What is Aconitic Acid?
Aconitic acid, or 1,2,3-tricarboxylic acid, is listed by the U.S. Department of Energy as one of the 30 most valuable chemicals due to its use in industry as a precursor to chemical raw materials and other important products. Chemicals and Polymers. Aconitic acid also plays a role in biological systems and has many applications.
Microbial conversion of aconitic acid to itaconic acid
The cost of producing itaconic acid remains high. However, economic feasibility can be improved by optimizing fermentation or chemical synthesis methods. Some options may include chemical or enzymatic decarboxylation or improved fermentation conditions using cane molasses (a feedstock rich in sugar and aconitic acid). Optimized fermentation conditions may include the use of inexpensive feedstocks, such as agricultural waste, or metabolic engineering of microbial strains to eliminate undesired pathways and directly increase carbon flux for itaconic acid production.
Some fungi naturally convert aconitic acid into itaconic acid through decarboxylation. For example, Aspergillus terreus is used in the fermentative production of itaconic acid. In Aspergillus terreus, aconitic acid (CAA) is first transported from the tricarboxylic acid (TCA) cycle in mitochondria to the cytosol by the mitochondrial transporter At_MttA, where CAA is transported by aconitic acid decarboxylase Cad- A is decarboxylated to itaconic acid. Subsequently, itaconic acid is transported out of the mycelium via a specific transporter belonging to the major facilitator superfamily (MFS) type transporter (MfsA). The genes involved in itaconic acid production in Kojima also include transcription factors. These four genes are called the "itaconic acid gene cluster." However, in Ustilago maydis basidiomycete, Ustilago maydis mitochondrial transporter Um_Mtt1 also transports CAA from mitochondria to the cytoplasm, but CAA is first isomerized to TAA by aconitic acid-Δ isomerase Adi1 , and then decarboxylated by trans-aconitate decarboxylase. , Tad1, itaconic acid. The "itaconate gene cluster" in maize is induced under nitrogen-limiting conditions. Overall, the metabolic differences in itaconic acid production between these two fungi are noteworthy as it demonstrates the importance of producing itaconic acid from the trans or cis isomers of aconitic acid in different organisms. possibility. Differential microbial conversion of CAA and TAA to itaconate may also provide insight into ex vivo enzymatic conversion options for recombinantly expressed decarboxylase enzymes.
The use of microorganisms as carbon sources
Microorganisms such as the soil bacterium Pseudomonas. WU-0701 encodes an aconitate isomerase that catalyzes the reversible isomerization between TAA and CAA, an intermediate in the conversion of citric acid to isocitrate in the TCA cycle . This allows organisms to grow on TAA as the sole carbon source, isomerize TAA to CAA, and then feed back into the TCA cycle. Interestingly, others have reported the presence of various Pseudomonas species in the sugarcane rhizosphere, while others have reported that some rhizosphere-associated bacteria may improve plant growth and photosynthesis under certain conditions . The utilization of TAA as a carbon source by some of these bacteria may indicate a possible symbiotic relationship between sugarcane and rhizosphere microorganisms involving TAA.
Aconitic acid as a fermentation inhibitor
There are some reports that aconitic acid may act as an inhibitor in the fermentation of sugarcane juice, molasses, molasses, and sweet sorghum syrup. In a study of fermentation of sweet sorghum juice as a function of harvest time, ethanol production from sake and wine yeasts decreased later in the harvest season, despite an increase in sugar content. Aconitic acid was hypothesized to be responsible for the reduced ethanol yield. When fermentable sugars are increased by concentrating sweet sorghum juice. reported that fermentation efficiency decreased with increasing water removal, and it was speculated that an increase in aconitic acid levels due to water removal may be the cause. In the fermentation of sweet sorghum juice, the ethanol yield of Saccharomyces cerevisiae is low, depending on the sweet sorghum variety. Aconitic acid is hypothesized to be partially responsible for this suppression, as aconitic acid varies between species. This inhibitory effect was still evident in the fermentation of a mixture of sweet sorghum juice and hydrolyzed grain mash. The inhibitory effect of corn mash is greater than that of wheat mash. Another report showed a 29% decrease in fermentation rate compared to ethanol production from sweet sorghum juice containing 0.114% and 0.312% aconitic acid. These authors attributed the reduced rate to aconitic acid and showed that the intracellular acid concentration of yeast increased 2- to 4-fold when the pH was changed from pH 5.0 to pH 3.5 to pH 2.0. In a detailed study, it was shown that it is the undissociated form of aconitic acid that inhibits ethanol production by S. cerevisiae during the fermentation of sweet sorghum sugar. By controlling the pH during the fermentation process, the inhibitory effect can be overcome. When the pH is controlled above 4.5, the presence of aconitic acid (5 g/L) becomes slightly more favorable for ethanol titer (+4%) and yield. (+3%), confirming the results of previous synthetic media studies. Fermentation of dilute sweet sorghum syrup to butanol by C. beijerinckii does not appear to be inhibited by aconitic acid at fermentation pH > 4.5.
