**5. The role of SDH in human disease**

220 Dehydrogenases

disodium salt (WST-5) [19, 22, 23].

them (Figures 8-10 A, B).

Many hundreds of tetrazolium salts and formazans were prepared in the following years, but only a handful have found applications in biological research.There is a wide range of tetrazolium salts commonly used in the field of microbiology from the classical ones to the new generation of its derivatives. Among them are: blue tetrazolium chloride (BT), 2,3,5 triphenyl tetrazolium chloride (TTC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), 2,3-bis(2-methoxy-4-nitro-5 sulphophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), 4-[3-(4 idophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST1), 2-(piodophenyl)-3(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) or 2,2'-dibenzothiazolyl-5,5'-[4-di(2-sulfoethyl)carbamoylphenyl]-3,3'-(3,3'-dimethoxy-4,4' biphenyl) ditetrazolium,

In the case of enzymatic reaction conducted *in situ* the plasma membrane forms a barrier with low degree of penetration. Therefore, cell permeabilization, e.g. by digitonin, is recommended as an alternative method for the study of intracellular enzyme activities. According to the results obtained by Berlowska et al. [23] digitonin was effective in membrane permeabilization without negative influence on cell morphology. After digitonin treatment, the visible formazan crystals were observed inside the yeast cells, but not outside

The formazan products are water-insoluble, but readily diffuses out of yeast cells after solubilization in DMSO. Good correlation (R2=0,97) between BTf absorbance intensity after DMSO extraction and number of yeast cells was seen. Linear correlation was observed in the concentration range of yeast cells from 9107 to5108 per sample. For yeast cell concentrations below 1107 per sample the formazan color intensity signals were too low to detect with good precision. The results obtained for SDH activity were in good agreement

**Figure 8.** Yeast cells after reaction with blue tetrazolium chloride (BT). A – without permeabilization; B

(A) (B)

– with permeabilization by 0.05% digitonin. Images of light microscopy.

*Saccharomyces cerevisiae* is a simple eukaryotic organism, with a complete genome sequence. Many genetic tools that have been created during these years, including the complete collection of gene deletions and a considerable number of mechanisms and pathways existing in higher eukaryotes was first studied and described in yeast. Moreover, about 40% of human genes whose mutations lead to diseases have an orthologue in yeast and genomic screens have been extended to mitochondrial diseases. The study of mitochondrial functions and dysfunction is of special interest in yeast because it is in this organism that mitochondrial genetics and recombination have been discovered and that nucleomitochondrial interactions have been studied in-depth. There are also specific reasons for choosing *S. cerevisiae* for mitochondrial studies. This organism is petite-positive, which can successfully grow in the absence of oxygen. Therefore it can lose its mitochondrial genome provided it is supplied with a substrate for fermentation. Consequently, all mutations of the mitochondrial genome can be studied without cell lethality. The frequency of homologous recombination is very high (1% recombination is considered to correspond to about 100 bp in the mitochondrial genome). It is genetically easy to transfer mitochondria from one nuclear genetic background to another via karyogamy. Additionally, mitochondria can be transformed making in vitro mutation analysis possible. The richness and ease of yeast molecular genetics opens big opportunities, and even the major difference existing between human and yeast mitochondrial genomes, i.e. the predominant heteroplasmy of human and the homoplasmy of yeast, can result in the easier definition of the pathogenic mutations. To review mitochondrial diseases may be a very difficult task because the definition might include different kinds of metabolic disorders or degenerative syndromes [24]. Moreover, some important aspects have been extensively reviewed and the reader might refer to very good recent articles by DiMauro and Garone [25] for historical aspects, by Wallace et al. [26, 27] for bioenergetics, by Spinazzola and Zeviani [28] for nucleo– mitochondrial intergenomic cross-talk. The previous review by Schwimmer et al. [29] was given an important outline of yeast models of mitochondrial diseases. SDH in yeast and human are very similar. They are composed of four subunits (SDHA-D, like SDH1-4 in yeast), all encoded by nuclear genes.

