PRODUCTS

ITACONIC ACID

CAS No. : 97-65-4
EC No. : 202-599-6

Synonyms:
itakonik asit; 2-Methylidenebutanedioic acid; 2-Methylenesuccinic acid; Methylenesuccinic acid[1]; itakonic asit; itaconik acid; itakonik acid; itaconic asit; 1-Propene-2-3-dicarboxylic acid; IA; Itaconic acid; 97-65-4; 2-Methylenesuccinic acid; METHYLENESUCCINIC ACID; Methylenebutanedioic acid; itaconate; Propylenedicarboxylic acid; 2-methylidenebutanedioic acid; Butanedioic acid, methylene-; 2-Propene-1,2-dicarboxylic acid; Succinic acid, methylene-; 2-methylenebutanedioic acid; Poly(itaconic acid); Itaconic acid polymers; Itaconic acid polymer; butanedioic acid, 2-methylene-; Methylenesuccinic acid polymers; Poly(2-methylenesuccinic acid); EINECS 202-599-6; MFCD00004260; 2-Methylene-Succinic Acid; Butanedioic acid, methylene-, homopolymer; CHEMBL359159; CHEBI:30838; NSC3357; Succinic acid, methylene- (8CI); Q4516562YH; Itaconic acid, 99+%; DSSTox_CID_6608; DSSTox_RID_78161; 2-methylenebutanedioate; DSSTox_GSID_26608; 25119-64-6; CAS-97-65-4; Methylenesuccinate; Methylenebutanedioate; ITACONIC ACID; Succinic acid, methylene-, polymers; 2-Methylenesuccinate; Methylensuccinic Acid; Propylenedicarboxylate; ACMC-1CFCS; Itaconic acid, >=99%; bmse000137; Probes1_000076; Probes2_000247; EC 202-599-6; 2-Methylenesuccinic acid #; NCIStruc1_001783; 2-Methylidenebutanedioic acid; 2-Methylenesuccinic acid; Methylenesuccinic acid[1]; 1-Propene-2-3-dicarboxylic acid; IA; Itaconic acid; 97-65-4; 2-Methylenesuccinic acid; METHYLENESUCCINIC ACID; Methylenebutanedioic acid; itaconate; Propylenedicarboxylic acid; 2-methylidenebutanedioic acid; Butanedioic acid, methylene-; 2-Propene-1,2-dicarboxylic acid; Succinic acid, methylene-; 2-methylenebutanedioic acid; Poly(itaconic acid); Itaconic acid polymers; Itaconic acid polymer; butanedioic acid, 2-methylene-; Methylenesuccinic acid polymers; Poly(2-methylenesuccinic acid); 2-methylene-butanedioic acid; NCIOpen2_004822; SCHEMBL21523; 2-Propene-1,2-dicarboxylate; DTXSID2026608; CTK3I6894; Itaconic acid, analytical standard; ZINC895261; LMFA01170063; Propylenedicarboxylic acid 97-65-4; s3095; STL163322; AKOS000118895; 2-Hydroxy-3-Naphthoyl-2-Naphthylamine; Butanedioic acid,ethylidene-,(E)-(9ci); Succinic acid, methylene-, polymers (8CI); 2-METHYLENE,1,4-BUTANEDIOIC ACID (ITACONIC ACID)

Itaconic Acid

Itaconic acid, or methylidenesuccinic acid, is an organic compound. This dicarboxylic acid is a white solid that is soluble in water, ethanol, and acetone. Historically, itaconic acid was obtained by the distillation of citric acid, but currently it is produced by fermentation. The name itaconic acid was devised as an anagram of aconitic acid, another derivative of citric acid.

Production
Since the 1960s, it is produced industrially by the fermentation of carbohydrates such as glucose or molasses using fungi such as Aspergillus itaconicus or Aspergillus terreus.
For A. terreus the itaconate pathway is mostly elucidated. The generally accepted route for itaconate is via glycolysis, tricarboxylic acid cycle, and a decarboxylation of cis-aconitate to itaconate via cis-aconitate-decarboxylase.

The smut fungus Ustilago maydis uses an alternative route. Cis-aconitate is converted to the thermodynamically favoured trans-aconitate via aconitate-Δ-isomerase (Adi1). trans-Aconitate is further decarboxylated to itaconate by trans-aconitate-decarboxylase (Tad1).
Itaconic acid is also produced in cells of macrophage lineage. It was shown that itaconate is a covalent inhibitor of the enzyme isocitrate lyase in vitro. As such, itaconate may possess antibacterial activities against bacteria expressing isocitrate lyase (such as Salmonella enterica and Mycobacterium tuberculosis).
However, cells of macrophage lineage have to "pay the price" for making itaconate, and they lose the ability to perform mitochondrial substrate-level phosphorylation.

Laboratory synthesis
Dry distillation of citric acid affords itaconic anhydride, which undergoes hydrolysis to itaconic acid.

