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Heterologous Protein Expression in Pichia Thermomethanolica

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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 (Bo¨er 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 (Bo¨er 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-man- nose 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 mod- ification 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 contain- ing 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
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