Heterologous Protein Expression in Pichia Thermomethanolica

Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein Sutipa Tanapongpipat1, Peerada Promdonkoy1, Toru Watanabe2, Witoon Tirasophon3, Niran Roongsawang1, Yasunori Chiba2 & Lily Eurwilaichitr1

1Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathum Thani, Thailand; 2Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan; and 3The Institute of Molecular Biosciences, Mahidol University, Nakhonpathom, Thailand

Correspondence: Sutipa Tanapongpipat, Abstract Bioresources Technology Unit, National Center for Genetic Engineering and This study describes Pichia thermomethanolica BCC16875, a new methylotrophic Biotechnology, National Science and yeast host for heterologous expression. Both methanol-inducible alcohol Technology Development Agency, 113 oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase Phahonyothin Road, Khlong Nueng, Khlong (GAP) promoters from Pichia pastoris were shown to drive efficient gene Luang, Pathum Thani 12120, Thailand. expression in this host. Recombinant phytase and xylanase were expressed from Tel.: + 66 2 5646700 ext. 3472; both promoters as secreted proteins, with the former showing different patterns fax: + 66 2 5646707; e-mail: [email protected] of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of Received 15 March 2012; revised 21 June cell wall mannoproteins. The major glycoprotein oligosaccharide species 2012; accepted 22 June 2012. produced from P. thermomethanolica BCC16875 is Man8-12GlcNAc2, which is Final version published online 18 July 2012. similar to that from other methylotrophs. Moreover, mannosylphosphate and a-1,6- and a-1,2-linked mannose modifications of heterologous secreted DOI: 10.1111/j.1574-6968.2012.02628.x protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial Editor: Derek Jamieson yeasts makes this microorganism an attractive host for large-scale fermentation.

Keywords N-linked glycans; Pichia thermomethanolica; thermotolerant.

with minimal secretion of host endogenous proteins Introduction (Boër et al., 2007). Yeasts are efficient hosts for heterologous protein expres- One advantage of yeast as an expression host is that it sion, and Saccharomyces cerevisiae is the best characterized performs post-translational modification similar to higher yeast host for expression of eukaryotic proteins. However, eukaryotes, including glycosylation. As many therapeutic S. cerevisiae has drawbacks, including instability of the proteins are glycosylated, their production requires the expression plasmids and low level of protein production. most appropriate system, that is mammalian cells (De These drawbacks have driven efforts to investigate other Poureq et al., 2010). However, due to the high cost of yeast species for their potential as heterologous protein production and potential of viral contamination, alterna- expression hosts, for example Yarrowia lipolytica, Kluyver- tive expression systems are needed. Yeast, therefore, is an omyces lactis and, most importantly, methylotrophic attractive host. yeasts such as Pichia pastoris, Hansenula polymorpha, Both yeast and mammalian cells share the same initial Pichia methanolica and Ogataea minuta (Boër et al., 2007; steps of N-glycosylation which occur at the cytoplasmic Chiba & Akeboshi, 2009). The advantages of methylo- site of the endoplasmic reticulum. However, after entering trophic yeasts include the ability to grow to high cell the Golgi apparatus, the process of adding outer chains density, cheap media, and secretion of target proteins between yeasts and higher eukaryotes differs. In mam-

Reproduced from FEMS Microbiol. Lett. 334: 127-134 (2012).

