Mitochondrial NAD dependent aldehyde dehydrogenase either from yeast or human replaces yeast cytoplasmic NADP dependent aldehyde dehydrogenase for the aerobic growth of yeast on ethanol

Background

In a previous study, we deleted three aldehyde dehydrogenase (ALDH) genes, involved in ethanol metabolism, from yeast Saccharomyces cerevisiae and found that the triple deleted yeast strain did not grow on ethanol as sole carbon source. The ALDHs were NADP dependent cytosolic ALDH1, NAD dependent mitochondrial ALDH2 and NAD/NADP dependent mitochondrial ALDH5. Double deleted strain ΔALDH2+ΔALDH5 or ΔALDH1+ΔALDH5 could grow on ethanol. However, the double deleted strain ΔALDH1+ΔALDH2 did not grow in ethanol.

Methods

Triple deleted yeast strain was used. Mitochondrial NAD dependent ALDH from yeast or human was placed in yeast cytosol.

Results

In the present study we found that a mutant form of cytoplasmic ALDH1 with very low activity barely supported the growth of the triple deleted strain (ΔALDH1+ΔALDH2+ΔALDH5) on ethanol. Finding the importance of NADP dependent ALDH1 on the growth of the strain on ethanol we examined if NAD dependent mitochondrial ALDH2 either from yeast or human would be able to support the growth of the triple deleted strain on ethanol if the mitochondrial form was placed in cytosol. We found that the NAD dependent mitochondrial ALDH2 from yeast or human was active in cytosol and supported the growth of the triple deleted strain on ethanol.

Conclusion

This study showed that coenzyme preference of ALDH is not critical in cytosol of yeast for the growth on ethanol.

General significance

The present study provides a basis to understand the coenzyme preference of ALDH in ethanol metabolism in yeast.     

Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C₄ to C₁₄), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.     

Engineering of the Pyruvate Dehydrogenase Bypass in Saccharomyces cerevisiae: Role of the Cytosolic Mg2+ and Mitochondrial K+ Acetaldehyde Dehydrogenases Ald6p and Ald4p in Acetate Formation during Alcoholic Fermentation

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate by Saccharomyces cerevisiae during alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressing ACS2 and displaying a four- to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies of ALD6, encoding the cytosolic Mg²⁺-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion of ALD4, encoding the mitochondrial K⁺-activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, the ald6 ald4 double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of the ALD family.     

Characterization of the Link between Ornithine,Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.     

Dre2, a conserved eukaryotic Fe/S cluster protein, functions in cytosolic Fe/S protein biogenesis

In a forward genetic screen for interaction with mitochondrial iron carrier proteins in Saccharomyces cerevisiae, a hypomorphic mutation of the essential DRE2 gene was found to confer lethality when combined with Δmrs3 and Δmrs4. The dre2 mutant or Dre2-depleted cells were deficient in cytosolic Fe/S cluster protein activities while maintaining mitochondrial Fe/S clusters. The Dre2 amino acid sequence was evolutionarily conserved, and cysteine motifs (CX₂CXC and twin CX₂C) in human and yeast proteins were perfectly aligned. The human Dre2 homolog (implicated in blocking apoptosis and called CIAPIN1 or anamorsin) was able to complement the nonviability of a Δdre2 deletion strain. The Dre2 protein with triple hemagglutinin tag was located in the cytoplasm and in the mitochondrial intermembrane space. Yeast Dre2 overexpressed and purified from bacteria was brown and exhibited signature absorption and electron paramagnetic resonance spectra, indicating the presence of both [2Fe-2S] and [4Fe-4S] clusters. Thus, Dre2 is an essential conserved Fe/S cluster protein implicated in extramitochondrial Fe/S cluster assembly, similar to other components of the so-called CIA (cytoplasmic Fe/S cluster assembly) pathway although partially localized to the mitochondrial intermembrane space.     

Role of ςB in Heat, Ethanol, Acid, and Oxidative Stress Resistance and during Carbon Starvation in Listeria monocytogenes

To determine the contribution of sigma B (ς^B) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50°C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45°C, 5% ethanol, or pH 4.5). The ΔsigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the ΔsigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the ΔsigB strain. These results suggest the existence in L. monocytogenes of both a ς^B-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. ς^B contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The ΔsigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the ΔsigB strain lost viability more rapidly than the parent strain. ς^B contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that ς^B plays a role in protecting L. monocytogenes against environmental adversities.     

Extensive DNA End Processing by Exo1 and Sgs1 Inhibits Break-Induced Replication

Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination.     

Sap6, a secreted aspartyl proteinase,participates in maintenance the cell surface integrity of Candida albicans

Background

The polymorphic species Candida albicans is the major cause of candidiasis in humans. The secreted aspartyl proteinases (Saps) of C. albicans, encoded by a family of 10 SAP genes, have been investigated as the virulent factors during candidiasis. However, the biological functions of most Sap proteins are still uncertain. In this study, we applied co-culture system of C. albicans and THP-1 human monocytes to explore the pathogenic roles and biological functions of Sap proteinases.

