Pathway transfer in fungi.

Itaconic acid is an important building block for the chemical industry. Currently, Aspergillus terreus is the main organism used for itaconic acid production. Due to the enormous citric acid production capacity of Aspergillus niger, this host is investigated as a potential itaconic acid production host. Several strategies have been tried so far: fermentation optimization, expression of cis-aconitate decarboxylase (cadA) alone and in combination with aconitase targeted to the same compartment, chassis optimization, and the heterologous expression of two transporters flanking the cadA gene. We showed that the heterologous expression of these two transporters were key to improving itaconic acid production in an A. niger strain that was unable to produce oxalic acid and gluconic acid. The expression of transporters has increased the production levels of other industrially relevant processes as well, such as ß-lactam antibiotics and bioethanol. Thus far, the role of transporters in production process optimization is a bit overlooked.

Expression of the Aspergillus terreus itaconic acid biosynthesis cluster in Aspergillus niger.

BACKGROUND: Aspergillus terreus is a natural producer of itaconic acid and is currently used to produce itaconic acid on an industrial scale. The metabolic process for itaconic acid biosynthesis is very similar to the production of citric acid in Aspergillus niger. However, a key enzyme in A. niger, cis-aconitate decarboxylase, is missing. The introduction of the A. terreus cadA gene in A. niger exploits the high level of citric acid production (over 200 g per liter) and theoretically can lead to production levels of over 135 g per liter of itaconic acid in A. niger. Given the potential for higher production levels in A. niger, production of itaconic acid in this host was investigated. RESULTS: Expression of Aspergillus terreus cis-aconitate decarboxylase in Aspergillus niger resulted in the production of a low concentration (0.05 g/L) of itaconic acid. Overexpression of codon-optimized genes for cis-aconitate decarboxylase, a mitochondrial transporter and a plasma membrane transporter in an oxaloacetate hydrolase and glucose oxidase deficient A. niger strain led to highly increased yields and itaconic acid production titers. At these higher production titers, the effect of the mitochondrial and plasma membrane transporters was much more pronounced, with levels being 5-8 times higher than previously described. CONCLUSIONS: Itaconic acid can be produced in A. niger by the introduction of the A. terreus cis-aconitate decarboxylase encoding cadA gene. This results in a low itaconic acid production level, which can be increased by codon-optimization of the cadA gene for A. niger. A second crucial requirement for efficient production of itaconic acid is the expression of the A. terreus mttA gene, encoding a putative mitochondrial transporter. Expression of this transporter results in a twenty-fold increase in the secretion of itaconic acid. Expression of the A. terreus itaconic acid cluster consisting of the cadA gene, the mttA gene and the mfsA gene results 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.

Overexpression of a modified 6-phosphofructo-1-kinase results in an increased itaconic acid productivity in Aspergillus niger.

A modified 6-phosphofructo-1-kinase was expressed in a citrate producing Aspergillus niger strain in combination with cis-aconitate decarboxylase from Aspergillus terreus to study the effect on the production of itaconic acid. The modified pfkA gene was also expressed in combination with the itaconic acid biosynthetic cluster from A. terreus, which consists of cis-aconitate decarboxylase cadA, a putative mitochondrial transporter mttA and a putative plasmamembrane transporter mfsA. 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 was 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.