The production cost of itaconic acid remains high. However, economic feasibility may be improved through optimization of either fermentation or chemical synthesis methods. Some options may include using sugarcane molasses, a sugar- and aconitic acid-rich feedstock, for chemical or enzyme-based decarboxylation or for improved fermentation conditions. Optimized fermentation conditions may include the use of inexpensive feedstocks such as agricultural waste products or through metabolic engineering of microbial strains to eliminate unwanted pathways and directly increase carbon flux toward itaconic acid production.

Some fungi naturally convert aconitic acid to itaconic acid through decarboxylation. For example, Aspergillus terreus is used in fermentations to produce itaconic acid ^[5][6][7][8][43,44,45,71]. In A. terreus, CAA is first transported by a mitochondrial transporter, At_MttA, from the tricarboxylic acid (TCA) cycle in the mitochondria to the cytosol, where CAA is decarboxylated to itaconic acid by the cis-aconitic acid decarboxylase Cad-A ^[5][9][10][43,46,72]. Subsequently, itaconic acid is transported out of the mycelia by a specific transporter, belonging to the major facilitator superfamily (MFS) type transporter (MfsA) ^[11][73]. The genes involved in itaconic acid production in A. terreus also include a transcription factor, and the four genes are termed the “itaconate gene cluster” ^[11][73]. However, in the corn smut basidiomycete, Ustilago maydis, the mitochondrial transporter Um_Mtt1, also transports CAA from the mitochondria to the cytoplasm, but CAA is first isomerized to TAA by aconitate-Δ-isomerase, Adi1, then decarboxylated by trans-aconitic acid decarboxylase, Tad1, to itaconic acid ^{[5][9][12][13]}[43,46,47,74]. The “itaconate gene cluster” in U. maydis is induced upon nitrogen limitation ^[14][75]. Overall, the metabolic differences in itaconic acid production by these two fungi is notable because it demonstrates the possibility of producing itaconic acid from either the trans or cis isomer of aconitic acid in different organisms ^[12][47]. Differential microbial conversions of CAA and TAA to itaconic acid may also provide insight into ex vivo enzymatic conversion options with recombinantly expressed decarboxylases.

Microorganisms such as the soil bacterium Pseudomonas sp. WU-0701 encodes an aconitate isomerase that catalyzes the reversible isomerization between TAA and CAA, an intermediate in the conversion of citrate to isocitrate in the TCA cycle ^[15][49]. This enables the organism to grow on TAA as a sole carbon source by isomerizing TAA to CAA, which feeds back into the TCA cycle. Interestingly, others have reported the presence of various Pseudomonas species in the rhizosphere of sugar cane, while others have reported that some rhizosphere-associated bacteria may improve plant growth and photosynthesis under certain conditions ^[16][76]. The utilization of TAA as a carbon source by some of these bacteria could suggest a possible symbiosis between sugar cane and microorganisms in the rhizosphere involving TAA.

There are some reports that aconitic acid may act as an inhibitor in the fermentation of sugar cane juice, syrup, molasses, and sweet sorghum syrup. When studying the fermentation of sweet sorghum juice as a function of harvesting time, Day and Sarkar ^[17][77] noted that the yield of ethanol produced by sake and wine yeasts dropped later in the harvest season, even though the sugar content increased. They speculated that the aconitic acid was responsible for the reduction in ethanol yield. When increasing the fermentable sugars by concentrating sweet sorghum juice, Wu et al. ^[18][78] reported that the fermentation efficiency decreased with increasing water removal and speculated that the increase in aconitic acid levels due to the water removal may have been the reason ^[18][78]. In fermentation of sweet sorghum juice, Gibbons and Westby ^[19][79] reported low ethanol yields by Saccharomyces cerevisiae depending on sweet sorghum variety. They speculated that aconitic acid was partially responsible for the inhibition, as it varied between varieties. The inhibition also remained noticeable in the fermentation of mixtures of sweet sorghum juice with hydrolyzed cereal mash. More inhibition was noted with corn mash than with wheat mash. Another report showed that the fermentation rate decreased by 29% when comparing the production of ethanol from sweet sorghum juices containing 0.114 and 0.312% aconitic acid ^[20][50]. These authors attributed the rate reduction to aconitic acid and showed that the intracellular acid concentration of the yeast increased by a factor of 2 and 4 when the pH is changed from pH 5.0 to pH 3.5 to pH 2.0. In a detailed study, Klasson ^[21][52] showed that it was the undissociated form of aconitic acid that was responsible for inhibition of ethanol production by Distiller’s yeast, S. cerevisiae, in the fermentation of sweet sorghum sugars. By controlling the pH during the fermentation, the inhibition could be overcome, and when the pH was controlled above 4.5, the presence of aconitic acid (5 g/L) became slightly advantageous, and ethanol titer (+4%) and yield (+3%) increased slightly, confirming results of a previous study in synthetic media ^[22][51]. At a fermentation pH > 4.5, the fermentation of diluted sweet sorghum syrup to butanol by Clostridium beijerinckii did not appear to be inhibited by aconitic acid ^[21][52].