Results

After 1 hr of co-culture of C. albicans strains and THP-1 human monocytes at 37°C, more than 60% of the THP-1-engulfed wild type and Δsap5 Candida cells were developing long hyphae. However, about 50% of THP-1-engulfed Δsap6 Candida cells were generating short hyphae, and more dead Candida cells were found in Δsap6 strain that was ingested by THP-1 cells (about 15% in Δsap6 strain vs. 2 ~ 2.5% in SC5314 and Δsap5 strains). The immunofluorescence staining demonstrated that the Sap6 is the major hyphal tip located Sap protein under THP-1 phagocytosis. The sap6-deleted strains (Δsap6, Δsap4/6, and Δsap5/6) appeared slower growth on Congo red containing solid medium at 25°C, and the growth defect was exacerbated when cultured at 37°C in Congo red or SDS containing medium. In addition, more proteins were secreted from Δsap6 strain and the β-mercaptoethanol (β-ME) extractable surface proteins from Δsap6 mutant were more abundant than that of extracted from wild type strain, which included the plasma membrane protein (Pma1p), the ER-chaperone protein (Kar2p), the protein transport-related protein (Arf1p), the cytoskeleton protein (Act1), and the mitochondrial outer membrane protein (porin 1). Moreover, the cell surface accessibility was increased in sap6-deleted strains.

Conclusion

From these results, we speculated that the cell surface constitution of C. albicans Δsap6 strain was defect. This may cause the more accessible of β-ME to disulfide-bridged cell surface components and may weaken the resistance of Δsap6 strain encountering phagocytosis of THP-1 cells. Sap6 protein displays a significant function involving in maintenance the cell surface integrity.     

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

The Role of Djp1 in Import of the Mitochondrial Protein Mim1 Demonstrates Specificity between a Cochaperone and Its Substrate Protein

A special group of mitochondrial outer membrane proteins spans the membrane once, exposing soluble domains to both sides of the membrane. These proteins are synthesized in the cytosol and then inserted into the membrane by an unknown mechanism. To identify proteins that are involved in the biogenesis of the single-span model protein Mim1, we performed a high-throughput screen in yeast. Two interesting candidates were the cytosolic cochaperone Djp1 and the mitochondrial import receptor Tom70. Our results indeed demonstrate a direct interaction of newly synthesized Mim1 molecules with Tom70. We further observed lower steady-state levels of Mim1 in mitochondria from djp1Δ and tom70 tom71Δ cells and massive mislocalization of overexpressed GFP-Mim1 to the endoplasmic reticulum in the absence of Djp1. Importantly, these phenotypes were observed specifically for the deletion of DJP1 and were not detected in mutant cells lacking any of the other cytosolic cochaperones of the Hsp40 family. Furthermore, the djp1Δ tom70Δ tom71Δ triple deletion resulted in a severe synthetic sick/lethal growth phenotype. Taking our results together, we identified Tom70 and Djp1 as crucial players in the biogenesis of Mim1. Moreover, the involvement of Djp1 provides a unique case of specificity between a cochaperone and its substrate protein.     

Engineering Neurospora crassa for Improved Cellobiose and Cellobionate Production

We report engineering Neurospora crassa to improve the yield of cellobiose and cellobionate from cellulose. A previously engineered strain of N. crassa (F5) with six of seven β-glucosidase (bgl) genes knocked out was shown to produce cellobiose and cellobionate directly from cellulose without the addition of exogenous cellulases. In this study, the F5 strain was further modified to improve the yield of cellobiose and cellobionate from cellulose by increasing cellulase production and decreasing product consumption. The effects of two catabolite repression genes, cre-1 and ace-1, on cellulase production were investigated. The F5 Δace-1 mutant showed no improvement over the wild type. The F5 Δcre-1 and F5 Δace-1 Δcre-1 strains showed improved cellobiose dehydrogenase and exoglucanase expression. However, this improvement in cellulase expression did not lead to an improvement in cellobiose or cellobionate production. The cellobionate phosphorylase gene (ndvB) was deleted from the genome of F5 Δace-1 Δcre-1 to prevent the consumption of cellobiose and cellobionate. Despite a slightly reduced hydrolysis rate, the F5 Δace-1 Δcre-1 ΔndvB strain converted 75% of the cellulose consumed to the desired products, cellobiose and cellobionate, compared to 18% converted by the strain F5 Δace-1 Δcre-1.     

