CHEMICALLY INDUCED MUTAGENESIS IN THE KING OYSTER MUSHROOM Pleurotus eryngii TO GENERATE HIGH-TEMPERATURE TOLERANT STRAIN

In this study, a high-temperature-tolerant strain of the king oyster mushroom (Pleurotus eryngii) was generated by chemical mutagenesis. Cultivation of P. eryngii generally involves incubating the mycelia at 25°C and then moving the spawns for further incubation at 18°C for fruitification. However, in tropical countries, the temperature is a major concern in the production of oyster mushroom where the average temperature is 32°C. In the current study, the mycelia were treated with ethyl methane sulphonate (EMS) or methyl methane sulphonate (MMS) for chemical-induced mutation. Seven mutants (EMS 1, 2, 6, 26, 35, 36, and 38) from EMS mutagenesis exhibited higher growth rates than the wild-type strain at 32°C. However, mutant strains from MMS mutagenesis showed a low growth rate when compared with wild-type. On sawdust substrate, the spawn running conditions for these strains were performed at 32°C, and fruitification occurred at 18°C. The yield and biological efficiency of EMS 36 and 38 mutants were higher than those of the wild-type strain. The activities of cellulase and xylanase of EMS 36 and 38 mutants showed that both these mutants had higher activities than the wild-type strain which may influence mushroom production. Therefore, these EMS 36 and 38 mutants can be cultivated in tropical countries, which could provide a high yield and reduce the cost during spawn running step.

Disruption of YCP4 Enhances Freeze-Thaw Tolerance in Saccharomyces Cerevisiae

Objective The aim of this study was to identify genes related to a freeze-thaw tolerance and to elucidate the tolerance mechanism in yeast Saccharomyces cerevisiae as an appropriate eukaryote model. Results In this study, one tolerant strain under exposure to freeze-thaw stress was isolated by screening a transposon-mediated mutant library and the disrupted gene was identified to be YCP4. In addition, this phenotype related to freeze-thaw tolerance was comfirmed by deletion and overexpressing of this corresponding gene. This mutant strain showed a freeze-thaw tolerance by the reduction in the intracellular level of reactive oxygen species (ROS) and the activation of the MSN2/4 and STRE-mediated genes such as CTT1 and HSP12. Conclusions Disruption of YCP4 in S. cerevisiae results in increased tolerance to freeze-thaw stress.

LncRNAs of Saccharomyces cerevisiae dodge the cell cycle arrest imposed by the ethanol stress

Ethanol impairs many subsystems of Saccharomyces cerevisiae, including the cell cycle. Cyclins and damage checkpoints drive the cell cycle. Two ethanol-responsive lncRNAs in yeast interact with cell cycle proteins, and here we investigated the role of these RNAs on the ethanol-stressed cell cycle. Our network dynamic modeling showed that the higher and lower ethanol tolerant strains undergo a cell cycle arrest during the ethanol stress. However, lower tolerant phenotype arrest in a later phase leading to its faster population rebound after the stress relief. Two lncRNAs can skip the arrests mentioned. The in silico overexpression of lnc9136 of SEY6210 (a lower tolerant strain), and CRISPR-Cas9 partial deletions of this lncRNA, evidenced that the one induces a regular cell cycle even under ethanol stress; this lncRNA binds to Gin4 and Hsl1, driving the Swe1p, Clb1/2, and cell cycle. Moreover, the lnc10883 of BY4742 (a higher tolerant strain) interacts to the Mec1p and represses Bub1p, circumventing the DNA and spindle damage checkpoints keeping a normal cell cycle even under DNA damage. Overall, we present the first evidence of the direct roles of lncRNAs on cell cycle proteins, the dynamics of this system in different ethanol tolerant phenotypes, and a new cell cycle model.

Analysis of lipid composition reveals mechanisms of ethanol tolerance in the model yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae is an important unicellular yeast species within the biotechnological and food and beverage industries. A significant application of this species is the production of ethanol, where concentrations are limited by cellular toxicity, often at the level of the cell membrane. Here, we characterize 61 S. cerevisiae strains for ethanol tolerance and further analyse five representatives with varying ethanol tolerances. The most tolerant strain, AJ4, was dominant in co-culture at 0% and 10% ethanol. Unexpectedly, although it does not have the highest NIC or MIC, MY29 was the dominant strain in co-culture at 6% ethanol, which may be linked to differences in its basal lipidome. Whilst relatively few lipidomic differences were observed between strains, a significantly higher PE concentration was observed in the least tolerant strain, MY26, at 0% and 6% ethanol compared to the other strains that became more similar at 10%, indicating potential involvement of this lipid with ethanol sensitivity. Our findings reveal that AJ4 is best able to adapt its membrane to become more fluid in the presence of ethanol and lipid extracts from AJ4 also form the most permeable membranes. Furthermore, MY26 is least able to modulate fluidity in response to ethanol and membranes formed from extracted lipids are least leaky at physiological ethanol concentrations. Overall, these results reveal a potential mechanism of ethanol tolerance and suggests a limited set of membrane compositions that diverse yeast species use to achieve this. Importance Many microbial processes are not implemented at the industrial level because the product yield is poorer and more expensive than can be achieved by chemical synthesis. It is well established that microbes show stress responses during bioprocessing, and one reason for poor product output from cell factories is production conditions that are ultimately toxic to the cells. During fermentative processes, yeast cells encounter culture media with high sugar content, which is later transformed into high ethanol concentrations. Thus, ethanol toxicity is one of the major stresses in traditional and more recent biotechnological processes. We have performed a multilayer phenotypic and lipidomic characterization of a large number of industrial and environmental strains of Saccharomyces to identify key resistant and non-resistant isolates for future applications.

Complete Genome Sequence of Sinorhizobium meliloti S35m, a Salt-Tolerant Isolate from Alfalfa Rhizosphere in Soil Native to the Caucasus Region

The genome of a symbiotically effective salt-tolerant strain, Sinorhizobium meliloti S35m, isolated from alfalfa rhizosphere in soil native to the Caucasian region, was sequenced. Genomic islands, prophages, and elements of a potential CRISPR/Cas I type (Cas3_0_I) system were identified in the genome.