Reactions
Upon heating, itaconic anhydride isomerizes to citraconic acid anhydride, which can be hydrolyzed to citraconic acid (2-methylmaleic acid).
Steps in conversion of citric acid to citraconic acid via itaconic and aconitic acids.
Partial hydrogenation of itaconic acid over Raney nickel affords 2-methylsuccinic acid.
Itaconic acid is primarily used as a co-monomer in the production of acrylonitrile butadiene styrene and acrylate latexes with applications in the paper and architectural coating industry.

Properties and Application of Itaconic Acid
Itaconic acid is a white crystalline powder having a hygroscopic property and a specific odor. Its melting point is 167–168 °C and the boiling point is 268 °C. Water solubility is 83.1 g l−1, and a solution (80 mg l−1) of itaconic acid in pure water has a pH of 2.0. The density of itaconic acid is 1.63 (20 °C). The pKa values of itaconic acid, its two dissociation steps, are 3.84 and 5.55 (25 °C). The equilibrium constants are K1 = 1.4 × 10−4 and K2 = 3.6 × 10−6 (25 °C).

Itaconic acid is mainly used in the plastic and paint industry. It is an unsaturated dicarbonic acid, and can readily be incorporated into polymers and used at a concentration of 1–5% (w/w) as a comonomer in polymers. The polymerized methyl, ethyl, or vinyl esters of itaconic acid are used as plastics, adhesives elastomers, and coatings. Styrene butadiene copolymers containing itaconic acid yield rubber-like resins of excellent strength and flexibility and water-proofing coatings with good electrical insulation. Other fields for use are synthetic fibers, lattices, detergents, and cleaners. On the other hand, several mono- and diesters of partially substituted itaconic acid possess anti-inflammatory or analgesic activities, and a special new market has opened for the use of itaconic acid pharmaceutical fields. A small quantity of itaconic acid is used as acidulant.

Itaconic acid (2-methylenesuccinic acid, 1-propene-2–3-dicarboxylic acid) is an unsaturated, weak dicarboxylic acid (pKa =3.83 and 5.41), discovered in 1837 as a thermal decomposition product of citric acid. The presence of the conjugated double bond of the methylene group allows polymerization both by addition and condensation. Esterification of the two carboxylic groups with different co-monomers is also possible (Kuenz et al., 2012). These diverse properties have led to a variety of applications in the pharmaceutical, architectural, paper, paint, and medical industries such as plastics, resins, paints, synthetic fibers, plasticizers, and detergents. Recently, itaconic acid applications have penetrated the dental, ophthalmic and drug delivery fields (Hajian and Yusoff, 2015). Itaconic acid polymers could even replace the petroleum-based polyacrylic acid, which has a multi-billion dollar market (Saha et al., 2019). Not surprisingly, the US Department of Energy assigned itaconic acid as one of the top 12 most promising building block chemicals for bio-based economy in 2004 (Werpy and Petersen, 2004).

Little is known about the reasons why fungi produce itaconate. Like the other organic acids, as outlined above, also itaconic acid might serve as acidifier of the environment and thus provide selective advantage for the acid-tolerant A. terreus over other micro-organisms. However, itaconic acid also has clear inhibitory properties: in macrophages of mammals, bacterial infection prompts the induction of a gene encoding a cis-aconitate decarboxylase, resulting in itaconic acid formation that inhibits bacterial metabolism as part of the immune response. The effect has been attributed to the inhibition of succinate dehydrogenase and isocitrate lyase (McFadden et al., 1971), the latter being a key enzyme of the glyoxylate cycle, required for the survival of pathogens inside a host. In turn, a few strains of these bacteria have evolved to be capable of degrading itaconate (Sasikaran et al., 2014). Itaconic acid also induces a transcription factor which is essential for protection against oxidative and xenobiotic stresses, and to attenuate inflammation (Kobayashi et al., 2013; Bambouskova et al., 2018). Whether a similar function of itaconate exists in the fungi producing it has not yet been studied.

The biosynthetic pathway of itaconic acid resembles that of citric acid, the latter acid being a direct precursor of the former. The only difference is that citric acid in A. terreus is further metabolized via cis-aconitate to itaconate by cis-aconitate decarboxylase (Bonnarme et al., 1995). To this end, cis-aconitate is transported out of the mitochondria by a specific antiporter in exchange for oxaloacetate (Li et al., 2011a,b). Itaconic acid – formed upon cis-aconitate decarboxylation – is finally secreted out of mycelia by a specific cell membrane transporter. Genes encoding these three enzymes, and a fourth one encoding a transcription factor, constitute the “itaconate gene cluster” in the A. terreus genome, while the cluster is notably absent in A. niger. Although several itaconate producers have been tested, the plant pathogenic Basidiomycete Ustilago maydis (the corn smut fungus) – and particularly its low pH-stable relative Ustilago cynodontis (Hosseinpour Tehrani et al., 2019b) – seems to be the only one with a reasonable chance to become another industrial platform organism (Hosseinpour Tehrani et al., 2019a). Ustilago has developed an alternative biochemical pathway to synthetize itaconate inasmuch as cis-aconitate is converted to the thermodynamically favored trans-aconitate by aconitate-delta-isomerase. Trans-aconitate is then decarboxylated to itaconate by trans-aconitate-decarboxylase.

