**6. Metabolic engineering of yeast SDH for the biotechnological processes**

Metabolic engineering, i.e., the intentional redirection of metabolic fluxes, plays an exceptional role in improving yeast strains for all industrial applications. In contrast to classical methods of genetic strain improvement such as selection, mutagenesis, mating, and hybridization, metabolic engineering confers two major advantages: (1) the directed modification of strains without the accumulation of unfavorable mutations and (2) the introduction of genes from foreign organisms to equip *S. cerevisiae* with novel traits. The latter is particularly crucial for industrial biotechnology to provide pathways that extend the 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 biotechnological production processes [43].

226 Dehydrogenases

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

enzymes that generate succinate as a by-product [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

**6. Metabolic engineering of yeast SDH for the biotechnological processes** 

Metabolic engineering, i.e., the intentional redirection of metabolic fluxes, plays an exceptional role in improving yeast strains for all industrial applications. In contrast to classical methods of genetic strain improvement such as selection, mutagenesis, mating, and hybridization, metabolic engineering confers two major advantages: (1) the directed modification of strains without the accumulation of unfavorable mutations and (2) the introduction of genes from foreign organisms to equip *S. cerevisiae* with novel traits. The latter is particularly crucial for industrial biotechnology to provide pathways that extend the 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, e.g. enhanced production of heterologous protein [44].

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 engineering [45].

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

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

produced from different, including renewable sources, a number of which were organic acids [45]. One example of such a chemical is succinic acid. Succinic acid is used as a surfactant, detergent or foaming agent, as an ion chelator, and also in the food industry as an acidulant, flavoring agent or anti-microbial agent, as well as in health-related products (such pharmaceuticals and antibiotics). Currently, it is produced from petrol and is too expensive to be used as a general building-block chemical. However, provided that its price becomes competitive, succinic acid could replace petrol-derived maleic anhydride in chemical synthesis processes in the future [46-47]. Similar chemical derivatizations can be applied to malic and fumaric acid, so that they can also be considered interesting C4 building blocks [48-53].

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

Replacement of fossilderived BDO by bio-based BDO in polybutylene tereftalate (PBT) Replacement of fossilderived BDO by bio-based BDO in polybutylene succinate (PBS)

production of polyamides

as green solvents

Replacement of fossilderived NMP by bio-based NMP; NMP is an important, versatile solvent for the chemical industry

Replacement of fossilderived BGL by bio-based BGL ; BGL is important as an intermediate in the manufacture of pyrrolidone derivatives and as a solvent

for polymers and agrochemicals

Replacement of fossilderived THF by bio-based THF ; THF is mainly used as

a solvent and as an intermediate in the

etc.

production of thermoplastic polyurethanes, elastic fibers

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 229

**No Name Chemical structure Examples of uses** 

4 Succinonitrile Precurser for industrial

8 2-Pyrolidone As above

5 Dimethylsuccinate Used as fuel oxygenates and

**Table 2.** Examples of various substances that can be derived from succinic acid by chemical conversion

3 1,4 – Butanediol (BDO)

<sup>6</sup>N-methyl-pyrolidone (NMP)

7 -Butyrolactone (GBL)

9 Tetrahydrofuran (THF)

[47].

The chemical behavior of the dicarboxylic acid – succinic acid is determined principally by its two carboxyl groups. This substance is either directly utilized in the pharmaceutical or chemical industry or represent building block or precursor for further chemical or enzymatic syntheses. The following reactions and derivatives are considered interesting: (1) reductions of succinic acid to 1,4-butanediol, -butyrolactone, tetrahydrofuran and its derivatives; (2) reductive amination of succinic acid or -butyrolactone to pyrrolidiones; (3) polymerization of succinic acid with diols (building block of polyesters); (4) polymerisation of succinic acid with diamines to form polyamides, etc. The examples of the substances that can be derived from succinic acid are shown in table 2.

Butanediol, tetrahydrofuran and -butyrolactone, are standard substances for the chemical industry. These are used as solvents, as well as for fiber and polymer production. Dimethylsuccinate is one of the so-called dibasic esters that have great potential as solvents with environmentally benign characteristics. Thus, the potential market volume for succinic acid is high, fuelling substantial efforts to establish a microbial process for succinic acid production [47].

The chemical synthesis of succinic acid is predominantly based on maleic anhydride and requires heavy metal catalysts, organic solvents, high temperatures and high pressures. It makes the conversion of maleic anhydride to succinic acid costly and ecologically questionable [49]. On the other hand, succinate is produced naturally by many microorganisms as an intermediate of the central metabolism or as a fermentation end product.