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

[30] [31]

[37] [38]

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 223

Enzyme Clinical presentation References

Hereditary leiomyomatosis and renal cell

depletion [36]

Progressive encephalopathy

Malate dehydrogenase Alzheimer's disease [32, 33]

Isocitrate dehydrogenase Low-grade gliomas [34]

dehydrogenase Congenital lactic acidosis [35]

Encephalopathy (Leigh syndrome) Pheochromocytoma and paraganglioma

assays that are both laborious and time-consuming. The limited set of assays allowing both measurement of all TCA enzyme activities and detection of abnormalities in enzyme activity ratios were developed. These assays were used successfully to detect severe and partial isolated deficiencies in several TCA enzymes. The first assay measures succinyl-CoA ligase, SDH, glutamate dehydrogenase (GDH), fumarase, and malate dehydrogenase. This assay was performed in medium containing 50 mM KH2PO4 (pH 7.2) and 1 mg/ml BSA. The reduction of DCPIP was measured using two wavelengths (600 nm and 750 nm) with various substrates and the electron acceptors decylubiquinone and phenazine methosulfate. The second assay measured -ketoglutarate dehydrogenase, aconitase, and isocitrate dehydrogenase activities. The pyridine nucleotide (NAD+/NADP+) reduction is measured with various substrates using wavelengths of 340 nm and 380 nm. In the third assay, citrate synthase was measured by monitoring dithionitrobenzene (DTNB; Ellman's reagent)

The deleterious mutations in any of the SDH genes should invariably result in a decreased SDH activity [13]. Hence, SDH 'inactivation' induces abnormal stimulation of the hypoxiaangiogenesis pathway. Therefore, the striking phenotypic differences associated with mutations in the four subunits raise puzzling questions. It has been reported that inherited deficiencies of SDH associated with presence of mutant protein Fp are always associated with relatively high residual activities, ranging from 25-50% of control mean values. As a comparison, less than 5% residual activity is frequently measured in patients with severe defect of complex IV or complex I. The mutations in any of the SDH cause the complex II to fully disassemble. When complex II is absent, it can be disregarded as a source of additional superoxide production. Thus, the superoxide overproduction would lead to tumour formation that should be ascribed to the decreased ability of the SDH to adequately reduce

cancer

Succinyl-CoA ligase Encephalomyopathy with mtDNA

**Table 1.** Primary deficiencies in TCA cycle enzymes in humans [22].

reduction at wavelengths of 412 nm and 600 nm [22].

the Q pool, a necessary condition to resist oxidative stress [8].

Citrate synthase No disease identified so far Aconitase No disease identified so far

Fumarase


Succinate dehydrogenase

In the last ten years, deficiencies in TCA cycle enzymes have been shown to cause a wide spectrum of human diseases. For instance, mutation in the gene encoding fumarase is a rare cause of encephalomyopathy and a far more common cause of leiomyomas of the skin and uterus and of renal cancer (Table 1).

The TCA path dysfunction may also result from concurrent impairments in several steps of the cycle. The combined deficiencies in SDH and aconitase was observed in Friedreich's ataxia [22, 39]. Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity [33]. The ratios between TCA enzymes are consistent for each mammalian tissues presumably reflecting their metabolic demand. Consequently, in addition to the determination of residual absolute activities, estimation of ratios between enzyme activities is an effective means of detecting partial but potentially harmful deficiencies. When used to assess respiratory chain activities, this approach enabled the identification of several gene mutations, even in patients with partial respiratory chain deficiencies. At present, TCA enzyme activities are measured using a series of independent


**Table 1.** Primary deficiencies in TCA cycle enzymes in humans [22].

222 Dehydrogenases

yeast), all encoded by nuclear genes.

uterus and of renal cancer (Table 1).

collection of gene deletions and a considerable number of mechanisms and pathways existing in higher eukaryotes was first studied and described in yeast. Moreover, about 40% of human genes whose mutations lead to diseases have an orthologue in yeast and genomic screens have been extended to mitochondrial diseases. The study of mitochondrial functions and dysfunction is of special interest in yeast because it is in this organism that mitochondrial genetics and recombination have been discovered and that nucleomitochondrial interactions have been studied in-depth. There are also specific reasons for choosing *S. cerevisiae* for mitochondrial studies. This organism is petite-positive, which can successfully grow in the absence of oxygen. Therefore it can lose its mitochondrial genome provided it is supplied with a substrate for fermentation. Consequently, all mutations of the mitochondrial genome can be studied without cell lethality. The frequency of homologous recombination is very high (1% recombination is considered to correspond to about 100 bp in the mitochondrial genome). It is genetically easy to transfer mitochondria from one nuclear genetic background to another via karyogamy. Additionally, mitochondria can be transformed making in vitro mutation analysis possible. The richness and ease of yeast molecular genetics opens big opportunities, and even the major difference existing between human and yeast mitochondrial genomes, i.e. the predominant heteroplasmy of human and the homoplasmy of yeast, can result in the easier definition of the pathogenic mutations. To review mitochondrial diseases may be a very difficult task because the definition might include different kinds of metabolic disorders or degenerative syndromes [24]. Moreover, some important aspects have been extensively reviewed and the reader might refer to very good recent articles by DiMauro and Garone [25] for historical aspects, by Wallace et al. [26, 27] for bioenergetics, by Spinazzola and Zeviani [28] for nucleo– mitochondrial intergenomic cross-talk. The previous review by Schwimmer et al. [29] was given an important outline of yeast models of mitochondrial diseases. SDH in yeast and human are very similar. They are composed of four subunits (SDHA-D, like SDH1-4 in

