Transformation and electrophoretic karyotyping of Coniochaeta ligniaria NRRL30616.

Coniochaeta ligniaria NRRL30616 is an ascomycete that grows with yeast-like appearance in liquid culture. The strain has potential utility for conversion of fibrous biomass to fuels or chemicals. Furans and other inhibitory compounds in lignocellulosic biomass are metabolized by NRRL30616, facilitating subsequent microbial fermentation of biomass sugars. This study undertook initial characterization of the genetic system of C. ligniaria NRRL30616. Transformation using hygromycin as a dominant selectable marker was achieved using protoplasts generated by incubating cells in 1% (v/v) ß-mercaptoethanol, followed by cell wall-digesting enzymes. Thirteen chromosomes with an estimated total size of 30.1 Mb were detected in C. ligniaria. The GC content of chromosomal DNA and of coding regions from cDNA sequences were 49.2 and 51.9%, respectively. This study is the first report of genome size, electrophoretic karyotype, and transformation system for a member of the Coniochaetales.

Culture nutrition and physiology impact the inhibitor tolerance of the yeast Pichia stipitis NRRL Y-7124.

Pichia stipitis NRRL Y-7124 is one of the natural yeasts best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to economically recoverable concentrations of ethanol. To test the impact of culture conditions on inhibitor tolerance, inhibitors were spiked to growing or stationary-phase P. stipitis supplied either glucose or xylose and varying nitrogen and mineral compositions; then the ensuing specific death rate response was measured. Resistance of glucose- or xylose-grown cells to inhibitors was generally greater in stationary-phase cells than log-phase cells, despite a greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products, furan methanol or furan-2,5-dimethanol, from respective spikes of furfural or HMF increased as cultures progressed into stationary phase. However, when xylose was the substrate, ethanol resistance behaved uniquely and was greater for log- than stationary-phase cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose supplied carbon. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions tested had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate was excessive. Observed tolerance optimization based on specific death rate as a function of culture physiological state, carbon source, nitrogen source and mineral composition provides new knowledge supporting process designs to convert biomass to ethanol using P. stipitis.

Importance of mitochondrial dynamics during meiosis and sporulation.

Opposing fission and fusion events maintain the yeast mitochondrial network. Six proteins regulate these membrane dynamics during mitotic growth-Dnm1p, Mdv1p, and Fis1p mediate fission; Fzo1p, Mgm1p, and Ugo1p mediate fusion. Previous studies established that mitochondria fragment and rejoin at distinct stages during meiosis and sporulation, suggesting that mitochondrial fission and fusion are required during this process. Here we report that strains defective for mitochondrial fission alone, or both fission and fusion, complete meiosis and sporulation. However, visualization of mitochondria in sporulating cultures reveals morphological defects associated with the loss of fusion and/or fission proteins. Specifically, mitochondria collapse to one side of the cell and fail to fragment during presporulation. In addition, mitochondria are not inherited equally by newly formed spores, and mitochondrial DNA nucleoid segregation defects give rise to spores lacking nucleoids. This nucleoid inheritance defect is correlated with an increase in petite spore colonies. Unexpectedly, mitochondria fragment in mature tetrads lacking fission proteins. The latter finding suggests either that novel fission machinery operates during sporulation or that mechanical forces generate the mitochondrial fragments observed in mature spores. These results provide evidence of fitness defects caused by fission mutations and reveal new phenotypes associated with fission and fusion mutations.

Phenotypic characterization and comparative transcriptomics of evolved Saccharomyces cerevisiae strains with improved tolerance to lignocellulosic derived inhibitors.