The succinate producers include bacterial strains, e.g. *Mannheimia succiniproducens* [54]. However, none of these microorganisms are currently used in industry. Some prokaryotes



building blocks [48-53].

production [47].

product.

2 Succindiamide

can be derived from succinic acid are shown in table 2.

produced from different, including renewable sources, a number of which were organic acids [45]. One example of such a chemical is succinic acid. Succinic acid is used as a surfactant, detergent or foaming agent, as an ion chelator, and also in the food industry as an acidulant, flavoring agent or anti-microbial agent, as well as in health-related products (such pharmaceuticals and antibiotics). Currently, it is produced from petrol and is too expensive to be used as a general building-block chemical. However, provided that its price becomes competitive, succinic acid could replace petrol-derived maleic anhydride in chemical synthesis processes in the future [46-47]. Similar chemical derivatizations can be applied to malic and fumaric acid, so that they can also be considered interesting C4

The chemical behavior of the dicarboxylic acid – succinic acid is determined principally by its two carboxyl groups. This substance is either directly utilized in the pharmaceutical or chemical industry or represent building block or precursor for further chemical or enzymatic syntheses. The following reactions and derivatives are considered interesting: (1) reductions of succinic acid to 1,4-butanediol, -butyrolactone, tetrahydrofuran and its derivatives; (2) reductive amination of succinic acid or -butyrolactone to pyrrolidiones; (3) polymerization of succinic acid with diols (building block of polyesters); (4) polymerisation of succinic acid with diamines to form polyamides, etc. The examples of the substances that

Butanediol, tetrahydrofuran and -butyrolactone, are standard substances for the chemical industry. These are used as solvents, as well as for fiber and polymer production. Dimethylsuccinate is one of the so-called dibasic esters that have great potential as solvents with environmentally benign characteristics. Thus, the potential market volume for succinic acid is high, fuelling substantial efforts to establish a microbial process for succinic acid

The chemical synthesis of succinic acid is predominantly based on maleic anhydride and requires heavy metal catalysts, organic solvents, high temperatures and high pressures. It makes the conversion of maleic anhydride to succinic acid costly and ecologically questionable [49]. On the other hand, succinate is produced naturally by many microorganisms as an intermediate of the central metabolism or as a fermentation end

The succinate producers include bacterial strains, e.g. *Mannheimia succiniproducens* [54]. However, none of these microorganisms are currently used in industry. Some prokaryotes

production

Used as anticonvulsant drugs and to form covalent bonds between proteins or peptides and plastics

**No Name Chemical structure Examples of uses**  1 1,4 -Diaminobutane Used for polyamide

**Table 2.** Examples of various substances that can be derived from succinic acid by chemical conversion [47].

have complex medium requirements and they are generally unable to grow and produce organic acids at the low pH values. These restrictions provide strong incentives to integrate and optimize succinate production pathways in other microorganisms via metabolic engineering approaches.

Succinate Dehydrogenase of *Saccharomyces cerevisiae* –

The Unique Enzyme of TCA Cycle – Current Knowledge and New Perspectives 231

The constructed yeast strains with disruptions in the TCA cycle produced succinic acid up

*Saccharomyces cerevisiae* is one of the most highly researched model organisms in different biological studies. Using this yeast we can effectively re-examine long-standing and fundamental questions regarding regulation of metabolism and prediction of dynamic models in various cells, including mammalian tissues. This is a considerable knowledge about the composition, enzymology and membrane binding of the enzyme and relatively new discoveries about its genetics and biosynthesis. Through such efforts, we are able to identify key features of cellular metabolic pathways which can be use both in medicine and

*Institute of Fermentation Technology and Microbiology, Technical University of Lodz, Poland* 

I wish to thank dr Joanna Berlowska from Technical University of Lodz for her help in the

[1] McCammon MT, Epstein CB, Przybyla-Zawislak B, McAlister-Henn L, Butow RA (2003) Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes. Mol.

[2] Kruckeberg AL, Dickinson JR (2004) Carbon metabolism. In: Dickinson JR, Schweizer M, editors. The metabolism and molecular physiology of *Saccharomyces cerevisiae*.

