A Combination of Flow Cytometry and Traditional Screening Using Chemicals to Isolate High Glutathione-Producing Yeast Mutants

Nishiuchi, Hiroaki; Tabira, Yukiko; Yamagishi, Kazuo

doi:10.1271/bbb.110883

Cited by 10 publications

(11 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…starkeyi DSM 70296 was not so evident than those reported in these other studies (Wang et al 2009; Nishiushi et al 2012). Further studies should be performed to adapt this strain to the conditions of fermentation of hemicelluloses hydrolysates of sugarcane bagasse, as well as to characterize metabolic changes using the tools of genomics in order to identify which genes/enzymes were modified during mutagenesis procedures.…”

Section: Discussionmentioning

confidence: 51%

“…Recent studies have employed this molecule as nutritional supplement for obtaining improvements on the native production of poly-unsaturated fatty acids by Moritella marina and Shewanella marinintestina (Morita et al 2005), as well as a selective agent in the screening of high lipid production by Rhodotorula glutinis mutants (Wang et al 2009) and high gluthatione production by Saccharomyces cereviseae and Candida utilis mutants (Nishiushi et al 2012). …”

Section: Discussionmentioning

confidence: 99%

“…cereviseae and C . utilis , authors obtained a > 100% increase in glutathione production when associating cerulenin to the screening of mutants (Nishiushi et al 2012). …”

Section: Discussionmentioning

confidence: 99%

“…starkeyi is of major importance. In such cases, it is preferred to employ methods to increase the natural rates of mutation of their DNA through the action of mutagens, such as UV light, ionizing radiation or others mutagenic agents, as already determined for other microorganisms of industrial interest (Keller et al 2004, Patnayak and Sree 2005, Wang et al 2009, Nishiuchi et al 2012). …”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening

et al. 2012

View full text Add to dashboard Cite

In this work we performed assays for the genetic improvement of the oleaginous yeast Lipomyces starkeyi DSM 70296 focusing on its utilization for lipid biosynthesis from renewable sources. The genetic optimization was carried out by random mutagenesis by ultraviolet irradiation and mutant selection by cerulenin, a compound displaying inhibitory effects on lipid biosynthesis. Mutants demonstrating normal growth in presence of cerulenin were considered as good candidates for further studies. Using this strategy, we selected 6 mutants for further studies, in which their productivities were evaluated by fermentation in shaken flasks and bioreactor. The evaluation of the fermentative performance of mutants was carried out using xylose as sole carbon source; the fermentation of wild-type strain was used as reference. Using this strategy it was possible to identify one mutant (termed A1) presenting a significant increase in the productivity rates of both biomass and lipid in comparison to wild-type strain. A1 mutant was further studied in bioreactor using the same fermentation parameters optimized for L. starkeyi lipid production from a mixed carbon source (xylose:glucose), as previously determined by other studies in our laboratory. A1 presented a productivity increase of 15.1% in biomass and 30.7% in lipid productivity when compared to the wild-type strain with a similar fatty acid composition, despite a slight increase (approx. 7%) on the unsaturated fraction. Our work demonstrates the feasibility of the random mutagenesis strategy coupled with mutant selection based on cerulenin screening for the genetic improvement of the oleaginous yeast L. starkeyi.

show abstract

Section: Discussionmentioning

confidence: 51%

Section: Discussionmentioning

confidence: 99%

“…cereviseae and C . utilis , authors obtained a > 100% increase in glutathione production when associating cerulenin to the screening of mutants (Nishiushi et al 2012). …”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening

et al. 2012

View full text Add to dashboard Cite

show abstract

“…We then verified if the stress response and delay in growth were observed as in vivo [23]. Intracellular levels of glutathione, a well-established marker of stress response in various mammalian cell types [26], fungi [27] including C. neoformans [23], were assessed kinetically by using 5-chloromethylfluorescein diacetate (CMFDA) staining at day 0 (STAT) and up to day 8 (D4, D6, D8). A decrease in stress response was observed over time with an apparently homogenous population harboring a low CMFDA intensity in 8D-HYPOx, as opposed to 8D-NORMOx for which a double population (low and high CMFDA intensity corresponding to low and high stress response) was observed (Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

Cryptococcus neoformansresist to drastic conditions by switching to viable but non-culturable cell phenotype

Hommel

Sturny-Leclère

Volant

et al. 2019

Preprint

View full text Add to dashboard Cite

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30-40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial masse increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast. Authors Summary Quiescence/dormancy in microorganism is a common feature that enables survival at the population level. In fungi, quiescence has been studied in the baker yeast Saccharomyces cerevisiae. In Cryptococcus neoformans, dormancy is of great interest since it is known from the natural history of cryptococcosis that dormancy in yeast can last decades exists before possible reactivation upon immunosuppress...

show abstract

Novel method for screening Saccharomyces cerevisiae mutants with increased sulfur-containing compounds: Color-based selection of ade1 or ade2 mutants

Tarutina

Dutova

Yezhova

et al. 2012

Journal of Bioscience and Bioengineering

View full text Add to dashboard Cite

A Combination of Flow Cytometry and Traditional Screening Using Chemicals to Isolate High Glutathione-Producing Yeast Mutants

Cited by 10 publications

References 22 publications

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening

Optimization of lipid production by the oleaginous yeast Lipomyces starkeyi by random mutagenesis coupled to cerulenin screening

Cryptococcus neoformansresist to drastic conditions by switching to viable but non-culturable cell phenotype

Novel method for screening Saccharomyces cerevisiae mutants with increased sulfur-containing compounds: Color-based selection of ade1 or ade2 mutants

Contact Info

Product

Resources

About