Respiration-deficient Mutants Research Articles

Expression level of methanol-inducible peroxisomal proteins and peroxisome morphology are affected by oxygen conditions and mitochondrial respiratory pathway function in the methylotrophic yeastCandida boidinii

In the methylotrophic yeast, Candida boidinii, methanol-inducible peroxisomal proteins, for example alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), and peroxisomal glutathione peroxidase (Pmp20), were induced only under aerobic conditions, while expression of PMP47 encoding peroxisomal integral membrane protein Pmp47 was independent of oxygen conditions. Expression of the methanol-inducible peroxisomal enzymes was repressed by inhibition of the mitochondrial respiratory chain. In the respiratory-deficient (ρ0) mutant strain, their induction was at very low levels despite the presence of oxygen, whereas the expression of PMP47 was unaffected. Taken together, these facts indicate that C. boidinii can sense oxygen conditions, and that mitochondrial respiratory function may have a profound effect on induction of methanol-inducible gene expression of peroxisomal proteins. Peroxisome morphology was also affected by oxygen conditions and respiratory function. Under hypoxic conditions or respiration-inhibited conditions, cells induced by methanol contained small peroxisomes, indicating that peroxisome biogenesis and the protein import machinery were not affected by oxygen conditions but that peroxisome morphology was dependent on induction of peroxisomal matrix proteins.

Partial suppression of Oxa1 mutants by mitochondria‐targeted signal recognition particle provides insights into the evolution of the cotranslational insertion systems

The biogenesis of hydrophobic membrane proteins involves their cotranslational membrane integration in order to prevent their unproductive aggregation. In the cytosol of bacteria and eukaryotes, membrane targeting of ribosomes that synthesize membrane proteins is achieved by signal recognition particles (SRPs) and their cognate membrane-bound receptors. As is evident from the genomes of fully sequenced eukaryotes, mitochondria generally lack an SRP system. Instead, mitochondrial ribosomes are physically associated with the protein insertion machinery in the inner membrane. Accordingly, deletion of ribosome-binding sites on the Oxa1 insertase and the Mba1 ribosome receptor in yeast leads to severe defects in cotranslational protein insertion and results in respiration-deficient mutants. In this study, we expressed mitochondria-targeted versions of the bacterial SRP protein Ffh and its receptor FtsY in these yeast mutants. Interestingly, Ffh was found to bind to the large subunit of mitochondrial ribosomes, and could relieve, to some degree, the defect of these insertion mutants. Although FtsY could also bind to mitochondrial membranes, it did not improve membrane protein biogenesis in this strain, presumably because of its inability to interact with Ffh. Hence, mitochondrial ribosomes are still able to interact physically and functionally with the bacterial SRP system. Our observations are consistent with a model according to which the protein insertion system in mitochondria evolved in three steps. The loss of genes for hydrophilic polypeptides (step 1) allowed the development of ribosome-binding sites on membrane proteins (step 2), which finally made the existence of an SRP-mediated system dispensable (step 3).

Identification of Gold Nanoparticle-Resistant Mutants of Saccharomyces cerevisiae Suggests a Role for Respiratory Metabolism in Mediating Toxicity

Positively charged gold nanoparticles (0.8-nm core diameter) reduced yeast survival, but not growth, at a concentration of 10 to 100 μg/ml. Among 17 resistant deletion mutants isolated in a genome-wide screen, highly significant enrichment was observed for respiration-deficient mutants lacking genes encoding proteins associated with the mitochondrion.