Production of Itaconic Acid by Fermentation Processes
Itaconic acid is produced in batch fermentation in a process largely similar to that of citric acid. The carbon source should be in an easily metabolizable form (glucose syroup, molasses, and crude starch hydrolysates) and diluted to approximately 10% wt. Phosphate limitation is necessary for growth restriction. Some trace metals should also be in limited amounts and this is usually achieved by treating the media with hexacyanoferratl or addition of copper. The pH is kept between 2.8 and 3.2. Lower pH values favor the formation of byproducts. Yields of 50–60% of the theoretical yield are obtained in 8–10 days [5].

For many years, there seems to be almost no research interest for the production of itaconic acid and the process remained unchanged since its introduction. The situation is different today. Itaconic acid is listed by the US Department of Energy (DOE) as one of the 12 building blocks with the highest potential to be produced by industrial biotechnology [11]. Its current low production limits its uses. Metabolic engineering strategies, as an approach for yield improvement, have not yet been applied with A. terreus, as they were restricted by the poor knowledge of the genetics of itaconic acid biosynthesis. Recently, however, three genes – crucial in itaconic acid production by A. terreus – were identified by researchers in Toegepast Natuurwetenschappelijk Onderzoek (TNO), the Netherlands [15]. Apart from the new knowledge on the genetics of biosynthesis, the development of new fermentation technologies and more sophisticated bioprocess control has led to renewed interest in improving itaconic acid production. Novel fed-batch strategies and continuous processes using immobilized cultures are being developed and investigated.

Itaconic acid is a dicarboxylic acid, which is used in industry as a precursor of polymers used in plastics, adhesives, and coatings. New uses of itaconic acid-derived polymers are under active investigation. The production of itaconic acid for 2001 was quoted as 15 000 tons. There is a renewed interest in this chemical as industry searches for substitutes of petroleum-derived chemicals. Virtually all itaconic acid produced is by fermentation by specific strains of A. terreus. Itaconic acid production is a further perversion of the Krebs cycle, citrate is converted as normally into cis-aconitate, which for reasons unknown is, in some organisms, decarboxylated into itaconitate, which has no known metabolic role in the cell.

The fact that different strains of Aspergillus and more generally of fungi can divert metabolic pathways to the overproduction and secretion of useful chemicals, coupled with the fact that these organisms can grow on residues of processes such as sugar and ethanol production, open the possibility of engineering pathways to produce high value chemicals through ‘green’, low polluting, waste-eliminating procedures.

Production Itaconic Acid
Itaconic acid is an example of a di-carbonic unsaturated acid. These acids are used as building blocks for large numbers of compounds, such as resins, paints, plastics, and synthetic fibers (acrylic plastic, super absorbants, and antiscaling agents) [67]. The CAC intermediate cis-aconitate is enzymatically processed by cis-aconitate dehycarboxylase (CadA) to produce itaconic acid [68]. At the industrial scale the most explored organism for the fermentative production of itaconic acid is Aspergillus terrus. The biosynthetic pathway of itaconic acid is like citrate biosynthesis, where the flux of the CAC is used in the catalytic conversion of cis-aconitate into itaconic acid. Thus citrate is synthesized from oxaloacetate and acetyl CoA, while oxaloacetate is synthesized from pyruvate by anaplerosis, which starts from the pyruvate that is the end product of glycolysis (Fig. 13.17).

Itaconic acid (methylenesuccinic acid, C5H6O4) (Figure 17) is a white colorless crystalline, hygroscopic powder soluble in water, ethanol, and acetone. It is an unsaturated diprotic acid, which derives its unique chemical properties from the conjugation of one of its two carboxylic acid groups with its methylene group.
Itaconic acid was discovered by Baup in 1837 as a product of pyrolytic distillation of citric acid. The name itaconic was devised as an anagram of aconitic.

Itaconic acid is formed in fermentation of some sugars. In 1929, Kinoshita first showed the acid to be a metabolic product of Aspergillus itaconicus. A derivative of itaconic acid (trans-phenylitaconic acid) was isolated from another natural source (Artemisia argyi).
The biosynthetic pathway of itaconic acid from glucose is similar to that of citric acid, which occurs via the glycolytic pathway and anaplerotic formation of oxaloacetate by CO2 fixation and via the TCA cycle (Figure 2). Itaconic acid is formed by the cytosolic enzyme aconitate decarboxylase from cis-aconitic acid. Another biosynthetic pathway from pyruvate through citramalic acid, citraconic acid, and itartaric acid also results in itaconic acid (Figure 18).
In contrast to several other organic acids (e.g., citric, isocitric, lactic, fumaric, and l-malic acid) itaconic acid is used exclusively in nonfood applications, especially in the polymer industry. Itaconic acid derivatives are used in medicine, cosmetics, lubricants, thickeners, and herbicides (e.g., substituted itaconic acid anilides).