In the last ten years, deficiencies in TCA cycle enzymes have been shown to cause a wide spectrum of human diseases. For instance, mutation in the gene encoding fumarase is a rare cause of encephalomyopathy and a far more common cause of leiomyomas of the skin and

The TCA path dysfunction may also result from concurrent impairments in several steps of the cycle. The combined deficiencies in SDH and aconitase was observed in Friedreich's ataxia [22, 39]. Measures in autopsied brains from Alzheimer's Disease (AD) patients reveal a decrease in the activity of α-ketoglutarate dehydrogenase complex (KGDHC) and an increase in malate dehydrogenase (MDH) activity [33]. The ratios between TCA enzymes are consistent for each mammalian tissues presumably reflecting their metabolic demand. Consequently, in addition to the determination of residual absolute activities, estimation of ratios between enzyme activities is an effective means of detecting partial but potentially harmful deficiencies. When used to assess respiratory chain activities, this approach enabled the identification of several gene mutations, even in patients with partial respiratory chain deficiencies. At present, TCA enzyme activities are measured using a series of independent assays that are both laborious and time-consuming. The limited set of assays allowing both measurement of all TCA enzyme activities and detection of abnormalities in enzyme activity ratios were developed. These assays were used successfully to detect severe and partial isolated deficiencies in several TCA enzymes. The first assay measures succinyl-CoA ligase, SDH, glutamate dehydrogenase (GDH), fumarase, and malate dehydrogenase. This assay was performed in medium containing 50 mM KH2PO4 (pH 7.2) and 1 mg/ml BSA. The reduction of DCPIP was measured using two wavelengths (600 nm and 750 nm) with various substrates and the electron acceptors decylubiquinone and phenazine methosulfate. The second assay measured -ketoglutarate dehydrogenase, aconitase, and isocitrate dehydrogenase activities. The pyridine nucleotide (NAD+/NADP+) reduction is measured with various substrates using wavelengths of 340 nm and 380 nm. In the third assay, citrate synthase was measured by monitoring dithionitrobenzene (DTNB; Ellman's reagent) reduction at wavelengths of 412 nm and 600 nm [22].

The deleterious mutations in any of the SDH genes should invariably result in a decreased SDH activity [13]. Hence, SDH 'inactivation' induces abnormal stimulation of the hypoxiaangiogenesis pathway. Therefore, the striking phenotypic differences associated with mutations in the four subunits raise puzzling questions. It has been reported that inherited deficiencies of SDH associated with presence of mutant protein Fp are always associated with relatively high residual activities, ranging from 25-50% of control mean values. As a comparison, less than 5% residual activity is frequently measured in patients with severe defect of complex IV or complex I. The mutations in any of the SDH cause the complex II to fully disassemble. When complex II is absent, it can be disregarded as a source of additional superoxide production. Thus, the superoxide overproduction would lead to tumour formation that should be ascribed to the decreased ability of the SDH to adequately reduce the Q pool, a necessary condition to resist oxidative stress [8].

Ubiquinone, beside its function in the respiratory chain as an electron carrier mediating electron transfer between the various dehydrogenases and the cytochrome path, is working as a powerful antioxidant in biological membranes [13]. It is possibly for this exact reason in much larger amounts compared to other electron carriers of the respiratory chain, including the sum of the dehydrogenases. When it is defective, the respiratory chain can produce an abnormal amount of superoxides involving additional respiratory chain components such as flavin radicals of complex I. Delivering electrons for the full reduction of Q to QH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria. Therefore, the SDH is a key enzyme to control Q pool redox poise under these conditions, due to its unique redox properties [8].

