and nutritional compositions. These problems can be solved by providing heterologously expressed bromelain. It has been shown that the recombinant production of pineapple proteases with P. pastoris generates more stable proteins when compared to native bromelain (Mueller et al., 2009).

127

(Vandamme and Derycke, 1983). It can be found in Jerusalem artichoke (Helianthus tuberosus), chicory (Cichorium intybus), dahlia (Dahlia pinnata) and dandelion (Taraxacum officinale) (Gupta and Kaur, 1997; Trojanová et al., 2004). Since Inulin is abundantly available in nature, it is a suitable substrate for the production of fructooligosaccharides (FOS) and high fructose syrup, which can be used in the food, beverage and pharmaceutical industries. Due to their prebiotic properties, fructooligosaccharides are of extraordinary importance as functional food ingredients. Inulinases (EC 3.2.1.7) cleave at the ␤-2,1 linkage of inulins yielding fructose and glucose and can be divided into exoinulinase and endoinulinase (Roberfroid, 2000). Exoinulinase hydrolyses at the terminal fructose from the non-reducing end of the inulin molecule. Endoinulinase hydrolyses the internal linkages of inulin and produces inulotriose, inulotetraose, and inulopentose as essential products (Chi et al., 2009). Recently P. pastoris was used to produce high levels of recombinant endoinulinase from Aspergillus niger (He et al., 2014). The usage of a codon optimized version of the gene resulted in the secretion of recombinant endoinulinase activity that reached 1.3 U/mL, which is 4.18 times that observed in expressions with the native gene. The protein concentration in the supernatant increased from 0.7 to 2.21 g/L with codon optimisation (He et al., 2014). In an 8 h biotransformation at 50 ◦ C and pH 6.0 with 400 g/l inulin and an enzyme concentration of 40 U per 1 g of substrate, the FOS yield was about 91%. 11.4. Laccase Laccases (EC 1.10.3.2) can be used to oxidise phenolic substrates, aromatic amines or polycyclic aromatic hydrocarbons (Johannes and Majcherczyk, 2000). They occur in bacteria, insects, and plants (Arakane et al., 2005; Claus, 2004; Gavnholt and Larsen, 2002), but most laccases are of fungal origin (Hoegger et al., 2006). Due to its broad substrate range, the industrial usage is widespread. Up to now there are only few commercial applications of laccase, with most of its applications being in the textile industry (Osma et al., 2010). Laccases have been evaluated for use in the food and feed industry for different applications, such as stabilisation of beverages, off-flavour reduction and improvement of wheat dough (Kirk et al., 2002; Osma et al., 2010; Rodríguez Couto et al., 2006). Microbial conversion of phenolic compounds in wine or even in the cork stoppers can cause off-flavours. Suberase, a commercial laccase from Novozymes, is used to polymerise phenolic compounds in the cork that can be precursors for malodours. The application of laccase in the production of apple juice can reduce off-flavour caused from phenolic compounds like 2,6-dibromophenol, guaiacol, and ␣-terpineol (Schroeder et al., 2008). In the baking industry, laccase is applied in combination with xylanases or proteases to improve the dough quality and to increase the loaf-specific volume of oat flour-based bread (Flander et al., 2008; Renzetti et al., 2010). So far, different Laccases have been expressed in P. pastoris (Kittl et al., 2012; Bohlin et al., 2006; Hong et al., 2002; Soden et al., 2002). The production of recombinant Botrytis aclada Laccase with P. pastoris yield 51 U/mL and a protein concentration of 0.495 g/L (Kittl et al., 2012). The production of a recombinant, blood-tolerant, thermostable laccase from Basidiomycete sp. PM1 (Mate et al., 2013b), with P. pastoris under the control of the AOX1 promoter, yield 3.2 U/mL. To adapt this protein, the gene was first subjected to in vitro evolution for functional expression in S. cerevisiae and activity in human blood (Mate et al., 2010, 2013a).

11.3. Inulinase

11.5. Lactase

Inulin is a plant polyfructan consisting of linear ␤-(2,1)linked fructose chains attached to a terminal sucrose molecule

Lactase (EC 3.2.1.23) occurs naturally in the intestinal tract in sufficient quantities. This enzyme converts the milk sugar in