Phosphatidylserine Decarboxylase 1 (Psd1) Promotes Mitochondrial Fusion by Regulating the Biophysical Properties of the Mitochondrial Membrane and Alternative Topogenesis of Mitochondrial Genome Maintenance Protein 1 (Mgm1)

Non–bilayer-forming lipids such as cardiolipin, phosphatidic acid, and phosphatidylethanolamine (PE) are proposed to generate negative membrane curvature, promoting membrane fusion. However, the mechanism by which lipids regulate mitochondrial fusion remains poorly understood. Here, we show that mitochondrial-localized Psd1, the key yeast enzyme that synthesizes PE, is required for proper mitochondrial morphology and fusion. Yeast cells lacking Psd1 exhibit fragmented and aggregated mitochondria with impaired mitochondrial fusion during mating. More importantly, we demonstrate that a reduction in PE reduces the rate of lipid mixing during fusion of liposomes with lipid compositions reflecting the mitochondrial membrane. This suggests that the mitochondrial fusion defect in the Δpsd1 strain could be due to the altered biophysical properties of the mitochondrial membrane, resulting in reduced fusion kinetics. The Δpsd1 strain also has impaired mitochondrial activity such as oxidative phosphorylation and reduced mitochondrial ATP levels which are due to a reduction in mitochondrial PE. The loss of Psd1 also impairs the biogenesis of s-Mgm1, a protein essential for mitochondrial fusion, further exacerbating the mitochondrial fusion defect of the Δpsd1 strain. Increasing s-Mgm1 levels in Δpsd1 cells markedly reduced mitochondrial aggregation. Our results demonstrate that mitochondrial PE regulates mitochondrial fusion by regulating the biophysical properties of the mitochondrial membrane and by enhancing the biogenesis of s-Mgm1. While several proteins are required to orchestrate the intricate process of membrane fusion, we propose that specific phospholipids of the mitochondrial membrane promote fusion by enhancing lipid mixing kinetics and by regulating the action of profusion proteins.     

Lipid Biosynthetic Genes Affect Candida albicans Extracellular Vesicle Morphology,Cargo, and Immunostimulatory Properties

Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase (CHO1) and phosphatidylserine decarboxylase (PSD1 and PSD2) are Candida albicans genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The cho1Δ/Δ mutant releases EVs comparable in size to wild-type EVs, but EVs from the psd1Δ/Δ psd2Δ/Δ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-κB in bone marrow-derived macrophage cells. While wild-type and psd1Δ/Δ psd2Δ/Δ mutant-derived EVs activated NF-κB, the cho1Δ/Δ mutant-derived EV did not. These studies indicate that the presence and absence of these C. albicans genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.     

Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.     

Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h⁻¹ at 100 g of glucose·liter⁻¹). In aerated batch cultures grown on 400 g of glucose·liter⁻¹, this engineered S. cerevisiae strain produced over 200 g of glycerol·liter⁻¹, corresponding to a molar yield of glycerol on glucose close to unity.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

Involvement of Acyl Coenzyme A Oxidase Isozymes in Biotransformation of Methyl Ricinoleate into γ-Decalactone by Yarrowia lipolytica

We reported previously on the function of acyl coenzyme A (acyl-CoA) oxidase isozymes in the yeast Yarrowia lipolytica by investigating strains disrupted in one or several acyl-CoA oxidase-encoding genes (POX1 through POX5) (H. Wang et al., J. Bacteriol. 181:5140–5148, 1999). Here, these mutants were studied for lactone production. Monodisrupted strains produced similar levels of lactone as the wild-type strain (50 mg/liter) except for Δpox3, which produced 220 mg of γ-decalactone per liter after 24 h. The Δpox2 Δpox3 double-disrupted strain, although slightly affected in growth, produced about 150 mg of lactone per liter, indicating that Aox2p was not essential for the biotransformation. The Δpox2 Δpox3 Δpox5 triple-disrupted strain produced and consumed lactone very slowly. On the contrary, the Δpox2 Δpox3 Δpox4 Δpox5 multidisrupted strain did not grow or biotransform methyl ricinoleate into γ-decalactone, demonstrating that Aox4p is essential for the biotransformation.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.     

Impaired Cellular Bioenergetics Causes Mitochondrial Calcium Handling Defects in MT-ND5 Mutant Cybrids

Mutations in mitochondrial DNA (mtDNA) can cause mitochondrial disease, a group of metabolic disorders that affect both children and adults. Interestingly, individual mtDNA mutations can cause very different clinical symptoms, however the factors that determine these phenotypes remain obscure. Defects in mitochondrial oxidative phosphorylation can disrupt cell signaling pathways, which may shape these disease phenotypes. In particular, mitochondria participate closely in cellular calcium signaling, with profound impact on cell function. Here, we examined the effects of a homoplasmic m.13565C>T mutation in MT-ND5 on cellular calcium handling using transmitochondrial cybrids (ND5 mutant cybrids). We found that the oxidation of NADH and mitochondrial membrane potential (Δψ_m) were significantly reduced in ND5 mutant cybrids. These metabolic defects were associated with a significant decrease in calcium uptake by ND5 mutant mitochondria in response to a calcium transient. Inhibition of glycolysis with 2-deoxy-D-glucose did not affect cytosolic calcium levels in control cybrids, but caused an increase in cytosolic calcium in ND5 mutant cybrids. This suggests that glycolytically-generated ATP is required not only to maintain Δψ_m in ND5 mutant mitochondria but is also critical for regulating cellular calcium homeostasis. We conclude that the m.13565C>T mutation in MT-ND5 causes defects in both mitochondrial oxidative metabolism and mitochondrial calcium sequestration. This disruption of mitochondrial calcium handling, which leads to defects in cellular calcium homeostasis, may be an important contributor to mitochondrial disease pathogenesis.