RRC ID 48175
Author Watanabe T, Morita T, Koike H, Yarimizu T, Shinozaki Y, Sameshima-Yamashita Y, Yoshida S, Koitabashi M, Kitamoto H.
Title High-level recombinant protein production by the basidiomycetous yeast Pseudozyma antarctica under a xylose-inducible xylanase promoter.
Journal 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.
Volume 100(7)
Pages 3207-17
Published 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
Times Cited 12
General Microbes JCM 10317 JCM 10318 JCM 10323 JCM 10324