Itaconic acid production in Aspergillus niger

Abstract

Itaconic acid (2-methylidenebutanedioic acid) is an important building block chemical that can be produced from sugars via chemical or biological conversions. Itaconic acid can be processed into a polymer, which can subsequently be used to replace the petroleum-based polyacrylic acids. Itaconic acid is naturally produced by Aspergillus terreus, certain Ustilago and Candida species and Pseudozyma antarctica. Also in mammalian cells itaconic acid is found during macrophage activation. Aspergillus niger is a filamentous fungus that is the current host of choice for the production of citric acid on an industrial scale, whereas A. terreus is the main natural producer of itaconic acid. The biosynthesis of itaconic acid is very similar to the biosynthesis of citric acid in A. niger. Citric acid concentrations of 200 g/L are nowadays obtainable with A. niger. Theoretically, this allows itaconic acid concentrations of over 135 g/L to be obtained with A. niger, which is higher that the concentrations currently obtained with A. terreus (80 g/L). However, in A. niger, cis-aconitate decarboxylase (CadA), the key enzyme for itaconic acid biosynthesis, is missing. Expression of A. terreus CadA in A. niger results in the production of a low itaconic acid concentration (0.05 g/L). The itaconic acid concentration was increased by the expression of a putative mitochondrial transporter (MttA) and a putative plasmamembrane transporter (MfsA). Expression of the MttA transporter in an itaconic acid producing A. niger strain resulted in a twenty-fold increase in itaconic acid secretion. Expression of the A. terreus itaconic acid cluster consisting of the cadA gene, the mttA gene and the mfsA gene resulted in A. niger strains that produce over twenty five-fold higher levels of itaconic acid and show a twenty-fold increase in yield compared to a strain expressing only CadA. To further increase itaconic acid production a modified 6-phosphofructo-1-kinase, pfkA, was expressed in a citrate producing A. niger strain in combination with cis-aconitate decarboxylase, cadA, from A. terreus. The combined expression of pfkA and cadA resulted in increased citrate levels, but did not show increased itaconic acid levels. The combined expression of pfkA with the itaconic acid biosynthetic cluster resulted in significantly increased itaconic acid production at earlier time points. Also the itaconic acid productivity increased significantly. The maximum itaconic acid productivity that was reached under these conditions equaled 0.15 g/L/h, which is only a factor 17 lower than the 2.5 g/L/h that, according to the US Department of Energy, should be achieved to have an economically feasible production process. With the requirement of an increasing number of different genes that needed to be integrated in the genome of A. niger, there appeared a need for an efficient flexible pathway transfer system. The developed Funbrick system enables easy and quick construction of expression vectors for integration of pathways and the construction of localization vectors. A major advantage of this system is the ability to freely alter the so- called Funbrick after its construction. The Funbrick system was successfully applied by integration of the itaconic acid biosynthesis gene cluster of A. terreus in the genome of A. niger. The highest itaconic acid concentration obtained equaled 169 mg/L. In addition, the Funbrick system was used to localize the itaconic acid biosynthesis proteins. In order to further increase the efficiency of pathway transfer in A. niger and other filamentous fungi, we developed a non-laborious way for the rapid selection of transformants resulting from integration at a defined locus in the genome. In A. niger, homologous or targeted integration is relatively rare. The A. niger genome contains a spore color formation gene fwnA, which was investigated as a selective marker for homologous integration. Transformants resulting from homologous recombination at the fwnA locus can be rapidly distinguished on the basis of their spore color, which is fawn. Overall, itaconic acid production has been successfully established in A. niger, albeit in low concentrations, by expressing the gene encoding the key enzyme CadA. Expression of the transporters MttA and MfsA from A. terreus in A. niger showed that especially the mitochondrial MttA transporter has a major effect on the production of itaconic acid in the host strain A. niger NW186. Overall, transport processes play a major role in itaconic acid production and the focus thereon has led to large improvements in itaconic acid production by A. niger.