Possible Roles of the Mitochondria in Sulfur Dioxide Production by Lager Yeast

Sulfur dioxide is one of the key lager yeast metabolites that brewers seek to control, as it has multiple effects on the quality of lager beers. It has been widely reported that yeast cells, when fermented in high glucose worts, are susceptible to producing elevated sulfur dioxide, yet the exact mechanisms are not fully understood. It is known that when yeast cells utilize glucose as a primary carbon source to fuel yeast growth, this negatively affects mitochondrial development, specifically in its unique membrane lipid, cardiolipin. In this study, we show the results of incubation experiments aimed at altering cardiolipin formation and mitochondrial development with stimulators of cardiolipin synthesis (L-thyroxine and glycerol) and an inhibitor (inositol). These results demonstrated significantly lower sulfite levels (P < 0.05), adjusted for ethanol production, under conditions promoting cardiolipin formation. In addition, fermentations with respiratory deficient mutants also revealed significantly lower efficiency in the conversion of sulfate, as measured by the ratio of the amount of sulfite produced to the amount of sulfate taken up, as compared to the parental strain (P < 0.05). These findings suggest that this overlooked organelle may have a vital role in sulfite production, which we speculate is related to a key prosthetic group in the enzyme complex, sulfite reductase. This group, namely the [4Fe-4S] iron-sulfur cluster, provides the catalytic site in sulfite reductase electrons to reduce sulfur dioxide to sulfide. It is postulated that limitations in mitochondrial ATP levels affect [4Fe-4S] cluster formation or export of the cluster for cytosolic apoprotein modification.

The Putative GTPase Encoded by MTG3 Functions in a Novel Pathway for Regulating Assembly of the Small Subunit of Yeast Mitochondrial Ribosomes

Very little is known about biogenesis of mitochondrial ribosomes. The GTPases encoded by the nuclear MTG1 and MTG2 genes of Saccharomyces cerevisiae have been reported to play a role in assembly of the ribosomal 54 S subunit. In the present study biochemical screens of a collection of respiratory deficient yeast mutants have enabled us to identify a third gene essential for expression of mitochondrial ribosomes. This gene codes for a member of the YqeH family of GTPases, which we have named MTG3 in keeping with the earlier convention. Mutations in MTG3 cause the accumulation of the 15 S rRNA precursor, previously shown to have an 80-nucleotide 5' extension. Sucrose gradient sedimentation of mitochondrial ribosomes from temperature-sensitive mtg3 mutants grown at the permissive and restrictive temperatures, combined with immunobloting with subunit-specific antibodies, indicate that Mtg3p is required for assembly of the 30 S but not 54 S ribosomal subunit. The respiratory deficient growth phenotype of an mtg3 null mutant is partially rescued by overexpression of the Mrpl4p constituent located at the peptide exit site of the 54 S subunit. The rescue is accompanied by an increase in processed 15 S rRNA. This suggests that Mtg3p and Mrpl4p jointly regulate assembly of the small subunit by modulating processing of the 15 S rRNA precursor.

Respiratory Chain Analysis of Zymomonas mobilis Mutants Producing High Levels of Ethanol

We previously isolated respiratory-deficient mutant (RDM) strains of Zymomonas mobilis, which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochrome bd-type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within the ndh gene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

Stable expression of antibiotic-resistant gene ble from Streptoalloteichus hindustanus in the mitochondria of Chlamydomonas reinhardtii.

The mitochondrial expression of exogenous antibiotic resistance genes has not been demonstrated successfully to date, which has limited the development of antibiotic resistance genes as selectable markers for mitochondrial site-directed transformation in Chlamydomonas reinhardtii. In this work, the plasmid pBSLPNCB was constructed by inserting the gene ble of Streptoalloteichus hindustanus (Sh ble), encoding a small (14-kilodalton) protective protein into the site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA (mtDNA) of C. reinhardtii. The fusion DNA-construct, which contained TERMINVREP-Left, Sh ble, cob, and partial nd4 sequence, were introduced into the mitochondria of the respiratory deficient dum-1 mutant CC-2654 of C. reinhardtii by biolistic particle delivery system. A large number of transformants were obtained after eight weeks in the dark. Subsequent subculture of the transformants on the selection TAP media containing 3 ìg/mL Zeomycin for 12 months resulted in genetically modified transgenic algae MT-Bs. Sequencing and Southern analyses on the mitochondrial genome of the different MT-B lines revealed that Sh ble gene had been integrated into the mitochondrial genome of C. reinhardtii. Both Western blot, using the anti-BLE monoclonal antibody, and Zeomycin tolerance analysis confirmed the presence of BLE protein in the transgenic algal cells. It indicates that the Sh ble gene can be stably expressed in the mitochondria of C. reinhardtii.