179 mals, N-glycans are processed to sialic acid, galactose and on Luria–Bertani agar supplemented with zeocin À fucose, whereas in yeast, mannose is the sole sugar unit (25 lgmL 1). (De Poureq et al., 2010). Yeast mannose chains contain a conserved core structure of a-1,6-mannose backbone and Transformation in P. thermomethanolica the first a-1,2-mannose branches, while the rest of the BCC16875 outer chain structure varies between species. Saccharomy- ces cerevisiae extends its core with long a-1,6-linked man- Yeast competent cells were prepared according to Faber nose residues, which are then further extended by a-1,2 (1993). To electroporate DNA into yeast cells, 1 lg of line- and a-1,3-linked mannose chains. In addition, another arized DNA was mixed with 60 lL of yeast competent cells. À type of glycan modification, phosphomannan, is also The electroporation apparatus was set at 5 kV cm 1, 400 Ω found in this yeast (Jigami & Odani, 1999). Among the and 25 lF. The cell culture was resuspended in 1 mL of methylotrophic yeasts, P. pastoris produces mannopro- YPD (1% yeast extract, 2% peptone and 2% dextrose) and teins with shorter N-glycans and negatively charged incubated at 30 °C for 1–2 h and then spread on YPD agar À mannosylphosphate oligosaccharides (Hirose et al., 2002). plate containing 100 lgmL 1 of zeocin and incubated at Hansenula polymorpha also produces glycoproteins with 30 °C for 2–3 days until colonies were observed. short a-1,6-mannose linkages elongated with a-1,2-mannose additions (Kim et al., 2004). Neither P. pastoris nor Expression of fungal enzymes in H. polymorpha contain the terminal immunogenic a-1,3- P. thermomethanolica BCC16875 linked mannose residues. As yeast post-translational modification is similar to higher eukaryotes, yeasts have been A single colony of the recombinant yeast was inoculated exploited as alternative heterologous systems for produc- in 5 mL of YPD and incubated at 30 °C overnight with tion of human-like glycoproteins (Choi et al., 2003; Kim vigorous shaking. A 10-lL aliquot of starter culture was et al., 2006; Kuroda et al., 2006; Song et al., 2007; Chiba transferred to 10 mL of BMGY (buffered glycerol-com- & Akeboshi, 2009; Ohashi et al., 2009). plex medium; Invitrogen) and the culture was grown Although methylotrophic yeast heterologous expression overnight under the same conditions. After the culture systems are well established, there is scope for improve- reached an OD600 nm of 6–10, the cells were resuspended ment, especially development of thermotolerant or ther- in 1 mL of BMMY (buffered methanol-complex medium; mophilic yeasts better suited for industrial processes. The Invitrogen) containing 3% methanol as an inducer. To methylotrophic yeast Pichia thermomethanolica BCC16875 maintain the induction, methanol was added every 24 h was shown to utilize methanol as a sole carbon source to give a final concentration of 3% (v/v). A 20-lL sample and it can tolerate a broad range of growth temperatures of the induction medium containing the secreted recom- (Limtong et al., 2005). Therefore, in this study, we fur- binant phytase from each day was analyzed by SDS- ther explored its potential as a new expression host. PAGE. For constitutive expression of enzyme, a single Recombinant enzyme was expressed in P. thermomethano- colony of recombinant yeast was inoculated into 5 mL of lica BCC16875 under the control of P. pastoris AOX1 and YPD and incubated at 30 °C overnight with vigorous GAP promoters. In addition, the N-glycosylation pattern shaking. A 40-lL starter culture was transferred to 20 mL of proteins expressed in this yeast was investigated. of YPD and the culture was grown overnight under the same conditions. A 20-lL sample of the medium containing the secreted recombinant phytase from each day was Materials and methods analyzed by SDS-PAGE. Phytase activity was determined as described by Promdonkoy et al. (2009). rPHY pro- Yeasts, plasmids and media duced from both AOX1 and GAP promoters in P. pastoris Yeast strains were obtained from the BIOTEC Culture KM71 and P. thermomethanolica BCC16875 was degly- Collection (BCC, Bioresources Technology Unit, National cosylated using PNGaseF according to the manufacturer’s Center for Genetic Engineering and Biotechnology, instructions (New England Biolabs). Thailand). Recombinant plasmid, pPICZaA-rPhyA170 (Promdonkoy et al., 2009) was used for expression of Preparation of pyridylaminated glycans and phytase under methanol induction in P. thermomethanolica analysis of mannoproteins BCC16875. To express phytase constitutively, pPICZaA- rPhyA170 was digested with EcoRI and XbaI and then Pichia thermomethanolica BCC16875 was grown in YPD at ligated into pGAPZaA (Invitrogen) which had been 20, 30 and 37 °C for 72 h. Cells were harvested and resus- digested with EcoRI and XbaI. Ligation was transformed pended in 100 mM sodium citrate buffer (pH 7.0) and into Escherichia coli DH5a. Transformants were selected autoclaved at 121 °C for 2 h. Supernatants were recovered