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 225

Mutations in the *SDHA* gene were identified in a small number of people with Leigh syndrome, a progressive brain disorder that typically appears in infancy or early childhood. Affected children may experience vomiting, seizures, delayed development, muscle weakness, and problems with movement. Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder. The one child died suddenly at the age of five months from a severe deterioration of neuromuscular, cardiac, and hepatic symptoms after an intermittent infection. The *SDHA* gene mutations responsible for Leigh syndrome change single amino acids in the SDHA protein or result in an abnormally short protein. These genetic changes disrupt the activity of the SDH enzyme, impairing the ability of mitochondria to produce energy. Further studies showed that several patients with complex II deficiency do not have mutations of *SDHA*. This suggested a role of additional nuclear genes involved in synthesis, assembly, or maintenance of SDH. It is not known, however, how mutations in the *SDHA* gene are related to the specific features of Leigh

Two plausible hypotheses have been proposed to explain the peculiar linkage between disruption of electron flow through mitochondrial complex II and tumorigenesis in neuroendocrine cells. In the reactive oxygen species (ROS) hypothesis, it is proposed that an intact, catalytically active SDHA subunit generates genotoxic ROS by uncoupled electron flow from succinate to oxygen or water in cells where one of the electron-carrying subunits (SDHB, SDHC or SDHD) is missing or inactive. The ROS model implies that genotoxic ROS mutagenize nuclear proto-oncogenes or tumor suppressors (Figure 11). This model predicts that ROS should be increased in cells lacking SDHB, SDHC or SDHD, but not when SDHA is missing. Although certain mutations in these genes result in ROS production in *Saccharomyces cerevisiae* and mammalian cell lines, it is not clear that ROS accumulate to

In the succinate accumulation hypothesis, the loss of SDHB results in loss of SDH activity

syndrome [41, 42].

levels that are mutagenic.

**Figure 11.** ROS model [18].

and causes succinate accumulation (Figure 12).

Iron-sulfur (Fe-S) proteins facilitate multiple functions, including redox activity, enzymatic function, and maintenance of structural integrity. More than 20 proteins are involved in the biosynthesis of iron-sulfur clusters in eukaryotes. Defective Fe-S cluster synthesis not only affects activities of many iron-sulfur enzymes, such as aconitase and succinate dehydrogenase, but also alters the regulation of cellular iron homeostasis, causing both mitochondrial iron overload and cytosolic iron deficiency. Fe-S cluster biogenesis takes place essentially in every tissue of humans, and products of human disease genes have important roles in the process [40].

Succinate is an oxygen sensor in the cell and can help turn on specific pathways that stimulate cells to grow in a low-oxygen environment (hypoxia). In particular, succinate stabilizes a protein called hypoxia-inducible factor (HIF) by preventing a reaction that would allow HIF to be broken down. HIF controls several important genes involved in cell division and the formation of new blood vessels in a hypoxic environment. Mutations in genes encoding SDH subunits have been linked to severe encephalopathy and, more recently, to familial paraganglioma (PGL) and pheochromocytoma (PHEO) (PHEO: adrenal gland PGL). Isolated complex II deficiency is a very rare condition, occurring in approximately 2–4% of all respiratory chain enzyme deficiencies. At least three mutations in the SDH genes have been identified in people with PGL or PHEO, which are noncancerous (benign) tumors associated with the nervous system. *SDHB-D* gene mutations are seen most commonly in people with PGL, but they were found in people with PHEO. However, a single mutation in the *SDHA* gene increases the risk that an individual will develop the condition, and additional mutation that deletes the normal copy of the gene is needed to cause tumor formation. This second mutation, called a somatic mutation, is acquired during a person's lifetime and is present only in tumor cells. The SDH genes mutations associated with nonsyndromic PGL or PHEO change single protein building blocks (amino acids) in the SDH protein sequence or result in a shortened protein. As a result, there is little or no SDH enzyme activity. Because the mutated SDH enzyme cannot convert succinate to fumarate, succinate accumulates in the cell. The excess succinate abnormally stabilizes HIF, which also builds up in cells. Excess HIF stimulates cells to divide and triggers the production of blood vessels when they are not needed. Rapid and uncontrolled cell division, along with the formation of new blood vessels, can lead to the development of tumors.

