Production of Itaconic Acid in Microorganisms

By Dr. Maho Yokoyama, Ph.D.

Itaconic acid (2-methylidenebutanedioic acid) can be produced by microorganisms and has commercial value as a precursor of polymers, chemicals and fuels. There is interest in using microorganisms to convert biomass into useful products such as itaconic acid. The main itaconic acid producer is the fungal species Aspergillus terreus, which can produce as much as 86g/L.

Image Credit: Kateryna Kon / Shutterstock.com

What is itaconic acid?

Itaconic acid, also known as 2-methylidenebutanedioic acid, is an unsaturated dicarbonic acid. It is considered to be a value-added product from biomass by the US Department of Energy, as it can be utilized as a precursor of polymers and chemical intermediates.

These include synthetic fibers, resins, paint, acrylic plastics, acrylate latexes, super-absorbent materials, anti-scaling agents, styrene, 2-methyl-1,4-butanediol, and 3-methytetrahydrofuran. Itaconic acid has also shown some antimicrobial capabilities.

How is itaconic acid produced by microorganisms?

Some microorganisms have the capability of producing itaconic acid by fermentation. These are mainly fungal species and include Ustilago zeae, Ustilago maydis, species of Candida and Rhodotorula. The main producer is the fungal species Aspergillus terreus, which can produce as much as 86g/L. While this may seem high, it is not as high as levels of citric acid which can be produced, which is as much as 200g/L.

Biosynthetic pathway

Itaconic acid is produced from a citric acid cycle intermediate, cis-aconitate, which is formed from citric acid. As it is linked to the citric acid cycle, itaconic acid can be produced from sugars such as glucose.

Initially, glucose is broken down into pyruvate by glycolysis, which is further converted into acetyl-CoA with the release of CO₂. Acetyl-CoA then joins the citric acid cycle, where the first steps form cis-aconitate. Alternatively, pyruvate can be converted to oxalate by the addition of CO₂, which is further converted to malate, another molecule involved in the citric acid cycle.

In A. terreus, the gene cadA encodes for the enzyme cis­-aconitate decarboxylase, known as CadA in this species. CadA is a 490 amino acid, 55kDa protein, and is the enzyme that converts cis­­-aconitate to itaconic acid. Studies have shown that CadA catalyzes the allylic rearrangement and decarboxylation of cis-aconitate.

Interestingly, studies have shown that while a strain which overproduces itaconic acid had increased transcription of cadA, there were no mutations in the resulting protein. Further experiments using the fungus Aspergillus niger, which does not have CadA naturally, using various constitutively expressing promoters showed that levels of itaconic acid produced correlates with the levels of cadA transcription.

CadA is not the only component required for itaconic acid production in A. terreus. Two other genes mttA, which encodes for a putative mitochondrial tricarboxylic transporter (MTTA), and mfsA, which encodes for a putative major facilitator superfamily transporter (MFSA), is also involved. MTTA is thought to transport cis-aconitate from the mitochondria to the cytosol, while MFSA is thought to transport itaconic acid into the extracellular environment.  

Another microorganism that produces itaconic acid, U. maydis, utilizes trans-aconitate, the isomer of cis-aconitate, to produce itaconic acid. Here, cis-aconitate is isomerized to trans-aconitate by aconitate-Δ-isomerase first, which is subsequently converted to itaconic acid by trans-aconitate decarboxylase.

Producing itaconic acid on an industrial scale

Once itaconic acid is produced, it needs to be separated from the fermentation culture, typically by filtration and crystallization, before it is decolorized and dried. Crystallization is where the bulk of itaconic acid is recovered, and the yield is typically around 80%. Other processes can be used in place of crystallization, including precipitation, liquid-liquid extraction, membrane separation and adsorption.

Can itaconic acid production by microorganisms be improved?

A. terreus is the main microbial species when it comes to itaconic acid production. However, the growth requirements of A. terreus mean that it is not the most efficient. For example, it requires a continuous source of O₂, which in turn leads to high NADH levels. High NADH levels are inhibitory to key enzymes involved in itaconic acid synthesis. A. terreus also grows mycelia, which can easily become damaged by intensively stirring during the fermentation process.

Other microorganisms have been genetically engineered to produce itaconic acid, including Saccharomyces cerevisiae and E. coli. It was observed that due to low-level activity of endogenous cis-aconitate decarboxylase in these engineered strains, the CadA from A. terreus needed to be over-expressed. Even so, the yield from these engineered strains were still low.

In a study by van der Straat et al., the authors attempted to produce itaconic acid in A. niger; this was chosen as it is capable of producing high levels of citric acid (200g/L), and the pathways for citric acid and itaconic acid production are linked. A. niger does not naturally produce itaconic acid due to not having cadA, therefore the authors took cadA from A. terreus and put it into A. niger.

Initially, the yield of itaconic acid from the engineered A. niger strain was low, so the authors looked to see if this could be improved. They codon-optimized the cadA gene for A. niger, as well as adding the mttA gene and the mfsA gene, which lead to a 20 fold increase in yield. This suggests that MTTA and MFSA are key components in itaconic acid production.

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