Tools for the genetic manipulation of<i>Zygosaccharomyces rouxii</i>

Přibylová, Lenka; Montigny, Jacky de; Sychrová, Hana

doi:10.1111/j.1567-1364.2007.00308.x

Cited by 24 publications

(36 citation statements)

References 36 publications

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“…The primers (obtained from Sigma-Aldrich) used for the cassette amplifications are listed in Table S1 in the supplemental material (ZrFFZ1-Kan-F and ZrFFZ1-Kan-R for ZrFFZ1; ZrFFZ2-Kan-F2 and ZrFFZ2-Kan-R2 for ZrFFZ2), and the pUG6 plasmid (21) was used as a template. The pZCRE plasmid expressing the Cre recombinase was used to remove the integrated kanMX marker (20).…”

Section: Methodsmentioning

confidence: 99%

“…XM_002499318, GeneID 8204919) and ZrFFZ2 (ZYRO0F02090g; GenBank accession no. XM_ 002497242, GeneID 8205043) genes in strain UL4 (a ura3 auxotrophic strain derived from CBS 732 T ) (19) was performed with PCR-amplified loxP-kanMX-loxP deletion cassettes (20). The primers (obtained from Sigma-Aldrich) used for the cassette amplifications are listed in Table S1 in the supplemental material (ZrFFZ1-Kan-F and ZrFFZ1-Kan-R for ZrFFZ1; ZrFFZ2-Kan-F2 and ZrFFZ2-Kan-R2 for ZrFFZ2), and the pUG6 plasmid (21) was used as a template.…”

Section: Methodsmentioning

confidence: 99%

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The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732^T

et al. 2014

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Zygosaccharomyces rouxii is a fructophilic yeast that consumes fructose preferably to glucose. This behavior seems to be related to sugar uptake. In this study, we constructed Z. rouxii single-, double-, and triple-deletion mutants in the UL4 strain background (a ura3 strain derived from CBS 732 T ) by deleting the genes encoding the specific fructose facilitator Z. rouxii Ffz1 (ZrFfz1), the fructose/glucose facilitator ZrFfz2, and/or the fructose symporter ZrFsy1. We analyzed the effects on the growth phenotype, on kinetic parameters of fructose and glucose uptake, and on sugar consumption profiles. No growth phenotype was observed on fructose or glucose upon deletion of FFZ genes. Deletion of ZrFFZ1 drastically reduced fructose transport capacity, increased glucose transport capacity, and eliminated the fructophilic character, while deletion of ZrFFZ2 had almost no effect. The strain in which both FFZ genes were deleted presented even higher consumption of glucose than strain Zrffz1⌬, probably due to a reduced repressing effect of fructose. This study confirms the molecular basis of the Z. rouxii fructophilic character, demonstrating that ZrFfz1 is essential for Z. rouxii fructophilic behavior. The gene is a good candidate to improve the fructose fermentation performance of industrial Saccharomyces cerevisiae strains.T he most problematic spoilage yeasts in the food and beverage industry belong to the genus Zygosaccharomyces. Zygosaccharomyces bailii and Zygosaccharomyces rouxii are the main spoilage yeasts of sugar syrups, fruit juices, honey, and sweet sauces, due to their unique properties, such as high resistance to weak-acid preservatives, extreme osmotolerance (some Z. rouxii strains are able to grow on 90% [wt/vol] glucose), ability to vigorously ferment hexoses (which can cause swelling or bursting of packaging, due to gas accumulation), ability to adapt to high temperatures, and growth at low pH (1, 2). Z. rouxii is also industrially used in the fermentation of some salted condiments, such as soy sauce and miso paste, and in the production of balsamic vinegar (3, 4).Z. rouxii and Z. bailii are fructophilic yeasts, meaning that they consume fructose faster than glucose, a behavior opposite to that of the glucophilic Saccharomyces cerevisiae, which prefers glucose over fructose (5). In 1956, Sols (6) showed that phosphorylation of sugars was not involved in fructophily of a "sauternes yeast" (probably Z. bailii [7]), suggesting that another step preceding phosphorylation was the main regulator of the fructophilic behavior. This was further supported by later studies that indicated that hexokinase activities were similar in Z. bailii and S. cerevisiae and that sugar transport should be involved in Z. bailii fructophily (5).The transport across the plasma membrane is the first step at which the utilization of nutrients by cells can be controlled and regulated in response to extracellular conditions and intracellular requirements. The majority of sugar transporters belong to the sugar porter (SP) family, t...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732^T

et al. 2014

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“…The primers that were used in the gene disruption are indicated in Table S1. The removal of the G418 and Zeocin resistance markers from the genomes were performed as previously described (Pribylova et al 2007b) with little modification. The cells were transformed with pZCRE, and the transformants were grown selectively on SCD ura -plates for 2 days and then nonselectively in liquid YPD supplemented with 0.17% uracil (YPDU) for 24 hr.…”