Mutations in the *SDHA* gene were identified in a small number of people with Leigh syndrome, a progressive brain disorder that typically appears in infancy or early childhood. Affected children may experience vomiting, seizures, delayed development, muscle weakness, and problems with movement. Heart disease, kidney problems, and difficulty breathing can also occur in people with this disorder. The one child died suddenly at the age of five months from a severe deterioration of neuromuscular, cardiac, and hepatic symptoms after an intermittent infection. The *SDHA* gene mutations responsible for Leigh syndrome change single amino acids in the SDHA protein or result in an abnormally short protein. These genetic changes disrupt the activity of the SDH enzyme, impairing the ability of mitochondria to produce energy. Further studies showed that several patients with complex II deficiency do not have mutations of *SDHA*. This suggested a role of additional nuclear genes involved in synthesis, assembly, or maintenance of SDH. It is not known, however, how mutations in the *SDHA* gene are related to the specific features of Leigh syndrome [41, 42].

Two plausible hypotheses have been proposed to explain the peculiar linkage between disruption of electron flow through mitochondrial complex II and tumorigenesis in neuroendocrine cells. In the reactive oxygen species (ROS) hypothesis, it is proposed that an intact, catalytically active SDHA subunit generates genotoxic ROS by uncoupled electron flow from succinate to oxygen or water in cells where one of the electron-carrying subunits (SDHB, SDHC or SDHD) is missing or inactive. The ROS model implies that genotoxic ROS mutagenize nuclear proto-oncogenes or tumor suppressors (Figure 11). This model predicts that ROS should be increased in cells lacking SDHB, SDHC or SDHD, but not when SDHA is missing. Although certain mutations in these genes result in ROS production in *Saccharomyces cerevisiae* and mammalian cell lines, it is not clear that ROS accumulate to levels that are mutagenic.

**Figure 11.** ROS model [18].

224 Dehydrogenases

due to its unique redox properties [8].

important roles in the process [40].

Ubiquinone, beside its function in the respiratory chain as an electron carrier mediating electron transfer between the various dehydrogenases and the cytochrome path, is working as a powerful antioxidant in biological membranes [13]. It is possibly for this exact reason in much larger amounts compared to other electron carriers of the respiratory chain, including the sum of the dehydrogenases. When it is defective, the respiratory chain can produce an abnormal amount of superoxides involving additional respiratory chain components such as flavin radicals of complex I. Delivering electrons for the full reduction of Q to QH2 might then be of a tremendous importance for the control of oxygen toxicity in the mitochondria. Therefore, the SDH is a key enzyme to control Q pool redox poise under these conditions,

Iron-sulfur (Fe-S) proteins facilitate multiple functions, including redox activity, enzymatic function, and maintenance of structural integrity. More than 20 proteins are involved in the biosynthesis of iron-sulfur clusters in eukaryotes. Defective Fe-S cluster synthesis not only affects activities of many iron-sulfur enzymes, such as aconitase and succinate dehydrogenase, but also alters the regulation of cellular iron homeostasis, causing both mitochondrial iron overload and cytosolic iron deficiency. Fe-S cluster biogenesis takes place essentially in every tissue of humans, and products of human disease genes have

Succinate is an oxygen sensor in the cell and can help turn on specific pathways that stimulate cells to grow in a low-oxygen environment (hypoxia). In particular, succinate stabilizes a protein called hypoxia-inducible factor (HIF) by preventing a reaction that would allow HIF to be broken down. HIF controls several important genes involved in cell division and the formation of new blood vessels in a hypoxic environment. Mutations in genes encoding SDH subunits have been linked to severe encephalopathy and, more recently, to familial paraganglioma (PGL) and pheochromocytoma (PHEO) (PHEO: adrenal gland PGL). Isolated complex II deficiency is a very rare condition, occurring in approximately 2–4% of all respiratory chain enzyme deficiencies. At least three mutations in the SDH genes have been identified in people with PGL or PHEO, which are noncancerous (benign) tumors associated with the nervous system. *SDHB-D* gene mutations are seen most commonly in people with PGL, but they were found in people with PHEO. However, a single mutation in the *SDHA* gene increases the risk that an individual will develop the condition, and additional mutation that deletes the normal copy of the gene is needed to cause tumor formation. This second mutation, called a somatic mutation, is acquired during a person's lifetime and is present only in tumor cells. The SDH genes mutations associated with nonsyndromic PGL or PHEO change single protein building blocks (amino acids) in the SDH protein sequence or result in a shortened protein. As a result, there is little or no SDH enzyme activity. Because the mutated SDH enzyme cannot convert succinate to fumarate, succinate accumulates in the cell. The excess succinate abnormally stabilizes HIF, which also builds up in cells. Excess HIF stimulates cells to divide and triggers the production of blood vessels when they are not needed. Rapid and uncontrolled cell division, along with the formation of new blood vessels, can lead to the development of tumors.

