Recombinant Protein Secretion in<i>Pseudozyma flocculosa</i>and<i>Pseudozyma antarctica</i>with a Novel Signal Peptide

Cheng, Yan; Avis, Tyler J.; Bolduc, Sébastien; Zhao, Yingyi; Anguenot, Raphaël; Neveu, Bertrand; Labbé, Caroline; Belzile, François; Bélanger, Richard R.

doi:10.1271/bbb.80340

Cited by 5 publications

(2 citation statements)

References 26 publications

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“…In P. flocculosa , random mutations were accomplished by insertional mutagenesis and screened for their antagonistic properties (Cheng et al , 2003). Recently, development of the gene expression vectors and the procedure for transformation in the genus Pseudozyma were reported (Morita et al , 2007a; Avis et al , 2005, 2008; Cheng et al , 2008). Further genetic study of the genus Pseudozyma should enable us not only to understand the biological function of MELs but also to facilitate the practical use of the glycolipids on an industrial scale.…”

Section: Discussionmentioning

confidence: 99%

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

Morita

Ito

Kitamoto

et al. 2010

Yeast

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The yeast Pseudozyma antarctica produces a large amount of glycolipid biosurfactants known as mannosylerythritol lipids (MELs), which show not only excellent surfaceactive properties but also versatile biochemical actions. To investigate the biosynthesis of MELs in the yeast, we recently reported expressed sequence tag (EST) analysis and estimated genes expressing under MEL production conditions. Among the genes, a contiguous sequence of 938 bp, PA 004, showed high sequence identity to the gene emt1, encoding an erythritol/mannose transferase of Ustilago maydis, which is essential for MEL biosynthesis. The predicted translation product of the extended PA 004 containing the two introns and a stop codon was aligned with Emt1 of U. maydis. The predicted amino acid sequence shared high identity (72%) with Emt1 of U. maydis, although the amino-terminal was incomplete. To identify the gene as PaEMT1 encoding an erythritol/mannose transferase of P. antarctica, the gene-disrupted strain was developed by the method for targeted gene disruption, using hygromycin B resistance as the selection marker. The obtained PaEMT1 strain failed to produce MELs, while its growth was the same as that of the parental strain. The additional mannosylerythritol into culture allowed PaEMT1 strain to form MELs regardless of the carbon source supplied, indicating a defect of the erythritol/mannose transferase activity. Furthermore, we found that MEL formation is associated with the morphology and low-temperature tolerance of the yeast.

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Section: Discussionmentioning

confidence: 99%

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

Morita

Ito

Kitamoto

et al. 2010

Yeast

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“…Drug‐resistance genes have been used as transformation markers in Pseudozyma yeast. For example, the hygromycin resistance gene consisting of an E. coli hygromycin phosphotransferase gene and Ustilago maydis HSP70 promoter and terminator has been used to introduce recombinant green florescent protein genes into P. flocculosa and P. antarctica (Cheng et al ., ). The construct was also used to disrupt a gene essential for MEL synthesis (Pa EMT1 ) in P. antarctica T‐34 using homologous recombination (Morita et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

Yarimizu

Shimoi

Sameshima–Yamashita

et al. 2017

Yeast

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The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 μg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley& Sons, Ltd.

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Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme

Masaki

Tsuchioka

Hirano

et al. 2011

Appl Microbiol Biotechnol

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Yeast host-vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host-vector system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host-vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5 times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately 15 times greater than that of the wild type.

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Recombinant Protein Secretion inPseudozyma flocculosaandPseudozyma antarcticawith a Novel Signal Peptide

Cited by 5 publications

References 26 publications

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

Identification of the gene PaEMT1 for biosynthesis of mannosylerythritol lipids in the basidiomycetous yeast Pseudozyma antarctica

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

Construction of a new recombinant protein expression system in the basidiomycetous yeast Cryptococcus sp. strain S-2 and enhancement of the production of a cutinase-like enzyme

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