Pathogenic nematodes can be a significant problem for some crops such as sugar beets and cotton. For instance, the beet cyst nematode, Heterodera schachtii, is especially problematic for sugar beets, which supply about one-third of the world’s supply of sugar ^[23][80]. Finding a sustainable, plant-based source of nematocide that is nontoxic to humans is of considerable interest. Interestingly, the soil bacterium, Bacillus thuringiensis, produces TAA as a virulence factor against soil nematodes ^[24][53]. Studies with the thuringiensin-producing strain, B. thuringiensis CT-43 in particular, revealed a chemical product called CT-A, with nematocidal activity against the major pest root-knot nematode, Meloidogyne incognita ^[24][53]. Further study revealed that CT-A contains TAA and that TAA exhibits a significantly higher nematocide activity than the cis isomer, CAA, in a survival bioassay with M. incognita J2s after 72 h. A plasmid-encoded operon for TAA biosynthesis was described in B. thuringiensis CT-43 that encodes an aconitate isomerase, named TAA biosynthesis-related gene A (tbrA), and a membrane-bound transporter tbrB that transports TAA out of the cell ^[24][25][53,81].

Anti-leishmanial activity has also been attributed to TAA against the protozoan pathogen, Leishmania donovani, the causative agent of visceral leishmaniasis, also known as kala-azar, thirty years ago, which is potentially fatal and difficult to treat ^[26][27][54,55]. During the lifecycle of this protozoan, the promastigote form is present in the vector during disease transmission, while the amastigote form is found intracellularly within infected macrophages of the host. Anti-leishmanial drugs can be problematic due to toxicity. TAA was investigated as an alternative and in combination with conventional chemotherapy since TAA is an inhibitor of the aconitase enzyme in the TCA cycle ^[28][56]. The L. donovani amastigote relies on mitochondrial β−oxidation of fatty acids as an important energy source. During β-oxidation, fatty acids are converted to acetyl-CoA, which feeds into the TCA cycle to generate ATP for energy, so TAA was of particular interest as an inhibitor of aconitase in the TCA cycle ^[29][57]. Interestingly, 20 mM TAA significantly attenuated promastigote replication that could be reversed by the addition of 20 mM CAA at 72 h, indicating divergent biological activity of the two aconitic acid isomers. Furthermore, 2 mM TAA reduced parasitic liver burden in infected hamsters in a dose-dependent manner ^[26][27][54,55]. A dose of 2 mM TAA reduced the number of amastigotes within a macrophage model by 60% ^[26][54]. Five (5) mM TAA, together with anti-leishmanial drugs, sodium stibogluconate, pentamidine, or allopurinol, completely inhibited amastigote transformation to promastigote ^[27][55]. These reports from L. donovani may provide insight into the mechanism of action against other organisms such as nematodes.

The production of TAA by sugarcane, sweet sorghum, and other plants may confer a survival advantage against pests and help to regulate metabolic processes during rapid plant growth. Stout et al. ^[30][82] assayed 94 species of grasses and non-grasses from rangeland and found that 47% of grasses and 17% of non-grasses accumulated TAA to high levels. Furthermore, aconitic acid has also been detected in grasses such as oats, rye, wheat, barley, and maize ^[31][83].

In higher plants, trans-aconitate is produced and stored as a “tricarboxylic acid pool” ^[32][7]. TAA is produced via two mechanisms connected to the TCA cycle. The first occurs via the citrate valve and citrate hydratase to form TAA ^[32][7]. The second mechanism occurs with aconitase conversion of citrate to isocitrate via a cis-aconitate intermediate, which can then be isomerized to TAA via aconitate isomerase ^[32][7]. Accumulation of TAA may play a role in regulating the TCA cycle by inhibition of aconitase ^[28][56]. Moreover, this inhibition can be alleviated by monomethyl esterification by trans-aconitate methyltransferase TMT1, which has been described in Escherichia coli, Saccharomyces cerevisiae, and Ashbya gossypii ^[33][34][35][84,85,86].

Aconitic acid may be an inhibitor of biofilm formation. Pestana-Nobles et al. ^[49][70] reported a computational study based on molecular docking and molecular dynamic simulation that screened 224,205 molecules from the natural products ZINC15 database. The results predicted TAA as a possible ligand and inhibitor of the PleD protein involved in bacterial biofilm formation. PleD and its homologs are diguanylate cyclases that contain a GGDEF domain involved in cyclic di-GMP second messenger formation, a critical signaling molecule involved in quorum sensing needed for biofilm formation. As such, PleD homologs are often the target of high-throughput screens for biofilm inhibitors ^[50][51][92,93]. While TAA was identified computationally as an inhibitory ligand for PleD, it has yet to be experimentally validated.

TAA has also been reported as an anti-inflammatory treatment for conditions such as arthritis with mucoadhesive microspheres containing TAA ^[52][53][63,64]. For instance, the medicinal plant Echinodorus grandifloras contains high levels of TAA and is used to treat rheumatoid arthritis in Brazil. TAA, along with other fractions extracted from Echinodorus grandiflorus leaves, acts as an anti-inflammatory by inhibiting tumor necrosis factor-alpha (TNF-α) release during in vitro assay of lipopolysaccharide (LPS)-stimulated, THP-1 human monocyte cells ^[54][65]. Furthermore, the lipophilicity of TAA can be improved by Fisher esterification with an alcohol to form mono- di- or tri-esters of TAA ^[53][64]. Improving lipophilicity of TAA by esterification was used as a strategy to improve the pharmacokinetics and transport of TAA across biological membranes ^[55][94]. The TAA esters were administered orally and tested in a mouse model of lipopolysaccharide (LPS)-induced arthritis. TAA diesters were found to be the most biologically active, and the anti-inflammatory activity increased the longer the aliphatic chain of the alcohol was used for esterification ^[53][64].