In the succinate accumulation hypothesis, the loss of SDHB results in loss of SDH activity and causes succinate accumulation (Figure 12).

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 227

spectrum of usable industrial media (e.g., lignocellulosic biomass) and/or to produce compounds not naturally formed by *S. cerevisiae*. Since the first introduction of metabolic engineering, there have been tremendous enhancements of its toolbox, and several related disciplines have emerged, such as inverse metabolic engineering and evolutionary engineering. These developments have strongly influenced yeast strain improvement programs in the past few years and have greatly enhanced the potential for using yeast in

The main goals of metabolic engineering can be summarized in the following four categories: (1) improvement of yield, productivity and overall cellular physiology, (2) extension of the substrate range, (3) deletion or reduction of by-product formation and (4) introduction of pathways leading to new products. Commonly these goals can be achieved by a three-step procedure. Firstly, a genetic modification is proposed, based on metabolic models. After genetic modification, the recombinant strain is analysed and the results are then used to identify the next target for genetic manipulation, if necessary. Thus, the construction of an optimal strain involves a close interaction between synthesis and analysis, usually for several consecutive rounds. The rapid development and frequent success in this field is demonstrated by the large number of reviews about the theoretical and practical aspects of metabolic engineering. Knowledge of cellular and microbial physiology, as well as the underlying metabolic networks or enzymes, is an important prerequisite for successful engineering. A new term, 'inverse metabolic engineering' (IME) coins to encompass the construction of strains with a particularly desirable physiological phenotype,

Recently, a computational approach for the identification of every possible biochemical reaction from a given set of enzyme reaction rules was reported. This analysis suggested that the native pathways are thermodynamically more favorable than the alternative possible pathways. The pathways generated involve compounds that exist in biological databases, as well as compounds that exist in chemical databases and novel compounds, suggesting novel biochemical routes for these compounds and the existence of biochemical compounds that remain to be discovered or synthesized through enzyme and pathway

Due to its importance in traditional biotechnology such as baking, brewing, and wine making, research activities historically have focused on the yeast *Saccharomyces cerevisiae.* It is relatively tolerant to low pH values and high sugar and ethanol concentrations, i.e., properties which lower the risk of contamination in industrial fermentation. These features are the major reasons for increasing *S. cerevisiae* exploration in industrial ("white") biotechnology, focusing on the fermentative production of industrially relevant biochemicals, e.g., glycerol, propanediol, sugar alcohols, organic acids, etc. Among these compounds, several organic acids may fulfill a role as platform molecules using their

In the United States were identified 10 organic acids as key chemical building blocks [44]. Similarly, the European focus group BREW identified 21 key compounds that can be

(multiple) functional groups as a target for enzymic or chemical catalysis [43].

biotechnological production processes [43].

e.g. enhanced production of heterologous protein [44].

engineering [45].

**Figure 12.** Succinate accumulation model [18].

Excess succinate is shuttled from the mitochondrial matrix to the cytoplasm, where it inhibits any of several aKG-dependent enzymes (E) that regulate levels or activities of important regulatory proteins (black box). The loss or inactivation of SDHB, C or D proteins yields a catalytically inactive SDHA subunit, resulting in blockade of the TCA cycle and diffusion of accumulated succinate to the cytoplasm. Succinate can then act as an inhibitor of a-ketoglutarate-dependent enzymes that use ferrous iron and molecular oxygen as cofactors to hydroxylate their substrates and generate succinate as a product. It has been demonstrated that two a-ketoglutarate -dependent enzymes, the prolyl hydroxylases, are inhibited by succinate accumulation in cells that have lost SDHD function. Smith et al [18] reported that yeast cells disrupted in *SDH2* (*sdh2*Δ) show increased in ROS production and protein oxidation without detectable increase in DNA damage. More strikingly, *sdh2*Δ cells dramatically accumulate succinate resulting in inhibition of at least two aKG-dependent enzymes that generate succinate as a by-product [18].
