| RRC ID |
48175
|
| 著者 |
Watanabe T, Morita T, Koike H, Yarimizu T, Shinozaki Y, Sameshima-Yamashita Y, Yoshida S, Koitabashi M, Kitamoto H.
|
| タイトル |
High-level recombinant protein production by the basidiomycetous yeast Pseudozyma antarctica under a xylose-inducible xylanase promoter.
|
| ジャーナル |
Appl Microbiol Biotechnol
|
| 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.
|
| 巻・号 |
100(7)
|
| ページ |
3207-17
|
| 公開日 |
2016-4-1
|
| DOI |
10.1007/s00253-015-7232-7
|
| PII |
10.1007/s00253-015-7232-7
|
| PMID |
26695155
|
| MeSH |
Batch Cell Culture Techniques
Biodegradable Plastics / metabolism*
Biodegradation, Environmental
Bioreactors
Chromosomes, Fungal / chemistry
Chromosomes, Fungal / metabolism
Endo-1,4-beta Xylanases / genetics
Endo-1,4-beta Xylanases / metabolism*
Escherichia coli / enzymology
Escherichia coli / genetics
Fungal Proteins / biosynthesis*
Fungal Proteins / genetics
Gene Expression
Neomycin
Oxo-Acid-Lyases / genetics
Oxo-Acid-Lyases / metabolism*
Plasmids / chemistry
Plasmids / metabolism
Promoter Regions, Genetic
Protein Engineering
Recombinant Proteins / biosynthesis
Recombinant Proteins / genetics
Saccharomyces cerevisiae / enzymology
Saccharomyces cerevisiae / genetics
Transgenes
Ustilaginales / enzymology*
Ustilaginales / genetics
Xylose / metabolism*
|
| IF |
3.53
|
| 引用数 |
12
|
|
WOS 分野
|
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
| リソース情報 |
| 一般微生物 |
JCM 10317
JCM 10318
JCM 10323
JCM 10324 |