The abc1 − /coq8 − respiratory-deficient mutant of Schizosaccharomyces pombe suffers from glutathione underproduction and hyperaccumulates Cd2+

The abc1(-)/coq8(-) gene deletion respiratory-deficient mutant NBp17 of fission yeast Schizosaccharomyces pombe displayed a phenotypic fermentation pattern with enhanced production of glycerol and acetate, and also possessed oxidative stress-sensitive phenotypes to H(2)O(2), menadione, tBuOOH, Cd(2+), and chromate in comparison with its parental respiratory-competent strain HNT. As a consequence of internal stress-inducing mutation, adaptation processes to restore the redox homeostasis of mutant NBp17 cells were detected in minimal glucose medium. Mutant NBp17 produced significantly increased amounts of O(2)•- and H(2)O(2) as a result of the decreased internal glutathione concentration and the only slightly increased glutathione reductase activity. The Cr(VI) reduction capacity and hence the •OH production ability were decreased. The mutant cells demonstrated increased specific activities of superoxide dismutases and glutathione reductase (but not catalase) to detoxify at least partially the overproduction of reactive oxygen species. All these features may be explained by the decreased redox capacity of the mutant cells. Most notably, mutant NBp17 hyperaccumulated yellow CdS.

Respiration-deficient mutants of Zymomonas mobilis show improved growth and ethanol fermentation under aerobic and high temperature conditions

Respiration-deficient mutant (RDM) strains of Zymomonas mobilis were isolated from antibiotic-resistant mutants. These RDM strains showed various degrees of respiratory deficiency. All RDM strains exhibited much higher ethanol fermentation capacity than the wild-type strain under aerobic conditions. The strains also gained thermotolerance and exhibited greater ethanol production at high temperature (39°C), under both non-aerobic and aerobic conditions, compared with the wild-type strain. Microarray and subsequent quantitative PCR analyses suggest that enhanced gene expression involved in the metabolism of glucose to ethanol resulted in the high ethanol production of RDM strains under aerobic growth conditions. Reduction of intracellular oxidative stress may also result in improved ethanol fermentation by RDM strains at high temperatures.

An in silico approach combined with in vivo experiments enables the identification of a new protein whose overexpression can compensate for specific respiratory defects in Saccharomyces cerevisiae

BackgroundThe mitochondrial inner membrane contains five large complexes that are essential for oxidative phosphorylation. Although the structure and the catalytic mechanisms of the respiratory complexes have been progressively established, their biogenesis is far from being fully understood. Very few complex III assembly factors have been identified so far. It is probable that more factors are needed for the assembly of a functional complex, but that the genetic approaches used to date have not been able to identify them. We have developed a systems biology approach to identify new factors controlling complex III biogenesis.ResultsWe collected all the physical protein-protein interactions (PPI) involving the core subunits, the supernumerary subunits and the assembly factors of complex III and used Cytoscape 2.6.3 and its plugins to construct a network. It was then divided into overlapping and highly interconnected sub-graphs with clusterONE. One sub-graph contained the core and the supernumerary subunits of complex III, it also contained some subunits of complex IV and proteins participating in the assembly of complex IV. This sub-graph was then split with another algorithm into two sub-graphs. The subtraction of these two sub-graphs from the previous sub-graph allowed us to identify a protein of unknown function Usb1p/Ylr132p that interacts with the complex III subunits Qcr2p and Cor1p. We then used genetic and cell biology approaches to investigate the function of Usb1p. Preliminary results indicated that Usb1p is an essential protein with a dual localization in the nucleus and in the mitochondria, and that the over-expression of this protein can compensate for defects in the biogenesis of the respiratory complexes.ConclusionsOur systems biology approach has highlighted the multiple associations between subunits and assembly factors of complexes III and IV during their biogenesis. In addition, this approach has allowed the identification of a new factor, Usb1p, involved in the biogenesis of respiratory complexes, which could not have been found using classical genetic screens looking for respiratory deficient mutants. Thus, this systems biology approach appears to be a fruitful new way to study the biogenesis of mitochondrial multi-subunit complexes.