Itaconic acid is produced solely by batch submerged fungal fermentation. Aspergillus terreus has been used from the 1940s in the fermentation process, which is similar to that of citric acid (see ‘Citric acid’), that is, it requires an excess of readily metabolizable sugar (glucose syrup, crude starch hydrolysates, and decationized molasses – up to 200 g l−1 sugar), continuous aeration, a low initial pH (between 3 and 5), sufficient nitrogen, high magnesium sulfate concentration (0.5%), low phosphate to limit biomass production, and a limitation in metal ions (zinc, copper, and iron). However, there exists one significant difference in that the sensitivity of this fungus to the formed acid, in contrast to A. niger, necessitates maintaining of the pH at 2.8–3.1 throughout the fermentation, in order to obtain high amounts of the acid. At present, the published production yield of itaconic acid is about 85% of theoretical, accompanied by product concentrations of about 80 g l−1 during a cultivation at 39–42 °C for 8–10 days. Recovery of itaconic acid is accomplished by first separating the fungal biomass by filtration followed by evaporation, treatment with active carbon, and crystallization and recrystallization. Actual markets for itaconic acid are currently limited because the fungal fermentation is carried out at a relatively high cost. New biotechnological approaches, such as published immobilization techniques, screening programs for other producing organisms (such as yeast), and genetic engineering of A. terreus (the annotated genome sequence of A terreus strain NIH 2624 has been publicly released), or of A. niger, could lead to higher production of itaconic acid. Also, the use of alternative substrates may reduce costs and thus open the market for new and expanded applications of this acid.

This valuable acid can be produced by several organisms, such as Candida sp., Pseudozyma antarctica, and several species of Aspergillus [49], but the two most common microorganisms used are Aspergillus terreus, used in industrial processes, and Ustilago maydis, which is currently being actively investigated as a possible industrial product. The acid is used commercially as a comonomer in some synthetic rubbers (styrene-butadiene and nitrilic) and as a plasticizer in the formulation of other polymers. Its production is traditionally done using sugars as raw materials, in a technology that was developed in the first half of the 20th century [50], but that was not developed due to the low competitivity of the acid with the petrochemical acrylic acid. With the development of integrated and sustainable processes, the interest in the bioproduction of itaconic acid is renewed.

Itaconic acid is produced using A. terreus, from simple sugars. The production can be done using submerged solid fermentation, and the typical substrates are derived from sugar production, such as molasses. The accepted mechanism for itaconic acid production consists of the conversion of cis-aconitate to itaconate by an enzymatically catalyzed decarboxylation [53] (Fig. 18.6).

Cis-aconitate is part of the Krebs cycle, so that the process is aerobic—actually extremely oxygen dependent, as determined by Gyamerah [54]. Calcium and zinc are important [55], as well as copper [56], and the maintenance of a low phosphate level is essential [53]. The ideal temperature is 40°C, and pH must be reduced to 2 to start the production. The process is extremely aerobic for the first 72 h of the process, with yields around 60%w/w (product/substrate) [55]. The final concentration ranges between 30 and 60 g/L depending on the substrate [56–58]. After fermentation, the broth is clarified and the free acid can be concentrated and crystallized, but if a base is used for partial neutralization during the process (which can increase the yield), it is necessary to remove the cations used in the crystallization.

The production of itaconic acid in SSF is still elusive: reports describe productions on the order of 5–40 g/kg dry substrate [59]. Some of the reports that describe higher yields, around 60%, actually use a support soaked with a nutritive solution [60,61]. A comparison between synthetic liquid and solid media showed that the process in SSF has a lower conversion (16%–23%) than that of the submerged process (around 60%). There is no definite explanation for the lower production in solid-state yet, but there seems to be an excess of phosphate or the lack of essential nutrients in most solid substrates tested for itaconic acid reduction.

First obtained from the distillation of citric acid, since 1960 itaconic acid has been produced by fermentation of carbohydrates by A. terreus (Mitsuyasu et al., 2009; Hajian and Yusoff, 2015). Itaconic acid has been applied in a numerous range of industries with the larger producers in the world being the USA, Japan, Russia, and China (Global Industry Analysts Inc., 2011).