Section: Strains Media and Genetic Methodsmentioning

confidence: 99%

“…The specific signal from URA3 coded on the P subgenome was detected at 2.2 kb in the BglIIdigested ATCC46251 genome. The band was recovered and cloned into the same site of pZEAK, which was constructed from pZEU (Pribylova et al 2007b) by removing the URA3 marker genes and disrupting the BamHI recognition site derived from pSRI. S. cerevisiae BY4743 (a uracil auxotrophic mutant) was transformed with the resulting plasmid and selected on an SCD ura -plate.…”

mentioning

confidence: 99%

“…The Z3 genomic DNA was partially digested with Sau3AI, and the resulting fragments were separated by agarose gel electrophoresis. The 6-to 9-kb DNA fragments were recovered using a PCR and Gel Purification Kit (Promega, Fitchburg, WI) and cloned into the BamHI site of pZEUneo, which was constructed from pZEU (Pribylova et al 2007b) by disrupting the BamHI recognition site derived from pSRI. Escherichia coli DH5a cells were transformed with the resulting plasmids, and the transformants were selected by Luria broth medium containing carbenicillin.…”

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Adaptation of the Osmotolerant Yeast Zygosaccharomyces rouxii to an Osmotic Environment Through Copy Number Amplification of FLO11D

2013

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Copy number variations (CNVs) contribute to the adaptation process in two possible ways. First, they may have a direct role, in which a certain number of copies often provide a selective advantage. Second, CNVs can also indirectly contribute to adaptation because a higher copy number increases the so-called "mutational target size." In this study, we show that the copy number amplification of FLO11D in the osmotolerant yeast Zygosaccharomyces rouxii promotes its further adaptation to a florformative environment, such as osmostress static culture conditions. We demonstrate that a gene, which was identified as FLO11D, is responsible for flor formation and that its expression is induced by osmostress under glucose-free conditions, which confer unique characteristics to Z. rouxii, such as osmostress-dependent flor formation. This organism possesses zero to three copies of FLO11D, and it appears likely that the FLO11D copy number increased in a branch of the Z. rouxii tree. The cellular hydrophobicity correlates with the FLO11D copy number, and the strain with a higher copy number of FLO11D exhibits a fitness advantage compared to a reference strain under osmostress static culture conditions. Our data indicate that the FLO gene-related system in Z. rouxii has evolved remarkably to adapt to osmostress environments.A N organism adapts to adverse environments to survive and produce progeny. Adaptive evolution is the process through which a population becomes better suited to its environment via mutation, selection, and random drift (Bürger 2000;Cressman 2003;Ewens 2004;Nowak and Sigmund 2004). Types of mutations include nucleotide changes (Steiner et al. 2007;Barrick and Lenski 2009), transposition events (Wilke and Adams 1992;Aminetzach et al. 2005), and copy number variations (CNVs), including gene amplifications (duplication) and deletions (Brown et al. 1998;Perry et al. 2007;Gresham et al. 2010). CNVs can have a phenotypic impact and can consequently alter the fitness of an allele through the following mechanisms: (i) changing the coding sequence of a gene (Yamanaka et al. 2009;Schlattl et al. 2011), (ii) creating paralogs that can diverge from each other and take on new or specialized functions (neofunctionalization or subfunctionalization, respectively) (Ohno 1970;Innan and Kondrashov 2010), and (iii) altering the expression level of a gene (gene dosage effect) (Stranger et al. 2007;Yamanaka et al. 2009;Iskow et al. 2012). In humans, most CNVs overlapping genes are under purifying (negative) selection (Conrad et al. 2010), but a handful of CNVs are thought to be under positive selection (Cooper et al. 2007;Hurles et al. 2010;Iskow et al. 2012) such as AMY1 (Perry et al. 2007). In yeast, there are several examples of how the (increased) copy number can provide a direct selective advantage (Gresham et al. 2008;Voordeckers et al. 2012).In nature, budding yeast exhibits a number of adaptive responses, such as filamentation, invasive growth, flocculation, and biofilm formation, to overcome a deleterious enviro...

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Current awareness on yeast

2008

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Reviews; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals ‐ search completed 5th. Mar. 2008)

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Tools for the genetic manipulation ofZygosaccharomyces rouxii

Cited by 24 publications

References 36 publications

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732^T

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732^T

Adaptation of the Osmotolerant Yeast Zygosaccharomyces rouxii to an Osmotic Environment Through Copy Number Amplification of FLO11D

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Tools for the genetic manipulation ofZygosaccharomyces rouxii

Cited by 24 publications

References 36 publications

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732T

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732T

Adaptation of the Osmotolerant Yeast Zygosaccharomyces rouxii to an Osmotic Environment Through Copy Number Amplification of FLO11D

Current awareness on yeast

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The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732^T

The High-Capacity Specific Fructose Facilitator ZrFfz1 Is Essential for the Fructophilic Behavior of Zygosaccharomyces rouxii CBS 732^T