180 by centrifugation at 6000 g for 10 min. After three volumes strains and zeocin-sensitive, and were therefore further of ethanol were added, the pellets were collected by centri- investigated for their potential as heterologous expres- fugation at 23 000 g,4°C for 15 min. Mannoprotein pel- sion hosts. The AOX1 promoter from P. pastoris in lets were finally dissolved in distilled water. N-linked pPICZaA was first exploited for heterologous protein glycans were removed from glycoproteins (both secreted expression in these yeast strains. The recombinant plas- recombinant enzymes and extracted cell wall mannopro- mid, pPICZaA-rPhyA170 was integrated into the yeast teins) by digestion with PNGaseF according to the manu- genome by electroporation as described. However, only facturer’s instructions (Takara Bio). Oligosaccharides were one strain, identified as P. thermomethanolica BCC16875, then fluorescence-labeled with 2-aminopyridine (PA) exhibited stable transformation and integration of DNA according to the manufacturer’s instructions (Takara Bio). insert (data not shown). In addition, this strain tolerates The linkage structures were further analyzed by exogly- a wide temperature range from 10 to 37 °C (Limtong cosidase digestion using a-1,2-mannosidase (from Asper- et al., 2005). Further investigation demonstrated that this gillus saitoi; Seikagaku Corp.), jack bean a-mannosidase strain was able to grow in temperatures as high as 40 °C (Seikagaku Corp.) and b-mannosidase (from Achatina fulica; (data not shown). Seikagaku Corp.) according to the manufacturer’s instructions. Pichia thermomethanolica BCC16875 has the ability to À be transformed with efficiency of 1 9 104 CFU lg 1 DNA. Recombinant phytase (rPHY) was readily Acid treatment of P. thermomethanolica expressed from both AOX1 and GAP promoters as phosphomannan secreted functional proteins (Fig. 1a). rPHY expressed Mannosylphosphorylated oligosaccharide samples were from both systems was larger than its predicted molecu- resuspended in 0.1 M HCl and heated at 100 °C for 2 h. lar weight of 51 kDa, suggesting that the enzyme is The reaction was dried and dissolved in 50 mM Tris-HCl post-translationally modified. It should be noted that pH 9.5, 3 units of alkaline phosphatase (Takara Bio) were the level of rPHY expressed under the control of AOX1 added, and the reaction was incubated overnight at 37 °C. promoter from P. thermomethanolica BCC16875 was relatively lower than that reported from P. pastoris (Promdonkoy et al., 2009). This is unlikely to be due to High performance liquid chromatography proteolytic degradation of the recombinant protein pro- High performance liquid chromatography (HPLC) analy- duced from the new yeast strain because extracellular sis of N-linked oligosaccharides was performed using a protease activity was not detected (data not shown). TSK-gel Amide-80 column (4.6 mm inner diameter by Intriguingly, rPHY expressed from the two promoters À 15 cm; Tosoh Corp.) at a flow rate of 1.0 mL min 1 with showed different mobility patterns in SDS-PAGE. rPHY solvent A (acetronitrile) and solvent B (200 mM triethyl- produced from AOX1 showed a major molecular mass amine acetate buffer). The HPLC column was equili- (MW) of c. 66 kDa, although a small variation of sizes brated with solvent A. After injecting the sample, the still occurred. On the other hand, rPHY produced from concentration of solvent B was increased from 30% to the GAP promoter showed a higher and more heteroge- 62% over 40 min. For phosphomannan analysis, HPLC neous MW (Fig. 1a). After PNGaseF digestion to elimi- profiling was performed using a Shodex Asahipak NH2P- nate the N-linked glycan moiety, rPHY expressed in 50 4E column (4.6 mm inner diameter by 25 cm; Showa P. thermomethanolica BCC16875 from the two different À Denko K.K) at a flow rate of 1.0 mL min 1. The HPLC expression conditions exhibited the same SDS-PAGE column was equilibrated with solvent A. After sample mobility of 51 kDa (Fig. 1b). We infer from this result injection, the proportion of solvent B was increased line- that N-linked oligosaccharides were assembled on rPHY arly up to 70% over 60 min. PA-oligosaccharides were to different extents depending on the expression detected by measuring fluorescence (320 nm excitation promoter used. wavelength and 400 nm emission wavelength). The efficiency of P. thermomethanolica BCC16875 for producing heterologous proteins was also tested for expression of xylanase, a fungal non-glycosylated Results protein. It was found that xylanase was efficiently produced as secreted protein with similar mobility in Expression of recombinant fungal enzymes SDS-PAGE to that produced in P. pastoris (Ruanglek from thermotolerant yeast et al., 2007). The levels of constitutive expression of P. thermomethanolica BCC 16875 phytase and xylanase from both P. thermomethanolica Among 47 isolates of Pichia spp. available from BCC, BCC16875 and P. pastoris KM71 were comparable (0.2 À 11 were found to be rapid-growing methanol-utilizing –0.5 mg mL 1).

181 (a)

(b) Fig. 1. (a) SDS-PAGE analysis of secreted recombinant phytase from Pichia thermomethanolica BCC16875 under the control of AOX1 and GAP promoters. M is protein marker. Cultures were grown in appropriate media for 3 days and 10 lgof proteins was analyzed. (b) SDS-PAGE analysis of deglycosylated rPHY produced as secreted proteins from Pichia pastoris KM71 and Pichia thermomethanolica BCC16875 by PNGaseF. À, the samples with no addition of PNGaseF. +, rPHY digested with PNGaseF. The arrow indicates the 51-kDa deglycosylated phytase. In each lane, 2 lg of proteins was loaded.

of 1,4-b-linked core oligosaccharides, as found in all Glycosylation pattern of rPHY expressed from eukaryotes. No further conversion of other remaining AOX1 and GAP promoters by N-glycans was observed, suggesting that no additional P. thermomethanolica BCC16875 b-inkage was present in the oligosaccharides (Fig. 2c).