During the 1950s, itaconic acid was used in industrial adhesives. In that period, itaconic acid was used at an industrial scale and large amounts of it were required. It has been employed as a detergent and in shampoos, as well as in plastics, elastomers, fiberglass, and in the coating process of carpets and book covers (Mitsuyasu et al., 2009; Jin et al., 2010). Besides that itaconic acid may also be used as artificial gems and synthetic glasses (Kin et al., 1998). Lately, the applications of the compound have reached the biomedical fields, such as the ophthalmic, dental and drug delivery fields (Hajian and Yusoff, 2015).

Several studies have focused on improving and optimizing the production of itaconic acid from A. terreus in recent years. The biotechnological aspects involved in the metabolic pathways of itaconic acid and the production process parameters have been reviewed by Klement and Büchs (2013). Regarding the production, Amina et al. (2013) obtained itaconic acid using oil byproduct jatropha curcas seed cake, while Li et al. (2011), Huang et al. (2014), and van der Straat et al. (2014) studied the itaconic acid production by using genetic engineering techniques. In this process the relevant pathways have been revealed and new microbial production platforms designed, contributing to an enhanced production of itaconic acid. Furthermore, the reduction of its production costs is an important aspect for itaconic acid producers, either by optimizing processes or by using cost-favorable raw materials.

Itaconic acid or methylene succinic acid is a high-value platform chemical that finds application in polymer industry, wastewater treatment, and ion-exchange chromatography sector (Willke and Vorlop, 2001). It can be converted to 3-methyltetrahydrofuran that has superior emission and combustion properties when compared to gasoline. Industrial production of itaconic acid is carried out with A. terreus using glucose as the sole carbon source. Itaconic acid production by metabolically engineered Neurospora crassa using lignocellulosic biomass was evaluated by Zhao et al. (2018). Cis-aconitic acid decarboxylase gene was heterologously expressed in N. crassa to synthesize itaconic acid. The engineered strain was capable of producing itaconic acid (20.41 mg/L) directly from lignocellulosic biomass.

Itaconic acid production from biomass hydrolyzate using Aspergillus strains was reported by Jiménez-Quero et al. (2016). Acid and enzymatic hydrolyzates were evaluated for the production of itaconic acid. Maximum itaconic acid production (0.14%) was observed when submerged fermentation was carried out with corncob hydrolyzate by A. oryzae. The study reveals the possibility of SSF of biomass for the production of itaconic acid.

Klement et al. (2012) evaluated itaconic acid production by Ustilago maydis from hemicellulosic fraction of pretreated beech wood. One of the advantages of U. maydis is that the strain grows as yeast-like single cells, and it can survive under high osmotic stress. The study revealed that under mild pretreatment conditions, U. maydis would be a promising candidate for itaconic acid production. Fine tuning of pretreatment conditions should be carried out for the improved production of itaconic acid.

Itaconic acid (IA) can be used:
• As a comonomer in the polymerization of polyacrylonitrile (PAN) to promote the thermo-oxidative stabilization of polymer.[1]
• In combination with acrylamide to form (poly[acrylamide-co-(itaconicacid)]) to synthesize biodegradable superabsorbent polymers.[2]
• To synthesize biobased polyester composite in fabric industry.

Itaconic acid is an unsaturated dicarbonic acid which has a high potential as a biochemical building block, because it can be used as a monomer for the production of a plethora of products including resins, plastics, paints, and synthetic fibers. Some Aspergillus species, like A. itaconicus and A. terreus, show the ability to synthesize this organic acid and A. terreus can secrete significant amounts to the media (>80 g/L). However, compared with the citric acid production process (titers >200 g/L) the achieved titers are still low and the overall process is expensive because purified substrates are required for optimal productivity. Itaconate is formed by the enzymatic activity of a cis-aconitate decarboxylase (CadA) encoded by the cadA gene in A. terreus. Cloning of the cadA gene into the citric acid producing fungus A. niger showed that it is possible to produce itaconic acid also in a different host organism. This review will describe the current status and recent advances in the understanding of the molecular processes leading to the biotechnological production of itaconic acid.

Itaconic acid (2-methylidenebutanedioic acid) is an unsaturated di-carbonic acid. It has a broad application spectrum in the industrial production of resins and is used as a building block for acrylic plastics, acrylate latexes, super-absorbents, and anti-scaling agents (Willke and Vorlop, 2001; Okabe et al., 2009). Since the 1960s the production of itaconic acid is achieved by the fermentation with Aspergillus terreus on sugar containing media (Willke and Vorlop, 2001). Although also other microorganisms like Ustilago zeae (Haskins et al., 1955), U. maydis, Candida sp. (Tabuchi et al., 1981), and Rhodotorula sp. (Kawamura et al., 1981) were found to produce itaconic acid, A. terreus is still the dominant production host, because so far only bred strains of this species can reach levels of up to 80–86 g/L (Okabe et al., 2009; Kuenz et al., 2012). Since the 1990s, itaconic acid as a renewable material is attracting a lot of interest. Currently, the worldwide production capacity of itaconic acid is expected to be about 50 kt per year, facing a demand of about 30 kt (Shaw, 2013, Itaconix Corporation, personal communication). Especially, for the production of polymers it is of interest, because in the future it can function as a substitute for acrylic and methacrylic acid used for the production of plastics (Okabe et al., 2009). However, these applications require an even lower price of the starting material. The current knowledge about the biotechnological production of itaconic acid was recently reviewed (Willke and Vorlop, 2001; Okabe et al., 2009). The latter review covers the industrial production of itaconic acid and the applications of this product. Therefore, we focus in this report on the recent advances with an emphasis on the biochemistry of the process and new genetic engineering targets. For rational strain improvement, it is essential to understand the underlying biological concepts and biochemical pathways leading to the production of this important organic acid in microorganisms.