[3] Sass E, Blachinsky E, Karniely S, Pines O (2001) Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino

[4] Steffan JS, McAlister-Henn L (1992) Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase. J Biol Chem 267: 24708–24715. [5] DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of

[6] Gombert AK, Moreira dos Santos M, Christensen B, Nielsen J (2001) Network identification and flux quantification in the central metabolism of *Saccharomyces cerevisiae* under different conditions of glucose repression. J Bacteriol 183: 1441–1451. [7] Modica-Napolitano JS, Kulawiec M, Singh KK 2007 Mitochondria and human cancer.

to 3.62 g/L at a yield of 0.11 mol /mol glucose in shake flask cultures.

in different biotechnological processes.

making of figures and selection of images of yeast cells.

**Author details** 

**Acknowledgement** 

Biol. Cell 14: 958–972.

London: CRC, pp. 42–103.

Curr Mol Med 7: 121-131.

termini. J Biol Chem 276: 46111–46117.

gene expression on a genomic scale. Science 278: 680–668.

Dorota Kregiel

**7. References** 

The popularity of *S. cerevisiae* in basic and applied research is undoubtedly influenced by its classification as GRAS (generally regarded as safe) by the U.S. Food and Drug Administration (FDA). Baker's yeast *S. cerevisiae* was the first eukaryotic organism in which complete genomic sequence was determined. Several databases such as the *Saccharomyces*  Genome Database (http://www.yeastgenome.org/) and the Comprehensive Yeast Genome Database (http://mips.gsf.de/genre/proj/yeast/) contain an enormous amount of information concerning *S. cerevisiae* genes, open reading frames, and gene products. The yeast *S. cerevisiae* became a well established eukaryotic model organism to study fundamental biological processes such as aging, mRNA transport, the cell cycle, and many more. *Saccharomyces cerevisiae* grows well in a simple chemically defined medium, under acidic conditions, even at pH values equal 3. At such low pH values, many weak acids, including succinate, occur predominantly in their undissociated form. This is advantageous for industrial production, as it reduces the need for titration with alkali and allows for direct recovery of undissociated acids. Consequently, there is no need for large quantities of acidifying agents, and the formation of salt byproducts (e.g. gypsum) is strongly reduced. In addition, *S. cerevisiae* robust tolerance in acidic conditions represents a major advantage in that it lowers the risk of contamination in industrial fermentation [44, 49].

The yeast-based fermentation process, which operates at a much lower pH than competing processes, allows succinic acid to be produced with a significantly higher energy efficiency compared to the traditional method. This compound is not accumulated intracellularly. It is also one of the first bio-based processes that sequesters carbon dioxide in the production process [47, 53]. This makes the yeast *Saccharomyces cerevisiae* a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.

The metabolic engineering strategy was used for the oxidative production of succinic acid by deletion SDH1, SDH2 genes in the genome. Arikawa et al. [55] reported an increased succinic acid productivity in sake yeast strains with deletions of TCA cycle genes. In comparison to the wild-type, succinate levels were increased up to2.7 fold in a strain with simultaneous disruption of a subunit of succinate dehydrogenase (SDH1) and fumarase (FUM1) under aerobic conditions. The single deletion of gene SDH1 led to a1.6-fold increase of succinic acid production. In another study on sake yeast strains, the deletion of genes encoding for succinate dehydrogenase subunits (SDH1, SDH2, SDH3, and SDH4) also resulted inincreased succinate productivity in aerobic conditions. Raab et al. [48] reported the construction of yeast strains for the biotechnological production of succinic acid. The genes *SDH1, SDH2, IDH1* and *IDP1*, which encode mitochondrial enzymes were deleted with the aim to disrupt succinate and isocitrate dehydrogenase activity to redirect the carbon flux and to allow succinate to accumulate as an end-product. This study showed that the yeast *S. cerevisiae* is capable of synthesizing significant amounts of succinic acid, which is exported quantitatively into the culture broth and not being accumulated intracellularly. The constructed yeast strains with disruptions in the TCA cycle produced succinic acid up to 3.62 g/L at a yield of 0.11 mol /mol glucose in shake flask cultures.

*Saccharomyces cerevisiae* is one of the most highly researched model organisms in different biological studies. Using this yeast we can effectively re-examine long-standing and fundamental questions regarding regulation of metabolism and prediction of dynamic models in various cells, including mammalian tissues. This is a considerable knowledge about the composition, enzymology and membrane binding of the enzyme and relatively new discoveries about its genetics and biosynthesis. Through such efforts, we are able to identify key features of cellular metabolic pathways which can be use both in medicine and in different biotechnological processes.