From the phytase amino acid sequence, eight potential The resistant oligosaccharides corresponded to Man4-7 N-glycosylation sites were predicted (Promdonkoy et al., GlcNAc2, and small peaks of larger glycans might result 2009). Glycosylation patterns of rPHY produced from from the presence of negatively charged mannose units both promoters were analyzed and compared. rPHY attached to the neutral glycan linkages, which are resis- glycosylation mainly consisted of Man8GlcNAc2 to Man12 tant to mannosidases. Similar results were obtained with GlcNAc2, as shown in peaks detected at 20–30 min reten- rPHY expressed from the AOX1 promoter (data not tion time. However, for constitutively expressed rPHY, shown). larger sized N-glycan fractions (> Man15GlcNAc2) were observed after 30 min, consistent with high molecular Analysis of P. thermomethanolica BCC16875 weight glycosylated rPHY expressed from the GAP phosphomannan promoter as detected by SDS-PAGE (Fig. 2a and b). The N-glycans from both rPHY were then digested with The N-glycans of rPHY expressed from GAP and AOX1 a-1,2-mannosidase. Large oligosaccharide structures were promoters were separated by HPLC on an NH2P-50 partially converted to Man5GlcNAc and Man6GlcNAc, column to investigate the presence of negatively charged suggesting that the outer chain oligosaccharides contained mannose residues (Fig. 3). It was clearly shown that a-1,2 mannose linkages (data not shown). Digestion with N-glycans of rPHY from both expression systems exhib- jack bean mannosidase converted most of N-glycans ited different oligosaccharide structures. N-glycans of produced from GAP to Man1GlcNAc2, although small rPHY produced from the AOX1 promoter were separated fractions of Man4-7 and larger N-glycans remained into three distinct peaks. The ﬁrst group of peaks (Fig. 2c). After digesting with b-mannosidase, the peak detected at 10–20 min corresponded to neutral glycan, corresponding to Man1GlcNAc2 was converted to give a whereas the other two possibly represent mono and peak corresponding to GlcNAc, indicating the presence di-mannosylphosphorylated glycans (Fig. 3b, retention

182 (a) (a)

(b)

(c)

Fig. 3. Mild acid treatment and alkaline phosphatase digestion of N-glycans from rPHY produced under the control of GAP (a) and AOX1 (b) promoters. The top trace represents the non-treated sample and the bottom trace represents N-glycans treated with HCl and subsequent alkaline phosphatase. HPLC analysis was performed using an NH2P-50 column, which separated oligosaccharides according to the combination of size and charge difference.

extracted from rPHY were treated with mild acid and subsequent alkaline phosphatase, which converts phos- Fig. 2. Size fractionation HPLC of N-linked oligosaccharides phorylated glycans to neutral oligosaccharides. The peaks assembled on rPHY produced under the control of GAP (a) and AOX1 corresponding to negatively charged N-glycans detected at Pichia thermomethanolica (b) promoters from BCC16875. (c) HPLC 20–50 min retention time from both rPHYs were analysis of oligosaccharides obtained from secreted rPHY under the control of GAP promoter with mannosidase digestions. Peaks completely shifted to 10 min retention time after treat- corresponding to PA-labeled oligomannose standards are also shown. ment, indicating that these samples were phosphorylated oligosaccharides (Fig. 3). time 20–50 min; Wang et al., 1997). On the other hand, N-linked oligosaccharide proﬁles of cell wall rPHY produced from the GAP promoter contained mannoproteins from P. thermomethanolica neutral glycans as a major fraction with small populations BCC16875 grown at different temperatures of negatively charged mannans (Fig. 3a). To conﬁrm that these negatively charged glycans were Pichia thermomethanolica BCC16875 is a thermotolerant of the mannosylphosphorylated type, the N-glycans yeast that can grow at temperatures from 10 °Cupto