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dairy products to glucose and galactose. Biotechnologically produced enzymes are applied to produce “lactose-free” nutrition. From a technological point of view, the milk sugar is hydrolysed to achieve an increase in sweetness and a reduction in the susceptibility of facilitating crystallization during spray drying of milk and whey (Burin et al., 2004; Sabioni and Pinheiro, 1984). Commercial enzymes are derived mainly from fungi like Kluyveromyces sp. and Aspergillus sp. (Panesar et al., 2006). Although many enzymes are commercially available, new efforts are still being made using recombinant engineering techniques (Wu et al., 2013) and new enzymes are being discovered by screening metagenome databases. In industrial processes for the hydrolysis of milk or whey products, lactase is applied by immobilisation onto carriers, such as alginate, cellulose or other polymers (Czermak et al., 1990; Czermak, 1992; Panesar et al., 2010a). Using appropriate immobilisation methods, the conversion rates of lactose can reach 95% (Roy and Gupta, 2003). The production of lactase with Kluyveromyces lactis yields an activity of 18 U/mL (4.3 g/L) (Dagbagli and Goksungur, 2008), whereas the recombinant production of lactase with P. pastoris under the control of the AOX1 promoter yields 9500 U/mL (22 g/L) (Katrolia et al., 2011a). 11.6. Lipase In the food industry, Lipases (3.1.1.x) contribute to the distinctive flavour development in cheese production to produce characteristic flavours, which range from the distinct flavours of blue and Roquefort cheeses to the piquant flavour typical of Romano. Additionally, the formation of trans fatty acids (TFAs) during fat hardening can be avoided. Naturally, TFAs occur in meat and milk. Due to the fact that the consumption of TFAs increases the risk of coronary heart diseases, the concentrations of TFAs in food products should be reduced (Filip et al., 2010). For example, various lipases have been applied to the production of table margarine from fat-oil blends. For the transesterification of fat blends of palm stearin and vegetable oil, the lipases from Thermomyces lanuginosus, Rhizomucor miehei and of Pseudomonas sp. were applied (Ming et al., 1999; Zhang et al., 2001). The activity of T. lanuginosa lipase in culture supernatant, for example, was found to be about 0.3 U/mg. The cloning and expression of Aspergillus tamarii lipase gene in P. pastoris, under control of the AOX1 promoter, yields 20 U/mL in the supernatant of shake flasks (Shi et al., 2010). 11.7. Phytase The major form of phosphorus in plant-based feeds is phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate). Simple-stomached species such as pigs (Lenis and Jongbloed, 1999; Pallauf and Rimbach, 1997), poultry (Selle and Ravindran, 2007) and fish (Cao et al., 2007; Kumar et al., 2012) require extrinsic phytases to liberate inorganic phosphate. Additionally, phytase are used in the food industry to improve the nutritional value of cereal food products (Kumar et al., 2010). Phytases are phosphatases that are able to hydrolyse O–P bonds. Consequently, this enzyme is supplemented in these species’ diets to decrease their phosphorus excretion. Phytases have emerged as one of the most effective and lucrative feed additives. According to the attack on the hexaphosphoric ester, phytases are divided in two groups; 3-phytase (EC 3.1.3.8, myo-inositol-hexakisphosphate 3-phosphohydrolase) and 4-phytase (EC 3.1.3.26, myo-inositolhexakisphosphate 4-phosphohydrolase), which release the phosphate at the corresponding position of the inositol ring (cf. Fig. 1). The most commercial phytases are recombinant proteins produced in ascomycetes (AMFEP, 2014; Haefner et al., 2005). The

Fig. 1. Hydrolysis of phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate) by 3phytase and 4-phytase.

origin of the phytase genes varies between different phyla including: ascomycetes and basidiomycetes. In addition, the cultivation of phytase producing filamentous fungi has also been studied (Awad et al., 2014; Roopesh et al., 2006). Also, transgenic plants, such as maize, rice, soybean, and wheat have been used to produce phytase (Chen et al., 2008b; Gao et al., 2007; Hamada et al., 2014). The Escherichia coli phytase, AppA2 has been expressed and secreted in three inducible yeast systems: S. cerevisiae, Schizosaccharomyces pombe and P. pastoris. During 8-day batch fermentation in shaking flasks, P. pastoris produced the highest activity (272 U/mL), whereat the AppA2 phytase expressed in S. pombe had the lowest Km for sodium phytate and the highest heat-stability at 65 ◦ C (Lee et al., 2005b). The production of modified Aspergillus niger phytase with P. pastoris yielded 865 U/mL and 6.1 g/L purified phytase (Xiong et al., 2005). 11.8. Rhamnosidase The fruit industry has had to deal with wide varieties of raw material. While trying to maintain organoleptic quality and stability of the finished product, fruits have to be processed at the lowest possible cost. To improve pulp washing, increase the recovery yield of essential oils, and to debitter and clarify orange and grapefruit juices, the enzyme ␣-l-Rhamnosidase (EC 3.2.1.40) is applied (Aehle, 2007; Godfrey and West, 1996; Whitaker, 1974). Naringin, limonin, and neohesperidin are bitter compounds present in citrus juices (Kefford, 1970; Marwaha et al., 1994). The bitterest component is Naringin with a taste threshold in water of approximately 20 ppm. For a long time, the bitterness of grapefruit juices has been a major limitation in the commercial acceptance. The naringin level can be reduced by technologies such as adsorptive debittering, chemical methods, treatment with polystyrene divinyl benzene styrene (DVB) resins and ␤-cyclodextrin treatment. Acid hydrolysis, for example, produces not only rhamnose and glucose from naringin, but also naringenin, a very bitter aglycon. The usage of ␣-l-Rhamnosidases enabled the specific debittering of juices (cf. Fig. 2). The production of ␣-l-Rhamnosidase with Aspergillus sp. yields relative activities about 0.25 U/mL (Abbate et al., 2012). The production of recombinant secreted A. terreus ␣-l-Rhamnosidase with P. pastoris under the control of the AOX1 promoter yields 8.5 U/mL in shake flasks. In a later project, the production of this recombinant ␣-l-rhamnosidase in a high-cell-density fermentation resulted in yields of up to 0.627 U/mL/h (Winter et al., 2013). Recently, we used ␣-l-Rhamnosidase from A. terreus to trim rhamnose from Dulcoside A (cf. Fig. 3) to improve the taste quality of steviol glycoside mixtures (Spohner et al., 2014). The constitutive production of secreted recombinant ␣-l-Rhamnosidase in

