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

Abstract

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.

Figure 1

Metabolic pathway for itaconic acid production via the different compartments in the specific A. niger strain.

Figure 2

Itaconic acid production in A. niger strains expressing cis-aconitate decarboxylase. Production of itaconic acid (mM) in strains expressing cadA cDNA (cCAD) (A) and production of itaconic acid (mM) in strains expressing codon-optimized cadA synthetic DNA (sCAD) (B).

Figure 3

Itaconic acid production in fermenter cultures. The itaconic acid production is given in gram per gram dry weight at T = 78 hours after induction of the transformants carrying the complete itaconic acid biosynthesis cluster from A. terreus compared to the best performing CadA, CadA + MfsA and CadA + MttA transformants.

Figure 4

Itaconic acid and citric acid production during fermentation of the different strains. A) sCAD4 strain, B) cadA + mfsA 2.5, C) cadA + mttA 1.4 and D) cadA + mttA + mfsA 4. The black lines represent citric acid concentration and the blue line represents the itaconic acid concentration. The time is shown in hours (x-axis) and the concentrations are shown in mM (y-axis).