Development and application of co-culture for ethanol production by co-fermentation of glucose and xylose: a systematic review

This article reviews current co-culture systems for fermenting mixtures of glucose and xylose to ethanol. Thirty-five co-culture systems that ferment either synthetic glucose and xylose mixture or various biomass hydrolysates are examined. Strain combinations, fermentation modes and conditions, and fermentation performance for these co-culture systems are compared and discussed. It is noted that the combination of Pichia stipitis with Saccharomyces cerevisiae or its respiratory-deficient mutant is most commonly used. One of the best results for fermentation of glucose and xylose mixture is achieved by using co-culture of immobilized Zymomonas mobilis and free cells of P. stipitis, giving volumetric ethanol production of 1.277 g/l/h and ethanol yield of 0.49-0.50 g/g. The review discloses that, as a strategy for efficient conversion of glucose and xylose, co-culture fermentation for ethanol production from lignocellulosic biomass can increase ethanol yield and production rate, shorten fermentation time, and reduce process costs, and it is a promising technology although immature.

Repeated-batch fermentations of xylose and glucose–xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g −1) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L −1 h −1) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L −1 h −1). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g −1) and productivities (0.52 g L −1 h −1) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g −1) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g −1). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.

Dried Yeast: Impact of Dehydration and Rehydration on Brewing Yeast DNA Integrity

As a consequence of fluidized bed drying, yeast cells may be exposed to a series of physiological stresses, of which the most evident is water loss. Desiccation can lead to cell wall crenellation, cytoplasmic crowding, loss of DNA supercoiling, membrane disruption, phase transitions, and ultimately cell death. Although the dehydrated phenotype has been well characterized, the sequence of events that causes damage to the cell has not been widely reported. Here we investigate the impact of dehydration and rehydration on the stability of the brewing yeast genome, including both the chromosomal and mitochondrial DNA (mtDNA). Analysis of rehydrated and control yeast populations for chromosome length polymorphisms (karyotyping), DNA sequence changes (PCR analysis of interdelta sequences), gross mitochondrial damage (presence of respiratory deficient mutants), and mitochondrial sequence changes (mtDNA RFLP [restriction fragment length polymorphisms]) indicated that the genetic constitution of dried yeast is not affected by the drying process. Active dried yeast (ADY) populations also demonstrated an enhanced tolerance to mutagen challenge (targeted at mtDNA), suggesting that the susceptibility of mtDNA to change is not associated with fluidized bed drying. These results demonstrate that the genetic stability of ADY is comparable to control populations.

Mitochondrial genome from the facultative anaerobe and petite-positive yeast Dekkera bruxellensis contains the NADH dehydrogenase subunit genes

The progenitor of the Dekkera/Brettanomyces clade separated from the Saccharomyces/Kluyveromyces clade over 200 million years ago. However, within both clades, several lineages developed similar physiological traits. Both Saccharomyces cerevisiae and Dekkera bruxellensis are facultative anaerobes; in the presence of excess oxygen and sugars, they accumulate ethanol (Crabtree effect) and they both spontaneously generate respiratory-deficient mutants (petites). In order to understand the role of respiratory metabolism, the mitochondrial DNA (mtDNA) molecules of two Dekkera/Brettanomyces species were analysed. Dekkera bruxellensis mtDNA shares several properties with S. cerevisiae, such as the large genome size (76 453 bp), and the organization of the intergenic sequences consisting of spacious AT-rich regions containing a number of hairpin GC-rich cluster-like elements. In addition to a basic set of the mitochondrial genes coding for the components of cytochrome oxidase, cytochrome b, subunits of ATPase, two rRNA subunits and 25 tRNAs, D. bruxellensis also carries genes for the NADH dehydrogenase complex. Apparently, in yeast, the loss of this complex is not a precondition to develop a petite-positive, Crabtree-positive and anaerobic nature. On the other hand, mtDNA from a petite-negative Brettanomyces custersianus is much smaller (30 058 bp); it contains a similar gene set and has only short intergenic sequences.

