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

Abstract

BackgroundAspergillus 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.ResultsExpression 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.ConclusionsItaconic 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.

Itaconic acid production from glycerol using Escherichia coli harboring a random synonymous codon‐substituted 5′‐coding region variant of the cadA gene

“…Previous reports have shown that the expression level of CadA is closely related to the increase in IA production (Klement et al, 2012;van et al, 2014). However, the cadA gene isolated from A. terreus is mainly expressed as insoluble aggregates in surrogate hosts.…”

Itaconic Acid: The Surprising Role of an Industrial Compound as a Mammalian Antimicrobial Metabolite

“…However, compared to the citric acid synthesis capacity, very low itaconic acid concentrations (0.05 g · l −1 ) were achieved. Only concomitant overexpression of a mitochondrial transporter (mttA) in combination with a plasma membrane transporter (mfsA) led to a 20-fold increased yield compared to the overexpression of cadA alone (101). The authors of this article suggest that MfsA is a membrane transporter required for itaconic acid secretion (for more details on subcellular compartmentalization of itaconic acid biosynthesis, see Section 2).…”

P gas , a Low-pH-Induced Promoter, as a Tool for Dynamic Control of Gene Expression for Metabolic Engineering of Aspergillus niger

“…Because A. niger produced large amounts of citrate (180 g/liter), the precursor of itaconate, there was great interest in transforming the natural citrate producer into an itaconate producer; however, constitutive expression of the CAD gene alone led to only small amounts of itaconate production. Four strategies have been successfully used to improve itaconic acid production: (i) enhancement of transporter systems (37,38), including the mitochondrial aconitate transporter MttA (14) and a putative membrane major facilitator superfamily protein, MfsA; (ii) mitochondrial targeting of key enzymes (23); (iii) overexpression of aconitase (aco) (14, 23), which not only catalyzes citrate to form aconitic acid but also further transforms aconitic acid to isocitrate, necessitating improvement of the enzyme-substrate affinity of the three acids; (iv) increasing cytosol citrate formation through expression of the cytosolic citrate synthase gene citB (39). In this study, we reported a useful strategy for improving itaconate production through dynamic expression of the CAD gene during fermentation.…”