BACKGROUND: Lignocellulosic biomass continues to be investigated as a viable source for bioethanol production. However, the pretreatment process generates inhibitory compounds that impair the growth and fermentation performance of microorganisms such as Saccharomyces cerevisiae. Pinewood specifically has been shown to be challenging in obtaining industrially relevant ethanol titers. An industrial S. cerevisiae strain was subjected to directed evolution and adaptation in pretreated pine biomass and resultant strains, GHP1 and GHP4, exhibited improved growth and fermentative ability on pretreated pine in the presence of related inhibitory compounds. A comparative transcriptomic approach was applied to identify and characterize differences in phenotypic stability of evolved strains. RESULTS: Evolved strains displayed different fermentative capabilities with pretreated pine that appear to be influenced by the addition or absence of 13 inhibitory compounds during pre-culturing. GHP4 performance was consistent independent of culturing conditions, while GHP1 performance was dependent on culturing with inhibitors. Comparative transcriptomics revealed 52 genes potentially associated with stress responses to multiple inhibitors simultaneously. Fluorescence microscopy revealed improved cellular integrity of both strains with mitochondria exhibiting resistance to the damaging effects of inhibitors in contrast to the parent. CONCLUSIONS: Multiple potentially novel genetic targets have been discovered for understanding stress tolerance through the characterization of our evolved strains. This study specifically examines the synergistic effects of multiple inhibitors and identified targets will guide future studies in remediating effects of inhibitors and further development of robust yeast strains for multiple industrial applications.

Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae.

BACKGROUND: Biofuels offer a viable alternative to petroleum-based fuel. However, current methods are not sufficient and the technology required in order to use lignocellulosic biomass as a fermentation substrate faces several challenges. One challenge is the need for a robust fermentative microorganism that can tolerate the inhibitors present during lignocellulosic fermentation. These inhibitors include the furan aldehyde, furfural, which is released as a byproduct of pentose dehydration during the weak acid pretreatment of lignocellulose. In order to survive in the presence of furfural, yeast cells need not only to reduce furfural to the less toxic furan methanol, but also to protect themselves and repair any damage caused by the furfural. Since furfural tolerance in yeast requires a functional pentose phosphate pathway (PPP), and the PPP is associated with reactive oxygen species (ROS) tolerance, we decided to investigate whether or not furfural induces ROS and its related cellular damage in yeast. RESULTS: We demonstrated that furfural induces the accumulation of ROS in Saccharomyces cerevisiae. In addition, furfural was shown to cause cellular damage that is consistent with ROS accumulation in cells which includes damage to mitochondria and vacuole membranes, the actin cytoskeleton and nuclear chromatin. The furfural-induced damage is less severe when yeast are grown in a furfural concentration (25 mM) that allows for eventual growth after an extended lag compared to a concentration of furfural (50 mM) that prevents growth. CONCLUSION: These data suggest that when yeast cells encounter the inhibitor furfural, they not only need to reduce furfural into furan methanol but also to protect themselves from the cellular effects of furfural and repair any damage caused. The reduced cellular damage seen at 25 mM furfural compared to 50 mM furfural may be linked to the observation that at 25 mM furfural yeast were able to exit the furfural-induced lag phase and resume growth. Understanding the cellular effects of furfural will help direct future strain development to engineer strains capable of tolerating or remediating ROS and the effects of ROS.

Nitrogen source and mineral optimization enhance D: -xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124.

Nutrition-based strategies to optimize xylose to ethanol conversion by Pichia stipitis were identified in growing and stationary-phase cultures provided with a defined medium varied in nitrogen, vitamin, purine/pyrimidine, and mineral content via full or partial factorial designs. It is surprising to note that stationary-phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source, such as amino acids. Ethanol accumulation increased with arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Ethanol production from 150 g/l xylose was maximized (61+/-9 g/l) by providing C:N in the vicinity of approximately 57-126:1 and optimizing the combination of urea and amino acids to supply 40-80 % nitrogen from urea and 60-20 % from amino acids (casamino acids supplemented with tryptophan and cysteine). When either urea or amino acids were used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/l, respectively, from the maximum of 46 g/l for the optimal nitrogen combination. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary-phase cultures. In nongrowing cultures supplied with nitrogen as amino acids, ethanol concentration increased from 24 to 54 g/l with the addition of an optimized mineral supplement of Fe, Mn, Mg, Ca, Zn, and others.

Mitochondrial GFA2 is required for synergid cell death in Arabidopsis.

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.