Strategy for simultaneous saccharification and fermentation using a respiratory-deficient mutant of Candida glabrata for bioethanol production

High temperature and agitation are advantageous for ethanol production from insoluble feedstock when using the simultaneous saccharification and fermentation (SSF) technique. To construct an effective SSF system, a respiratory-deficient mutant was isolated from the thermotolerant yeast Candida glabrata. Our results suggest that this respiratory-deficient mutant has higher ethanol production abilities in SSF.

Serial Repitching of Dried Lager Yeast

To demonstrate the suitability of active dried yeast (ADY) for serial repitching, dried and wet yeast slurries derived from the same strain were analyzed for five consecutive fermentations (G0–G4). In each instance, cell viability was determined prior to pitching, while yeast performance was assessed by analyzing fermentation profiles and the characteristics of the final products. In addition, beers produced using wet and dried G0 and G4 yeast were analyzed and compared for their concentrations of esters and higher alcohols. Yeast obtained at the end of each fermentation was screened for the presence of respiratory-deficient mutants and flocculent variants. Yeast slurries recovered from G4 beer were examined for genetic changes, as characterized by movement of Ty elements and chromosome length polymorphisms. This study shows that serially repitched wet and dried yeasts perform in a similar fashion and that repitched cultures exhibit more typical fermentation and flavor profiles than fresh slurries. In addition, the study demonstrates that the accumulation of genetic and phenotypic variants is not associated with serial repitching of dried yeast cultures. Consequently, it is proposed that ADY can be reused in a similar fashion to wet yeast without any adverse effects on fermentation performance, yeast quality, or flavor production.

The effect of pre-irradiation culture conditions on the yield of X-ray-induced respiratory-deficient mutants in haploid Saccharomyces cerevisiae*

The effect of pre-irradiation culture conditions on the yield of X-ray-induced respiratory-deficient mutants in haploid Saccharomyces cerevisiae*

Strategy for Adapting Wine Yeasts for Bioethanol Production

The Saccharomyces cerevisiae wine yeast strains 71B-1122 and K1-V1116 were used to derive strains that could tolerate and produce higher ethanol yields. Respiratory-deficient mutants resistant to 500 μg/mL lycorine were isolated. Two mutants, 71B-1122 YEBr L3 and K1-V1116 YEBr L4, were shown to achieve about 10% and 18% improvement in their glucose-to-ethanol conversion efficiency compared to their respective parent strains. The K1-V1116 YEBr L4 in particular can tolerate an ethanol yield of 18.8 ± 0.8% at 3.5 weeks of fermentation and continued to consume most of the sugar until less than 1% glucose was left.

Genome-wide deletion mutant analysis reveals genes required for respiratory growth, mitochondrial genome maintenance and mitochondrial protein synthesis in Saccharomyces cerevisiae

A genome-wide deletion mutant analysis in budding yeast reveals genes required for respiratory growth, mitochondrial genome maintenance and mitochondrial protein synthesis.

Slow Growth Induces Heat-Shock Resistance in Normal and Respiratory-deficient Yeast

Yeast cells respond to a variety of environmental stresses, including heat shock and growth limitation. There is considerable overlap in these responses both from the point of view of gene expression patterns and cross-protection for survival. We performed experiments in which cells growing at different steady-state growth rates in chemostats were subjected to a short heat pulse. Gene expression patterns allowed us to partition genes whose expression responds to heat shock into subsets of genes that also respond to slow growth rate and those that do not. We found also that the degree of induction and repression of genes that respond to stress is generally weaker in respiratory deficient mutants, suggesting a role for increased respiratory activity in the apparent stress response to slow growth. Consistent with our gene expression results in wild-type cells, we found that cells growing more slowly are cross-protected for heat shock, i.e., better able to survive a lethal heat challenge. Surprisingly, however, we found no difference in cross-protection between respiratory-deficient and wild-type cells, suggesting induction of heat resistance at low growth rates is independent of respiratory activity, even though many of the changes in gene expression are not.