Biosynthesis Pathway
Kinoshita (1932) recognized that a filamentous fungus was able to produce itaconic acid and consequently described this species as A. itaconicus. The biosynthesis of itaconic acid was for a long time hotly debated, because it was not clear whether itaconic acid arises from a pathway including parts of the tricarboxylic acid (TCA) cycle or an alternative pathway via citramalate or the condensation of acetyl-CoA.

Bentley and Thiessen (1957a) proposed a pathway for the biosynthesis of itaconic acid, which is depicted in Figure 1. Starting from a sugar substrate like glucose the carbon molecules are processed via glycolysis to pyruvate. Then the pathway is split and part of the carbon is metabolized to Acetyl-CoA releasing a carbon dioxide molecule. The other part is converted to oxaloacetate so that the previously released carbon dioxide molecule is again incorporated. In the first steps of the citric acid cycle, citrate and cis-aconitate are formed. In the last step, the only itaconic acid pathway dedicated step, cis-aconitate decarboxylase (CadA) forms itaconic acid releasing carbon dioxide. This pathway was confirmed by tracer experiments with 14C and 13C labeled substrates (Bentley and Thiessen, 1957a; Winskill, 1983; Bonnarme et al., 1995) and also the necessary enzymatic activities have been all determined (Jaklitsch et al., 1991).

The formation of carboxylic acids, like citric and itaconic acid, involves the shuttling of intermediate metabolites between different intracellular compartments and utilizes the different enzymatic capabilities of the respective compartment. In case of itaconic acid the compartmentalization of the pathway was analyzed by fractionized cell extracts distinguishing the enzymatic activity of a mitochondrial from a cytosolic enzyme. It was found that the key enzyme of the pathway, CadA, is not located in the mitochondria but in the cytosol (Jaklitsch et al., 1991), whereas the enzymes preceding in the pathway, namely citrate synthase and aconitase, are found in the mitochondria. However, a residual level of aconitase and citrate synthase activity is also found in the cytosolic fraction. The proposed mechanism is that cis-aconitate is transported via the malate–citrate antiporter into the cytosol (Jaklitsch et al., 1991). However, so far it was not shown whether cis-aconitate makes use of the mitochondrial malate–citrate antiporter or uses another mitochondrial carrier protein to be translocated to the cytosol.

Besides A. terreus, itaconic acid is known to be produced also by other fungi like U. zeae (Haskins et al., 1955), U. maydis (Haskins et al., 1955; Klement et al., 2012), Candida sp. (Tabuchi et al., 1981), and Rhodotorula sp. (Kawamura et al., 1981). No further investigations exist about the underlying reaction principles leading to itaconic acid formation in those species. However, recent evidence (Strelko et al., 2011; Voll et al., 2012) points into the direction that CadA activity constitutes the general pathway toward the formation of itaconic acid in nature. Very recently, itaconic acid was detected in mammalian cells, where it was found in macrophage-derived cells (Strelko et al., 2011). Those cells also possess a CadA activity and have the ability to form itaconic acid de novo. But, up to now no specific gene encoding this enzymatic activity was identified in mammalian cells.

However, the physiological role of itaconic acid in mammalian cells is still unknown. Strelko et al. (2011) speculate on the role of itaconic acid as an inhibitor of metabolic pathways, because it is described as an enzymatic inhibitor. On the one hand, itaconic acid is known to inhibit isocitrate lyase (Williams et al., 1971; McFadden and Purohit, 1977), which is the crucial part of the glyoxylate shunt, and thus can act as an antibacterial agent. On the other hand, itaconic acid can inhibit fructose-6-phosphate 2-kinase (Sakai et al., 2004) and thus have a direct influence on the central carbon metabolism. In rats it was shown that a itaconate diet leads to a reduced visceral fat accumulation, because of a suppressed glycolytic flux (Sakai et al., 2004).

