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.
Laura
L
van der Straat
Marloes
M
Vernooij
Marieke
M
Lammers
Willy
W
van den Berg
Tom
T
Schonewille
Jan
J
Cordewener
Ingrid
I
van der Meer
Andries
A
Koops
Microbial Systems Biology, Laboratory of Systems and Synthetic Biology, Wageningen University, Dreijenplein 10, Wageningen 6703 HB, Netherlands. leo.degraaff@wur.nl.
Leo H
LH
de Graaff
D001230:Aspergillus / Q000235:genetics*; D001234:Aspergillus niger / Q000235:genetics* / Q000378:metabolism*; D019149:Bioreactors; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism; D003001:Cloning, Molecular; D056915:DNA Copy Number Variations; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D013386:Succinates / Q000378:metabolism*
D005656:Fungal Proteins; D026901:Membrane Transport Proteins; D013386:Succinates; D019343:Citric Acid; D006867:Hydrolases; C054172:oxaloacetase; D002262:Carboxy-Lyases; C018742:aconitate decarboxylase; C005229:itaconic acid