Nematicidal activity of trans-aconitic acid
Pathogenic nematodes can be a serious problem for certain crops such as sugar beet and cotton. For example, the sugar beet cyst nematode Heterodera schachtii is particularly problematic for sugar beets, which supply about one-third of the world's sugar. There is considerable interest in finding a sustainable, plant-based source of nematicides that is non-toxic to humans. Interestingly, the soil bacterium Bacillus thuringiensis produces TAA as a virulence factor against soil nematodes. Studies of thuringin-producing strains, specifically Bacillus thuringiensis CT-43, revealed a chemical product called CT-A with nematicidal activity against the major pest Meloidogyne incognita, Meloidogyne incognita. Further studies showed that CT-A contains TAA and that TAA exhibited significantly higher nematicidal activity than the cis-isomer CAA in a survival bioassay of Meloidogyne incognita J2s after 72 hours. A plasmid-encoded operon for TAA biosynthesis has been described in Bacillus thuringiensis CT-43, which encodes an aconitate isomerase, termed TAA biosynthesis-related gene A (tbrA), and a membrane-bound TAA transporter out of the cell. Transporter tbrB.
Anti-Leishmania activity of trans-aconitic acid
Anti-Leishmania activity has also been attributed to TAA against the protozoan pathogen Leishmania donovani, the causative agent of visceral leishmaniasis (also known as kala-azar) three decades ago, which can be fatal and difficult to treat. During the life cycle of this protozoa, the promastigote form is present in the vector, while the amastigote form is present intracellularly in infected macrophages of the host. Anti-leishmaniasis drugs can be problematic due to toxicity. Since TAA is an inhibitor of aconitase in the TCA cycle, TAA has been studied as an alternative and in combination with conventional chemotherapy. Lactobacillus donovani amastigotes rely on mitochondrial β-oxidation of fatty acids as an important energy source. During the β-oxidation process, fatty acids are converted into acetyl-CoA, which enters the TCA cycle to generate ATP to provide energy, so TAA is particularly interesting as an inhibitor of aconitase in the TCA cycle. Interestingly, 20 mM TAA significantly attenuated promastigote replication, which could be reversed by the addition of 20 mM CAA at 72 h, indicating that the two aconitic acid isomers have different biological activities. Furthermore, 2 mM TAA reduced parasitic liver burden in infected hamsters in a dose-dependent manner. A dose of 2 mM TAA reduced the number of amastigotes by 60% in a macrophage model. Five (5) mM TAA, together with anti-leishmanial drugs, sodium gluconate, pentamidine, or allopurinol, completely inhibits the conversion of amastigotes to promastigotes. These reports from L. donovani may provide insights into the mechanisms of action in other organisms such as nematodes.
Aconitic acid production confers survival advantage
TAAs produced by sugarcane, sweet sorghum, and other plants may confer a survival advantage against pests and help regulate metabolic processes during rapid plant growth. Stout et al. Ninety-four species of grassy and non-graminic plants in the pasture were tested and found that 47% of the grassy species and 17% of the non-graminicaceous species accumulated high levels of TAA. In addition, aconitic acid has been detected in grasses such as oats, rye, wheat, barley and corn.
In higher plants, trans-aconitic acid is produced and stored as a "tricarboxylic acid pool". TAA is produced through two mechanisms related to the TCA cycle. The first process is the formation of TAA through the citric acid valve and citrate hydratase. The second mechanism is that aconitase converts citric acid into isocitrate through the cis-aconitic acid intermediate, and then isomerizes it into TAA through aconitate isomerase. The accumulation of TAA may play a role in regulating the TCA cycle by inhibiting aconitase. Furthermore, this inhibition can be alleviated by monomethylesterification by the anti-aconitate methyltransferase TMT1, which has been described in E. coli, Saccharomyces cerevisiae, and Ashbya gossypii.