Itaconic Acid Pathway Specific Enzymes and Genes
The reaction catalyzed by the cis-aconitic acid decarboxylase was already described in 1957 (Bentley and Thiessen, 1957a,b). Subsequently performed 13C and 14C labeling experiments (Winskill, 1983; Bonnarme et al., 1995) confirmed the reaction scheme depicted in Figure 2. Itaconic acid is formed by an allylic rearrangement and decarboxylation from cis-aconitic acid removing either carbon C1 or C5 from the starting citric acid molecule (because of the symmetry of the molecule).

Catabolization of Itaconic Acid
Much is known about the biosynthesis of itaconic acid and the underlying enzymatic mechanisms, but for a complete biochemical picture of a certain metabolite, also the knowledge about its degradation is necessary. Unfortunately, the information about the degradation pathway of itaconic acid is scarce. In mammalian cells (guinea pig and rat liver) it was found that itaconate is converted to itaconyl-CoA (Adler et al., 1957) and is further processed via citramalyl-CoA (Wang et al., 1961) to pyruvate and acetyl-CoA. Hereby, it was found that malonate has an inhibitory effect and an addition prevents the degradation of itaconic acid (Adler et al., 1957). The first step of this degradation pathway can be catalyzed by the ubiquitous succinyl-CoA synthetase (Adler et al., 1957; Nagai, 1963; Schürmann et al., 2011). The third step of the pathway is catalyzed by a citramalyl-CoA lyase, where genes from Chloroflexus aurantiacus (Friedmann et al., 2007) and Pseudomonas putida (Jain, 1996) have been cloned. However, no protein and gene sequence was identified so far, which can catalyze the second step of the degradation pathway, which is an itaconyl-CoA hydratase (Cooper and Kornberg, 1964).

Metabolic Engineering of the Itaconic Acid Pathway in A. terreus and A. niger
The levels of itaconic acid which were reached with A. terreus are currently limited to about 85 g/L. Although this is already a substantial amount it cannot be compared with the production of citric acid where titers over 200 g/L are steadily obtained in industrial processes. Transferred to the itaconic acid production a maximal theoretical titer of about 240 g/L should be achievable (Li et al., 2011). This goal could be reached by further breeding of currently existing strains or targeted genetic engineering.

In A. terreus, a gene was shown to influence the performance of itaconic acid production, which is a key enzyme of glycolysis. 6-phosphofructo-1-kinase is known to be inhibited by citrate and adenosine triphosphate (ATP). However, a truncated version of the A. niger pfkA gene was shown to exhibit a higher citric acid yield due to a reduced inhibition by citrate and ATP (Capuder et al., 2009). This truncated pfkA version had also a positive impact on the itaconic acid accumulation when expressed in A. terreus (Tevz et al., 2010). Another engineering approach deals with the intracellular oxygen supply. The production of itaconic acid requires continuous aeration and already a short interruption of oxygen decreases the itaconic acid yield. In order to reduce the sensitivity to oxygen a hemoglobin gene from Vitreoscilla was expressed in A. terreus. Indeed, the expression of this gene leads to an increased itaconic acid production. Furthermore, the strains exhibited a better recovery after the aeration was interrupted (Lin et al., 2004).

There is the possibility that the genetic make-up of A. terreus is not efficient enough to support the production of higher titers of organic acids. Therefore, a strategy is to genetically engineer the itaconic acid biosynthesis pathway into another host organism, which is already known to support the production of high titers of organic acids. As already mentioned, A. niger is such a candidate. The unique and crucial step in the biosynthesis pathway is the decarboxylation of cis-aconitic acid toward itaconic acid. When the cadA gene (Kanamasa et al., 2008) was characterized in A. terreus genetic engineering of the pathway into another organism became possible. Li et al. (2011) expressed the A. terreus cadA gene in A. niger strain AB 1.13. For this purpose, the cadA gene was placed under the control of the A. niger gpdA promoter, which enables a strong and constitutive expression. An A. niger strain which expresses the cadA gene alone has the ability to produce about 0.7 g/L itaconic acid. This level is not comparable with current production strains of A. terreus, but is a promising starting point for further engineering steps. Further attempts to rise the yield are to express genes like the above mentioned mitochondrial carrier protein together with the cadA gene (Jore et al., 2011; van der Straat et al., 2012).

