title: High-level recombinant protein production by the basidiomycetous yeast Pseudozyma antarctica under a xylose-inducible xylanase promoter.
abstract: Yeast host-vector systems are useful tools for the production of recombinant proteins. Here, we report the construction of a new high-level expression plasmid pPAX1-neo for the basidiomycetous yeast, Pseudozyma antarctica. pPAX1-neo harbours a xylose-inducible expression cassette under control of the xylanase promoter and terminator of P. antarctica T-34, a selection cassette of neomycin/G418 with an Escherichia coli neomycin resistance gene under control of the homocitrate synthase promoter of strain T-34, and an autonomously replicating sequence fragment of Ustilago maydis (UARS). Biodegradable plastic (BP)-degrading enzymes of P. antarctica JCM10317 (PaE) and Paraphoma-related fungal strain B47-9 (PCLE) were used as reporter proteins and inserted into pPAX1-neo, resulting in pPAX1-neo::PaCLE1 and pPAX1-neo::PCLE, respectively. Homologous and heterologous BP-degrading enzyme production of transformants of P. antarctica T-34 were detected on agar plates containing xylose and emulsified BP. Recombinant PaE were also produced by transformants of other Pseudozyma strains including Pseudozyma aphidis, Pseudozyma rugulosa, and Pseudozyma tsukubaensis. To improve the stability of transformed genes in cells, the UARS fragment was removed from linearized pPAX1-neo::PaCLE1 and integrated into the chromosome of the P. antarctica strain, GB-4(0), which was selected as a PaE producer in xylose media. Two transformants, GB-4(0)-X14 and X49, had an 11-fold higher activity compared with the wild type strain in xylose-containing liquid media. By xylose fed-batch cultivation using a 3-L jar fermentor, GB-4(0)-X14 produced 73.5 U mL(-1) of PaE, which is 13.4-fold higher than that of the wild type strain GB-4(0), which produced 5.5 U mL(-1) of PaE.
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Research Institute for Innovation in Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5-2, 1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
authors.forename: Tomotake
authors.initials: T
authors.lastname: Morita
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Koike
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yarimizu
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan.|Research Fellow of the Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo, 102-8472, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Shinozaki
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan.|Research Fellow of the Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo, 102-8472, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Sameshima-Yamashita
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan.
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Yoshida
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan.
authors.forename: Motoo
authors.initials: M
authors.lastname: Koitabashi
authors.affiliation: National Institute for Agro-environmental Sciences (NIAES), 3-1-3 Kannondai, Tsukuba, Ibaraki, 305-8604, Japan. kitamoto@affrc.go.jp.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Kitamoto
mesh_terms: D061249:Batch Cell Culture Techniques; D063566:Biodegradable Plastics / Q000378:metabolism*; D001673:Biodegradation, Environmental; D019149:Bioreactors; D015825:Chromosomes, Fungal / Q000737:chemistry / Q000378:metabolism; D043364:Endo-1,4-beta Xylanases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000235:genetics; D015870:Gene Expression; D009355:Neomycin; D007652:Oxo-Acid-Lyases / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D019076:Transgenes; D014587:Ustilaginales / Q000201:enzymology* / Q000235:genetics; D014994:Xylose / Q000378:metabolism*
chemical_list: D063566:Biodegradable Plastics; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014994:Xylose; C021365:homocitrate synthase; D043364:Endo-1,4-beta Xylanases; D007652:Oxo-Acid-Lyases; D009355:Neomycin
keywords: Biodegradable plastic-degrading enzyme; Expression vector; Jar fermentor; Pseudozyma antarctica; Xylose-inducible promoter