Development of resources for the analysis of gene function in Pucciniomycotina red yeasts

Ianiri, Giuseppe; Wright, Sandra A. I.; Castoria, Raffaello; Idnurm, Alexander

doi:10.1016/j.fgb.2011.03.003

Cited by 26 publications

(41 citation statements)

References 60 publications

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“…strain IAM 13481. The genome of this strain has been sequenced, and it can be transformed to generate mutants (35). In this study, we first demonstrate that this fungus is able to degrade patulin in a similar way to R. kratochvilovae LS11.…”

mentioning

confidence: 77%

“…IAM 13481 was used as the wild type for gene function studies. This strain has a complete genome sequence available at the Joint Genome Institute of the U.S. Department of Energy, and forward and reverse genetic tools to mutate genes were recently developed (35). The strain DH5␣ of Escherichia coli was used in a bioassay previously developed for preliminary evaluation of patulin degradation (18) in a collection of Sporobolomyces sp.…”

Section: Methodsmentioning

confidence: 99%

“…This plasmid pAIS4 contains the Sporobolomyces sp. URA5 gene between the right and the left borders of the T-DNA (35). Overnight cultures of cells of A. tumefaciens (grown in Luria Bertani plus 50 mg/liter kanamycin) and cells of the auxotroph AIS2 (grown in yeast extract-peptone-dextrose [YPD], 20 g/liter Bacto peptone, 10 g/liter yeast extract, 20 g/liter glucose) were adjusted to an optical density at 600 nm (OD 600 ) of 0.6.…”

Section: Methodsmentioning

confidence: 99%

“…Inverse PCR was used to determine regions flanking the T-DNA insertions in strains of interest as previously described (35). Briefly, 2 to 5 g of genomic DNA was digested with an individual restriction enzyme (see below) and self-ligated with T4 DNA ligase, and then the ligation reaction products were used as templates in PCR.…”

Section: Methodsmentioning

confidence: 99%

“…The 5= and 3= fragments of the VPS8 gene were amplified from genomic DNA with primer pairs GI0005-GI0002 and GI0003-GI0006, respectively, and the URA5 gene was amplified with primer pair ALID0562-ALID0564 from plasmid pAIS4 (35). The three fragments were fused together with plasmid pRS426 using the S. cerevisiae in vivo recombination system and strain FY834 (43), which were provided by the Fungal Genetics Stock Center (44).…”

Section: Methodsmentioning

confidence: 99%

See 4 more Smart Citations

Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin

Ianiri

Idnurm

Wright

et al. 2013

Appl Environ Microbiol

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f Patulin is a mycotoxin that contaminates pome fruits and derived products worldwide. Basidiomycete yeasts belonging to the subphylum Pucciniomycotina have been identified to have the ability to degrade this molecule efficiently and have been explored through different approaches to understand this degradation process. In this study, Sporobolomyces sp. strain IAM 13481 was found to be able to degrade patulin to form two different breakdown products, desoxypatulinic acid and (Z)-ascladiol. To gain insight into the genetic basis of tolerance and degradation of patulin, more than 3,000 transfer DNA (T-DNA) insertional mutants were generated in strain IAM 13481 and screened for the inability to degrade patulin using a bioassay based on the sensitivity of Escherichia coli to patulin. Thirteen mutants showing reduced growth in the presence of patulin were isolated and further characterized. Genes disrupted in patulin-sensitive mutants included homologs of Saccharomyces cerevisiae YCK2, PAC2, DAL5, and VPS8. The patulin-sensitive mutants also exhibited hypersensitivity to reactive oxygen species as well as genotoxic and cell wall-destabilizing agents, suggesting that the inactivated genes are essential for tolerating and overcoming the initial toxicity of patulin. These results support a model whereby patulin degradation occurs through a multistep process that includes an initial tolerance to patulin that utilizes processes common to other external stresses, followed by two separate pathways for degradation.

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mentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin

Ianiri

Idnurm

Wright

et al. 2013

Appl Environ Microbiol

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Highly‐efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants

Lin

Yang

Zhou

et al. 2012

Yeast

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Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplification of genes from red yeast samples remains technically challenging. Here a highly efficient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualified as PCR templates. Single-copied gDNA fragments o up to 2.8 kb were successfully amplified. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identification of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts.

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Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola

Lodens

Graeve

Roelants

et al. 2018

Methods in Molecular Biology

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In this chapter, a step-by-step approach on how to transform non-conventional yeasts or fungi into platform organisms is described. The non-conventional glycolipid producing yeast Starmerella bombicola (and in some cases also Pseudohyphozyma bogoriensis) is used as a case study. And more specifically how to engineer it toward production of new-to-nature glycolipids like bola sophorolipids. When starting genetic engineering efforts for non-lab strains, one should start at the very basis: identifying selection markers and possibly developing auxotrophic strains. Once this is done, the quest for the development of an optimal transformation method can be started. After optimization thereof, knock-out and knock-in strains can be generated based upon the specific strategy/aim. Sometimes this can lead to unexpected, but yet very interesting findings. To fully and efficiently expand the potential as a production platform of these yeast strains, a range of additional molecular tools are required. A well-equipped molecular toolbox should contain a set of characterized promotors, terminators, and defined genomic landing paths. The availability of an episomal system greatly facilitates engineering and screening efforts, but also offers the possibility of developing more advanced engineering techniques such as Crispr-Cas. InBio.be is a world leading pioneer to do this for the yeast S. bombicola and combined, these efforts will result in the commercialization of new types of glycolipids in the next few years.

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Development of resources for the analysis of gene function in Pucciniomycotina red yeasts

Cited by 26 publications

References 60 publications

Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin

Searching for Genes Responsible for Patulin Degradation in a Biocontrol Yeast Provides Insight into the Basis for Resistance to This Mycotoxin

Highly‐efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants

Transformation of an Exotic Yeast Species into a Platform Organism: A Case Study for Engineering Glycolipid Production in the Yeast Starmerella bombicola

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