183 tion in promoter function and gene regulatory mechanism with P. pastoris is unknown. It is possible, however, that heterologous protein expression in P. thermomethanolica could be higher when expressed under the control of a P. thermomethanolica promoter. Recombinant phytase expressed and secreted as heterologous protein in P. thermomethanolica showed different N-glycan proﬁles, depending on the promoter used to drive expression. It was clearly seen that N-glycans on rPHY expressed constitutively contained longer sugar chains than those expressed from an inducible promoter. This phenomenon was also observed in P. pastoris (data not shown). The AOX1-inducible promoter is stronger than the constitutive GAP promoter and thus the high rate of protein production from AOX1 might cause an imbalance in the glycosylation process such that the attached N-glycans on the recombinant proteins contain smaller sugar chains. Different culture media can also Fig. 4. Size fractionation HPLC of N-linked oligosaccharides of cell affect the production of N-glycans. In H. polymorpha, dif- wall mannoproteins extracted from Pichia thermomethanolica ferent glycosylation patterns were found when grown in BCC16875. Yeast cells were grown at 20, 30 and 37 °C for 72 h and rich, fast-growing or slow-growing media (So-Young N cell wall mannoproteins were extracted. -glycans were digested by et al., 2007). PNGaseF and labeled with PA. HPLC analysis was performed using an We further investigated the pattern of N-glycans Amide-80 column. The positions of Man8GlcNAc2 to Man10GlcNAc2 are indicated. assembled on the recombinant protein. After digestion with a-1,2-mannosidase, the fractions of Man6GlcNAc2 and Man5GlcNAc2 were detected, indicating the presence 40 °C (data not shown). Different growth temperatures of a-1,2 mannose linkage, which is common among yeast might affect the structure of oligosaccharides produced in glycosylated proteins (De Poureq et al., 2010). After jack the host cells. Therefore, we next investigated the bean mannosidase digestion, Man1GlcNAc2 was found N-linked sugar chain structures of cell wall mannoproteins together with large glycans longer than Man8GlcNAc2. from P. thermomethanolica BCC16875 grown at various This suggests that N-glycans produced from P. thermo- temperatures (Fig. 4). Yeast grown at 20 and 30 °C methanolica BCC16875 consist of a-1,2, a-1,3 and a-1,6 exhibited a similar pattern of N-glycan structures, in mannose linkages. However, it should be noted that which there were comparable ratios of long- and short- P. pastoris lacks a-1,3 mannosyltransferase (Trimble et al., chain N-linked mannoproteins, whereas cell wall manno- 1991). Given that two other methylotrophs, O. minuta proteins from the 37 °C culture tended to produce more and H. polymorpha, also lack a-1,3-mannose extension in short-chain N-linked glycans (Fig. 4). the outer chains (Kim et al., 2004; Kuroda et al., 2006), it is unlikely that P. thermomethanolica BCC16875 glycopro- a Discussion teins contain -1,3 mannose linkages. Nevertheless, further analysis is needed to exclude the possibility of a-1,3- Pichia thermomethanolica BCC16875 (recently renamed linked mannose structures in P. thermomethanolica. Ogataea thermomethanolica), was isolated from soil in Oligosaccharides attached to secreted recombinant pro- southern Thailand (Limtong et al., 2005, 2008). Since this teins from both AOX1 and GAP exhibited negatively strain is methylotrophic, we reasoned that P. pastoris charged properties. Although not common, negatively expression vectors would be functional. Recombinant charged N-glycans are found in some yeast strains. plasmid vectors with P. pastoris GAP and AOX1 Phosphomannoproteins are produced in S. cerevisiae, promoters driving expression of recombinant phytase O. minuta, Y. lipolytica and P. pastoris (Jigami & Odani, were integrated into the P. thermomethanolica genome 1999; Hirose et al., 2002; Kuroda et al., 2006; Park et al., and the proteins were secreted as functional enzymes, 2011). Although the functions of negatively charged although the level of protein expression was not as high mannoproteins are not fully understood, genes involved as when expressed in P. pastoris (Promdonkoy et al., in mannosylphosphate transfer are regulated in response 2009). Pichia thermomethanolica BCC16875 has not been to growth phase and are affected by environmental characterized genetically and so the degree of conserva- change (Jigami & Odani, 1999). From our study, phytase

184 produced in methanol-containing media had a higher Advanced Industrial Science and Technology (AIST), phosphomannan content, which is in line with a previous Japan, are greatly appreciated. report that different culture media affect the production of phosphorylated glycans (Montesino et al., 1999). In References S. cerevisiae, two genes involved in mannosylphosphate transfer, MNN4 and MNN6, have been identified and Ahn WS, Jeon JJ, Jeong YR, Lee SJ & Yoon SK (2008) Effect characterized and both genes are regulated by a subunit of culture temperature on erythropoietin production and of the RSC (Remodels the Structures of Chromatin) chro- glycosylation in a perfusion culture of recombinant CHO matin-remodeling complex (Conde et al., 2007). As there cells. 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