Outlook
Itaconic acid as a renewable organic acid is of growing interest for the chemical industry, because of its potential to replace crude oil based products like acrylic acid. Up to now, the microorganism based processes were improved by classical strain breeding and optimizations of the fermentation strategies and conditions. Especially the knowledge about the biotechnological process including oxygen supply, media compositions, and different bioreactor systems was significantly expanded (Kuenz et al., 2012). Regarding the media composition, it was found that copper ions positively influence the itaconic acid production in a genetically engineered A. niger strain (Li et al., 2012). However, it is not understood which biochemical reactions are responsible or involved in such an effect. As already mentioned above, the biochemical reactions and effects of itaconic acid in the production hosts are not fully described. The catabolization pathway of itaconic acid requires further investigations in order to engineer a production host with a disabled degradation pathway. The effect of itaconic acid on other metabolic pathways is also of interest because the understanding of its physiological role can prevent undesired side effects (toxicity, health risk, pathway inhibition) and increase the safety of its use. Furthermore, it can be an interesting target for medical research because in mammalian cells it was detected in a metastatic tumor cell line (Strelko et al., 2011). Further knowledge about its role as an enzyme inhibitor can help to develop less-resistant enzyme varieties like in the case of the phosphofructokinase 2. Another target for further engineering is the CadA enzyme, which is described as an unstable protein. Prolonging its in vivo stability can help to increase the efficiency of existing production hosts. Also the genetic regulation of the itaconic acid pathway in A. terreus requires a profound analysis. Li et al. (2011) have shown that genes involved in the biosynthesis pathway (cadA) can be identified by transcriptomic approaches. However, nothing is known so far about the regulatory mechanisms leading to the expression of those genes.
The investigations on the molecular principles of itaconic acid synthesis revealed that cis-aconitic acid decarboxylase is the dedicated step in its biosynthesis in A. terreus. Genetic engineering of this enzymatic step also renders other microbial hosts like A. niger to producers of itaconic acid.

Itaconic acid is well known as a precursor for polymer synthesis and has been involved in industrial processes for decades. In a recent surprising discovery, itaconic acid was found to play a role as an immune-supportive metabolite in mammalian immune cells, where it is synthesized as an antimicrobial compound from the citric acid cycle intermediate cis-aconitic acid. Although the immune-responsive gene 1 protein (IRG1) has been associated to immune response without a mechanistic function, the critical link to itaconic acid production through an enzymatic function of this protein was only recently revealed. In this review, we highlight the history of itaconic acid as an industrial and antimicrobial compound, starting with its biotechnological synthesis and ending with its antimicrobial function in mammalian immune cells.

Itaconic acid or methylenesuccinic acid is a five-carbon unsaturated dicarboxylic acid with one carboxyl group conjugated to the methylene group. According to the annual forecast, market is predicted to exceed 410,000 t by 2020 (Choi et al., 2015). Itaconic acid has broad applications manufacture of absorbents, phosphate-free detergents, cleaners, and bioactive compounds. Itaconic acid is sought as a compound for replacement of petroleum-based chemicals such as acrylic acid or methyl acrylic acids which are used presently in the polymer industry. The polymerized esters of Itaconic acid (IA) are used widely in adhesive and paints/coating industries. Itaconic acid is also used in the polymer industry and is also used in the synthesis of 3-methyltetrahydrofuran. A company called Itaconix is working on the use of wood biomass as a feedstock for fermenting itaconic acid.

Itaconic acid is an important building block in the chemical industry. It is a white crystalline powder and readily biodegrades in soil. Hence, it is an optimum substitute for petro-derived chemicals such as acrylic acid, maleic anhydride, or acetone cyanohydrin in various end-user industries. The demand for itaconic acid is high in the manufacturing of superabsorbent polymers, mainly used in diapers, adult incontinence, and feminine hygiene products. Itaconic acid is used as a cross-linking agent due to its ability to efficiently take part in addition polymerization. It also finds large application in seed coating, root dipping, ornamental gardens, food packaging, and artificial snow. Moreover, increasing demand for unsaturated polyester resins in pipes, artificial stones, electrical cabinets, and laminating resins is expected to increase the demand for itaconic acid. High price of itaconic acid is the major factor hampering the growth of itaconic acid market. Polyitaconic acid (a derivative of itaconic acid) has the potential to replace sodium tripolyphosphate in detergents.

Itaconic acid is a biobased product mainly produced by fermentation using certain filamentous fungi (e.g. Ustilago, Helicobasidium, and Aspergillus). A mixture of itaconic acid, citraconic acid, and citraconic anhydride is also obtained by reaction of succinic anhydride with formaldehyde at 200–500°C in the presence of alkali or alkaline earth hydroxides (could at least partially be biobased if biobased succinate is used as raw material for the production of succinic anhydride). Other methods involve carbonylation of propargyl chloride with metal carbonyl catalysts and thermal decomposition of citric acid, which is also a biobased chemical. Aspergillus terreus is the strain commonly used for the industrial production of itaconic acid. A significant amount of research has been put into the reduction of the production costs: the replacement of sugar, used as the carbon source, by cheaper alternative substrates such as cellulolytic biomass; optimizing the bioreactor type and configuration; deriving innovations by which the process becomes more energy saving; strain improvement by genetic and metabolic engineering, allowing the effective use of cheap alternative substrates, etc. Recent patent activity has particularly focused on the improvement of the producing strain, mainly by using recombinant DNA techniques, and several patents have been submitted worldwide in the last 10 years. There is a significant market opportunity for the development of biobased products from the C5 building block, itaconic acid. The major challenges are primarily associated with reducing the overall cost of the fermentation.