Aconitic acid may also play a role in some plants' antifungal defenses. For example, TAA may accumulate in wheat as part of a protective mechanism against the powdery mildew Blumeria graminis f. sp. wheat. In particular, potassium sulfate can induce significant accumulation of TAA and to a lesser extent CAA in wheat leaves. Furthermore, later studies showed that in wheat plants experimentally infected and fed silica, TAA was methylated to form methylTAA, which acted as a phytoalexin to limit disease.
Organic acid root secretions such as malic acid and tartaric acid also appear to inhibit blight caused by Fusarium oxysporum. sp. Broad bean (FOF), broad bean (Vicia faba). Under nitrogen-limiting conditions, TAA was only detected in root exudates, while tartaric acid and malic acid were detected after nitrogen application. So far, it is unclear what role TAA plays in antifungal defense of faba bean under nitrogen-limited conditions.
TAA also appears to have an antifeedant effect on certain plants, such as barnyard grass against the brown planthopper (Nilaparvata lugens). Other studies have further demonstrated that barnyardgrass and a resistant rice variety, Babawee, are resistant to brown planthopper feeding due to the presence of TAA but not the absence of CAA. In addition, TAA was not detected in the susceptible rice variety "Koyonishiki". High levels of aconitic acid production may also play a role in aphid resistance in some cereal crops such as corn, sorghum, and barnyardgrass]. For example, higher levels of TAA in sorghum leaves corresponded to reduced aphid burden and leaf damage, further suggesting that TAA functions as a defensive phytochemical.
Protect against aluminum poisoning
Aconitic acid and oxalic acid are the main organic acids produced in corn. Aconitic acid appears to protect corn from aluminum toxicity. Organic acid content in corn is higher during early harvests and decreases with each successive harvest. About 60% of aconitic acid is the trans isomer. TAA is found in the shoots and roots of corn and may help protect plants from aluminum toxicity by chelating organic acids. It was found that TAA accumulated at higher levels in roots due to Al3+ activity than in shoots.
Interestingly, because TAA-producing grasses such as sugarcane are often part of soybean rotation strategies, and because sugarcane distillers grains are sometimes applied in fields, the effects of TAA on soybean growth were studied. Studies have found that TAA inhibits soybean growth by inhibiting photosynthesis and increasing H2O2 in the roots, resulting in reduced water absorption. It is unclear whether residual TAA remains in the soil after sugarcane is harvested, or whether TAA quickly dissipates to negligible levels, but it is worth considering the impact TAA may have on soybean rotations.
Aconitic acid may be an inhibitor of biofilm formation. Pestana-Nobles et al. reported a computational study based on molecular docking and molecular dynamics simulations to screen 224,205 molecules from the natural product ZINC15 database. The results predict that TAA may be a ligand and inhibitor of PleD protein involved in bacterial biofilm formation. PleD and its homologs are diguanylate cyclases that contain a GGDEF domain involved in the formation of cyclic di-GMP second messenger, a key signaling molecule involved in quorum sensing required for biofilm formation. Therefore, PleD homologs are often targets for high-throughput screening of biofilm inhibitors. Although TAA was computationally identified as an inhibitory ligand for PleD, it has not been experimentally verified.
It has been reported that TAA can also treat diseases such as arthritis through mucoadhesive microspheres containing TAA. For example, the medicinal plant Echinodorus grandifloras contains high levels of TAA and is used in Brazil to treat rheumatoid arthritis. TAA, together with other components extracted from Echinodorus grandiflorus leaves, inhibits the release of tumor necrosis factor-alpha (TNF-alpha) during an in vitro assay of lipopolysaccharide (LPS)-stimulated THP-1 human monocytes. Acts as an anti-inflammatory. In addition, the lipophilicity of TAA can be improved by Fisher esterification with alcohols to form TAA mono-, di- or triesters. Increasing the lipophilicity of TAA through esterification has been used as a strategy to improve the pharmacokinetics and transport across biological membranes of TAA. TAA esters were administered orally and tested in a mouse model of lipopolysaccharide (LPS)-induced arthritis. TAA diesters were found to have the strongest biological activity, and the longer the fatty chain of the alcohol used for esterification, the greater its anti-inflammatory activity.