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Fig. 2. Conversion of Naringin to Prunin and Rhamnose catalysed by ␣-l-Rhamnosidase.

Fig. 3. Conversion of Dulcoside A to Rubusoside catalysed by ␣-l-Rhamnosidase.

shake flasks under the control of the GAP promoter yields 10 U/mL (0.2 g/L) in shake flasks. 12. Conclusion While S. cerevisiae is still the prevalent yeast species in protein production processes, and is the best genetically characterized eukaryotic organism of all, for quite some time the most frequently used yeast species for heterologous protein expression is P. pastoris (Schmidt, 2004). P. pastoris can be grown cheaply and rapidly even to high-cell-densities over 400 g/L. It offers the advantage of being able to do complex post-translational modifications, secrete more efficiently than other yeasts, and being neither pyrogenic nor pathogenic. Due to the possible production of a wide range of recombinant proteins in P. pastoris, and the fact that it is considered to be superior to any other known yeast species in secretion efficiency (Schmidt, 2004), it is an attractive expression platform. By employing different secretion signals (Vadhana et al., 2013) or mutating S. cerevisiae ␣-Mating Factor (Lin-Cereghino and Lin-Cereghino, 2007), there is still potential to increase secretion yields. Additionally, knockdown or knockout of undesired genes can decease proteolytic decay, resulting in increased product quality and process performance. Well-developed tools for strain engineering, such as marker-free integration and deletion of desired genes, will provide a powerful set of engineered designer host strains in the near future. The increased use of P. pastoris expression platforms to achieve high-level protein expression demonstrated the existence of certain limitations. The typical process design for P. pastoris is high cell density fermentation. Here the particular growth conditions deviate far from the natural environment. In induced expression

processes, the varying methanol concentrations cause high osmolality; also pH and temperature controls are typical stress factors. Additionally, the overexpression of many protein products can lead to severe stress on the host cell, limiting the potential yield. A more detailed understanding of the physiological responses to such stresses has already led to engineered host cells with stress responses, including the unfolded protein response (UPR) pathway (Guerfal et al., 2010) which has the aim to improve the folding and secretion of a product. To replace methanol in industrial scale fermentations, new effective alternatives for induction have to be found (Delic et al., 2013; Prielhofer et al., 2013; Stadlmayr et al., 2010). The glycoengineering of P. pastoris strains to produce short homogeneous or even humanised glycosylation pattern of recombinant glycoproteins has been a great benefit. This approach greatly improves the expression system and makes it an attractive platform for the production of proteins for pharmaceutical applications and enables the correct assembling of dimeric and oligomeric proteins. In some cases, dimerization is crucial for the activity. Since the bulky high-mannose-type N-glycan can result in an increased molecular weight by 30 kDa or more, these structures are often a steric hindrance for the formation of enzyme complexes or the interaction of proteins with the target receptors (Ito et al., 2010). As shown in Table 4, many of the relevant proteins in food technology can be produced with recombinant P. pastoris strains. Due to their great performance in recombinant protein secretion and the low levels of secreted host cell protein, the recombinant protein can easily be prepared from the supernatant. Thus, the preparation of high quality enzyme solutions with special abilities for the use in the food and feed industry can be realised with P. pastoris and achieved with a higher efficiency of labour and effort.

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Expression of enzymes for the usage in food and feed industry with Pichia pastoris.

The methylotrophic yeast Pichia pastoris is an established protein expression host for the production of industrial enzymes. This yeast can be grown t...
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